key: cord- -rb xdzro authors: zheng, xuexing; wong, gary; zhao, yongkun; wang, hualei; he, shihua; bi, yuhai; chen, weijin; jin, hongli; gai, weiwei; chu, di; cao, zengguo; wang, chong; fan, quanshui; chi, hang; gao, yuwei; wang, tiecheng; feng, na; yan, feihu; huang, geng; zheng, ying; li, nan; li, yuetao; qian, jun; zou, yong; kobinger, gary; gao, george fu; qiu, xiangguo; yang, songtao; xia, xianzhu title: treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: rb xdzro recent successes with monoclonal antibody cocktails zmapp(tm) and mil against ebola virus (ebov) infections have reignited interest in antibody-based therapeutics. since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. we produced purified equine antisera from horses hyperimmunized with ebov virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. balb/c mice were given up to mg of purified equine antisera per animal, at minutes, or days post-infection (dpi), in which all animals survived. to decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate f(ab′)( ) fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. full protection was achieved with when treatment was initiated at dpi, but the suboptimal protection observed with the minute and dpi groups demonstrate that in addition to virus neutralization, other fc-dependent antibody mechanisms may also contribute to survival. guinea pigs given mg of antisera or f(ab′)( ) at or starting at or dpi were also fully protected from ebov infection. these results justify future efficacy studies for purified equine products in nhps. of these reasons means that there are high personal risks involved with working on ebov in a laboratory setting, and as such it is classified as a biosafety level (bsl- ) agent. in the spring of , a new ebov variant emerged in the west african nation of guinea , an area in which the virus had not been previously reported. the outbreak soon spread to neighbouring sierra leone and liberia. occasionally, cases have been exported into other countries through travel; with countries located in africa, europe and north america all having recorded ebov cases imported by travel, or repatriation of infected citizens. as of mid-december , there are over , fatalities and , infections , the largest evd outbreak in history. although the outbreak is now largely under control with no reported cases since the week of november th , , the virus had shown itself at the peak of the outbreak to be extremely resistant to traditional containment methods designed to curb ebov transmission. passive immunotherapy with sera of animal origin has been used for over years to treat bacterial and viral infections, envenomations and drug intoxications. in , a report demonstrated that the passive transfer of igg from nonhuman primate (nhp) survivors of ebov disease to naive nhps was sufficient to confer post-exposure protection against ebov challenge in all animals . building on these findings, cocktails of monoclonal antibodies (mabs) raised against the ebov glycoprotein (gp) were soon shown afterwards to be effective in the treatment of ebov disease , . this culminated in the development of zmapp tm , a combination of three mabs produced in genetically modified tobacco plants, which were shown to reverse advanced ebov disease in experimentally-infected nhps , and may have provided a survival benefit when administered to ebov-infected patients . a second antibody cocktail (mil- ), which was based on zmapp tm and produced in modified cho cells, were later shown to have similar efficacy to zmapp tm in nhps . therefore, passive immunotherapy is an extremely promising approach to control ebov disease. due to the ease of management, high antibody yield and low risk of human contamination by virus or adventitious agents, horses are the most commonly used animal species in the production of hyperimmune sera. immunization itself is standardized and performed under optimal conditions for both personnel and animals. passive immunotherapy is still commonly used in countries that are still resource-poor medically, and treatment with immune globulin against rabies virus and clostridium tetani infections remains frequent in africa, asia and latin america. the lower manufacturing costs of hyperimmune equine antisera therefore represents an attractive alternate avenue of treatment, especially to developing and third-world countries, compared to the more costly production process of ebov gp-specific mabs. to investigate further, we first prepared purified anti-ebov antisera via the immunization of horses with ebov enveloped virus-like particles (evlp), which consists of ebov viral protein (vp ) and gp. the equine antisera was then tested in vitro using a neutralization assay with recombinant ebov expressing egfp (ebov-egfp), as well as in vivo against a lethal challenge with mouse-adapted ebov (ma-ebov) in immunocompetent balb/c mice. to investigate whether ebov infections can be controlled by virus neutralization alone, and to prevent the possible induction of serum sickness in humans that would be administered antisera, the post-exposure efficacy of f(ab′ ) (immunoglobulin treated with pepsin to remove the fc regions of the antibody) were also investigated side-by-side with equine antisera in all experiments as a potential alternate treatment. guinea pigs then were used to confirm the efficacy results from mouse studies, due to their status as a higher phylogenic species which more closely models hallmarks of evd in humans. additionally, prior to efficacy studies both the equine antisera and f(ab′ ) had been evaluated for safety in the peking union medical college center for new drug safety evaluation, chinese academy of medical sciences, which is certified by the food and drug agency of the people's republic of china. both equine-derived products were found to meet safety standards for clinical use in china. immunization of horses and production of equine antibody products. the horses were immunized with evlp produced from the infection of sf cells with rbv-vp -gp. the filamentous evlp were observed under an electron microscope (supplementary figure ) and confirmed to be morphologically similar to ebov. immunoblotting of rbv-vp -gp infected sf cell lysates demonstrated that the evlp contained ebov gp and vp (supplementary figure ) . three horses were immunized intramuscularly (im) with injections of evlp over weeks and the hyperimmune sera were collected from each animal at specified timepoints ( fig. a) to determine the serum titers by neutralization assay against a recombinant hiv- virus pseudotyped with ebov gp. a pseudotyped-virus was used for these studies to confirm the in vitro efficacy of the antisera preparations under biosafety level (bsl- ) conditions, before subsequent studies in the bsl- laboratory. neutralizing serum titers were detectable after the th immunization at week , and increased until the th immunization at week (fig. b) . the serum neutralizing antibody titers of two horses were : , after seven immunizations, and was : , for the third animal. the equine antisera (purified by ammonium sulphate-based precipitation) was then digested with pepsin at a concentration of - u/ml for to min and purified to generate f(ab′ ) fragments. the purity of the antisera and f(ab′ ) preparations, as determined by sds-page followed by thin layer chromatography, was determined to be % and %, respectively (fig. ) . approximately ml of purified antisera could be obtained from each horse during each collection, with the stock concentration between - mg/ml. up to - collections can be performed on each horse, therefore each hyperimmunized horse yields between to g of purified antisera. in vitro characterization of equine antisera and f(ab') . the equine antisera and f(ab′ ) products were first characterized against a laboratory generated ebov-egfp for their potential to neutralize ebov. equine antisera and f(ab′ ) were found to possess levels of similar neutralizing activity, with the half effective maximal concentration (ec ) of the products to be . μg/ml and . μg/ml, respectively (fig. ) . moreover, the neutralization activities of both the equine antisera and f(ab′ ) were complete at a concentration of . μg/ml, scientific reports | : | doi: . /srep indicating that the two products were indistinguishable from each other in terms of neutralization at high concentrations. ebov-specific igg in the equine antisera preparations were also determined by elisa. serial -fold the neutralizing activities of equine antisera and f(ab′ ) against ebov-egfp were compared over different concentrations (x-axis). total fluorescence from infected veroe cells at dpi were shown as a percentage of the fluorescence observed with the pbs control, which is set at % (y-axis). samples were processed in triplicate, and error bars indicate standard error. data shown in this figure are representative of three independent neutralization studies with f(ab′ ) or antisera. dilutions of stock antisera with a concentration of - mg/ml were assayed in triplicate over three independent experiments, and the titer was determined to be between , and , endpoint dilutions. were first assessed in guinea pigs, and found to be - hours and - days, respectively (supplementary table ). the protective efficacy of equine antisera and f(ab′ ) were assessed over three experiments in balb/c mice. the first experiment was to compare the efficacy of antisera and f(ab′ ) treatments side-by-side. groups of mice were given intraperitoneal (ip) injections of f(ab′ ) at μg per dose, twice daily for days (b.i.d. × d), starting at either or days post-infection (dpi) with a uniformly lethal dose of ma-ebov. for comparison, groups of mice were administered an ip injection of equine igg at μg per dose (q.d. × d), at either or dpi. control mice were given an equal volume of pbs in place of the treatment (q.d. × d) at dpi. pbs was used instead of non-specific immunoglobulin or f(ab′ ) , because previous studies involving passive transfer of non-specific antisera in mice and nhps did not result in protection , , and thus survival due to the non-specific effects of serum proteins is considered unlikely. control mice lost approximately % of its body weight over the course of the experiment and none survived, with a mean time to death (mtd) of . ± . dpi. five of eight mice given f(ab′ ) starting at dpi survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi; however, none of the mice given f(ab′ ) starting at dpi survived the challenge (p-value = . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. a,b) . five of eight mice given antisera at dpi survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. c ). three of eight mice given antisera at dpi survived (p-value = . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi (fig. d ). comparing groups with equal treatment times, there was no statistical difference between f(ab′ ) at or dpi (p-value . and . , respectively). however, given that multiple administrations of f(ab′ ) were required to achieve similar protection levels demonstrated by a single injection of antisera, the results suggest that equine antisera is a superior product to f(ab′ ) in terms of efficacy, possibly due to a longer in vivo half-life of equine antiseras. since f(ab′ ) appear to be promising early in ma-ebov infection, the dosage of this treatment was increased for a second experiment. groups of - mice were given ip injections of f(ab′ ) at or mg per dose, twice daily for days (b.i.d. × d), starting at either minutes or dpi with ma-ebov. control mice were treated with pbs (q.d. × d) at dpi. control mice lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. partial survival was observed when treatment began minutes after challenge. four of nine mice in the mg group survived (p-value < . , compared with pbs group), with an average weight loss of . % and a mtd of . ± . dpi, whereas four of ten mice in the mg group survived (p-value < . , compared with pbs group), with an average weight loss of . % and an mtd of . ± . dpi (fig. a,b) . in contrast, all mice survived if treatment was initiated at dpi, with negligible weight loss (less than %) observed in both the and mg groups (p-value < . , compared with pbs group), indicating that protection was complete. these results indicate that f(ab′ ) can contribute to protection from ma-ebov, but only within a certain timeframe after challenge. the efficacy of equine antisera at higher doses was then investigated in a third experiment. groups of mice were given ip injections of antisera at mg per dose (q.d. × d), at minutes, or dpi with ma-ebov. control mice were treated with pbs (q.d. × d) at dpi. control mice lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. all mice treated with antisera at minutes and at dpi survived with negligible weight loss (p-value < . , compared with pbs group). nine of ten mice treated with antisera at dpi also survived the challenge (p-value < . , compared with pbs group), with the lone non-survivor dying at dpi and the average peak weight loss determined to be . % within this treatment group (fig. a,b) . these results again indicate that equine antisera, compared to f(ab′ ) is able to offer a greater contribution to protection from ma-ebov in the mouse model due to the need for fewer administrations to achieve a similar level of protection. guinea pigs were then used to confirm the protective effects from antisera and f(ab′ ) in a higher phylogenic species for ebov challenge, since these animals better mimic hallmarks of human ebov infections, such as coagulation abnormalities . groups of animals were given a single ip injection of antisera (q.d. × d) starting at or dpi with ga-ebov, or twice-daily ip injections of f(ab′ ) for three days (b.i.d. × d), starting at or dpi. each injection dose contained mg antisera or f(ab′ ) . a group of five control animals were given a single injection of pbs at dpi. control guinea pigs lost approximately . % of its body weight over the course of the experiment and none survived, with a mtd of . ± . dpi. all guinea pigs that were given antisera at or dpi survived (p-values = . for both groups, compared with pbs group), with no clinical signs of disease or significant weight loss (fig. a,b) . in addition, animals that were given f(ab′ ) starting at or dpi survived ga-ebov challenge (p-values = . for both groups, compared with pbs group) without signs of disease or significant weight loss (fig. a,b) . the disparity between the efficacy of f(ab′ ) between mice and guinea pigs may be attributed to a much higher dosage of f(ab′ ) administered per weight: each f(ab′ ) dose for guinea pigs was × higher than those given to mice, but the guinea pigs used in this experiment were only - times bigger than the mice by weight. the lack of bsl- laboratories globally, trained personnel as well as the rigors of working under high biocontainment conditions have severely hampered basic research with ebov, leading to a dearth of vaccines and therapeutics for use in humans. these weaknesses were exposed with the - ebov crises, and highlight the value and need for basic and translational science that occurs prior to an impending threat. in an effort to save lives, several experimental candidate treatments that had been tested for efficacy in mice or nhps, in addition to approved or nearly-approved drugs developed against unrelated pathogens, were expedited for use in humans under compassionate circumstances, with varying degrees of success . of these, antibody-based treatments appear to hold the most promise in the clinic and thus should be further investigated for use in humans. passive immunotherapy against ebov had been tried in the past. towards the end of the ebov outbreak in kikwit, zaïre (presently democratic republic of the congo), the passive transfer of whole blood from ebov survivors to patients resulted in the survival of seven out of eight recipients . however, safety concerns with blood transfusions, such as the spreading of blood-borne diseases, allergic reactions and concerns of inefficacy mean that this approach is controversial and will likely not be used unless a better alternative was unavailable. in the s, russian investigators had prepared hyperimmunized antisera from horses vaccinated with inactivated ebov, which were shown to be effective when administered at a dose of mg/kg to baboons, with % survival ( survivors out of ) when given hours before challenge, and % ( survivors out of ) when given immediately after challenge , although baboons are known to be more resistant to ebov infection compared to other nhp species . the survival benefits of the equine antisera were limited to a delay in the onset of viremia clinical symptoms when tested in cynomolgus macaques immediately after challenge, at a dose of mg for an animal weighing between - kg . comparing to successful passive immunotherapy studies with purified igg from nhps ( mg/kg), as well as zmapp and mil- ( mg/kg), the dosage for the cynomolgus macaque experiment was likely too low at the time. use of equine antisera as an emergency post-exposure treatment against ebov has been approved in russia, and several investigators that had been accidentally exposed to the virus have been administered this treatment, although it is not clear if they had actually been infected . the results of this study show that both equine antisera and f(ab′ ) preparations, which can be rapidly produced in large quantities at a lower cost compared to mabs, are effective in the post-exposure treatment of ma-ebov challenged mice and ga-ebov infected guinea pigs. f(ab′ ) was developed in this study due to past documented issues with antisera administrations, in which equine botulinum toxin and anti-snake venom antisera was shown to produce serum sickness at high doses . however, mice that were given a single dose of antisera demonstrated more complete protection over a greater period of time compared to multiple injections of f(ab′ ) . this suggests that f(ab′ ) has a shorter half-life compared to igg (which has been confirmed in this study), and/or that virus neutralization plays a partial role in survival from ebov. the observation that administering f(ab′ ) at dpi is more efficacious than when the same treatment was given at minutes post-exposure (fig. a ) was also observed in past studies with therapeutic ebov gp-specific monoclonal antibodies , , and suggests that virus neutralization may play a bigger role in protection at a later timepoint after ebov challenge. with regards to neutralization, past reports have shown that while elevated antisera levels in nhps vaccinated against ebov correlated with survival, levels of specific neutralizing antibodies did not . it is a possible explanation as to why mab kz , which was originally selected for its ability to neutralize ebov , failed to protect nhps when given at a dose of mg/kg starting day before challenge , and suggests that in the future, the screening of potentially efficacious antibodies against ebov should not be based solely on the ability of the antibody to neutralize virus. fc-dependent antibody functions, which include antibody dependent cellular cytotoxicity, complement-dependent cytotoxicity, and neonatal fragment crystallizable receptor (fcr)-mediated cross-presentation, likely play a role in protection. for instance, studies in mice against yellow fever virus and west nile virus infections have shown that the protective mechanisms of monoclonal antibodies are dependent on fcr , . furthermore, complement component c q has been previously shown in influenza virus and west nile virus infections to directly enhance the neutralizing activities of antibodies , . these mechanisms are not mutually exclusive, but determining their relative importance of each proposed mechanism to efficacy and survival will yield further insight into the specific mechanisms in which antibodies help protect against ebov disease. furthermore, the findings of this study justify the testing of equine igg in a higher phylogenic species for ebov, such as nhps, and may result in the development of a safe and economical method for the production of an effective ebov therapeutic. ethics statement. mice animals. balb/c mice and hartley guinea pigs were purchased from a commercial supplier (charles river). the animals were kept in sterile, autoclaved cages and provided sterilized food and water ad libitum. animals were also provided red, plastic shelters inside the cages as an added source of stimulation. horses were purchased from a commercial supplier (red hill military horse farm) and provided food and water ad libitum. the sequences of ebov gp and vp , with lengths of , and bp, respectively, were derived from genbank (zaire ebolavirus strain zaire , complete genome; genbank accession no. ay ). the gp and vp genes were cloned into the puc- vector, named puc-gp and puc-vp , and then inserted into pfastbac dual vector (life technologies) under the polyhedrin promoter and p promoter respectively, resulting in plasmid pfastbac dual-vp -gp. the purified plasmid was transformed into dh ™ bac e. coli (life technologies) for transposition into a bacmid. a cellfection ® ii reagent (life technologies) was used according to manufacturer instructions to generate recombinant baculoviruses co-expressing ebola vp and gp (rbv-vp -gp). evlp production and inspection. to produce evlp, sf cells were infected with rbv-vp -gp at a multiplicity of infection (moi) of , and incubated at °c for days. culture supernatants were harvested and centrifuged at × g for min to remove cells, and then pelleted by ultracentrifugation at , × g for min at °c. the pellets were re-suspended in pbs and purified through a - - % discontinuous sucrose gradient at , × g for min at °c. the evlp band obtained between % and % density range was collected, washed, and re-suspended in pbs . for immunoblotting, evlp and control sample (cell culture supernatant) were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, transferred onto a polyvinylidene fluoride (pvdf) membrane (whatman, kent, uk) and then probed with mouse anti-ebov gp polyclonal antibody (prokaryotic expression gp protein immunized mice) or mouse anti-ebov vp monoclonal antibody (ab , abcam) at a dilution of : overnight at °c. the sample was then incubated with horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody at a dilution of : (millipore, boston, ma, usa) for min at °c. the pvdf membrane was colored with a chemiluminescence solution (pierce biotechnology, rockford, il, usa). for electron microscopy, evlp were applied onto a carbon-coated formvar grid, which was immediately stained with % phosphotungstic acid and then observed by a transmission electron microscope. horse immunizations. horses (n = ) were vaccinated intramuscularly (subcutaneous multi-point injection) with either or mg of evlps emulsified in freund's complete adjuvant/freund's incomplete adjuvant at weeks , , , , , and ( times). blood samples were collected from the jugular vein at week prior to the first immunization and week after each immunization, and the sera were stored at − °c until further analysis. fragmentation and purification of equine antibody products. the equine antisera and f(ab′ ) were produced at changchun institute of biological products co., ltd., using the large-scale, current good manufacturing practice compatible equine antiserum manufacturing platform. equine antisera and f(ab′ ) preparations were characterized according to guidelines set forth by chinese pharmacopoeia ( edition), including appearance, color, visible foreign matter, ph value, f(ab′ ) and immunoglobulin content, and sterility. for equine antisera, the blood was taken from the jugular vein of immunized horses, and the sera were separated h later. the horse sera were diluted -fold with pbs, centrifuged at , rpm for min, and supernatants were subjected to filtration through a . μm filter. the antisera were purified by ammonium sulphate precipitation , followed by salt column, and then stored in . % nacl solution. for f(ab′ ) : horse sera were diluted -fold and the ph was adjusted to . using m hcl, and then the antisera were subjected to shock digestion at °c for min with pepsin, and . m naoh was added to terminate digestion. the digestion products were subjected to salt column purification, followed by protein a column. the flow-through fluid was harvested and subjected to ultrafiltration to remove the pepsin and small molecular proteins, and the f(ab′ ) was stored in . % nacl solution. sds-page and thin layer chromatography. the purified equine antisera and f(ab′ ) samples were mixed with non-reducing (without β -mercaptoethanol) protein sample buffer, heated at °c for min, and then subjected to sds-page ( % gel, staining for h and destaining for h); and then the target fractions in the gel were examined by thin layer chromatography scanner (cs- , shimadzu), (transmission, zigzag scan, dual wavelength, swing width: mm, delta y: . mm) to determine the purity of equine antisera and f(ab′ ) . elisa. ebov gp expressed by e. coli bl- was diluted with . m bicarbonate buffer ph . to a final concentration of μg/ml, and coated on -well elisa plates overnight at o c ( μl/well). after blocking with angiotensin converting enzyme (ace) buffer (bio-rad, california, usa) at °c for h, the plates were incubated with μl of serial -fold dilutions of stock equine antisera in triplicate at °c for . h. after washing, μl of hrp-conjugated goat anti-horse igg (comwin biotech co., ltd. beijing, china) diluted : in pbs- . % tween was added to the wells and incubated at °c for min. after washing, μl of substrate ′, , ′ -tetramethylbenzidine (tmb, sigma) was added and incubated at room temperature for min. the reaction was stopped by adding μl of . m h so , and the absorbance was measured at nm. a dilution was considered positive if the absorbance reading was at least twice that of the negative control (pbs) at the same dilution. the igg endpoint titer was calculated as the highest dilution still showing a positive result. the assay was performed independently three times. half-life studies in guinea pigs. group of guinea pigs were administered an ip injection of ml purified equine antisera (neutralization titer : ) or f(ab′ ) (neutralization titer : ). blood samples were collected at , , , , , , , , , , h post-injection, and sera were used for determination of neutralization titer against a recombinant hiv- virus pseudotyped with ebov gp. the time range post-infection in which the titer decreases by % is considered the half-life. each sample was performed in triplicate. determining the protective efficacy of equine antisera. balb/c mice, - week old, female and weighing between - g, were randomly assigned into groups of - mice. all animals were challenged intraperitoneally (ip) with a dose of × ld mouse-adapted ebov (ma-ebov, usamriid/balb/c-lab/ cod/ /mayinga-ma-p ) in μl dmem. the treatment was given ip (q.d. × d) at or days post-infection (dpi) with . mg equine anti-ebov antisera per mouse, or in a subsequent experiment, given ip at minutes, or dpi with mg equine anti-ebov antisera per mouse. the control group was given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. female strain hartley guinea pigs, - week old and weighing between and g, were randomly assigned into groups of animals. all animals were challenged ip with × ld guinea pig-adapted ebov (ga-ebov, vector/c.porcellus-lab/cod/ / mayinga-gpa-p ) in ml dmem. the treatment was given ip (q.d. × d) at or dpi with mg equine anti-ebov igg per animal. a control group of guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. determining the protective efficacy of f(ab′) . balb/c mice, - week old, female and weighing between - g, were randomly assigned into groups of - mice. all mice were challenged ip with a dose of × ld ma-ebov in μl dmem. the treatment was given ip at or dpi with μg f(ab′ ) per mouse, or in a subsequent experiment, given ip at minutes, or dpi with either mg or mg of f(ab′ ) per mouse. the treatment was given via ip twice a day for days (b.i.d. × d). the control group was given the same volume of pbs as a mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. female strain hartley guinea pigs, - week old, were randomly assigned into groups of animals. all animals were challenged ip with × ld guinea pig-adapted ebov in ml dmem. the treatment was given ip at or dpi with mg f(ab′ ) per animal. the treatment was given via ip twice a day for days (b.i.d. × d). a control group of guinea pigs were given the same volume of pbs as mock treatment. all animals were monitored for signs of disease, survival and weight change for days, and survival was monitored for additional days. pseudotyped virus neutralization assay. titers of equine antisera and f(ab′ ) from horses was tested in an neutralization assay in huh- cells against recombinant hiv- virus pseudotyped with ebov gp. the method for generating pseudotyped viruses was described in a previous publication . briefly, pseudotyped virus containing supernatants were incubated either with serially diluted horse sera at °c for h, before addition to pre-plated target cells in -well culture plates (density of cells/well). cells were re-fed fresh medium h after addition, and followed by lysing cells at h using cell lysis buffer (promega) and transferring the lysates into -well luminometer plates. luciferase substrate (promega) was added to the plates, and the relative luciferase activity was determined. the inhibition of pseudotyped was presented as % inhibition. the highest serum dilution giving over % reduction of luciferase activity was regarded as the neutralizing antibody titer. ebov-egfp neutralization assay. serial two-fold dilutions of f(ab′ ) or antisera (between . to . μg/ml) were incubated with plaque forming units (pfu) of ebov expressing egfp (ebov-egfp, nml/h.sapiens-lab/cod/ /mayinga-egfp-p ) at °c for h, transferred to vero e cells and incubated at °c for h, and then replaced with dmem supplemented with % fetal bovine serum. control wells contained pbs instead of f(ab′ ) or antisera, and all samples were repeated in triplicate. the plates were fixed with % phosphate buffered formalin at h after infection, and scored for the intensity of egfp using the biotek synergy ht microplate reader. the results were expressed as a percentage of the fluorescence reading with the control (which is set at %), and fitted to a -parameter logistic curve (graphpad). statistical analysis. the p-values for rodent studies were determined using the log-rank (mantel-cox) test. calculated values of less than . were considered statistically significant. all in vivo studies were 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immunochemical measure of the primary interaction between i bsa and antibody structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus x.z., g.w., y.z., h.w., s.h., y.b., x.q., s.y. and x.x. performed the experiments and analyzed the results. x.z., g.w., g.k., g.f.g., x.q., s.y. and x.x. wrote the manuscript. w.c., h.j., w.g., d.c., z.c., c.w., q.f., h.c., y.g., t.w., n.f., f.y., g.h., y.z., n.l., y.l. and j.q. prepared the horse antisera. g.f.g., x.q., s.y. and x.x. jointly supervised the work. all authors reviewed the manuscript. key: cord- -zwkl ywk authors: yu, dong-shan; weng, tian-hao; wu, xiao-xin; wang, frederick x.c.; lu, xiang-yun; wu, hai-bo; wu, nan-ping; li, lan-juan; yao, hang-ping title: the lifecycle of the ebola virus in host cells date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: zwkl ywk ebola haemorrhagic fever causes deadly disease in humans and non-human primates resulting from infection with the ebola virus (ebov) genus of the family filoviridae. however, the mechanisms of ebov lifecycle in host cells, including viral entry, membrane fusion, rnp formation, gp-tetherin interaction, and vp -inner leaflet association remain poorly understood. this review describes the biological functions of ebov proteins and their roles in the lifecycle, summarizes the factors related to ebov proteins or rna expression throughout the different phases, and reviews advances with regards to the molecular events and mechanisms of the ebov lifecycle. furthermore, the review outlines the aspects remain unclear that urgently need to be solved in future research. filoviridae are from the order mononegavirales, include ebola virus (ebov), marburg virus (marv) and cuevavirus [ , ] , which are single-stranded, negativesense rna viruses that exhibit unique heterogeneous filamentous structure. filovirus was first reported and named marburg virus in during an outbreak of viral haemorrhagic fever (hf) in frankfurt (germany) and belgrade (yugoslavia) [ ] . in , ebov was determined to be the cause of outbreaks of viral hf in the sudan and congo [ ] . five different species of ebov have since been established: ) zaire virus (zaire ebov); ) sudan virus (sudan ebov); ) bundibugyo virus (bundibugyo ebov); ) taï forest virus (taï forest ebov); and ) reston virus (reston ebov) [ ] . filovirus hf is transmitted directly via contact with bodily fluids from infected patients or other species (e.g., gorillas and chimpanzees) [ ] . infection is characterized by high levels of inflammatory cytokines, coagulation disorders, poor immune response and lymphopenia, which results in septic shock andmultiorgan failure finally [ ] [ ] [ ] . ebov is composed of seven genes coding at least ten proteins from the ′ leader to the ′ trailer: ) nucleoprotein (np); ) viral protein (vp ); ) vp ; ) glycoprotein (gp); ) soluble gp (sgp); ) Δ-peptide; ) ssgp; ) vp ; ) vp ; and ) polymerase (l) [ , ] (figure ). until now, biological functions of these proteins and their roles in the ebov lifecycle in host cells have been largely clear. np forms a large complex with vp and vp that encapsulates the viral genome, represents the polymerase cofactor, and involve in synthesizing viral rnas [ , ] . vp is involved in the formation of the viral nucleocapsid and l cofactor, dissociates np-rna oligomers, and releases the genomic rna from np-rna complexes for further replication [ , ] . in addition, it is implicated in regulating the interferon response to ebov and modulating other aspects of the host immune response [ , ] . vp has an important role in the maintenance of viral integrity and aggregation at the cell membrane for virion budding and egress [ , ] . gp and gp are two subunits of the glycoprotein (gp , ) which produced by the cleavage of a precursor (gp ) obtained by the translation of an mrna review www.impactjournals.com/oncotarget derived from an editing process of the primary transcript that codify the gp soluble form [ ] . moreover, there is an alternate transcription editing site in the gp gene, which leads to the expression of additional proteins, including the soluble gp (sgp), the Δ-peptide, and the small soluble gp (ssgp) [ , ] . gp is responsible for interacting with one or more cellular receptors, gp contains a fusion loop that is critical for membrane fusion, while sgp is supposed leads to immune subversionan and acts as a decoy for antibodies directed against gp , [ ] [ ] [ ] . the Δ-peptide is suggested to regulates filovirus entry as its expression limits infection on filovirus-permissive cells [ , ] . yet, the function of ssgp in viral pathogenicity remains unclear. vp is an activation of transcription factor involved in -ssrna packaging and nucleocapsid construction [ ] . vp is suggested to block ifn-α/β/γ signalling, interact with the endosomal traffic protein, and is required for a fully functional nucleocapsid [ , ] . although extensive progress has been made in the knowledge of the mechanisms throughout the ebov lifecycle in host cells, there are still several aspects remain poorly understood. in this review, we discuss the unravelled mechanisms and the outstanding questions regarding the ebov lifecycle in host cells and the advanced strategies for further research. the detailed mechanisms of ebov attachment are currently partially explored. ebov infects a wide variety of mammals, which complicates the identification of cellular proteins required for viral attachment. previous studies have demonstrated that ebov attachment on target cells is mediated by the binding of the transmembrane virus envelope gp to cell surface factor (s) [ ] . gp has three distinct domains: ) the receptor binding domain (rbd); ) the glycan cap; and ) the heavily o-linked glycosylated mucin-like domain (mld) [ ] . rbd is responsible for interacting with one or more cellular receptors. the glycan cap could protect the receptor binding sites from antibodies, and interacts with the internal fusion loop of gp that is critical for gp -mediated membrane fusion for preventing pre-mature fusion events [ ] . glycosylation is extensive in gp mld, it probably shield gp receptor binding sites from immune recognition and contributes to gp maturation and function, although not required for virus entry [ , ] . x-ray crystallography structure showed that the glycosylated glycan cap and mld are surround the rbd, coated a thick layer of oligosaccharides. this conformation probably benefit the need to truncate the transmembrane and certain glycosylation sites in order to achieve crystallization [ ] . to date, there are several factors that have been reported as ebov receptors or co-receptors. the c-type lectin family contains carbohydrate recognition domains (crds) that bind the glycan cap, since gp is highly glycosylated with several types of sugar side chains [ ] . the family, including asialoglycoprotein receptor (asgp-r), dendritic cell-specific icam- -grabbing nonintegrin (dc-sign), human macrophage galactose and acetylgalactosamine-specific c-type lectin (hmgl), and lymph node sinusoidal endothelial cell c-type lectin (lsectin/clec g), have all been shown to interact with ebov gp and facilitate viral attchment. for example, asgp-r is specifically expressed in hepatocytes and is reported to bind and benefit endocytosis of gp containing a terminal galactose, dc-sign and its homolog, l-sign were found to recognize high-mannose carbohydrate moieties and mediate attachment to the cell surface [ , ] . hmgl expression on immature dcs and macrophages was reported to recognize galactosyl residues and act as an attachment factor for ebov and marv [ ] . lsectin/clec g was described to bind n-acetylglucosamine of gp and enhance filovirus infection [ ] . then, the tyro protein kinase (tam) family, including axl, dtk, and mer, which widely expressed in many cell types, span the plasma membrane and contain intracellular tyrosine kinase domains, are facilitate ebov gp-dependent attachment [ , ] . in contrast, shimojima m et al. [ ] reported tyro family-independent entry of gp-pseudotyped murine leukemia virus (mlv) in vero-e cells, which demonstrated the interaction of other unknown factors and the complexity of the filovirus entry mechanism. meanwhile, t-cell immunoglobulin mucin domain (tim), including tim- and tim- are demonstrated to bind the receptor binding domain of the ebov gp [ , ] . tam, tim- and tim- target phosphatidyl-serine (ptdser), which is exposed on the outer leaflet of the filovirus membrane, strengthening an interplay promoting efficient attachment [ , ] . later, β integrins, responsible for extracellular matrix attachment is thought to stimulate endosomal proteases required for ebov transduction and increase ebov gpmediated pseudovirion entry [ , ] . however, no direct interaction has been found between gp and the β integrin family. in addition, folate receptor-α (fr-α) was reported to be a co-receptor in binding cells that expressed marv or ebov gp, mediated syncytia formation triggered by gp, and facilitated cellular entry of the virus [ ] . however, other papers questioned the role of fr-α as an important factor for ebola virus entry, and presumed it was an additional alternative factor, as fr-α highly expressed cells were not conferred susceptibility to ebov gp compared with fr-α insufficient cells [ , ] . filoviruses virions are uptaked into host cells involve different endocytic pathways. first, the internalization mechanisms were controversial over a period of time as clathrin-dependent and caveolindependent uptakes have been shown to occur [ , , ] . however, later data supported that clathrin and caveolinmediated endocytosis was not important for ebov entry, but macropinocytosis and other factors on the host cell and virus particle size were critical factors [ ] [ ] [ ] . other studies using pseudotype viruses packaged with ebov proteins asserted that the endocytic pathway of ebov entry was dependent upon the endocytic enzymes cathepsin b/l and cholesterol, a major component of caveolae and lipid-rafts [ , ] . several gtpases, such as rhob, rac , and cdc , have been implicated in endocytosis and play an important role in ebov gpdependent transduction [ ] . low ph was shown helpful for gp-mediated membrane fusion. acidic conditions have no direct effect but ph-dependent cathepsin activity to affect gp-mediated fusion [ , ] . once ebov gp is cleaved by cathepsin, acidic conditions directly induce conformational changes in cleaved gp that lead to fusion. it's confirmed that cell-cell fusion exhibits a maximum at ph . ; the ph-dependence of fusion in later is eliminated when ebov gp is cleaved, and the extent of fusion was independent of ph [ ] . the studies indicated the ph-dependence of fusion is solely due to the ability of cathepsin to cleave ebov. after the virus has been internalized into the endosome, fusion induced by cleaved gp is fundamentally independent of ph, which indicated an unidentified host cell factor critical for filovirus entry is sensitive to an acidic ph [ ] . taken together, the mechanisms of ebov entry have been partially characterized. there is an ongoing debate as the appropriateness of the background used for pseudotype viruses and that ebov probably infect different cell types via other mechanisms. the details and mutual connections of those identified factors are currently poorly understood as there is a missing link to the mechanisms that have been found to date. therefore, several of these molecules lack effective integration and require further identification. following endocytosis, the next steps consist of the uncoating and fusion of the viral membrane with the endosomal membranes. precursor gp (gp ) is cleaved by the host enzyme, furin in the golgi apparatus, resulting in gp , gp , and additional proteins, including sgp, Δ-peptide, and ssgp [ , ] . gp is critical for membrane fusion, as it's composed of five domains: ) a fusion loop; ) an n-terminal heptad repeat region; ) a c-terminal heptad repeat region; ) a transmembrane region; and ) a short cytoplasmic tail [ ] . the glycan cap of gp can interact with the internal fusion loop of gp to restrict the availability of the fusion peptide and prevent premature fusion events [ ] . the fusion loop, which contains a core hydrophobic sequence of amino acids, is thought to insert into host endosomal membranes and initiate membrane fusion process [ , ] . however, unknown enzymes trigger and accelerate this fusion process. it's considered that an endosomal/ lysosomal factor (e.g., lysosomal thiol reductase) which inhibited by cysteine protease inhibitors and restricted by a low ph, triggers the fusion events [ ] . what's more, enzymes of the arf family of gtpases involved in membrane traffic machinery, especially the small gtpase rab related to the late endosomes was reported to accelerate virion fusion [ , ] . thus, further work is necessary to identify the factors required to trigger filovirus gp-mediated fusion. following the insertion of the gp fusion loop into the host membrane, the gp trimeric heptad repeats (hr) recombine and form a transmembrane six-helix bundle containing three hr and hr domains. this bundle triggers an opening through the membrane, as described elsewhere [ ] . then the viral rna and associated proteins can be released into the host cell cytoplasm for replication. niemann-pick c (npc ) is a ubiquitous protein with transmembrane helices domain, termed as "sterolsensing domain" (ssd), and two luminal domains, resides primarily in the late endosomes and lysosomes. the biological function of npc is mainly as cholesterol transporter and re-distribution to cellular membranes, as the membranes is endosomal-receptor of ebov and crucial for ebov membrane fusion and entry [ ] [ ] [ ] [ ] . the helical structure core of npc contains two extended loops and are surrounded by severalβstrands. the crystal structure revealed that npc domain c utilizes the two loops to engage a hydrophobic cavity at the head of the primed gp (gpcl). after conformational changes, the uplift of the short helix in the loop helps to release the n-terminal portion of the internal fusion loop, then triggering the membrane fusion [ , ] . the ssd domain includes a two-way cavity open to both the endosomal lumen and the luminal leaflet of the lipid bilayer, and is large enough to accommodate one cholesterol molecule, which is important for npc transports cholesterol across the lipid bilayer [ ] . meanwhile, the activity of the transport is regulated by the cholesterol concentration of the endosomal bilayer, cells disrupted by an npc inhibitor or lacking npc exhibited resistance to ebov infection [ , ] . so, npc could be a critical hub between external cholesterol uptake and internal biosynthetic pathways. the details of how npc influences ebov invasion has yet to be further research. similar to other −ssrna viruses, the rna genome of ebov is encapsulated by np and further form a ribonucleoprotein (rnp) complex together with rnadependent rna polymerase (rdrp) [ ] . after entry into the cytoplasm and membrane fusion, the rnp is released from the virion and serves as the template. complementary positive stranded rna (crna) is produced in the form of an rnp, and then generates viral genomic rna to be packaged into the virions. it is reported that in the entire viral replication cycle of a -ssrna virus, the genomic length viral rna (crna or viral genomic rna) is only present in the form of an rnp that either serves as a template for rna synthesis or is packaged in the virions [ , ] . so, the correct rnp formation and function is a key step for the transcription, replication, and assembly for −ssrna viruses. however, the molecular mechanism of ebov rnp formation is largely unclear. np is composed of a n-terminus, c-terminus, and np core domain (np core) in the centre, which possesses an n-lobe and c-lobe to clamp an rna binding groove [ ] . the n-and c-terminal extend in a tetrameric structure to reach the rna-binding groove, contribute to np oligomerization in rnp formation and binding with rna [ ] . however, the vp n-terminal peptide binds to a hydrophobic site on the np c-terminal domain with high affinity and specificity, inhibits np oligomerization and releases rna from np-rna complexes in vitro [ ] . the crystal structure of ebov vp reveals that it contains a coiled-coil domain, forms a tetramer state in solution, contributes to the oligomeric states and variations in rna binding preferences, and benefits it's connection with the blunt-ended rna termini in a cooperative manner [ , ] . yet, the driving force that directs the vp peptide to release rna from the rnp remains largely unknown. it is thought that np oligomerization and simultaneous rna binding at the rnp complex might provide the necessary force to displace the vp peptide [ ] . moreover, it's demonstrated that the vp n-terminal peptide is responsible for preventing premature np oligomerization and rna binding by maintaining the protein in an np-vp complex, and reversing the oligomerization of rna-free np oligomers, but has no effect on rna-bound np oligomers, which providing insight into np's role as a part of the viral rna synthesis machinery [ , ] . however, further details and mechanisms have yet to be firmly established. assembly of viral particles begins with the formation of nucleocapsids which accumulate in the perinuclear region and are transported to the budding sites at the plasma membrane. throughout this processes, gp, vp , np, and vp proteins play different roles. gp protein is synthesized in the endoplasmic reticulum (er) as a precursor and transported along the classical secretory pathway from the er via the golgi apparatus to the plasma membrane. precursor gp is processed by the acylation, oglycosylation, and maturation of n-glycans, and finally undergoes proteolytic cleavage by furin [ , , ] . acylation is another posttranslational modification of viral gp, involved in particle formation, including virus assembly and budding. after those processes, gp is partially recruited to the late endosome to meet with vp for assembly and budding [ ] . vp protein has been proved a secondary matrix protein and minor component of virions and contributing to virion assembly [ , ] . silence of vp rna resulted in a reduction in the number of released virions, but viral transcription and replication were not affected, implicating a role for vp in viral assembly and/or budding [ ] . however, the detailed mechanisms surrounding the necessity of vp for assembly and budding are largely unknown. the n terminus of np protein shoulders np-np and np-rna interaction, whereas the c terminus changes with the np-vp interaction [ , ] . at the core of the nucleocapsid, np helices are thought to physically interact with vp via the c-terminal amino acids, and be incorporated into the vp -induced vlps [ ] . it has been proved np assembles into helical tubes, forms a nucleocapsid-like structure with vp and vp , then migrate to the cell surface via microtubules mediated by vp , and is finally incorporated into virions through an np-vp interaction [ , ] . this process is essential for nucleocapsid transport to the plasma membrane and incorporation into virions. in addition, the flexibility of the np-np interaction in oligomer formation allows rnp to be packaged into viral particles with higher structures and density [ , ] . these results deepen our understanding of np functionality in assembly and budding. more details regarding the self-assembly of helical tubes and the transmission process should be explored for the development of antiviral compounds. vp is the most abundant viral protein located under the viral envelope, plays a vital role in maintaining structural integrity and maturation of the ebov virion [ , ] . vp contains two differently folded domains [i.e., n-terminal (ntd) and c-terminal (ctd)] [ ] . in cytoplasm, the ntd hydrophobic interface of vp forms homodimers through contact with some cellular proteins including mammalian ubiquitin ligase (nedd /rsp ), tsg , and vps , the protein-protein interaction causes translocation of the vp dimers to the plasma membrane [ , ] . in budding, the interaction of vp and inner leaflet is one of the major processes. the n-terminal domain of vp constitutes oligomerization whereas the c-terminal domains are a flexible hydrophobic loop [ ] . it's indicated that vp contains both electrostatic and hydrophobic components which are associated with plasma membrane phosphatidylserine (ps), vp binds ps-containing membranes with nanomolar affinity, while ps regulates vp localization and oligomerization on the inner leaflet of the plasma membrane [ , ] . the rearrangement of the flexible hydrophobic loop induces the penetration and docking of the ps, and allows vp to lock into the membrane [ ] . however, information is still lacking regarding how vp associates with the inner leaflet and further induces the orchestrated processes. the precise mechanisms are still unknown, although some peripheral proteins have been shown to be involved in the decrease of the desolvation penalty associated with hydrophobic membrane insertion [ , ] . meanwhile, the interaction of gp and tetherin is another major processes in budding. tetherin is an ifn-ainduced, cell-surfaceprotein-based tether which can induces virion retention on the cell membrance [ , , ] . gp contains a glycan cap and hydrophobic membrane spanning domain (msd) that is suggested to play a considerable (but not the sole determinant) role in tetherin antagonism [ , ] . however, how tetherin precisely induces virion retention, as well as the mechanism by which the gp glycan cap and msd antagonize the antiviral activity of tetherin remain unknown. as vp has many different locations within host cells, including the inclusions, late endosome, nucleocapsids, and mvbs. it is thought that vp may be transported to the site of budding either associated with nucleocapsid structures or with cellular membranes [ ] . for example, vp is accumulated in the late endosome in high amounts for oligomerization and the formation of the regular arrays the molecular mechanism of rnp and the mechanism by which vp releases rna from rnp remain unknown. assembly and budding: assembly is initiated by the nucleocapsids which accumulate in the perinuclear region, and are then transported to the budding sites at the plasma membrane. budding: occurs at the plasma membrane, intracellular membranes of the mvbs and late endosomes. vp and gp play critical roles in the budding process. abbreviations: er: endoplasmic reticulum; mvb: multivesicular body; npc : niemann-pick c . of vp , myosin could change the localization of intra-filopodia motility and release vp -induced vlps [ , ] . however, there was no direct evidence of an interaction between myosin and vp . therefore, vp has many complex roles in the processes of assembly and budding, although more detail is required with further study. filoviral budding, has not only been detected on the plasma membrane but on intracellular membranes of mvbs and late endosomes as well [ ] . intracellular viral particles might serve as a source of infectious units that can be delivered by exosomes combined with another signal-dependent process upon cell-to-cell contact. the cell-to-cell contact is supposed to promote ebov gpmediated infection, and increase the local concentration of retroviral pseudovirions and ebov vlps [ ] . this would result in relatively high mois, thus enhancing infection and spread. as reports of exosomes in the ebov lifecycle are limited, further investigation is required. in brief, glorious progress has been made in the mechanisms throughout the ebov lifecycle in host cells. however, there are still several aspects remain poorly understood. the eobv lifecycle is presented vividly in figure . in summary, although considerable amounts of researches have uncovered the mechanisms of the ebov lifecycle, there are still several aspects remain for further investigation (table ) . ebov entry into the cells is initiated by the interaction of the viral gp with receptors on the surface of host cells, and then internalized via macropinocytosis pathway. in uncoating and fusion, gp binds the endosome via rbd, and gp guides fusion via the fusion loop. several host enzymes which remain to be fully characterized are regarded to catalyse the reaction. this is a challenge for researchers as there are abundant enzymes involved within host cells. regarding replication, the key step is when vp releases rna from the np-rna complexes by inhibiting np oligomerization. however, the structural details and molecular mechanisms of ebov rnp, and the dynamic of vp releasing rna from rnp have not been completely defined. during assembly and budding, gp antagonizes the anchoring of tetherin via unarticulated mechanisms; vp regulates viral budding by associating with the inner leaflet of the plasma membrane with unknown detailed mechanisms. therefore, the aspects that remain unclear in the ebov lifecycle is waiting for profound research. this work was supported by grants from the major program of national natural science foundation of china (# ), and the science & technology key program of zhejiang china (# c - / ). no conflicts of interests. the molecular mechanism of rnp is unclear. the driving force directing the vp peptide to release rna from rnp is not clear. assembly and budding gp-tetherin interaction, vp -induced correct assembly, np-related nucleocapsid transport and the incorporation into virions, and vp inner leaflet association how does gp antagonise tetherin? how does np complete its function? how does vp contribute to assembly and budding? what is the mechanism used to control vp oligomerization? how does myosin- influence the localisation of intrafilopodia motility release? how does vp associate with the inner 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zaire ebolavirus tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein tyro family-mediated cell entry of ebola and marburg viruses t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus phosphatidylserine receptors: enhancers of enveloped virus entry and infection the tim and tam families of phosphatidylserine receptors mediate dengue virus entry role of the phosphatidylserine receptor tim- in enveloped-virus entry downregulation of beta integrins by ebola virus glycoprotein: implication for virus entry alpha beta -integrin controls ebolavirus entry by regulating endosomal cathepsins folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha analysis of 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importance of the coiled-coil of the ebola virus glycoprotein role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein genome-wide rnai screening identifies human proteins with a regulatory function in the early secretory pathway mutational analysis of the putative fusion domain of ebola virus glycoprotein core structure of the envelope glycoprotein gp from ebola virus at . -a resolution ebola viral glycoprotein bound to its endosomal receptor niemann-pick c ebola virus entry requires the cholesterol transporter niemann-pick c direct visualization of ebola virus fusion triggering in the endocytic pathway structure of human niemann-pick c protein vp of marburg virus influences formation of infectious particles nucleoproteins and nucleocapsids of negative-strand rna viruses structural perspective on the formation of ribonucleoprotein complex in negative-sense single-stranded rna viruses ebolavirus vp coats the backbone of double-stranded rna for interferon antagonism crystal structure of the marburg virus vp oligomerization domain assembly of the ebola virus nucleoprotein from a chaperoned vp complex an intrinsically disordered peptide from ebola virus vp controls viral rna synthesis by modulating nucleoprotein-rna interactions the ebola virus vp -np interaction is a regulator of viral rna synthesis effect of ebola virus proteins gp, np and vp on vp vlp morphology ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies tetherin inhibits retrovirus release and is antagonized by hiv- vpu contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles the assembly of ebola virus nucleocapsid requires virion-associated proteins and and posttranslational modification of nucleoprotein ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling elucidation of the cellular interactome of ebola virus nucleoprotein and identification of therapeutic targets assembly and budding of ebolavirus molecular architecture of the nucleoprotein c-terminal domain from the ebola and marburg viruses multivesicular bodies as a platform for formation of the marburg virus envelope the structure of the c-terminal domain of the zaire ebolavirus nucleoprotein ebola virus vp -induced particle formation and association with the lipid bilayer the ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress structural rearrangement of ebola virus vp begets multiple functions in the virus life cycle crystal structure of the matrix protein vp from ebola virus structural characterization and membrane binding properties of the matrix protein vp of ebola virus host cell plasma membrane phosphatidylserine regulates the assembly and budding of ebola virus investigation of ebola vp assembly and oligomerization in live cells using number and brightness analysis the ebola virus matrix protein vp selectively induces vesiculation from phosphatidylserineenriched membranes mapping of the vp -binding regions of the nucleoprotein of ebola virus phleboviruses encapsidate their genomes by sequestering rna bases the role of phosphatidylserine receptors in enveloped virus infection ebola virus glycoprotein counteracts bst- /tetherin restriction in a sequenceindependent manner that does not require tetherin surface removal requirements within the ebola viral glycoprotein for tetherin antagonism analysis of determinants in filovirus glycoproteins required for tetherin antagonism biochemical and functional characterization of the ebola virus vp protein: implications for a role in virus assembly and budding formation and function of phosphatdiylserine and phosphatdiylethanolamine in mammalian cells myosin-x is a molecular motor that functions in filopodia formation the matrix protein of marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg interaction cell-cell contact promotes ebola virus gp-mediated infection key: cord- -njzyu mv authors: hofmann-winkler, heike; gnirß, kerstin; wrensch, florian; pöhlmann, stefan title: comparative analysis of host cell entry of ebola virus from sierra leone, , and zaire, date: - - journal: j infect dis doi: . /infdis/jiv sha: doc_id: cord_uid: njzyu mv the ongoing ebola virus (ebov) disease (evd) epidemic in western africa is the largest evd outbreak recorded to date and requires the rapid development and deployment of antiviral measures. the viral glycoprotein (gp) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. however, it is unknown whether the gps of the currently and previously circulating ebovs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. here, we show that the gps of the ebovs circulating in and transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody kz . thus, the viruses responsible for the ongoing evd epidemic should be fully susceptible to established antiviral strategies targeting gp and cellular entry factors. ebolaviruses, from the family filoviridae, cause a severe and frequently fatal disease in humans [ ] . the first documented outbreak of ebola virus (ebov, formerly zaire ebolavirus) disease (evd) occurred in zaire and was associated with a case-fatality rate of % [ ] . subsequently, evd outbreaks were recorded in sub-saharan africa [ ] . these outbreaks generally occurred in relatively remote areas, were contained by quarantine measures, and caused a maximum of cases per outbreak (during the - evd outbreak in uganda). the ongoing evd epidemic, which originated in guinea [ ] , differs markedly from previous outbreaks: it affects western africa and is associated with a higher death toll than all previously recorded outbreaks jointly [ ] . the massive scale of the epidemic is likely due to the delayed public health response. however, the currently circulating viruses harbor > unique amino acid substitutions [ ] , and it is currently unclear whether these mutations allow for more-efficient spread in and between humans, compared with viruses responsible for past outbreaks. the ebov glycoprotein (gp) is inserted into the viral envelope and facilitates viral entry into target cells [ , ] . moreover, gp is the sole target for the neutralizing antibody response. gp is synthesized in the constitutive secretory pathway of infected cells and consists of a surface unit, gp , and a transmembrane unit, gp [ , ] . the gp subunit contains an n-terminal receptor binding domain (rbd) [ , ] , which binds to host cell receptors, and a c-terminal mucin-like domain (mld), while the gp subunit contains functional elements required for fusion of the viral envelope with a host cell membrane. cellular entry of ebovs commences with uptake of virions into host cell endosomes. this process can be promoted by recognition of phosphatidylserine in the viral envelope by tim proteins [ ] and, via bridging molecules, tam receptor tyrosine kinases (tyro , axl, and mer) [ ] on the host cell surface or by binding of gp to host cell lectins [ , ] . within the endosome, cysteine proteases remove the mld [ ] , which allows the subsequent interaction of gp with the cholesterol transporter npc- [ , ] , and drug-induced accumulation of endosomal cholesterol blocks infectious entry [ , ] . finally, an incompletely characterized stimulus triggers the membrane fusion reaction [ ] , which allows the release of the ribonucleoprotein complex into the host cell cytoplasm. all viral and cellular factors involved in the entry of ebovs into host cells are potential targets for antiviral intervention. however, the gp of the ebov, which is currently circulating in west africa harbors unique substitutions in gp [ ] , and it is unknown whether these changes alter the entry cascade and/or render the virus resistant to inhibitors or neutralizing antibodies. the present study addressed this question by comparing host cell entry mediated by gps of the ebovs circulating in and . the adherent human and simian cell lines cells were cultivated in dulbecco's modified eagle's medium (paa laboratories) and the suspension cells in roswell park memorial institute medium (paa laboratories), supplemented with % fetal bovine serum (biochrom) and antibiotics. the following cell lines were used: t (human embryonic kidney), huh (human hepatoma), u (human glioblastoma), hos (human osteosarcoma), hela (human cervix carcinoma), rpe (human retinal pigment epithelial), ea-hy (human endothelial hybrid), raji (human b cell), jurkat (human t cell), cemx (human t-cell/b-cell hybrid), smagi (rhesus macaque mamma carcinoma), llc-mk (rhesus macaque kidney), vero (african green monkey kidney), and cos (african green monkey kidney). differentiation of human thp- monocytes into thp- macrophages was achieved by incubation with phorbol- -myristate- -acetate (pma; ng/ml) for hours. bat cell lines were propagated as described elsewhere [ ] . all cells were grown in a humidified atmosphere at °c and % co . the nucleotide sequence encoding the gp of the currently circulating ebov (makona variant; genbank accession number km ; ebola virus/h.sapiens-wt/sle/ /makona-g ) was synthesized (geneart, life technologies) and inserted into pcdna . zeo via hindiii and xbai sites. expression plasmids for the gps of ebov strain mayinga from (ebov-gp ), lassa fever virus, influenza a virus (strain a/wsn/ ), and vesicular stomatitis virus (vsv), as well as -component human immunodeficiency virus type (hiv- )and mlv-based vector systems for analysis of gp-driven transduction have been described elsewhere [ , , [ ] [ ] [ ] . plasmids encoding the lectins dc-sign, dc-signr, lsectin, asgpr- , clec a, and folate receptor α were also described previously [ , [ ] [ ] [ ] . monoclonal antibodies kz and b directed against ebov-gp [ , ] were kindly provided by prof s. becker, marburg. monoclonal antibody -h - c recognizing hiv- gag was obtained from the national institutes of health (nih) aids reagent program. the generation of retroviral vectors pseudotyped with heterologous viral gps ( pseudotypes) was carried out as described before [ ] . in brief, t cells underwent calcium phosphate transfection with an expression plasmid encoding the gp of choice in combination with plasmids encoding hiv- gag-pol and plasmid pcswf-luc [ ] or plasmids encoding mlv gagpol and packaging plasmid mlv-luc [ ] . the culture supernatants were harvested hours after transfection, passed through filters ( pore size, . µm), aliquoted, and stored at − °c. for production of rhabdoviral particles, a protocol described elsewhere [ ] was followed. vlps were produced by expression of hiv- gag in combination with the gp of interest in t cells, followed by centrifugation of the vlp-containing supernatant through a % sucrose cushion [ ] . the presence of gag and gp in vlp preparations was analyzed by western blot, using antibodies against hiv-p (nih) and monoclonal antibody b directed against ebov-gp [ ] . for all transduction experiments, target cells were seeded in -well plates at a density of × cells/well. cells were then incubated for hours with µl of medium containing pseudoparticles bearing the gp of interest. transduction efficiency was determined by quantification of luciferase activities in cell lysates at hours (rhabdoviral pseudotypes) or hours after transduction (retroviral pseudotypes), using commercially available kits (promega, pjk). in some experiments, target cells were transduced to express interferon-induced transmembrane (ifitm) proteins or were transfected to express cellular lectins, as described previously [ ] , before transduction with gp-bearing pseudotypes. alternatively, expression of ebov entry factors was inhibited by small interfering rnas (sirnas). for this, sirna knock down in target cells was performed hours prior to transduction by transfection of pmol of sirna (all santa cruz), using lipofectamine (life technologies). to determine whether cationic amphiphiles (u a, merck; clomiphene or terconazole, sigma-aldrich) or protease inhibitors (catl inhibitor iii, merck; ca me, calbiochem; ca ; sigma-aldrich; aebsf, roth) influence transduction efficiency, the inhibitors were diluted in appropriate solvent as recommended by the manufacturer. target cells were preincubated with inhibitor for minutes at °c before addition of pseudotypes, and the culture medium was replaced by fresh culture medium without inhibitor after hours. to assess virion stability, pseudotypes bearing ebov-gp or the gp of vsv (vsv-g) were normalized for comparable infectivity. subsequently, the pseudotypes were incubated at defined temperatures for increasing periods, frozen at − °c at a given time point, and used for transduction of t target cells. seventy-two hours after transduction, transduction efficiency was quantified by a luciferase assay. pseudotypes carrying the gps of the respective ebov isolate or vsv-g as a control were normalized for comparable infectivity and incubated with monoclonal antibody kz at indicated dilutions for hour at °c. thereafter, the antibody/pseudotype mixtures were added to t cells, the cells were incubated for hours, and luciferase activities in cell lysates were determined. ebolaviruses infect a broad spectrum of cell types in cell culture [ ] , but macrophages and dendritic cells constitute early and sustained targets in the infected host [ ] . in contrast, lymphocytes are refractory to infection, both in vitro and in vivo [ , ] . to analyze whether previously and currently circulating ebovs show differences in host cell tropism and entry, we comparatively analyzed pseudotypes bearing the gps of ebov, strain mayinga, (ebov-gp ) and ebov variant makona (ebov-gp ) [ ] , which differ in amino acid residue in the rbd and amino acid residues in the mld (supplementary figure ) . pseudotypes bearing vsv-g served as positive control, while pseudotypes bearing no gp were used as negative control. both ebov gps were efficiently expressed in transfected cells and incorporated into retroviral particles ( supplementary figure ) , and both mediated entry into the human cell lines t, huh , u , rpe, hos, and ea-hy with comparable efficiencies ( figure a) . similarly, transduction of macrophages obtained upon pma treatment of thp- cells was comparable ( figure a ). in contrast, both gps were unable to facilitate transduction of a human b-cell line (raji), a human t-cell line (jurkat and a human t/b hybrid cell line (cemx ; figure b ). ebov is highly pathogenic for nonhuman primates (nhps), which are used as animal models for evd in humans [ ] . therefore, we also analyzed whether ebov-gp and ebov-gp transduce nhp-derived cell lines. transduction of the llc-mk and smagi cell lines was comparable to transduction of the vero cell line ( figure c ). in contrast, ebov-gp was more efficient than ebov-gp at transducing cos cells. finally, the ability of ebov-gp and ebov-gp to transduce bat cell lines (derived from rousettus aegyptiacus and hypsignathus monstrosus) was examined, since h. monstrosus, epomops franqueti, and myonycteris torquata can serve as natural reservoir for ebovs [ ] . however, no appreciable differences were observed ( figure d ). in sum, these results suggest that ebov variants circulating in and exhibit a comparable cell tropism. lectins, tim- , and tam kinase axl can augment entry of ebovs into certain target cells [ ] . in contrast, npc- is universally required for entry (dahlmann et al, submitted) . we addressed whether ebov-gp and ebov-gp show differential dependence on these factors for host cell entry. the lectins dc-sign, dc-signr, asgpr , and lsectin are known to augment gp-driven entry [ , , ] and promoted transduction driven by ebov-gp and ebov-gp to similar extents while expression of clec a, which binds dengue virus [ ] , had no effect (figure a) . moreover, both ebov-gp and ebov-gp used tim- and axl for entry into certain target cells ( figure b ), while expression of npc- was universally required for entry ( figure b and c) . finally, entry driven by both gps was comparably inhibited by u a, which induces an npc- knockout phenotype in cells [ ] , and related compounds ( figure c ), in the absence of unwanted cytotoxic effects (not shown). thus, our studies revealed no obvious difference in entry factor use by ebov-gp and ebov-gp . the enzymatic activity of the ph-dependent endosomal/lysosomal cysteine proteases cathepsin b (catb) and cathepsin l (catl) is required for cellular entry of pseudotypes bearing ebov-gp and authentic ebov [ ] . we used a panel of inhibitors previously employed to study ebov-gp protease use [ ] , to determine whether ebov-gp and ebov-gp exhibit differences in their protease requirements. entry driven by both proteins was comparably inhibited by the cysteine protease inhibitors ca me and ca , which efficiently inhibit catb but have little or no effect on catl and cats activity [ ] . in contrast, the serine protease inhibitor aebsf had no effect on gp-driven entry, and none of the compounds tested modulated entry driven by vsv-g ( figure a ), as expected [ ] . these observations indicate that ebov-gp and ebov-gp both depend on catb activity for efficient transduction of t cells. however, ebov-gp was less susceptible than ebov-gp to blockade by a third inhibitor, catl ( figure b ), which inhibits catb and catl activity to similar extents [ ] , suggesting subtle differences in the protease dependence of these gps. hiv-derived particles rapidly lose infectivity when stored at room temperature [ ] , and this loss might be due to inactivation of the viral envelope protein. therefore, we asked whether retroviral particles bearing ebov-gp and ebov-gp exhibit different stabilities. incubation of viral particles bearing vsv-g, ebov-gp , and ebov-gp for up to hours at °c, room temperature, and °c modestly and comparably reduced particle infectivity, and this loss occurred slightly more rapidly at room temperature and °c as compared to °c (figure ) . a more profound loss was observed upon incubation at °c, but again no differences were observed between ebov-gp and ebov-gp (figure ) , suggesting that the stability of these gps is comparable. comparably inhibited by ifitm proteins and neutralizing antibody kz ifitm proteins , , and inhibit cellular entry of ebov [ ] and several other viral pathogens [ , ] and might reduce ebov amplification in the infected host, raising the question of whether viruses circulating in are less susceptible to ifitm protein inhibition than those responsible for the outbreak in zaire. engineered expression of ifitm proteins in t cells had no appreciable impact on transduction mediated by lassa fever virus gpc but reduced influenza virus hadriven transduction, with inhibition by ifitm protein being most efficient ( figure ), in keeping with published data [ ] . transduction by the ebov-gps tested was also inhibited by ifitm protein expression, and no appreciable differences in inhibition efficiency were observed ( figure ) . thus, the virus host cell entry of ebovs can be inhibited by gp-specific antibodies, and some neutralizing antibodies were shown to exert antiviral activity in the host. we assessed inhibition of ebov-gp -driven and ebov-gp -driven entry by antibody kz (obtained from an evd survivor), which targets gp [ ] and displays antiviral activity in guinea pigs but not monkeys [ ] . entry driven by both gps was comparably inhibited by preincubation of particles with kz , while this antibody had no effect on entry driven by vsv-g (figure ) , indicating that the epitope recognized by kz is conserved between ebov and ebov . the ongoing evd epidemic in western africa is of unprecedented proportions and calls for the large-scale assessment of existing options for antiviral intervention and the development of new antiviral strategies. however, amino acid substitutions unique to the gp of the currently circulating ebov [ ] might render the viruses nonsusceptible to antivirals and cellular defenses targeting gp or cellular factors promoting gp-driven entry. the present study indicates that this scenario does not apply: no major differences in host cell entry driven by ebov-gp and ebov-gp were observed, and both gps were comparably susceptible to blockade by inhibitors and antiviral host cell proteins. ebov-gp contains a unique amino acid substitution in the rbd [ , ] , which may alter the requirement for host cell receptors and could modulate viral cell tropism. our results suggest that this is not the case: entry mediated by ebov-gp and ebov-gp was dependent on expression of the cholesterol transporter npc- and was inhibited by compounds that induce accumulation of endosomal cholesterol. moreover, expression of endogenous tim- and axl, which can augment gp-driven entry by binding directly (tim- ) or via a bridging molecule (axl) to ptdser in the viral envelope [ , ] , promoted entry driven by both ebov-gp and ebov-gp . the mld of gp is heavily modified with olinked glycans and is required for binding to certain host cell lectins [ ] . the observation that the mld of ebov-gp contains amino acid substitutions relative to the mld of ebov-gp suggested that engagement of cellular lectins might differ between these gps. however, again no appreciable differences were observed. in keeping with the comparable entry factor use, both gps facilitated entry into an identical spectrum of cell lines derived from bats, nhps, and humans, including thp- cell-derived macrophages. the only consistent difference observed was a reduction of ebov-gp -mediated transduction of cos cells, relative to that mediated by ebov-gp . this effect might point toward subtle differences in entry factor use, which may not be detectable in the assay system used in the present study. the removal of the mld by endosomal cysteine proteases, catb and l, is required for subsequent binding of gp to npc- [ , ] . several studies indicate that vectors bearing ebov-gp, as well as authentic ebov, depend on the activity of catb/l for infectious entry into cell lines [ , , ] , although catb/l dependence seems to vary between ebolavirus species [ , ] , and the activity of these particular proteases is dispensable for ebov spread in a murine model [ ] . the present study suggests that catb/l dependence extends to viruses currently circulating in west africa. however, reduced susceptibility of ebov-gp to the inhibitor catl, relative to that of ebov-gp , points toward minor differences in the protease requirements of the respective gps. the gps of ebovs are cleaved by proprotein convertases in the golgi apparatus of infected cells, and evidence for cleavage of ebov-gp was obtained (data not shown). the cleavage products, the surface unit gp and the transmembrane unit gp , remain covalently associated via a disulphide bond. one could speculate that the stability of this association might be different between ebov-gp and ebov-gp , which could translate into different stability of retroviral particles bearing these gps. however, a comparable time-and temperature- dependent loss of infectivity of ebov-gp -bearing and ebov-gp -bearing particles was observed, arguing against substantial differences in gp stability, although it cannot be disregarded that gp stability might differ in the context of retroviral and filoviral particles. interferon-induced antiviral host factors and neutralizing antibodies can contribute to viral control in the infected host. the present study suggests that ebov variants from and are susceptible to both defense mechanisms. expression of ifitm proteins comparably inhibited host cell entry driven by ebov-gp and ebov-gp , potentially by increasing endosomal cholesterol levels [ , ] . moreover, entry driven by both gps was blocked by neutralizing antibody kz , which was obtained from a human evd survivor and targets gp [ ] . collectively, these results and the observations discussed above suggest that previously and presently circulating ebov variants might use similar mechanisms for host cell entry, infect a similar spectrum of target cells, and might be comparably susceptible to inhibition by innate and adaptive immune responses. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. ebola haemorrhagic fever world health organization (who) emergence of zaire ebola virus disease in guinea ebola virus disease in west africathe first months of the epidemic and forward projections genomic surveillance elucidates ebola virus origin and transmission during the outbreak host cell factors in filovirus entry: novel players, new insights filovirus entry: a novelty in the viral fusion world conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor comprehensive analysis of ebola virus gp in viral entry t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus tyro family-mediated cell entry of ebola and marburg viruses c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterolindependent mechanisms ebola virus entry requires the host-programmed recognition of an intracellular receptor comparative analysis of ebola virus glycoprotein interactions with human and bat cells dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus proteolytic activation of the influenza virus hemagglutinin infectious hepatitis c virus pseudoparticles containing functional e -e envelope protein complexes lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection structural flexibility of the macrophage dengue virus receptor clec a: implications for ligand binding and signaling preand postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody complex of a protective antibody with its ebola virus gp peptide epitope: unusual features of a v lambda x light chain the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin the signal peptide of the ebolavirus glycoprotein influences interaction with the cellular lectins dc-sign and dc-signr characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection nomenclature-and databasecompatible names for the two ebola virus variants that emerged in guinea and the democratic republic of the congo in animal models for ebola and marburg virus infections fruit bats as reservoirs of ebola virus differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr clec a is critical for dengue-virusinduced lethal disease cholesterol synthesis inhibitor u a and the role of sterol metabolism and trafficking in numerous pathophysiological processes cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression survival of hiv- activity after disinfection, temperature and ph changes, or drying distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus structure of the ebola virus glycoprotein bound to an antibody from a human survivor tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine the role of phosphatidylserine receptors in enveloped virus infection human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry cathepsin b & l are not required for ebola virus replication role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences acknowledgment. we thank dr bruce chesebro (aids reagent program, division of aids, national institute of allergy and infectious diseases, national institutes of health) for the kind gift of hiv- p hybridoma ( -h - c).financial support. this work was supported by the german research foundation (grant po / - to s. p.), the german ministry for research and education (subproject within ebocon; to s. p.), the leibniz graduate school for emerging infectious diseases (to s. p.), and the leibniz foundation.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -tc z nm authors: zhang, yuan; wei, yanqiu; li, yunlong; wang, xuan; liu, yang; tian, deyu; jia, xiaojuan; gong, rui; liu, wenjun; yang, limin title: igy antibodies against ebola virus possess post-exposure protection and excellent thermostability date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: tc z nm ebola virus (ebov) is the most virulent pathogens that cause hemorrhagic fever with high mortality rates in humans and nonhuman primates. the postexposure antibody therapies to prevent ebov infection are considered efficient. however, due to the poor thermal stability of mammalian antibody, their application in the tropics has been limited. here, we developed a thermostable therapeutic antibody against ebov based on chicken immunoglobulin y (igy). the igy antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at °c for one year, in contrast to conventional polyclonal or monoclonal antibodies (mabs). we immunized laying hens with a variety of ebov vaccine candidates and confirmed that vsv Δ g/ebovgp encoding the ebov glycoprotein could induce high titer neutralizing antibodies against ebov. the therapeutic efficacy of immune igy antibodies in vivo was evaluated in the newborn balb/c mice model. lethal dose of virus challenged mice were treated or h post-infection with different doses of anti-ebov igy. the group receiving a high dose of nau/kg (neutralizing antibody units/kilogram) achieved complete protection with no signs of disease, while the low-dose group was only partially protected. in contrast, all mice receiving naïve igy died within days. in conclusion, the anti-ebov igy exhibits excellent thermostability and protective efficacy, and it is very promising to be developed as alternative therapeutic entities. author summary although several ebola virus therapeutic antibodies have been reported in recent years, however, due to the poor thermal stability of mammalian antibody, their application in tropical endemic areas has been limited. we developed a highly thermostable therapeutic antibody against ebov based on chicken immunoglobulin y (igy). the igy antibodies demonstrated excellent thermal stability, which retained their neutralizing activity at °c for one year. the newborn mice receiving passive transfer of igy achieved complete protection against a lethal dose of virus challenge indicating that the anti-ebov igy provides a promising countermeasure to solve the current clinical application problems of ebola antibody-based treatments in africa. ebola virus (ebov) belongs to the filoviridae family and the known cause of severe hemorrhagic fever in humans and nonhuman primates (nhps). since the epidemic of zaire the ongoing outbreak in the drc is the second-largest ebola epidemic on record, with lives lost and confirmed infections since august , which prompted who to declare this epidemic a public health emergency of international concern. pandemic potential, high mortality, high infectivity, and lack of preventive and therapeutic approaches make ebov a class a pathogen that seriously threatens public health. the intermittent and continuous outbreak of ebola disease (evd) poses a challenge for lethal challenge in newborn mice. our results suggest that the potent igy warrant further development as prophylactic and therapeutic reagents for evd. preparation of immunogens vaccine-elicited neutralizing antibodies (nabs) are associated with protection against filoviridae family mediated disease. in order to obtain the most potent anti-ebov antibody, we prepared several ebov immunogens based on multiple different platforms, including dna vaccine (pcaggs/ebovgp), recombinant protein (rebovgp) or virus-like particle (ebov- vlp) subunit vaccines, and two viral vector vaccines (vsvΔg/ebovgp, ad /ebovgp). western blot confirmed that these immunogens could express or contain ebov gp that can induce nabs in animals (fig ) . due to the differences in humoral immune responses induced by different vaccines, we need to screen for the most suitable immunogen for igy antibody production. to obtain high titer ebov nabs, -month-old laying hens were vaccinated with five different immunogens, including or tcid vsvΔ g/ebovgp, μ g rebovgp, μ g pcaggs/ebovgp, μg ebov-vlp, and virus particles (vp) ad /ebovgp (fig a) . thirty-five laying hens were randomly divided into seven groups, which were immunized four times with each immunogen or pbs control intramuscularly (i.m.) at a -day interval. eggs were collected at , , , , weeks, and igy antibodies were purified from egg yolk for elisa and nabs test. both titers in all groups were gradually increased after the first immunization. the results showed that all immunogens except dna vaccine induced potent gp-specific elisa antibodies (fig b) . for the nabs, the geometric mean titers (gmts) in tcid vsvΔ g/ebovgp group reached : (vsv pseudoneutralisation, vsv-psn) and : (lentiviral vectors pseudoneutralisation, lvv-psn) after the third boost, which significantly higher than other groups (fig c- passive transfer of igy protect newborn balb/c mice from lethal challenge to determine whether the anti-ebov igy antibodies are protective against ebov, passive protection experiment was performed in newborn balb/c mice (within the first days of life). forty newborn balb/c mice were divided into eight groups, which were challenged subcutaneously (s.c.) with tcid vsvΔg/ebovgp. two hours or day post-infection (dpi), each mouse was adoptively transferred with igy twice daily for days, and control group mice treated with naive igy (fig a) . to determine the correlation between the transferred igy dosage and therapeutic efficacy, three different dosages with , , or nau/kg (nab year. however, the antibody titer stored at ℃ gradually decreased from the second month, and only about % of the antibody activity remained by the end. in contrast, the activity of the igy stored at ℃ is lost faster, and the nabs titer cannot be measured by the third month. these results proved that the anti-ebov igy has excellent thermal stability, can be stored at room temperature (rt) for up to one year, and can maintain one month of activity at ℃ without significant changes. even at a high temperature of ℃, it still can short-term retention of activity (fig ) . it is suggested that this anti-ebov igy can be used as an emergency immunizations seven groups of -month-old laying hens (n = per group) were inoculated i.m with immunogens prepared as described above. the detailed scheme is or tcid vsvΔ g/ebovgp, μg rebovgp, μg pcaggs/ebovgp, μg ebov-vlp, vp ad /ebovgp, or an equivalent volume of pbs as a sham control at weeks , , , ( times). eggs were collected at weeks, , , , , for elisa and nabs test. purification of yolk igy antibody igy was isolated from the egg yolk using the water dilution method, a rapid and simple method was used to separate igy from the yolk. the separation method is improved based on previous for western blot analysis, the proteins were electrically transferred onto a polyvinylidene difluoride (pvdf) membrane using a semi-dry blotting apparatus ( v, min, rt), then blocked with tris-buffered saline containing . % tween (tbs-t) and % non-fat dry milk for h at rt and was incubated overnight at °c with a : dilution of 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in healthy adults ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies recombinant vesicular stomatitis viruses from dna generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines pseudoparticle neutralization assay for detecting ebola-neutralizing antibodies in biosafety level settings key: cord- -ndz oarf authors: ayithan, natarajan; bradfute, steven b.; anthony, scott m.; stuthman, kelly s.; bavari, sina; bray, mike; ozato, keiko title: virus-like particles activate type i interferon pathways to facilitate post-exposure protection against ebola virus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ndz oarf ebola virus (ebov) causes a severe hemorrhagic disease with high fatality. virus-like particles (vlps) are a promising vaccine candidate against ebov. we recently showed that vlps protect mice from lethal ebov infection when given before or after viral infection. to elucidate pathways through which vlps confer post-exposure protection, we investigated the role of type i interferon (ifn) signaling. we found that vlps lead to accelerated induction of ifn stimulated genes (isgs) in liver and spleen of wild type mice, but not in ifnar(-/-) mice. accordingly, ebov infected ifnar(-/-) mice, unlike wild type mice succumbed to death even after vlp treatment. the isgs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. importantly, proinflammatory cytokine/chemokine expression was much higher in wt mice without vlps than mice treated with vlps. in ebov infected ifnar(-/-) mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of vlp treatment, supporting the view that type i ifn signaling helps to limit viral replication and attenuate inflammatory responses. further analyses showed that vlp protection requires the transcription factor, irf known to amplify type i ifn signaling in dendritic cells and macrophages, the probable sites of initial ebov infection. together, this study indicates that vlps afford post-exposure protection by promoting expeditious initiation of type i ifn signaling in the host. ebola viruses (ebovs) are enveloped, negative-sense rna filoviruses that can cause a severe hemorrhagic fever in humans and non-human primates (nhps) [ , ] . mouse-adapted ebov causes similar acute disease in mice, offering a useful animal model to study ebov infection [ , ] . ebov infection is characterized by rapid viral replication and dysregulated innate and adaptive immune responses. the disease follows profound suppression of type i ifn signaling and a contrasting excess inflammation that leads to mucosal hemorrhages and multi-organ failure resembling septic shock syndrome [ , ] . virally encoded anti-ifn proteins, vp and vp play major roles in ebov virulence [ , ] . vp blocks type i ifn induction in dendritic cells (dcs) and macrophages, and acts as a virulence factor necessary for a recombinant virus to attain infectivity in the host [ ] [ ] [ ] [ ] . vp , on the other hand, blocks ifn signaling by interfering with ifn activated jak/stat pathways [ ] . lines of evidence support the critical importance of type i ifn signaling in providing resistance against ebov infection; mice deficient in stat , a transcription factor required for ifn induction, or ifnar , encoding the membrane receptor for type i ifns, are susceptible to wild type zaire ebov, against which wild type mice are resistant [ ] [ ] [ ] . a study of sudan ebov infection in humans showed that ifnα levels are significantly higher in surviving patients than those with fatal ebov infection, who had higher levels of proinflammatory cytokines/chemokines such as il- , and mip- β [ , ] . high ifnα production is reported to correlate with increased resistance against ebov in mice as well [ ] . administration of recombinant ifnα or ifnβ confers delayed time-to-death in nhps [ , ] . furthermore, ifnα, used as an adjunct therapy for monoclonal antibody treatment, is shown to enhance protection in nhps [ ] . ebov infection remains a potential threat to public health, which is compounded with the lack of effective prevention or treatment. to overcome this problem, various vaccine candidates have been developed, including various dna constructs, recombinant viruses, vlps, as well as treatment with anti-sense sirna [ ] [ ] [ ] . vlps are subunit-based vaccines, extensively studied for a variety of infectious pathogens [ , ] . vlps prepared from ebov and other filoviruses are composed of the matrix protein (vp ), glycoprotein (gp), and at times nucleoprotein (np) and represent a potentially promising candidate for ebov vaccine. ebov vlps have been shown to confer protection upon rodents and nhps when given prior to infection [ ] [ ] [ ] . in the accompanying paper, we show that post-exposure administration of trivalent vlps protects mice from lethal ebov infection, further crediting the potential of vlps as a possible vaccine [ ] . in that study, we show that vlp protection requires macrophages, dendritic cells (dcs) as well as b and either cd or cd t lymphocytes, indicating that both innate and adaptive immunity are involved in conferring protection. the aim of this study was to further investigate molecular bases of postexposure protection by vlps. based on our previous report that vlps stimulate type i ifn expression in dcs and macrophages, in vitro, we focused on the role of type i ifn signaling, and found that post-exposure vlp treatment leads to accelerated activation of ifn signaling, resulting in early induction of isgs. significantly, vlp stimulated isg induction coincided with the attenuation of proinflammatory cytokine surge in ebov infected mice. the reduced inflammatory responses was attributed to activation of type i ifn signaling, since vlp treated ifnar -/mice were unable to inhibit not only viral replication but proinflammatory responses, and succumbed to death. our results indicate that early type i ifn response is a major mechanism that contributes to vlp mediated protection against ebov infection. ifnar -/-, ifnar +/+ mice of balb/c background and irf -/and irf +/+ mice of c bl/ background were bred in the nichd animal facility and transferred to the facility of the united states army medical research institute of infectious diseases (usamriid) for ebov infection studies. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, . the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the iacuc committee approving this protocol is the united states army medical research institute of infectious diseases (usamriid) iacuc. animals were monitored at least once daily and their status was evaluated according to an intervention score sheet approved by usamriid iacuc. monitoring increased to three times daily if the animals were given a score of three or four. euthanization was by co inhalation followed by confirmatory cervical dislocation. analgesics and anesthetics were not used in this study and animals were euthanized for humane purposes if they reached a score of five or more, which would be indicated if the animals exhibited ruffled fur, weakness, unresponsiveness, and/or difficulty walking. otherwise, animals were euthanized at the end of the study. vlps were composed of ebov gp, np and vp and were generated in mammalian t cells as reported previously [ ] . vlp preparations used in this study contained < . endotoxin u/mg. mice were infected with~ pfu (~ , ld ) of mouse-adapted ebov via intraperitoneal (i.p.) route [ ] . mice were injected with vlps ( μg) diluted in pbs through i.p. h after ebov infection. morbidity and mortality of ebov infected mice were monitored twice daily for up to days. total rna from liver and spleen of ebov infected mice were extracted by trizol method (invitrogen) and cdna was synthesized from μg total rna by superscript ii reverse transcriptase (invitrogen). qpcr amplification was done with ng cdna in μl sybr green pcr master mix (applied biosystems) with μm of both reverse and forward primers used in the abi prism sequence detection system (applied biosystems). mrna of expression of indicated genes were analyzed as described in detail elsewhere [ ] . the primer pairs were used for ebov gp, '-tgggctgaaaactgctacaatc- ' and '-ctttgtgcacataccggcac- '; nlrp , '-tgctcttcactgctatcaagccct- ' and '-acaagcctttgctccagaccctat- '. all other gene primer sequences were followed from the previous publications [ , ] . transcript levels were normalized with hprt, and expressed as relative expression. statistical analysis was carried out by excel software using two-tail paired student's t test. data represent the mean of at least three independent assay ± sem. a p value < . was considered significant. to assess the role of type i ifn signaling in vlp-mediated protection against ebov infection, we tested ifnar -/mice for protection by vlps. in fig. a , wild type (wt) and ifnar -/mice (both balb/c background, n = ) were injected with μg of vlps h after infection by the mouse adapted (ma) ebov, and the morbidity and mortality were checked daily for the subsequent days. wt mice without vlp injection all died between day and day , whereas % of mice that received vlps survived after ebov infection, confirming that vlps protect mice even when they were given post-infection. in contrast, ifnar -/mice that received vlps all died before or at day as those without vlp injection (fig. a) . these results are in agreement with previous report on early death of ma-ebov infected ifnar -/mice [ ] . ebovs are thought to initially infect dcs and macrophages in liver and spleen, making these tissues the major sites of ebov replication in the mouse, although the virus infects many other organs later [ , ] . to ascertain whether vlps inhibit viral replication, we measured ebov glycoprotein (gp) mrna expression in liver and spleen from wt and ifnar -/mice with or without vlp administration. qrt-pcr analysis in fig. b and c, found that levels of gp mrna rose sharply in ifnar -/mice on day of infection when gp mrna was still at background in wt mice. ifnar -/mice that received vlps also expressed considerable amounts of gp mrna, although levels varied between liver and spleen in the vlp treated group. thus, ebov appeared to replicate faster and to a greater extent in ifnar -/mice than wt mice. it should be noted here that in wt mice, gp mrna levels began to increase rapidly after day , peaking on day to day , and that vlp injection inhibited gp mrna expression by more than half (s fig.) . pfu ma ebov, followed h later by injection i.p. with μg of ebov vlps. one group of ifnar +/+ (wt) mice infected with ebov without vlp served as control. mortality is expressed as percent survival of each group on indicated days. results are a representative of three independent experiments, which gave very similar outcomes. qrt-pcr detection of ebov gp mrna level on day post-ebov infection with or without vlps from liver (b) and spleen (c) of wt or ifnar -/mice. gp transcripts were normalized by hprt and values represent the mean ± sem of duplicate samples from three independent experiments. asterisk denotes significant differences compared to wt controls (*p . , **p . ). these results are in line with the results that ifnar -/and stat -/mice are more susceptible to ebov infection, suggesting the possibility that vlp mediated protection is linked to the activation of type i ifn signaling [ ] [ ] [ ] . however, vlp injection may not have prevented ebov pathogenesis in ifnar -/mice, possibly because the disease manifests more severely in these mice than in wt mice. on the other hand, it has been recently shown that adenovirus based vaccine can protect ifnar -/mice from lethal evob infection presumably through antibody responses, which indicates that ifnar -/mice are not universally vulnerable, and anti-ebov resistance can be attained in some cases [ ] . we recently reported that ebov vlps activate type i ifn transcription in dcs and macrophages in vitro, leading to induction of many isgs in these cells [ ] . here we asked whether vlps stimulate isg induction in vivo. wt mice were infected with ma ebov and received vlps h later, and induction of isg mrnas was tested on days . and . isgs encoding anti-viral proteins were first examined, as they may provide early protection against ebov infection. upper panels in fig. a and b compare induction of anti-viral isgs, ifit , mx , oas a and stat with or without vlp injection in liver and spleen. in this early stage, levels of these isgs were consistently higher in the vlp-injected groups than those without vlps. at later stages of infection, however, the situation reversed, in that mice without vlps had higher levels of isgs, as seen on day (s fig. for complete kinetics) . these results indicate that vlp administration accelerated type i ifn and isg induction, which presumably provide early anti-viral activity, not afforded without vlps. we next tested whether vlps induce other isgs, particularly those with negative regulatory activities. this question was of interest to us, since mice that did not receive vlps expressed higher levels of proinflammatory cytokines and chemokines, which raised the possibility that ifn signaling exerts negative regulatory activity towards proinflammatory responses, perhaps by controlling nf-κb activation [ ] . shown in the lower panels in fig. a and b is induction of irgm , usp , trim and trim . irgm is an ifn inducible gtpase that inhibits lps induced endotoxin shock in mice [ ] . usp is an isg deconjugating factor that negatively regulates tlr signaling and resultant cytokine induction [ ] . trim and trim are members of the tripartite motif family that downregulate tlr induced inflammatory responses [ ] [ ] [ ] [ ] . expression of these isgs was also higher in the vlp injected group than that without vlps both in liver and spleen. similar to anti-viral isgs, expression of these negative regulatory factors changed at the later stage (s fig). these data indicate that vlps accelerate induction of anti-viral and negative regulatory isgs, which may help suppress ebov's anti-ifn antagonism (see discussion). to confirm that vlp induction of isg is dependent on type i ifn signaling, we next tested isg induction in ifnar -/mice. as expected, none of the isgs tested in fig. were induced in ifnar -/mice after vlp treatment or ebov infection (s fig). vlps lower expression of proinflammatory cytokines in ebov infected mice ebov pathophysiology such as severe hemorrhagic symptoms and tissue damage is thought to be associated with dysregulated inflammatory cytokine production [ , ] . given that vlps accelerated induction of negative regulatory isgs, we next evaluated whether vlps modulate expression of proinflammatory genes. in fig. , expression of tnfα, il- and il- β, chemokines such as mcp- (ccl ), mip- α (ccl ), mip- β (ccl ), kc (cxcl ) and inflammasome gene nlrp was measured in ebov infected mice with or without vlps. these genes were all strongly induced upon ebov infection and peaked on day with a gradual decline on days in all cases, their expression was significantly attenuated in the vlp-treated group as compared to the group without vlps. the difference was most dramatic in the early stage on day , where the expression was reduced at least by %. in agreement with these results, we noted that serum levels of some of these proinflammatory cytokines were higher in ebov infected mice that were treated with vlps as compared those without vlps [ ] . these results support the view that limiting superfluous inflammatory responses contribute to vlp mediated protection. ifnar -/mice increasing evidence indicates that type i ifns antagonize inflammatory responses in a variety of settings [ ] [ ] [ ] . in light of the results that vlps stimulate those isgs known to suppress proinflammatory responses, it was of importance to determine whether type i ifn signaling by and of itself affects ebov induction of proinflammatory cytokines and chemokines. results in fig. and s fig compare expression of the above proinflammatory factors in ifnar +/+ and ifnar -/mice infected with ebov. all cytokines and chemokines tested were induced after ebov infection in both strains. importantly, their levels were much higher in ifnar -/mice than ifnar +/+ mice. these results indicate that type i ifn signaling downregulates ebov stimulated induction of proinflammatory factors, possibly through isgs with negative regulatory activities. the above results indicated that type i ifns attenuate proinflammatory responses during ebov infection. to explore whether type i ifns have a similar activity in settings other than ebov infection, we next tested lps and ifnβ induced inflammatory responses in macrophages in vitro. lps activates nf-κb mediated proinflammatory cytokine induction, which can result in endotoxin shock [ ] . as shown in fig. , combined treatment with lps and ifnβ led to hyper induction of tnfα, il- , il- β and a chemokine kc in ifnar -/macrophages as compared to wt cells. lps and ifnβ also induced negative feedback factors, trim and trim , with much lower expression in ifnar -/cells than ifnar +/+ cells. these results support a model in which type i ifns negatively regulate proinflammatory cytokine/chemokine responses at least in some situations. we found that mip- α, mip- β and mcp- were not hyperinduced in ifnar -/cells, suggesting that some proinflammatory genes are regulated not only by type i ifns but other factors (data not shown). alternatively, these differences may reflect variances between in vivo and in vitro conditions. to further define pathways downstream of ifnar activity, important for vlp protection, we directed our attention on irf , a transcription factor expressed in macrophages and dcs [ ] [ ] [ ] . irf is induced by ifns and tlr ligands in a stat dependent manner, and plays a pivotal role in facilitating innate immune responses. although irf is not involved in initial triggering of type i ifn induction, it amplifies ifn transcription in dcs and macrophages [ ] . irf promotes induction of multiple anti-microbial factors and is required for innate resistance against a variety of pathogens [ ] [ ] [ ] . irf stimulates expression of mhc and costimulatory molecules to boost antigen presentation [ , ] . we thus tested whether irf disruption affects vlp-mediated protection against ebov. survival data in fig. a show that approximately % of irf -/mice that received vlps died between day and , which is nearly identical to the mortality curve of wt mice without vlps. as expected, the majority of wt mice that received vlps survived against ebov infection. it is of note that ifnar -/mice died to days earlier than irf -/mice, which may be attributed to the difference in the mouse background. correlating with the lack of protection, ebola gp mrna levels were much higher role of type i ifn in vlp-mediated protection against ebov infection in ebov infected irf -/mice than irf +/+ mice with or without vlps (fig. b) . we next examined whether induction of anti-viral and negative feedback isgs is dependent on irf . data in fig. c illustrate that induction of these isgs was very meager in irf -/mice, in contrast to robust induction in irf +/+ mice. importantly, vlps did not rescue isg induction in irf -/mice. results were similar in liver and spleens ( fig. c and s fig). these results indicate that vlps, upon initial activation of type i ifn cascade, rely subsequently on the activation of downstream pathways represented by irf to confer protection against ebov. to gain insight into the pathways through which vlps confer resistance against ebov infection, we investigated the role of type i ifn signaling in vivo and found that it significantly contributes to vlp-mediated protection. this conclusion is supported by the observation that post-exposure vlp treatment accelerated isg induction in ebov infected mice, leading to reduced viral replication and inflammatory gene expression. further supporting the critical role of type i ifn signaling in the protection, vlps did not induce isgs in ifnar -/mice, and did not protect the mice from lethal ebov infection. these results are consistent with the report that post-exposure ifnβ or ifnα treatment increases protection against ebov infection in nhps [ , , , ] . it is likely that vlps initially stimulated type i ifn genes, which in turn led to early induction of isgs. in line with this notion, we recently showed that exogenous vlps stimulate transcription of ifnα and ifnβ in dcs and macrophages in vitro, an event coupled with immediate and robust isg induction [ ] . it may be reasonable to assume that ifnar -/mice were not protected by vlps primarily because isg induction was absent. however, ifnar -/mice may be susceptible to infection due to additional defects in innate immunity that are a secondary consequence of defective ifn signaling, which obliterates vlps protection. contouring this notion however, it is of note that ifnar -/mice can be protected against ebov by an adenovirus-based vaccine, indicating that ifnar -/mice are not totally without defense [ ] . rather, it is possible that ifnar -/mice are not protected by vlps that rely on isg induction for protection, whereas they are protected by the adenovirus vaccine that depends on antibody response. vlp-induced isgs included anti-viral proteins known to inhibit replication of rna viruses such as ifit , mx and oas a, as well as negative feedback factors that curb excess inflammatory responses, such as irgm , usp , trim and trim . although the question of which antiviral isgs are effective in inhibiting ebov replication awaits further research, it is anticipated that some of anti-viral isgs induced by vlps may interfere with ebov life cycle [ ] . what is the significance of accelerated ifn response in vlp mediated protection? available evidence suggests that vlps may overcome ebov's anti-ifn antagonism. the virally encoded vp and vp disable the entire ifn system in the host; while vp blocks the jak/stat pathway of ifn signaling, vp , an ebov virulence factor, inhibits type i ifn induction in many cell types [ , , , ] . we previously showed that vp inhibits type i ifn induction in murine dcs by premature sumoylation and inactivation of irf [ ] . it is thought that vp and vp have a decisive effect on the subsequent host resistance, since abated ifn signaling would impair proper innate immune responses, leading to deficiency in dc maturation, defective antigen presentation and aberrant inflammation. compromised innate immunity would consequently undermine development of adaptive immunity [ ] (see a model in fig. ) . it is remarkable that in the vlp treated mice, isg induction began early within . to days after ebov infection (which was only . to days after vlp treatment), when little to no isg induction was seen in mice without vlps. the delayed isg induction in ebov infected mice is reminiscent of the reports showing that influenza virus delays isg induction in lung epithelial cells through ns , an influenza anti-ifn protein that is linked to disease pathology [ , ] . an influenza virus strain deficient in ns is shown to induce isgs earlier than wild type virus, although the wild type strain does stimulate isgs later on [ , ] . supporting the view that viral anti-ifn factors stall isg induction, rather than completely abrogate the induction, we also observed isg induction on day and later in mice without vlps. it may be envisaged that vlps trigger ifn activation early on, thereby eluding the activity of the ebov anti-ifn proteins (a model in fig. ) . the most striking observation made in this study is the vlp-dependent suppression of proinflammatory responses. this suppression was a result of type i ifn signaling, as ifnar -/mice expressed higher levels of proinflammatory cytokines and chemokines, observed not only after ebov infection but also by ifnβ and lps stimulation. these results are in accordance with the growing recognition that type i ifns are linked to attenuation of inflammatory responses [ , ] . for example, pinto et al., reported, in the west nile virus infection model that ifnar -/mice express excess proinflammatory cytokines, including those found in this study, as compared to wt mice, which correlated with increased disease pathology. in this system, the overt inflammatory responses were attributed to ifn signaling in macrophages and dcs [ ] . induction of proinflammatory cytokines and chemokines may be negatively regulated by ifn signaling through a series of negative feedback factors [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . irgm , induced by ifn signaling restricts lps induced endotoxin shock without limiting ifnβ expression [ ] . trim and trim inhibit proinflammatory cytokine induction, at least in part by interfering with the nf-κb dependent arm of transcription [ ] [ ] [ ] . in addition, these factors may act by post-transcriptional mechanisms, affecting inflammasome activation [ ] . in this regard, guarda et al. [ ] reported that type i ifns inhibit production of il- by inhibiting activity of the nlrp and nlrp inflammasomes and by il- induction. thus, isgs with negative regulatory activity may preferentially attenuate proinflammatory pathways, while sparing ifn induction pathways. given our earlier observations that vp does not grossly affect nf-κb activation, while strongly inhibiting type i ifn activation, ebov may promote proinflammatory pathways at least in part through vp [ ] . lastly, we show that the transcription factor irf is required for vlp mediated post-exposure protection. our results offer an added mechanistic insight into the pathways through which vlps provide protection. irf is expressed predominantly in macrophages and dcs, and helps to amplify type i ifn gene induction and boosts ifns biological activities [ ] . given that macrophages and dcs are the putative early sites of ebov infection, vlps may exert a major impact on these cells to facilitate early innate immunity, in 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signalling and immune regulation the content of this publication does not necessarily reflect the views or policies of the us department of defense or the united states army medical institute of infectious diseases. we thank members of pgd for in depth discussions and critical reading of the manuscript. we also thank ms. monica gupta for breeding ifnar -/mice for this study. key: cord- - zy unz authors: long, jason; wright, edward; molesti, eleonora; temperton, nigel; barclay, wendy title: antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: zy unz emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. the former was the only available intervention when the current unprecedented ebolavirus (ebov) outbreak in west africa began. prior to this, the development of ebov vaccines and anti-viral therapies required time and resources that were not available. therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of ebov and could be used immediately. here we test three such medicines and measure their ability to inhibit pseudotype viruses (pvs) of two ebov species, marburg virus (marv) and avian influenza h (flu-h ). we confirm the ability of chloroquine (cq) to inhibit viral entry in a ph specific manner. the commonly used proton pump inhibitors, omeprazole and esomeprazole were also able to inhibit entry of all pvs tested but at higher drug concentrations than may be achieved in vivo. we propose cq as a priority candidate to consider for treatment of ebov. past outbreaks have been of limited size affecting a local population, however a strain of ebov-z is the causative agent of the current outbreak that began in late and has since become an unprecedented and devastating epidemic , , resulting in over , suspected cases, of which those confirmed had a case fatality rate of around % in the afflicted west african countries (http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary- ?lang=en and http://www.who.int/csr/disease/ebola/ situation-reports/en/). towards the end of the trend in case numbers reversed in liberia and the epidemic slowed in sierra leone and guinea, but the virus continues to transit in new geographical areas . this epidemic has triggered a significant global health response relying on primary hygiene and other containment measures that have proved successful in limiting the spread of the virus in previous outbreaks. given the scale of this outbreak and the fear that traditional containment measures may fail to prevent global spread, several vaccines have been fast-tracked into phase i clinical trials - although even if proved efficacious, the limited supply of sufficient quantities of vaccine will hinder their use in the current situation. for disease treatment, patients suffering a haemorrhagic fever have relied on the clinical management of symptoms (http://www.cdc.gov/vhf/ebola/treatment/). with a handful of patients in this outbreak receiving experimental therapies such as zmapp, tkm-ebola, brincidofovir and favipiravir (http://www.nature.com/news/ebola-trials-to-start-in-december- . ) - . alternatively antibody treatment by transfusion therapy using blood or plasma from ebola virus survivors has been approved , - ; although issues with safety and lack of resources for this method limit its suitability in west africa today. having no approved or widely available therapeutics for ebov, as with many other emerging viral diseases, focus has turned to possible re-purposing of drugs already licensed for other uses by the ema and fda. several clinically approved drugs have been identified by researchers - , including amiodarone, one of the several cationic amphiphiles found to inhibit filovirus entry which is currently being trialled in sierra leone . however reservations have been expressed about the complications that could be caused by side effects of the drug in ebov patients. the anti-malarial drug chloroquine (cq) has also been shown to inhibit ebov entry and protected mice from ebov infection , and has been previously highlighted as a possible drug to treat ebov infection . the possible difficulties that may arise with use of re-purposed drugs include unforeseen interactions between virus/drug and host causing exacerbation of disease. therefore it is important to try and understand the mechanism of virus inhibition by such drugs. to this end we re-examined the anti-viral properties of cq, and show here that it inhibited the ph-dependent endosomal entry of a pseudotyped virus (pv) bearing ebov glycoproteins, in the same way as did the potent and specific vacuolar-atpase (vatpase) inhibitor bafilyomycin a (bafa ) (a non-medical laboratory compound). we also show that licensed and widely used proton pump inhibitors (ppis) for treatment of gastric acid reflux, omeprazole (om) and esomeprazole (esom), inhibited pv ebov entry, likely by their off-target inhibitory activity on endosomal vatpase. human embryonic kidney ( t/ ) (atcc) and human lung adenocarcinoma epithelial cells (a ) (attc) were maintained in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented with % fetal calf serum (fcs) (biosera) and % penicillinstreptomycin (ps) (invitrogen). the cell lines were maintained at °c in a % co atmosphere. chloroquine diphosphate salt (cq), bafilomycin a from streptomyces griseus (bafa ), omeprazole (om) and esomeprazole magnesium hydrate (esom) (sigma) were resuspended as per manufacturer's instructions and aliquots stored at - °c: cq was prepared in sterile dh o; bafa , om and esom were prepared in sterile dmso (sigma). the bundibugyo ebolavirus (ebov-b) envelope glycoprotein (gp) (fj ) coding sequence was synthesised (bio basic inc.) and the ha glycoprotein of avian influenza a/turkey/england/ - / (h n ) (flu-h ) was amplified from the ha plasmid of the h n reverse genetics system . both were sub-cloned into the pcaggs expression vector. expression vectors containing the envelope glycoproteins of zaire ebolavirus (mayinga) (ebov_z), marburg virus (lake victoria isolate; marv) and gibbon ape leukemia virus (galv) (modified to contain the trans-membrane domain of amphotropic murine leukemia virus (a-mlv) envelope glycoprotein) are described previously , . the renilla luciferase gene was sub-cloned into pcaggs expressing vector from a minigenome reporter described previously . the generation of all lentiviral pseudotype viruses was based on the methods detailed previously [ ] [ ] [ ] . briefly, t/ cells were seeded into cm tissue culture plates (nunc™ thermo scientific). the hiv gag-pol plasmid, pcmv-Δ . and the firefly luciferase reporter construct, pcsflw, were transfected together with either influenza ha, galv, ebov or marburg gp expression constructs at a ratio of : . : (core:reporter:envelope) using fugene transfection reagent (promega). at h post-transfection, cells were washed and fresh media applied. for the generation of h the units for chloroquine in table have been corrected from nm to μm. pvs, u exogenous recombinant neuraminidase from clostridium perfringens (sigma-aldrich) was also added h after transfection to effect egress from the producer cells. pv supernatants were harvested at and h post-transfection and passed through a . m pore filter. ebov pvs were aliquoted and stored at °c; the remaining pvs were stored at - °c. entry inhibition assay t cells in cm plates were transfected with ug of renilla luciferase expressing plasmid using lipofectamine according to manufacturer's instructions (life technologies™). cq, bafa , om and esom were serially diluted in -well white-bottomed plates (nunc™ thermo scientific) to give the final described concentrations. after h the transfected cells were trypsinised and × cells were added to each well. after min cells were transduced with no more than × rlu of pv per well (estimated from raw rlu values of previously infected t cells), and to an equal volume per well. h later supernatant was removed and cells were lysed with µl of passive lysis buffer (promega), and firefly/renilla luciferase activity measured using a fluostar omega plate reader (bmg labtech) and the dual luciferase assay system (promega). a cells were pre-treated with drug h before nm of the ph sensitive lysotracker ® red dnd- (life technologies™) was added to the media of each well . after minutes in growth conditions, cells were analyzed for fluorescence using an axiovert confocal laser (cfl) microscope and an axiocam mrc camera (carl zeiss). statistical analysis pv transduction rlus were normalised to the renilla value in the corresponding wells. percent infection of each drug dilution was calculated compared to untreated cells. two-way anova with bonferroni's multiple comparisons test between untreated and treated mean values (α- . ) was performed to measure statistically significant differences. ic values were calculated using nonlinear regression analysis (log[inhibitor] vs normalised response). all manipulation of data was performed on graphpad prism (graphpad software). the envelope glycoproteins of several emerging viruses with high pathogenicity and pandemic potential were used to create lentiviral based pseudotype particles as previously described . pvs were generated bearing the envelope glycoproteins from zaire ebolavirus (mayinga strain) (ebov-z), bundibugyo ebolavirus (ebov-b), marburg (lake victoria isolate) virus (marv), h ha from a highly pathogenic avian influenza virus a/turkey/ england/ - / (h n ) (flu-h ), and gibbon ape leukaemia virus (galv). galv pvs were included because galv is a virus that does not require acidification of endosomes for its entry into cells. all the pvs generated were shown to transduce t cells and firefly luciferase expression from the packaged reporter gene was measured above mock infected cells (non-transduced cells) (dataset ). in order to assess the ability of cq, bafa , om and esom to inhibit pv entry, drugs were serially diluted in triplicate in white bottomed -well plates. next, t cells transfected hours previously with a renilla luciferase expression plasmid to allow monitoring of cell viability, were added to each well. appropriately diluted pvs were then added to each dilution, including a no-drug control. after hours incubation, the supernatant was removed and firefly and renilla luciferase rlus were recorded using the dual luciferase assay system (promega). pv rlus were normalised to the corresponding renilla values, which reduced the edge effect observed in the -well plates, and controlled for toxicity of the drugs. only bafa appeared to reduce expression of renilla at the highest concentrations, suggesting cellular toxicity, (dataset ) and visible cytopathic effect was not observed in cells treated by cq, om and esom at the concentrations used in figure . both bafa and cq reduced ebov-z, ebov-b, marv and flu-h entry in a dose dependent manner ( figure a and b). the ic value of bafa was in the nm range for ebov-z, ebov-b, flu-h and marv and inhibition of entry was statistically significant at the nm concentration compared to the untreated control (table ) . cq inhibited ebov-z, ebov-b, marv and flu-h with ic of . , . , . and . µm respectively, and inhibition was statistically significant (table ). in contrast, galv entry was augmented by both bafa and cq above that of the untreated cells to a maximum of . % ( . nm) and . % ( . µm) respectively. both om and esom reduced entry of all pvs tested at µm but galv pv was the least affected ( figure c and d). inhibition of entry for ebov-z, ebov-b, marv and flu-h pvs by esom was significant at µm, and galv pv was not significantly inhibited at this dose ( figure d and table ). increasing endosome ph as a mechanism of inhibiting virus ph-dependent entry bafa and cq are known endosomal acidification inhibitors (bafa being a potent and specific vatpase inhibitor and cq a licensed lysotropic agent) . the effects of om and esom on endosomal acidification have also been previously reported , . to confirm that endosomal ph was being affected at doses used here, a cells were treated with drug for hour before applying lysotracker ® red dnd- (lifetechnologies). a cells were chosen here because t cells are poorly imaged due to their morphology. the lysotracker probe specifically fluoresces in acidic organelles. fluorescence was decreased in cells treated with bafa and cq in a dose dependant manner, but was unaffected in cells treated with vehicle alone (figure ). om and esom appeared to decrease fluorescence, and therefore increase endosomal ph, only at a concentration of µm, higher than that required to inhibit pv entry. moreover cellular toxicity was observed at this concentration after hours. table . after attachment to cells, viruses require a mechanism of fusion to deliver the viral genome. preventing this action by fusion inhibitors has been successful approach for hiv antiviral therapy . unlike hiv, ebov and many other viruses are dependent on the naturally low ph of acidic endosomes to activate and trigger fusion by their envelope glycoproteins. in this instance, a 'fusion inhibitor' could target the host cell machinery preventing acidification of the endosome, working to inhibit virus entry of several different viruses. here we have reiterated that cell entry by pvs representing ebov, flu-h and marv can be inhibited by increasing the endosome ph using bafa and cq (figure ) , and this correlates with their ability to prevent the acidification of intracellular organelles (figure ). cq has shown antiviral activity against several viruses in vitro, including ebov, influenza, nipah, hendra, dengue and chikv - . disappointingly, this antiviral activity has not always translated into efficacy in vivo models or clinical trials, although cq was effective in a mouse model against ebov , , [ ] [ ] [ ] [ ] [ ] . the variability in in vivo results may depend on study design and strains of virus used. in one study bafa treated mice were not protected from influenza infection but treatment with a related compound, saliphe, was protective, even though both drugs were potent in vitro . inhibition of endosome acidification as a target for inhibiting ebov can be justified by the knowledge that the filoviruses depend on the low ph for two separate steps of their entry pathway. not only is the fusion by g protein triggered by low ph, but its cleavage into a fusogenic form is carried out by endosomal enzymes cathepsins b and l whose activation is also ph dependent . some have argued that g protein cleavage by cathepsin is less essential than previously thought , and that ebov species other than zaire together with closely related marv do not require cathepsin cleavage for entry , . nonetheless, entry of marv pvs was still inhibited in our assays suggesting that inhibiting fusion alone is sufficient. recently, using computational modelling, ekins et al. suggested the anti-ebov mechanism of cq may be by binding the vp protein of ebov . if this drug had activity on several steps of the replication cycle it may not only be more effective in vivo but it may be even less likely that the virus could mutate to escape inhibition. at first we were surprised that cq actually increased entry of galv pv (figure ). however this effect has been noted before for other retroviruses, including a-mlv and hiv- , and is accounted for by the inhibitory effect of cq on the autophagy pathway. cq prevents degradation of phagosomes that contain virus particles and prevents them from otherwise being degraded [ ] [ ] [ ] . cq has been used for many years as an anti-malarial drug, although it is now only effective in parts of central america and the caribbean due to accumulation of drug resistance by the plasmodium parasite . interestingly, compounds belonging to the omeprazole family have also been described as having anti-malarial properties in vitro, possibly via their reported ability to target vatpase in the plasma membrane of plasmodium parasite . soon after its discovery om was found to also inhibit intracellular vatpase at µm concentrations as opposed to its licensed target of gastric h+/ k+-atpase against which it is effective at much lower concentrations , . indeed there are a plethora of publications indicating use of om and esom in cancer therapy, as a means to inhibit the characteristic acidic intracellular environment, and thus permit sensitivity to cytotoxic therapies [ ] [ ] [ ] [ ] [ ] . a role of om and esom has also been noted in the suppression of bone resorption, another physiological process dependent on ph [ ] [ ] [ ] . given the volume of research suggesting these off target effects depend on an ability to affect intracellular ph, we hypothesised that these drugs would, like cq and bafa , inhibit ebov, marv and influenza virus ph dependent entry. we used galv as a control again since its entry is reportedly independent of ph. indeed, ebov, flu-h and marv were inhibited by lower doses of om or esom than galv ( figure and table ). galv entry was also inhibited at the highest concentration, but we cannot exclude that this was due to a toxic effect that was not measured by the renilla control we employed here. we did not observe as close a correlation between drug doses that mediated the inhibition of ebov or influenza pv entry and increase in ph of intracellular vesicles for om and esom as for cq and bafa, (figure and figure ). more recently, it has been reported that om and esom altered the localisation of vatpase in the cell as well as the ph of intracellular vesicles and this may explain their ability to inhibit pv entry more potently than the ph changes we observed would suggest. inhibition of influenza virus entry to cells by means of inhibiting acidification of endosomes has been known for decades , although no current antivirals for influenza have been licensed on this basis. some epidemiological evidence from population studies suggests that om could exert a protective effect against influenza-likeillness , but our studies suggest that doses required for potent inhibition might be difficult to achieve without significant toxicity. despite these drugs being readily available, even without prescription in some countries, the licensed dosing would generate a plasma concentration reportedly . - . µm for esom that falls short of the ic calculated in this study, although higher doses have been used clinically . therefore it seems unlikely that om and esom would be a suitable therapy for ebolavirus infection, but more specifically designed vatpase inhibitors may have potential as broad acting antivirals against several emerging viruses in the future. with regard to cq, the evidence suggests a more promising position for use against ebolavirus. standard adult dosing ( mg/kg) achieves plasma concentration of µm, close to our ic value against ebov pv entry. protection in the mouse model was previously shown with a mg/kg dosage , . using re-purposed drugs to treat outbreaks of emerging diseases must surely be approached with caution. in ebola patients with severe life-threatening disease it would be important to ensure that any side effects of a therapy did not enhance disease progression, particularly if higher doses of re-purposed drugs, as suggested here, were considered. on the other hand, cq has been taken prophylactically in a tropical setting for many years to prevent malaria and we suggest that, with little additional need for scale up of production of a new agent, this might represent a useful adjunct to the current antiviral strategies being trialled in west africa. we envisage that in contacts of ebov cases, cq might decrease the viral load that establishes in the early days after virus transmission. further work in in vivo models including guinea pig and primates should inform about doses and administration regimens. author contributions dr jason long, dr edward wright and dr eleonora molesti generated the pvs. jason long performed the drug entry assay and ph assay. this work was planned by prof wendy barclay, dr nigel temperton and dr jason long. all authors were involved in preparing and revising the manuscript. evolutionary history of ebola virus entry mediated by the ebola virus glycoprotein pubmed abstract | publisher full text | free full text cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change pubmed abstract | publisher full text | free full text cathepsin b & l are not required for ebola virus replication pubmed abstract | publisher full text | free full text cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression a common feature pharmacophore for fdaapproved drugs inhibiting the ebola virus toll-like receptor binds single-stranded cpg-dna in a sequence- and ph-dependent manner tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production autophagy in health and disease: a double-edged sword pubmed abstract | publisher full text | free full text ph regulation in the intracellular malaria parasite, plasmodium falciparum. h( + ) extrusion via a v-type h( + )-atpase effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs proton pump inhibitors may reduce tumour resistance v-atpase as an effective therapeutic target for sarcomas v-atpase is a candidate therapeutic target for ewing sarcoma proton pump inhibitors induce apoptosis of human b-cell tumors through a caspase-independent mechanism involving reactive oxygen species effect of omeprazole, an inhibitor of h + ,k( + )-atpase, on bone resorption in humans omeprazole, a specific inhibitor of h + -k + -atpase, inhibits bone resorption in vitro weiss division of infection and immunity the authors wish to thank caroline goujon for the kind provision of the galv construct and olivier moncorgé for generating the renilla expression plasmid. no competing interests were disclosed. this work was supported by the grants flupig eu fp and bbsrc bb/k / . this is an elegant study employing envelope pseudotypes of highly pathogenic viruses which demonstrates that certain inhibitors of low endosomal ph can inhibit viral entry. because some of these molecules such as chloroquine have been in clinical use for decades, and are inexpensive, they might tip the balance between survival and death during human infection.i have no criticism of the experimental work. however, i have been told by a reliable physician who has recently cared for patients with ebola infection that treatment with chloroquine offered no clinical benefit. thus it is possible that an observation may not translate into a useful treatment . so one in vitro in vivo should be wary about the conclusions. no competing interests were disclosed. the work was of particular interest especially in light of several viruses shown to be taken up in this rather non specific way. i would have perhaps also like some discussion of receptor and channel mediated virus uptake -there are several publications in this space. the interplay between such different mechanisms may point to multiple targets or need for combined approaches to block them.the authors describe amiodarone, but there are many molecules that have been found as ebola replication or pseudoviral entry inhibitors, had they looked at more molecules to see if the ph mechanism was common across them? i would likely suggest adding repurposing in the title of the article.the conclusion might benefit from comparison of the chloroquine data with that previously published the conclusion might benefit from comparison of the chloroquine data with that previously published (higher ec ), potential ocular toxicity etc.some discussion as to whether the ph effect is an in vitro specific effect or something of in vivo relevance -would also be worth mention.this study confirms the previous work on chloroquine and suggestions by others as to its potential utility. this begs the question why it is not used clinically. what other data would be needed to show that chloroquine could be clinically useful?the study is well designed and reported and adds to the growing literature on chloroquine and its potential as an antiviral.i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.no competing interests were disclosed. competing interests: key: cord- - grqif authors: wong, gary; liu, wenjun; liu, yingxia; zhou, boping; bi, yuhai; gao, george f. title: mers, sars, and ebola: the role of super-spreaders in infectious disease date: - - journal: cell host & microbe doi: . /j.chom. . . sha: doc_id: cord_uid: grqif super-spreading occurs when a single patient infects a disproportionate number of contacts. the mers-cov, sars-cov, and to a lesser extent – ebola virus outbreaks were driven by super-spreaders. we summarize documented super-spreading in these outbreaks, explore contributing factors, and suggest studies to better understand super-spreading. a number of recent virus outbreaks have resulted in rapid virus spread, placing demands on the affected health infrastructures and sparking global concern. in september , middle east respiratory syndrome coronavirus (mers-cov) emerged as a novel virus that can result in severe respiratory disease with renal failure, with a case fatality rate of up to %. primary infections typically occur in countries located in the middle east, where dromedary camels have been identified as one of the species harboring the virus. however, travel has contributed to several cases in other countries. notably, between may and july , an outbreak of mers-cov centered in south korea killed people out of confirmed cases (promedmail.org, ) , with thousands quarantined as health authorities attempted to control virus spread. severe acute respiratory syndrome coronavirus (sars-cov), a close relative of mers-cov, was the etiological agent responsible for an outbreak centered around guangdong province in the southeast of china. the sars-cov outbreak killed and infected , people between november and july , for a case fatality rate of . %, in which the virus was first traced to palm civets, and then bats. over % of the total cases and deaths were from mainland china and hong kong. more recently, a devastating ebola virus (ebov) outbreak has swept through western africa. ebov is a filovirus that causes severe hemorrhagic fever in hu-mans with up to a % case fatality rate if untreated. outbreaks of ebov are sporadic, unpredictable, and localized to sub-saharan africa. prior to the - outbreak, a total of , deaths out of , infections were documented dating back to , when ebov first emerged. the natural reservoir of ebov is currently unknown, but fruit bats are suspected to be the animal species harboring the virus. in addition to their significant impact, these outbreaks were all perpetuated by super-spreaders who disproportionately infect a high number of contacts and likely contribute to the speed and degree of the outbreak. as discussed in detail below, these outbreaks can be traced to one or more individuals who largely contributed to subsequent infections. the mers-cov outbreak in south korea began from an imported case, a -year-old male with a recent travel history to several middle eastern countries, including bahrain, the united arab emirates, saudi arabia, and qatar. the latter three countries also had reported human cases of mers-cov during the same period of time. after the patient started exhibiting disease symptoms, he sought medical assistance at several clinics before being admitted to a hospital, where he was confirmed to be infected with mers-cov (who, a). twentynine secondary infections have since been directly traced to this index patient. additionally, two of these secondary cases were shown to be responsible for subsequent infections, out of known cases at the time (cowling et al., ) . transmission was mostly within nosocomial settings, and fourth generation infections have been documented for the first time since the virus was identified in . thus, the mers-cov outbreak in south korea was driven primarily by three infected individuals, and approximately % of cases can be traced back to three super-spreaders who have each infected a disproportionately high number of contacts ( figure a ). this mers-cov outbreak is the second largest on record and the largest outside of the middle east. the mers-cov outbreak currently appears to be under control, with no additional infections reported in south korea since july , . the sars-cov outbreak retrospectively, several super-spreading events were also documented during the sars-cov outbreak in . the index patient of the hong kong epidemic was treated at prince of wales hospital and was associated with at least secondary cases (riley et al., ) . subsequent super-spreader events occurred at the hotel metropole ( cases) and the amoy gardens housing complex (over cases) in hong kong and aboard an air china flight traveling from hong kong to beijing ( cases) (braden et al., ) . notably, cases from hotel metropole were responsible for the spread of sars-cov to canada, vietnam, and singapore through travel ( figure b) , and the imported case to canada resulted in sars-cov cases at a toronto hospital (braden et al., ) . have super-spreaders played a role in other outbreaks? to a lesser extent, similar events were observed with the - ebov outbreak, centered in the western african countries of guinea, sierra leone, and liberia. epidemiological work has linked five infections with a -year-old toddler in the remote village of meliandou, guinea. one of the contacts, a midwife, infected at least three others, including a hospital worker at gué cké dou hospital. this worker then infected several family members in the gué cké dou farako district, as well as others at macenta hospital (baize et al., ) . one of the subsequent cases, a doctor, was known to have infected several others who traveled to kissidougou and nzé ré koré , further propagating virus transmission (baize et al., ) . the outbreak eventually spilled over to neighboring liberia and sierra leone. in sierra leone, the funeral of a traditional healer that died from ebov was shown to have directly infected others (gire et al., ) and was eventually linked to more than cases (who, b). the ebov outbreak has killed , people, with , infected as of july , en route to becoming the largest filovirus outbreak ever documented. in addition to ebov, superspreading has also been documented in outbreaks with other pathogens, including measles (paunio et al., ) and mycobacterium tuberculosis (kline et al., ) . the initial stages of the outbreaks mentioned above involved at least one super-spreading event. the ability of the pathogen to infect subsequent superspreaders who may export the disease through travel is likely the difference between an infection cluster and an outbreak or epidemic ( figure c ). differences in the initial response to mers-cov is why south korea reported clusters of infections before bringing the virus under control and why china, thailand, and the philippines have not reported any additional cases despite imported mers-cov infections. it was why sars-cov was so devastating in mainland china and hong kong from to . it was also why the - ebov outbreak spiraled out of control in western africa, with hundreds of new cases reported weekly, before a coordinated international effort (including contact tracing, isolation, diagnosis, and treatment of infected patients, as well as community education) brought the number down to a more manageable - weekly infections, with most of the cases diagnosed in guinea and sierra leone. early discovery, diagnosis, intervention, and quarantine of confirmed cases is crucial for preventing further disease transmission in humans, especially via super-spreaders. as evidenced by the decreased numbers of new cases in the mers-cov and ebov outbreaks, current responses are effective, provided that the measures are implemented properly and followed strictly. vigilance and patience will be necessary until the ongoing mers-cov and ebov outbreaks are officially at an end. currently, super-spreaders are only retrospectively categorized after epidemiological tracing. given their high propensity to spread infection, super-spreaders should be able to shed higher levels of pathogen and/or for a longer period of time after infection. this would increase the probability that the pathogen contacts and subsequently infects a naive host. to address whether there are factors or parameters that promote or indicate enhanced virus shedding in a laboratory setting, an animal species that closely recapitulates symptoms of the human disease is required, such that any findings can be translated to humans. animals could be experimentally infected with different virus strains/variants at varying doses via different challenge routes, in which virus shedding from the oral, nasal, and rectal cavities should be correlated with different parameters, including viremia or virus titers in the organs. however, these studies are currently challenging for sars-cov as there is currently not an appropriate animal model that replicates the severe disease seen in humans; infected nonhuman primates exhibit variable, but at most mild to moderate, respiratory disease (mcauliffe et al., ) (lawler et al., ) . nonetheless, these types of studies are feasible for mers-cov. a model of this infection was developed in the common marmoset that results in severe respiratory disease and partial lethality, in which live virus can be detected in nasal and throat swabs of infected animals (falzarano et al., ) . thus, the studies proposed above can be investigated with mers-cov in this animal model. for ebov, the cynomolgus and rhesus macaque animal models are well characterized and have been used over the past + years. ebov shedding is known to occur from the oral, nasal, and/or rectal cavities of nonhuman primates during the advanced stages of disease, but factors that influence virus shedding from these animals have not yet been investigated. interestingly, using a guinea pig model, it was shown that animals infected intranasally (i.n.) with guinea pig-adapted ebov were more contagious to naive contact animals than those that were infected intraperitoneally (i.p.) with the same dose . i.n. infected animals shed virus from their nasal cavity earlier than their i.p. counterparts, and i.p. challenged animals died earlier (and thus were removed from contact with naive animals) compared to i.n. inoculated guinea pigs. it was therefore concluded that the route of infection in addition to the duration of contact time with an infectious host may be factors that influence the efficiency of virus transmission. it will take time to elucidate all possible host and viral factors that contribute to virus shedding, which remains an understudied topic to date. therefore, it is not currently possible to predict with any level of confidence or statistical significance whether a person will become a super-spreader of a certain disease. large-scale genome-wide association studies (gwas) of super-spreaders might provide some clues about the genetic backgrounds of super-spreaders, but the feasibility and robustness of these analyses will depend on the number of super-spreaders in a given outbreak. the large amount of patient data and samples available from the recent mers-cov and ebov outbreaks provide examples from mers-cov, sars-cov, and ebov virus mutation: the virus may acquire mutations to replicate more efficiently and become highly pathogenic, which may result in higher levels of virus shedding for a longer period of time. duration of contact with an infectious host and route of infection: in a guinea pig model of ebov infection, animals that were infected mucosally were more infectious than those that were infected systemically. additionally, prolonged contact with a contagious animal resulted in a higher rate of virus transmission. genetic susceptibility: ebov is known to be more pathogenic in cynomolgus macaques compared to other nonhuman primate species, such as rhesus macaques. different nonhuman primate species also demonstrate variable susceptibility to sars-cov infection. hosts that are more susceptible to disease may exhibit higher levels of virus shedding. underlying medical conditions: certain conditions may decrease immunity and increase susceptibility to pathogens, leading to enhanced virus shedding. many fatal mers-cov patients in the recent outbreak in south korea had other pre-existing medical conditions, including pneumonia and kidney disease. air re-circulation in enclosed spaces: this would increase chances of pathogen encounter, especially those that are airborne. during the sars-cov outbreak, this was shown to be a factor behind super-spreading events at the hotel metropole, amoy gardens housing complex, and a flight between china and canada. it is likely a contributing factor to the mers-cov outbreak since super-spreading events occurred at a hospital or medical clinic. population density: increased numbers of people equally increases the chances to infect a naive host through inadvertent direct or indirect contact. traditional customs and beliefs conducive to infectious disease spread: the culture of ''doctor shopping,'' in which patients seek medical attention from multiple doctors at different clinics/hospitals allowed mers-cov to spread rapidly in south korea. unsafe burials and traditional funerals, which involve touching and washing infectious bodies, played a role in ebov spread in western africa. travel and trade: the global nature of today's world allows infectious diseases to easily bypass geographical barriers. for instance, mers-cov originated from the middle east, but imported cases were reported in north america, europe, and asia. sars-cov originated in asia, and imported cases were reported in north america, europe, and other parts of asia. ebov originated from africa, and imported cases were reported in north america and europe. knowledge and adherence to public health advice: during the mers-cov outbreak, a case was imported to china because a south korean patient did not follow the recommendations of health authorities and traveled despite being a symptomatic, high-risk contact. however, china handled the imported case properly, and the imported korean patient did not become a super-spreader. during the ebov outbreak, some patients were able to escape quarantine, thereby increasing the likelihood of infecting others. possible opportunities to investigate factors that may correlate with an increased risk for becoming a super-spreader. using the mers-cov outbreak as an example, detailed records were kept for the majority of patients who were confirmed to be infected with mers-cov. since the identities of the three super-spreaders are known as patients # , # , and # , studies can be performed to investigate whether these three patients have common traits (i.e., age, gender, pre-existing medical conditions or underlying co-morbidities, levels of virus shedding, etc.). any common characteristics can then be compared with non-super-spreader patients to provide insight into possible risk factors behind enhanced spreading of infectious diseases. another approach would be to isolate mers-cov from the three known superspreaders, perform sequence analysis of the viral genomes, and determine whether there are any shared mutations between these isolates. any mutations that are identified and are absent from the viral genomes of other patients could be indicators of enhanced virus shedding, allowing for super-spreading to occur. a complex combination of factors likely plays a role in the number of subsequent infections initiating from a single superspreader. in addition to host and virus factors, other important factors impacting the spread of infectious diseases are the environment and behavior of individuals. environmental factors include the close proximity of other susceptible hosts and the airflow dynamics within an enclosed area. a high population density represents a greater number of susceptible hosts for direct/indirect contact with an infected person, whereas air recirculation would especially facilitate the transmission of airborne viruses, such as mers-cov and sars-cov. hospitals, enclosed housing complexes, and mass transportation, such as airplanes, are documented sites of super-spreader events, especially during the mers-cov and sars-cov outbreaks (table ) . individual behaviors may also enhance disease spread. these include ''doctor shopping''-going to multiple hospitals to treat the same ailments or traveling to other countries after the appearance of disease symptoms, which was observed with the mers-cov outbreak (su et al., ) . traditional customs, such as unsafe funerals/burials in africa, which involve direct contact with infectious bodily fluids of patients that passed away from ebov disease, is another major reason why the outbreak persisted in guinea and sierra leone (table ) . a thorough study of host, virus, and environmental dynamics will be important to delineate the relative contribution of each factor to the phenomenon of super-spreading. findings from these studies, combined with changes in behavior conducive to the spread of disease, will be important for the effective management of and preparation for future outbreaks. emergence of zaire ebola virus disease in guinea progress in global surveillance and response capacity years after severe acute respiratory syndrome preliminary epidemiological assessment of mers-cov outbreak in south korea infection with mers-cov causes lethal pneumonia in the common marmoset genomic surveillance elucidates ebola virus origin and transmission during the outbreak outbreak of tuberculosis among regular patrons of a neighborhood bar cynomolgus macaque as an animal model for severe acute respiratory syndrome replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys explosive school-based measles outbreak: intense exposure may have resulted in high risk, even among revaccinees mers-cov ( ): south korea, saudi arabia. international society for infectious diseases transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions mers in south korea and china: a potential outbreak threat middle east respiratory syndrome coronavirus (mers-cov) -republic of korea. disease outbreak news sierra leone: a traditional healer and a funeral ebola virus transmission in guinea pigs key: cord- -btaxvmsr authors: di paola, nicholas; sanchez-lockhart, mariano; zeng, xiankun; kuhn, jens h.; palacios, gustavo title: viral genomics in ebola virus research date: - - journal: nat rev microbiol doi: . /s - - - sha: doc_id: cord_uid: btaxvmsr filoviruses such as ebola virus continue to pose a substantial health risk to humans. advances in the sequencing and functional characterization of both pathogen and host genomes have provided a wealth of knowledge to clinicians, epidemiologists and public health responders during outbreaks of high-consequence viral disease. here, we describe how genomics has been historically used to investigate ebola virus disease outbreaks and how new technologies allow for rapid, large-scale data generation at the point of care. we highlight how genomics extends beyond consensus-level sequencing of the virus to include intra-host viral transcriptomics and the characterization of host responses in acute and persistently infected patients. similar genomics techniques can also be applied to the characterization of non-human primate animal models and to known natural reservoirs of filoviruses, and metagenomic sequencing can be the key to the discovery of novel filoviruses. finally, we outline the importance of reverse genetics systems that can swiftly characterize filoviruses as soon as their genome sequences are available. infections with viruses of the mononegaviral family filoviridae (in particular, members of the genera ebolavirus and marburgvirus) are an increasing threat to mankind. until recently, the infrequent spillover of these viruses into humans and the fact that spillover often occurred in remote locations, coupled with a limited knowledge of non-human reservoir hosts, the use of low-output genomic sequencing, and biosafety and bio security restrictions on filovirus research, contributed to the paucity of publicly available data on filovirus genome sequences . in december , complete genome sequences for only ebolaviruses and marburgviruses were available despite the fact that outbreaks of natural filovirus disease had been recorded . since december , atypically extensive filovirus disease outbreaks, from to in western africa and from to present in the democratic republic of the congo, have profoundly impacted public health systems. at least , fatalities from filovirus disease were reported between december and april , (refs , ). by leveraging the continued development and improvement of next-generation sequencing technology, > complete filovirus genome sequences and over , draft genomes (that is, genomes with > % coverage) across classified and unclassified filovirus family members have become available since (ref. ). indeed, among high-consequence, risk group viruses, the genomic diversity of filoviruses is arguably becoming the best characterized. the impact and importance of genomics in pathogen characterization is routinely demonstrated, but the rapid prediction of, response to and mitigation of outbreaks requires more detailed genomic information than virus consensus-genome sequencing. indeed, as predicted , metagenomic sequencing has become a powerful tool for identifying novel viruses and, crucially, for predicting pathogen emergence . targeted or unbiased sequencing of individual clinical samples aids in the identification of outbreaks, the determination of outbreak aetiology and the definition of virus transmission chains by identifying chain-defining single nucleotide polymorphisms (snps). furthermore, field transcriptomics improves our understanding of host responses to virus infection and will be important in deciphering the differences between asymptomatic and symptomatic disease states and in predicting whether patients with acute and chronic disease will survive . functional genomics is becoming the tool of choice for the rapid characterization of patient-specific viruses that have not been isolated in culture or that cannot be equitably shared among laboratories across borders . finally, the genomic analysis of patient-specific viruses also enables precision medicine by predicting the efficacy of available medical countermeasures (mcms) against these individual viruses. here, we review how recent advances in genomic technologies have shaped past and current responses to outbreaks of ebola virus disease (evd), including insights into filovirus diversity and evolution. we emphasize the importance of accurate and rapid large-scale data generation and its implications for the development of mcms and outbreak response. we also examine the phenomena of ebola virus (ebov) persistence in human hosts and provide an overview of recent genomic advances in threat characterization, vaccine development a collection of continuously evolving technologies and techniques that allow for the digitalization of genomic material. the sequencing of genetic material recovered directly from an environmental or clinical sample that allows the identification of all organisms and mobile genetic elements represented in the sample. and immunotherapy. although we focus primarily on ebov, these practices can apply to all pathogenic filoviruses and other high-consequence viruses capable of sustaining human-to-human transmission. although the global distribution and diversity of filoviruses remains largely undefined, metagenomic sequencing is becoming a valuable tool for identifying filovirus reservoirs. until , disease outbreaks owing to infection by ebolaviruses (including ebov, sudan virus (sudv) and marburgviruses (including marburg virus (marv) and ravn virus (ravv)) had only been recorded on the african continent ( fig. ). as the natural reservoir hosts of all of these viruses remained unidentified, despite extensive ecological studies, filoviruses were thought to be african viruses . this view changed after , when reston virus (restv; of the genus ebolavirus) was discovered and repeatedly identified as a lethal pathogen of captive crab-eating macaques (macaca fascicularis) in non-human primate (nhp) breeding facilities in the philippines - ( fig. ). however, although restv can infect humans, it appears to be apathogenic . restv was subsequently considered to be an asian anomaly to the african filovirus dogma. classical filovirus-targeted genome sequencing and, later, unbiased broad-scale metagenomic sequencing, shed new light on filovirus ecology. in , the sequencing of samples obtained from egyptian rousettes (rousettus aegyptiacus) in africa revealed that these bats, which are cavernicolous and frugivorous pteropodids, are natural reservoir hosts of both marv and ravv. coding-complete or complete genomic sequences of both viruses were repeatedly obtained from egyptian rousette populations in uganda, sierra leone and south africa [ ] [ ] [ ] [ ] , and genomic fragments of these viruses were also detected in populations of these bats in the democratic republic of the congo and in zambia . around and after , sequence-based evidence obtained using a range of techniques began to support the hypothesis that restv is an asian virus. restv genome sequences were obtained from captive dome stic pigs (sus scrofa domesticus) in the philippines and china , , and restv genome fragments were sequen ced from samples from molossid, pteropodid and vespertilionid bats in the philippines . next-generation sequencing further enabled the discovery of a highly divergent filovirus, lloviu virus (llov, genus cuevavirus), in deceased schreibers's long-fingered bats (miniopterus schreibersii) in hungary and in spain . the ebolavirus genus was expanded owing to the discovery (via next-generation sequencing) of bombali virus (bomv) in molossid little free-tailed bats (chaerephon pumilus) and angolan free-tailed bats (mops condylurus) in guinea, kenya and sierra leone [ ] [ ] [ ] in short, genomics has clarified that highly divergent filoviruses, frequently with unknown pathogenic potential, are likely to be distributed widely over the african, asian and european continents in highly diverse host reservoirs ( fig. , box ). furthermore, expanded animal sampling and unbiased host virome sequencing is likely to enable this diversity and distribution to be described in more detail. of note, the natural host reservoirs of three ebolaviruses that are human pathogens, namely bundibugyo virus (bdbv), sudv and taï forest virus (tafv), are still unclear. furthermore, although bats are suspected to be hosts of ebov owing to the detection of short ebov genomic fragments and/or antibodies to ebov in certain bats , no complete ebov genome has yet been sequenced from any bat sample. the genomic investigation of archived or newly acquired samples could also support or refute the often-repeated hypothesis that middle african central chimpanzee (pan troglodytes troglodytes), duiker (cephalophus spp.) and western lowland gorilla (gorilla gorilla gorilla) populations are frequently decimated by ebov infection [ ] [ ] [ ] . thus, genomics may enable the prevention of future filovirus disease outbreaks by identifying filovirus natural hosts and by limiting host-human contacts as well as the initial introduction of filoviruses into the human population. identifying and characterizing outbreaks genomics-based techniques have been central in the identification and characterization of filovirus disease outbreaks. the largest known filovirus disease outbreak occurred from to in western africa and was caused by a novel ebov variant, makona (ebov/mak) ( fig. ). genomic sequencing efforts during this evd outbreak showcased various platforms and strategies to characterize thousands of human clinical samples containing ebov/mak . early efforts relied on exporting positive samples to high-complexity genomic centres abroad , (box ). however, in december , ebov/mak genome sequencing using benchtop sequencers, such as the miseq system (illumina) and the ion torrent system nature reviews | microbiology r e v i e w s (thermo fisher scientific) in-country (namely, in liberia and sierra leone), became standard practice [ ] [ ] [ ] . in addition, field laboratories used the iseq (illumina), a portable bench-top sequencer with low error rates that can be transported in a suitcase, to obtain complete ebov genome sequences to determine virus transmission in the democratic republic of the congo , . use of the portable nanopore sequencing technology minion (oxford nanopore technologies) markedly reduced the time required to obtain the genome sequence from patient samples and enabled the reintroduction of ebov into guinea and sierra leone to be rapidly confirmed . similarly, the ebov variants causing the Équateur province evd (ebov/"tum") and the ongoing nord-kivu/sud-kivu/ituri province evd outbreak caused by ebov/"itu" in the democratic republic of the congo were quickly identified by the use of minion , . the timing and establishment of in-country genomic sequencing capabilities determine which information can be captured and disseminated during an evd outbreak. early sequencing efforts can provide an informative 'snapshot' of the genomic epidemiology of ebov during the initial phase of the outbreak . the extent of the genomic diversity of the virus at the beginning of an outbreak can be used to determine whether single or multiple virus spillover events have occurred and to provide a crude estimate of the time at which a virus emerged in a human population , , , . highly accurate genomic data have been used to characterize intra-host ebov populations and genetic drift and even to evaluate, in silico and in real time, the available diagnostic measures and mcms , , . as an outbreak progresses and the sampling size increases, phylodynamic and spatiotemporal analyses reveal broader trends in the intra-outbreak evolutionary rate of the virus, its geographical migration and factors contributing to virus transmission, disease outcome and virus-host adaptation , [ ] [ ] [ ] [ ] [ ] [ ] . ideally, the viral agent is initially identified using highly portable sequencing platforms on site. after this identification, considerations other than sequencing speed (for example, sequencing accuracy and processivity) become paramount in determining virus transmission networks and in detecting changes in the viral genome (between cases in the current outbreak and between the current and previous outbreaks) that could subvert mcms. however, whereas unbiased sequencing approaches using high fidelity platforms can lead to the discovery of co-infections and reveal important clinical considerations during the treatment of patients near the point of need, targeted methods of pathogen characterization using the portable sequencing platforms iseq and miseq (which use bait-enrichment techniques) and minion (which uses amplicon sequencing) can still provide useful genomic data albeit with a lower sequencing output (that is, a lower number of reads) than unbiased sequencing. sequencing only a single target during an enduring and large outbreak of evd may result in the detection of co-infections and/or superinfections being missed. the earliest evidence of an ebov co-infection was obtained in gabon in , where a patient with evd also tested positive for human immunodeficiency virus (hiv- ; a lentivirus of the retroviridae family) the change in the frequency of an existing gene variant (allele) in a population owing to the occurrence of random mutations. of the family hepadnaviridae) and epstein-barr virus (a lymphocryptovirus of the family herpesviridae) were reported [ ] [ ] [ ] . additionally, ebov infections in patients with a gram-negative septicaemia or with bacterial translocation have been described [ ] [ ] [ ] . moreover, an extensive infectious disease due to one particular virus could plausibly conceal a simultaneous outbreak caused by a different pathogen . notoriously, there have been outbreaks of cholera, plague, measles and malaria, and sporadic cases of monkeypox and yellow fever, alongside the -present evd outbreak in the north-eastern region of the democratic republic of the congo. detecting and characterizing co-infections during a disease outbreak can provide clinicians with crucial point-of-care information and identify differences in patient outcomes that may result from co-occurring infections. the characterization of unexpected, co-circulating viruses during large-scale viral outbreaks requires established and reliable sequencing strategies that can be applied without knowledge of which virus is present. the discovery of novel viruses is ideally facilitated by metagenomic sequencing, which often relies on the pre-processing of clinical samples by depleting host-derived genomic material, followed by single or random primer amplification by pcr and deep sequencing [ ] [ ] [ ] . however, metagenomic sequencing is limited by the requirement for computational and bioinformatics resources, which are not always readily available under field conditions. instead, target-enrichment approaches using a wide breadth of bait probes for known pathogenic viruses, including filoviruses, have led to the successful characterization of known viruses in clinical samples from patients with disease of unknown etiology [ ] [ ] [ ] . moreover, target-enrichment sequencing is more cost-effective than metagenomic approaches. the percentage of sequencing data matching the target pathogen or pathogens can range from % to % with target enrichment, whereas with metagenomics approaches often < % of sequencing data matches the target , . punctual and highly accurate sequencing efforts have revealed the molecular genomic epidemiology of disease and thus enabled the characterization of pathogen transmission during disease outbreaks . the first application of in-country, real-time genomic epidemiology started well into the evd outbreak of - in western africa; portable sequencing was performed and data were analysed in tangent with up-to-date public health data . the abundance of ebov genome sequences determined during recent large-scale evd outbreaks (including the - outbreak and the -present outbreak in the democratic republic of the congo) enabled real-time and retrospective investigations, using median-joining haplotype network establishment and phylodynamic inferences, to reveal cryptic human ebov transmission chains in humans , , , . in coordination with classical epidemiological data (for example, that obtained by manual contact tracing), individual virus transmission events identified by genomic analysis such as median-joining haplotype networks can be temporally and spatially linked to determine likely transmission pathways, including the mode of virus diffusion and the identification of 'superspreaders' (as reviewed in ref. as the quality and quantity of avail able, complete genome sequences improves, new methods identifying intra-host snps may provide more granular analyses of person-to-person transmission than previous techniques . such snps can distinguish between almost identical consensus sequences of two or more patients . however, acute infections resulting from direct contact with a recently infected and symptomatic individual, and for which primary infection occurred < days before the onset of symptoms, is not the only route for sustained person-to-person transmission during filovirus disease outbreaks. ebov initially infects monocyte-derived macrophages and dendritic cells, which disseminate the virus through the circulatory system to all main target organs, including the liver, spleen and kidneys. after infecting and damaging the vascular endothelia, ebov infiltrates the parenchymata of these organs, resulting in focal necroses and inflammation. such damage can eventually lead to multi-organ dysfunction syndrome and ultimately death , . however, in some cases, the intrinsic, innate and adaptive immune responses can contain viral replication and dissemination, resulting in the survival of the patient . until recently, it was hypothesized that survivors of filovirus diseases effectively abolished filovirus infection. however, new evidence indicates that ebov can persist in certain sites of the body in the absence of viraemia and that this persistence could cause disease flare-ups (for example, see ref. ). some of these sites, including the brain, eyes and testes, are immune-privileged box | endogenous filovirus elements the bioinformatic analysis of higher animal genome sequences, the number of which is steadily increasing, reveals that a marked percentage of these sequences are derived from ancient retroviruses . many animal genomes are mosaics that are likely to have evolved through the accidental integration of retroviral genes or gene fragments into germ cell genomes and the subsequent inheritance of this genetic material by descendants. in some cases, these sequences were positively selected for and were (or are still) expressed and their functions were exapted by hosts for novel functions . a famous example of exaptation is the use of the human endogenous retrovirus w-derived syncytin, which was once a retrovirion surface glycoprotein that mediated virion host cell entry but is now essential for placental morphogenesis in pregnant women . during the past decade, scientists have been increasingly aware that such 'viral fossils' or 'paleoviruses' can derive from viruses other than retroviruses. indeed, non-retroviral integrated rna viruses (nirvs; also known as endogenous viral elements) were derived from the ancestors of numerous extant virus families . prominent examples of negative-sense rna virus-derived nirvs are bornavirus sequences, which are found in the genomes of bats, fish, hyraxes, marsupials, primates, rodents, ruminants and elephants , , and rhabdovirus sequences, which are found in the genomes of crustaceans, mosquitoes, ticks and plants , . interestingly, filovirus-derived nirvs also appear to be widespread as they have been located in the genome of afrosoricids, bats, eulipotyphlans, marsupials and rodents , , . the function of these stably inherited filovirus sequences remains to be determined. however, the existence of nirvs indicates that filoviruses are at least several million years old and that highly divergent mammals were exposed to, and at least occasionally infected by, these viruses and perhaps by the descendants of these viruses that exist today. the novel use of an evolved trait for a different function. a minimum spanning tree analysis of recombinant-free genomic sequences that infers ancestry-descendant relationships using haploid genotypes that can be visualized in a single unrooted, reticulate network. the study of how evolutionary processes interact with epidemiological and immunological factors to influence phylogenetic estimations. because they are physically separated from tissues and cells that are under immune surveillance ( fig. ) . thus, foreign antigens such as ebov particles are tolerated within these sites without eliciting an inflammatory immune response . prior to the outbreak of evd in western africa, evidence of persistent ebov infection had been sparse probably owing to the small number of spillover events arising from this persistence ( fig. ). nevertheless, infectious ebov and marv and/or filoviral rna had been detected in the eyes and semen of convalescent survivors prior to this outbreak [ ] [ ] [ ] [ ] . the large pool of survivors following the - outbreak of evd (specifically, , individuals) was different. disease flare-ups or re-emergences were reported in at least nine individuals and attributed to sexual transmission or breast-feeding . sexual transmission of persistent ebov was implicated in the initiation of new ebov transmission chains , , [ ] [ ] [ ] . genomic analyses revealed that the evolutionary rate of ebov persisting in testes during convalescence is reduced relative to the rates of ebov persisting in blood and plasma , . all these observations prompted a notable number of studies of the long-term effects of persistent ebov infection in evd survivors. indeed, ebov persistence can be accompanied by various sequelae, colloquially often referred to as 'post-ebola [virus] syndrome' . studies of ebov persistence in humans and experimentally in nhps have revolutionized our understanding of ebov infection and changed the guidelines of clinical operation as well as the recommendations of the world health organization for evd survivors. the impact of genomics on understanding the persistence of ebov is broader than next-generation sequencing; advances have also revolutionized the field of pathology by allowing the rapid exploration of transcriptional expression in a chosen site of filovirus infection. the development of multi-labelled and targeted 'probes' that allow multiplex immunopathological hybridizations in sites of interest have also boosted our knowledge of ebov persistence. indeed, studies using novel histopathological tools have benefited genomic research in immune-privileged and non-immune-privileged sites. immune-privileged sites. various neurological complications have been noted in survivors of evd . in experimentally infected rhesus monkeys (macaca mulatta), encephalitic ebov persistence is always accompanied by various degrees of encephalitis or meningoencephalitis . persistent infection of ebov in the brain may lead to evd relapse and late-onset encephalitis in human survivors several months after acute disease , . in experimentally infected nhps, ebov enters the brain by breaking down the blood-brain barrier by directly infecting and damaging endothelial cells (fig. a) . interestingly, ebov primarily infects and persists in microglia . ocular complications, including uveitis, are some of the most common findings during evd convalescence , and persistent ebov and persistent marv have been isolated from the aqueous humour of human survivors with uveitis , . in experimentally infected nhps, ebov infects blood vessels during the acute phase of infection and later infects parenchymal eye tissues. however, in rhesus monkeys surviving the experimental infection of ebov with various degrees of uveitis, retinitis and vitritis, ebov only persisted in cd + cells (monocytes or macrophages) in the vitreous chamber and in the inner limiting membrane of the retina to which it is adjacent (fig. b ). whether ebov isolated from the aqueous humour of human survivors originates from the vitreous chamber and its adjacent structures, as appears to be the case in nhps, remains unknown . the first recorded sexual transmission of a filovirus occurred in , when a male survivor of marburg virus disease transmitted marv to his wife . ebov genomic rna was repeatedly detected in the semen of evd survivors up to months after acute infection , , , [ ] [ ] [ ] , and infectious ebov was isolated from a few semen samples . ebov infects the seminiferous tubules of both human and nhps ( fig. c) , which are the immune-privileged sites of sperm production, during the acute phase of disease . persistent ebov infection was detected in the epididymis of a single rhesus monkey survivor with epididymitis, whereas marv persistence in seminiferous tubules was multifocal in of crab-eating macaques that survived , . recent studies indicate that testicular persistence is not restricted to filoviruses, as potential cases of the sexual transmission of crimean-congo haemorrhagic fever virus (cchfv; an orthonairovirus of the family nairoviridae) and uveitis inflammation of the uvea (the pigmented layer between retina and the fibrous layer composed of sclera and cornea of the eye). over the past two decades, high-throughput benchtop platforms (for example, sequencing (roche), miseq, nextseq and hiseq (illumina), sequel and rs (pacbio) and ion torrent (thermo fisher)) have been the backbone of metagenomics and targeted sequencing approaches for pathogen identification . genomic centres generally had to allocate a notable portion of space to house and maintain sequencers and transporting sequencers was impractical. for example, miseq, which is the smallest of the sequencers, weighs kg and occupies ~ , cm . recently, 'capacity building' efforts have established on-site genomics centres in low-resource settings , . however, the international shipping of oversized sequencers that rely on precise optical alignments for functionality can be challenging and cost-prohibitive and may cause irreparable damage to the sequencer. the maintenance and repair of internationally shipped sequencers may also be challenging as they do not come with in-country service contracts from the manufacturers. moreover, in low-resource settings, trained local staff may not be permanent, and they will periodically require guidance and retraining in sample handling and storage and in how to ensure the continued service and activity of the sequencers. the technological improvements in, and practicality of, portable sequencing technologies have been embraced in recent viral disease outbreaks, including in the -present ebola virus disease outbreak in the democratic republic of the congo and a lassa fever outbreak in nigeria. the iseq (illumina) and minion (oxford nanopore) platforms both conform to on-site field requirements as they are miniature, rapid and easy to use. the zibra zika virus (zikv; a flavivirus of the family flaviviridae) sequencing project in brazil also demonstrated the feasibility of a 'trailer laboratory', but produced limited complete genome sequences owing to the small amounts of zikv rna in clinical samples . several commercialized mobile laboratories incorporated into trucks or trailers shield equipment and temperature-sensitive reagents from austere conditions while providing ample power and laboratory workspace for sequencing. within such mobile laboratories, smaller pcr thermocyclers, such as the programmable mini thermal cycler with a smartphone (minipcr bio™), and miniature centrifuges further bolster portability and ease space requirements. other innovations that limit the need of a 'cold chain' for supplies will greatly improve on-site sequencing in hot climates and remote communities that are prone to outbreaks of ebola virus disease. arenaviridae) have been reported [ ] [ ] [ ] . sertoli cells, the supporting cells of spermatogenesis, are the main cellular reservoir of testicular marv and cchfv persistence in experimentally infected nhps , , and they may also be the reservoir of testicular lasv. the majority of cases of filovirus persistence have been associated with immuneprivileged sites. however, in several evd flare-ups originating from asymptomatic survivors of evd that were persistently infected with ebov, identifying the exact sites of viral persistence in the index cases was not possible , . thus, it is expected that sites of ebov persistence that are not immune-privileged will be discovered. ebov can be detected in breastmilk of female survivors of evd and in various tissues, including the blood and liver, of laboratory mice that have been experimentally infected with ebov and have partial immunity up to days post-exposure , . persistence in sites that are not immune-privileged has also been recently reported for cchfv and lasv , . interestingly, cchfv persists within granulomas of nhp survivors with latent tuberculosis . lasv persists in the smooth muscle cells of blood vessels with vasculitis in both crab-eating macaque and domesticated guinea pig survivors , , suggesting that a local altered immunological environment may sustain viral persistence. genomics is uniquely suited to study the pattern of transmission from patients who are asymptomatic or paucisymptomatic. genomic studies of persistent infections revealed distinct evolutionary dynamics that might result in patterns that can when viral diversity cannot be explained by spillover and spatial-temporal estimations, a secondary spillover may be possible (top left). persistent infections through sexual transmission present with low genetic diversity (that is, with a slow evolutionary rate) over periods (bottom right) that are much longer than expected for acute reintroduction at the expected evolutionary rate (top right). a similar analysis was performed during the first discovery of sexual transmission during the - western african evd outbreak , and a theoretical example is shown (bottom right). this example indicates the number of days after the initial presentation of symptoms at which an acutely infected male is sampled (day ) and the day at which he recovers (day ). on day , the sexual partner of this male becomes symptomatic owing to a very similar ebov genotype, confirmed with epidemiological information and visualized using a median-joining haplotype network. help identify the source of the flare-ups . during the - evd outbreak, an unexpectedly low genetic diversity and complementary epidemiological data provided the first evidence of ebov sexual transmission , . recent pathogenesis studies elucidated the persistent infection of ebov and marv in seminiferous tubules , and epididymides , but niche-specific genomic studies (or single-cell sequencing efforts) have yet to be reported. certain ebov mutations may be tissue-specific and may therefore be required to establish persistence; genomics will be key in identifying these mutations. for instance, the sequencing of ebov genomes present in the plasma and cerebrospinal fluid of a patient with evd during a relapse revealed that only two non-coding changes to the genome had occurred compared to the originally obtained ebov genomes sequenced from plasma during the acute phase of disease . multiple or even single nucleotide changes may permit ebov to transverse the blood-brain barrier by directly infecting endothelial cells and microglia cells and establishing a persistent infection . although ebov and marv have been detected and isolated from the aque ous humour of evd survivors with uveitis , and in breastmilk of survivors , genomic studies have not yet investigated the evolutionary dynamics in these niches. in nature, diverse biotic communities constantly interact and evolve within an ecosystem in response to environmental factors. microorganisms, pathogenic or not, participate in these relationships and constantly react and evolve to changes within the environment. in highly complex organisms such as mammals, immune responses against viruses are inherently complex and involve humoural and intracellular mediators and diverse cell types. in turn, viruses such as ebov evolve sophisticated and diverse strategies to evade the host immune response. by studying host-pathogen interactions, we may improve our understanding of the mechanisms that govern infection, immunity and immune evasion. even though ebov is highly virulent and lethal in humans, some individuals survive infection. furthermore, some individuals who were exposed to ebov or who tested seropositive for the virus never reported disease. the seroprevalence rate of these individuals, categorized as asymptomatic or paucisymptomatic, has been reported to be > % throughout africa [ ] [ ] [ ] [ ] [ ] [ ] . transcriptomics offer insight as to why certain factors, such as the source, viral load, and infectivity of ebov or host genetics, contribute to the range of disease severity and survivorship in patients. host 'immune gene signatures' in patients infected with ebov/mak have been associated with clinical prognosis , . indeed, data generated from patients infected with ebov/mak ( survivors and fatal cases) revealed that interferon response-related genes and acute-phase responses were dysregulated in patients who did not survive . however, natural killer cell populations were increased in evd survivors, suggesting a crucial role for natural killer cells in controlling ebov infection. furthermore, low levels of inflammation and robust t cell responses with an upregulation of cytotoxic t lymphocyte-associated protein (ctla ) and programmed cell death (pd ) expression in t cells also correlated with survival from evd . additional transcriptomic studies focusing on the population dynamics of ebov in infected humans and possibly in naive nhps will complement earlier characterization of host responses to evd and provide a greater understanding of the mechanisms of evd pathogenesis and disease outcome. bringing transcriptomics tools to the field, facilitated by our ability to perform genomic sequencing in outbreak areas, will allow the promises of precision medicine to be realized in an outbreak setting. the genomic characterization of an outbreak pathogen is only the first step in characterizing a threat, a process that can be facilitated by reverse functional genomics. classical reverse genetics has focused on the virus rescue of filoviruses based on cloning a reference (that is, a consensus) filovirus sequence or using a replicating filovirus isolate to clone filovirus sequences , . however, synthetic reverse genomics can rapidly rescue individual virus haplotypes or genotypes from a virus population in the absence of replicating isolates, using gene and genome synthesis based on sequence information alone. these rescued viruses can be used to evaluate the functional aspects of individual filovirus genome mutations. thus, functional genomics can facilitate the rapid and precise functional characterization of a newly emerging filovirus or filovirus mutant. given that natural filovirus isolates must be studied while fulfilling strict biosafety and biosecurity requirements that are frequently not available in areas of filovirus disease outbreaks, the availability of in-country and field-deployable sequencing platforms increases the ability of researchers to gather crucial information about the virus without the need to export biological specimens across national borders. to succeed, field sequencing needs to produce highly accurate finished filovirus genome sequences (for example, sequences that include the complete genomic leader and trailer regions of the virus) from a variety of sample sources , , . filoviruses rescued from synthetic reverse genetics systems can then be used to study host adaptation and attenuation as well as the efficacy of therapeutics - . the de novo generation of ebov from the ongoing evd outbreak in the democratic republic of the congo (that is, of ebov/"itu") demonstrates the value of synthetic reverse genetics (fig. ). after the development of modular reverse genetic systems that are even more efficient than those available today, even high-throughput phenotypical characterizations of large numbers of minimally divergent filoviruses are likely to be possible even in the absence of a biological isolate. multiple mcms against ebov infection have been developed, including small molecules, monoclonal antibodies (mabs), antibody cocktails and vaccines , and clinical trials have yielded promising results. however, the population dynamics and evolution of ebov are influenced by mutations in rna, rna recombination rates, virus population bottlenecks, natural selection and fitness (including diversifying and purifying selection), host range and mode of transmission . these traits re-adjust depending on the environment in a natural host, a naive accidental host, a previously exposed accidental host (owing to infection or vaccination) or an accidental host that received an mcm. therapeutic pressures will force the selection of individual ebov genotypes, and thereby adaption, to ensure virus survival or persistence. highly accurate genomic data can be used to evaluate available mcms in silico prior to in vitro and in vivo testing and to characterize host responses to specific therapeutic interventions in real time. the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. for example, accurate sequencing data from ebov/"itu" allowed for the rapid in silico assessment of the ability of the mab (national institutes of health) and zmapp (mapp biopharmaceutical) mabs to bind to the receptor-binding domain of the ebov spike glycoprotein gp , , predicting that these mabs should be effective against circulating ebov/"itu" . using genomic approaches to understand how mcms influence ebov population dynamics and to identify mutant viruses that escape mcms could augment future therapeutic designs and filovirus-targeting strategies. correlates of protection for different vaccines and vaccination regimens against ebov have not been fully determined. however, both humoural and cellular immunity independently correlate with the protection of nhps from ebov infection and disease , . the use of a recombinant vesicular stomatitis indiana virus vaccine expressing ebov gp , , rvsvΔg-zebov-gp (sold under the brand name ervebo), the first ebov vaccine to be approved by the fda , , diminished evd case fatality rates in a limited ring-vaccination trial performed in guinea in (ref. ) and it is currently administered to people living in the area of the current evd outbreak. however, unfortunately, some vaccinated individuals still present with clinical signs of mild evd. the exact reasons for these 'breakthrough cases' are not completely understood; however, they are thought to result from ebov infection within the first days after vaccination. a breakthrough could be due to the individual having an insufficient immune response to control the virus (that is, when the vaccine induces a low titre of anti-ebov antibodies and/or scarce effector cells) or to ebov adapting to escape the immunological pressures produced by the vaccine. the characterization of the genomic ebov population in patients who developed evd after vaccination could help address these questions. immunotherapy has also been successfully used as a therapy for evd, most notably during the ongoing outbreak in the democratic republic of the congo . the pamoja tulinde maisha (palm) randomized controlled trial recently evaluated four investigational therapeutics (zmapp, remdesivir (gilead sciences, inc.), mab and regn-eb (regeneron pharmaceuticals)) in the treatment of evd . mab (a single mab) and regn-eb (a cocktail of three mabs) were significantly more effective than zmapp (a cocktail of three mabs) reverse functional genomics facilitates external support in the response to outbreaks of ebola virus disease (evd) that occur in remote areas that lack in-house resources to test available medical countermeasures. rapid, high accuracy , complete genome sequences determined in-country are shared with out-of-country collaborators to evaluate key genomic and proteomic changes in ebola virus (ebov) that may affect the efficacy of available therapeutics (part a). for example, changes in the double-stranded rna (dsrna)-binding site of the ebov polymerase cofactor viral protein (vp ), a protein targeted by several therapeutics, can be identified from the sequencing data obtained for a new ebov isolate. changes in this region of vp may compromise the efficacy of treatments. indeed, if two available therapeutic agents (hypothetical treatment a and hypothetical treatment b) target vp , reverse genetic systems can produce replicative ebovs de novo that contain the changes identified in vp (part b). these replicative ebovs can then be used for the in vitro and in vivo therapeutic evaluation of both hypothetical treatments; data obtained using this approach can inform on which treatment is potentially more efficacious against the ebov isolate causing the outbreak. in treating evd (p = . and p = . , respectively) and were also more effective than remdesivir (a nucleoside analogue); the effect of zmapp and remdesivir on evd was not significantly different. mab , obtained from a evd survivor from the democratic republic of the congo, is one of several recently identified ebolavirus-neutralizing antibodies that are being developed as therapeutics for evd [ ] [ ] [ ] [ ] . this mab protected nhps from ebov-induced disease and death, even when administered days after inoculation with ebov . as mab binds to the ebov glycoprotein gp , , the risk of escape mutants arising might be constrained by viral fitness. regn-eb also showed promising results in ebov-exposed nhps, and no mutant viruses were reported to escape this mcm , . by contrast, ebov mutants that escape zmapp have been characterized . a mutation in amino acid residue of ebov gp , abrogated its binding to two of the mabs within zmapp, and a mutation in amino acid residue of ebov gp , abrogated its binding to the third mab . moreover, epitope mapping of ebov gp , using an alanine-scanning assay identified several residues that are crucial for antibodies to bind to ebov gp , , although none of these mutations was tested for fitness. ( ) . as ebov continually replicates, expanding intra-host diversity can create heterogeneous subpopulations ( , blue circles). once exposed to treatment ( , dashed line), ebov intra-host populations may be countered and eliminated, demonstrating an effective treatment without further intra-host replication or diversification (green circles). however, a small subpopulation surviving the therapeutic bottleneck may allow escape mutants to subvert treatment ( ) and continue diversifying into novel, treatment-resistant genotypes ( , red circles). (~ - ) reduces the analytical power of comparative virus-host interactions and inter-host population-level genomics. in addition, experiments aimed at understanding the appearance of antiviral resistance are restrictive; precise protocols to measure and evaluate ebov and the host immune response to infection without violating established policies are urgently required. furthermore, the possibility that a more virulent or less treatable virus may evolve during such experiments and be accidently released is a concern, which is why institutional biosafety committees are cautious in approving experiments aimed at creating such viruses. additionally, concerns may arise from other nations that are parties of the biological weapons and toxins convention. convincing national rivals that experiments resulting in ebov strains that are resistant to mcms were actually intended to strengthen mcms, and not to deliberately create biological weapons, is not straightforward. the advancement of targeted and unbiased genomic sequencing has profoundly impacted our biological knowledge of, epidemiological preparedness for, and response strategies to known and unknown high-consequence filovirus infections. greater investments in in-country genomic infrastructure and mobile sequencing platforms have reduced the time from sample to viral genome sequence and simultaneously informed local clinicians, epidemiologists and public health officials of genome-guided information on the virus responsible for a current outbreak. advances in large-scale and timely data generation encourage future genomic studies to go beyond consensus-level pathogen genome sequencing during an outbreak. for example, persistent infections in immune-privileged sites of survivors can be characterized using single-cell sequencing technologies. additionally, an increased focus on host responses in an infected individual and the contribution of host genome characteristics to disease outcome and transmission would potentially further benefit the development of mcms and improve the outbreak response. complete pathogen-host genomic investigations could be applied to infected individuals, nhps and known natural host reservoirs of filoviruses. the successful integration of current and future genomic tools will rely on the establishment of new long-term partnerships between government, academia and public health agencies and on maintaining in-country genomic capabilities where the threat of filovirus outbreaks is imminent. in silico modelling using the three-dimensional structure of ebov gp , was used to predict which mutations in ebov would enable it to escape antibody binding , . initially, mab kz was analysed, followed by mab , mab and mab f - - . a list of mutations predicted to interfere with kz -ebov gp , binding was expanded to mutations predicted to disrupt the binding of other mabs to ebov gp , . three of the predicted mutations were found in ebovs infecting humans or experimental animals , , showing that viruses may escape current mcms under investigation. despite the proven efficacy of two therapeutics assessed in the palm trial, namely mab and regn-eb , none of the treatments achieved complete protection against evd . we hypothesize that the characterization of the ebov population in individuals who did not benefit from treatment in the palm trial might help elucidate the reasons for this failure. a clear demonstration of the power of genomics for this purpose was the investigation of two nhps that succumbed to experimental ebov exposure despite experimental treatment with mb- , a cocktail of three mabs. two clusters of genomic mutations (a cluster of five non-synonymous changes in one animal and a cluster of four non-synonymous changes in the other) reduced the binding of the three mabs to ebov gp , ( fig. ). viral isolates obtained after mb- treatment recapitulated the observed 'escape' phenotype. the mutations, all located in the ebov glycoprotein (gp) gene, abrogated antibody binding to gp , . although this study was restricted in size, it highlights mechanisms by which ebov can evade immuno therapies. first, adaptation to a selection pressure can occur rapidly. second, methods to monitor the appearance and phasing of viral mutants that can escape immunotherapies are crucial. third, with the use of reverse genetics systems, scientists can acquire pertinent information on the therapeutic efficacy of sequence-based mcms during an ongoing outbreak of evd (fig. ). for example, phenotypical changes resulting from clade-defining non-synonymous mutations (for example, alanine to histidine substitution at residue in nucleoprotein or isoleucine to methionine substitution at residue in viral protein , both in ebov/"itu") or positively selected non-synonymous mutations identified in the ongoing outbreak can be investigated. unfortunately, not many studies have investigated the resistance of ebov to candidate vaccines or to mcm. cost is a consideration, but 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ancient and integrated into mammalian genomes sequence-based evidence of a long co-evolutionary history of filoviruses and mammalian filovirus hosts clinical metagenomic next-generation sequencing for pathogen detection establishment and cryptic transmission of zika virus in brazil and the americas we thank laura bollinger (nih/niaid integrated research facility at fort detrick, frederick, md, usa) for critically editing the manuscript and william discher (usamriid, fort detrick, frederick, md, usa) for the diagram used in fig. . in no event shall any of these entities have any responsibility or liability for any use, misuse, inability to use or reliance upon the information contained herein. the us departments do not endorse any products or commercial services mentioned in this publication. these authors researched data for the article, contributed to the writing and discussion of the content, and reviewed and edited the manuscript prior to submission, focusing on the following sections: introduction (n.d., j.h.k. and g.p.); identifying filovirus reservoirs (j.h.k.); identifying and characterizing outbreaks (n.d., j.h.k., g. p. and x.z.); genomics in threat characterization (n.d. and g.p.); and genomics in prevention and therapy (m.s.-l. and g.p.). all of the authors contributed to the conclusions. all authors declare no competing interests. nature reviews microbiology thanks miles carroll and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. fda news release on the first fda-approved vaccine for the prevention of ebola virus disease: https://www.fda.gov/ news-events/press-announcements/first-fda-approvedvaccine-prevention-ebola-virus-disease-marking-criticalmilestone-public-health key: cord- -on zu w authors: falcinelli, shane d.; chertow, daniel s.; kindrachuk, jason title: integration of global analyses of host molecular responses with clinical data to evaluate pathogenesis and advance therapies for emerging and re-emerging viral infections date: - - journal: acs infectious diseases doi: . /acsinfecdis. b sha: doc_id: cord_uid: on zu w [image: see text] outbreaks associated with emerging and re-emerging viral pathogens continue to increase in frequency and are associated with an increasing burden to global health. in light of this, there is a need to integrate basic and clinical research for investigating the connections between molecular and clinical pathogenesis and for therapeutic development strategies. here, we will discuss this approach with a focus on the emerging viral pathogens middle east respiratory syndrome coronavirus (mers-cov), ebola virus (ebov), and monkeypox virus (mpxv) from the context of clinical presentation, immunological and molecular features of the diseases, and omics-based analyses of pathogenesis. furthermore, we will highlight the role of global investigations of host kinases, the kinome, for investigating emerging and re-emerging viral pathogens from the context of characterizing cellular responses and identifying novel therapeutic targets. lastly, we will address how increased integration of clinical and basic research will assist treatment and prevention efforts for emerging pathogens. e merging and re-emerging viruses pose a significant threat to public health and global economies. moreover, outbreaks caused by emerging and re-emerging viruses continue to increase in frequency as a result of changing socio-economic, environmental, and ecological factors. notably, the zoonotic viral pathogens, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), ebola virus, chikungunya virus, and zika virus, have emerged on a global scale in recent years; although less widely publicized, other emerging viral pathogens such as monkeypox virus and andes virus have led to smaller recurrent outbreaks. a critical challenge for combating these outbreaks is often the discordant relationship between the economic status of outbreak "hotspots" and resource distribution or control capacity within these regions. in addition, the development and delivery of therapeutics for combating such outbreaks have been complicated by both the associated costs in design and development for novel antiinfective therapeutics and the requirements for regulatory approval and licensure. importantly, emerging infectious diseases present the additional inherent challenge that they are only "emerging", and thus limited resources are made available for research until they present a significant risk. for many emerging viral pathogens, the requirement for highcontainment facilities has further impeded widespread research. from the perspective of drug development, the limited knowledge and understanding of molecular pathogenesis for these agents is a daunting challenge to overcome when outbreaks emerge. in the face of an increasing burden of emerging and reemerging pathogens, it is necessary to overcome the barriers imposed by a paucity of information regarding molecular pathogenesis for these agents. omics-based approaches present a mechanism to rapidly generate large amounts of data in regard to host responses and assist in target identification for drug development efforts. in addition, omics-based analyses allow for the characterization of molecular events that mitigate cellular responses to viral pathogens from a global perspective across multiple levels of cellular complexity (individual cell types < tissues < organs). high-throughput global analyses of host gene expression, including microarrays and rna-seq, provide important information regarding transcriptional responses during infection. although these validated approaches are among the most widespread of the omics-based technologies for infectious disease investigations, they do not provide a direct measure of the activation status of the cell signaling pathways that regulate underlying cellular responses. in contrast, global investigations of cellular kinase activities (the kinome) are able to provide insight into the activation status of cell signaling networks (including those that mediate pathogen recognition and innate immune activation, cell cycle activities, metabolic status, wound healing and repair, and cell death) at the level of individual kinase-mediated phosphorylation events. in addition, kinome investigations allow for potential identification of kinase drug targets. kinases are currently one of the top targets for drug design and development, and there is potential for repurposing of kinase inhibitors with existing regulatory approval. as emerging viral infections often result in severe illness including respiratory failure [severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza] and multiorgan failure [ebola virus disease (evd)], understanding complex pathogenesis of these infections is required for effective vaccine and therapeutic design and for improved patient care. healthcare providers caring for patients with severe emerging viral infections are generally focused on clinical care and biosafety as compared to the complex molecular events that underlie pathogenesis. in contrast, basic researchers typically focus on discrete aspects of pathogenesis through a variety of in vitro and in vivo analyses rather than the complex interplay between these events and the clinical, physiologic, and pathologic abnormalities observed by the clinician. integrating basic and clinical research is needed to accelerate the translation of knowledge for emerging infections toward vaccine development and therapeutic discovery. specifically, detailed natural history studies merging multiple data streams including omics approaches (high-throughput gene expression and kinomics) and focused translational investigations utilizing relevant models that can be validated to human disease are needed to clarify disease pathogenesis, advance therapeutic discovery, and facilitate regulatory approval. although an integrated approach between basic and clinical research is ideal for investigating the connections between molecular and clinical pathogenesis, there has been a paucity of investigations for which this has been undertaken. here, we will discuss emerging pathogens for which there is available information regarding the clinical course of disease, host immune responses during natural infection, and molecular information regarding the global cellular responses to infection, with particular attention on host kinome investigations. in this regard we will focus on the emerging viral pathogens mers-cov, ebola virus (ebov), and monkeypox virus (mpxv) (figure ). we will review the clinical presentation and immunological and molecular features of the diseases and summarize available omics data informing pathogenesis of these pathogens. lastly, we will discuss the benefit of improved integration of available clinical knowledge or data regarding the pathologic manifestations of disease with basic research investigations to advance treatment and prevention of severe emerging viral infections. global gene expression investigations have provided information regarding host response to emerging and re-emerging review pathogens at the level of individual genes or gene clusters. however, there is a paucity of information regarding the relationship of cell signaling networks, and in particular their activation status, with the biological/pathological events that occur throughout infection. it has been well established that many biological processes can be regulated independent of transcriptional or translational changes through post-translational modification (ptm) events. indeed, kinase-mediated phosphorylation of proteins, in which kinases catalyze the transfer of the γ phosphate group from atp to the hydroxyl group of a specific ser, thr, tyr residue, is figure . generation of kinome peptide array targets and kinome peptide arrays: ( ) species-specific proteomic or genomic information from a diverse range of species can be used to identify kinase recognition motifs that are composed of a central phosphorylation target and the surrounding amino acids (normally + and − amino acids from the central phosphorylated residue); ( ) peptides that comprise the kinase recognition motifs identified in ( ) are synthesized and covalently linked to a glass surface. peptide targets are spotted in replicates of three to nine spots on each array to account for intra-array variability. individual amino acids of the peptides are represented by orange, red, purple, and green spheres. ( ) biological samples are processed to generate cell lysates that are activated with atp and applied to the kinome peptide array. ( ) following the application of the cell lysate, activated kinases in the cell lysate will recognize their respective kinase recognition motifs and phosphorylate the central phosphorylated residue of the peptide. ( ) kinome peptide arrays are subsequently stained with a phospho-specific fluorescent stain and imaged followed by comparative bioinformatics analyses. tissue and cell images were derived and/or modified from servier medical arts under a creative commons attribution . unported license. among the most thoroughly characterized ptm. virtually all cell signal transduction events are regulated by kinases independent of biological complexity of the host (i.e., prokaryotes and eukaryotes). lending further credence to the biological importance of kinases, > kinases have been identified in the human genome, and ∼ % of the human proteome is modulated by kinase-mediated phosphorylation events. , thus, considering the central role of kinases in a broad range of cellular processes (including growth and development, metabolism, and immune responses), it has been postulated that the activities of individual kinases may represent more reliable predictors of cellular phenotypes than transcriptional or translational changes. indeed, transcriptional or translational-based omics approaches are often unable to account for regulatory events including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/ substrate subcellular sequestration, or protein activation/ repression ptms. given the central role of kinases in the regulation of biological processes, kinases are a logical drug target. as a testament to this, kinase inhibitors have been granted licensure by the u.s. food and drug administration (fda) for a broad range of malignancies, and there are a continually increasing number of kinase inhibitors that are in various stages of preclinical trials. furthermore, kinases are the second most frequently targeted gene class in cancer therapy after the g protein coupled receptors. , the recent prioritization for the repurposing of approved therapeutics for alternative malignancies by the national institutes of health center for advancing translational sciences (ncats) , also provides considerable impetus for the investigation of licensed kinase inhibitors as infectious disease therapeutics. concerns exist regarding the therapeutic application of kinase inhibitors as novel therapeutics for infectious disease, in particular to the potential immunosuppressive effects following prolonged treatment. however, it should be appreciated that the application of kinase inhibitors in such cases would need to be targeted in terms of timing and dose, with appropriate molecular biomarkers guiding initiation and cessation. it should also be appreciated that the clinical symptoms associated with many emerging and re-emerging pathogens have been associated with dysregulated host immune responses (in particular pro-inflammatory responses). thus, the global analysis of the activation state of host kinases (the kinome) can provide critical insight into the specific activation state of individual kinases, cell signaling pathways, or larger biological networks. in addition, kinome investigations may offer important, and predictive, insight into the cellular mechanisms that regulate phenotypic changes within cells. as many kinases recognize a particular phosphorylation motif composed of the central phosphoacceptor site and the amino acids + and − residues from the central phosphorylation site, peptides representing this kinase target motif can be synthesized with relatively high efficiency and low expense. indeed, kinase target motif peptides have been shown to be appropriate substrates for their respective kinases with v max and k m values approaching those of the fulllength protein. thus, peptide kinome arrays can be constructed in an analogous manner to traditional dna microarrays where kinase target motif peptides are spotted onto a glass slide representing hundreds to thousands of unique peptide targets for kinases ( figure ). following this, samples in the form of cellular lysates from whole organs, tissues, or individual cell types can be applied to the kinome peptide arrays, allowing for the phosphorylation of specific peptide targets by kinases within the lysate (figure ). the development of kinome-specific bioinformatics analysis software, including the platform for intelligent, integrated kinome analysis (piika), has provided a mechanism to identify the complex patterns of kinase-mediated phosphorylation events and quantitate the differences between compared conditions. clinical findings during mers-cov infection. syndromic case-definition for mers infection requires a compatible clinical syndrome and an epidemiologic risk factor including travel to an affected region or contact with a known or suspected case. initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of days, which can range from to days. mildly symptomatic or possibly asymptomatic infections have been reported, and progression to severe disease is associated with pre-existing medical conditions including cardiopulmonary disease, obesity, and diabetes. most ( %) of reported mers cases are among adults, with a median age of years. in severe cases respiratory failure requiring mechanical ventilation typically occurs within days of symptom onset. laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes. − high lactate levels and consumptive coagulopathy have also been reported. , chest radiographic abnormalities are observed in most cases consistent with viral pneumonitis, secondary bacterial pneumonia, or acute respiratory distress syndrome. cytokine levels in serum and bronchoalveolar lavage (bal) from two mers patients, one with fatal disease and one who survived. higher levels of retinoic acid-inducible gene (rig- ), melanoma differentiation-associated protein (mda ), interferon regulatory factor (irf)- and - , interleukin (il) a, and il- and lower levels of il- and ifnγ were observed in the fatal case compared with the survivor. more recently, min et al. performed a temporal analysis of cytokine, chemokine, and growth factor blood levels from patients during the recent outbreak of mers in south korea. the patients were subcategorized into four groups on the basis of disease severity: group i patients developed fever and recovered. group ii patients developed mild pneumonia without hypoxemia. group iii patients had prolonged and severe pneumonia. group iv patients had severe pneumonia and acute respiratory distress syndrome. group iv patients included five fatal cases of mers; all patients in groups i−iii fully recovered from illness. ifnα was elevated in all groups and largely peaked during the second week of illness. granulocyte-colony stimulating factor (g-csf) and granulocyte macrophage (gm)-csf were similarly elevated across all patient groups; however, patients with fatal disease had reduced gm-csf responses following antiviral treatment as compared to patients that recovered. patients with pneumonia had relative elevations of il- , tumor necrosis factor (tnf)-α, il- , and il- during the second and third weeks of illness. elevated il- and il- appeared to trend positively with the severity of illness. a robust induction of multiple chemokines was found in most patients. notably, eotaxin and regulated on activation, normal t expressed and secreted (rantes) was elevated in all patients. in contrast, il- , monocyte chemotactic protein (mcp)- and macrophage inflammatory protein (mip)- β were more prominent in groups ii and iii as compared to groups i and iv. furthermore, elevated interferon gamma induced protein (ip)- correlated with the development of pneumonia (groups i−iii). multiple growth factors, including epidermal growth factor (egf), fibroblast growth factor (fgf)- , vascular endothelial growth factor (vegf), and tgf-α, were significantly elevated across all patients; however, egf was significantly higher in patients that recovered from disease as compared to the fatal cases. further evaluations are needed to characterize the natural history of immune response during acute mers-cov infection and recovery. transcriptome analyses of mers-cov. in an effort to better characterize mers-cov pathogenesis in the absence of available samples from human patients and, in particular, address pathologic changes associated with infection, multiple animal species have been employed in mers-cov investigations. while multiple small animals are not susceptible to mers-cov infection, rhesus macaques and marmosets develop mild to severe lung pathology following experimental infection. transcriptome analysis in mers-cov-infected rhesus macaques revealed that genes related to antiviral immunity, chemotaxis, and inflammation were overexpressed in lesional versus grossly normal lung tissue at days postinfection. a significantly smaller number of differentially expressed genes was found on day postinfection with no obvious trends following pathway enrichment analysis. significant changes in the transcriptome profiles of peripheral blood mononuclear cells (pbmcs) were observed at only day postinfection. this global analysis suggests a key role for an initial rapid innate immune and inflammatory response (through pattern recognition receptors) followed by rapid resolution. a study of mers-cov-infected marmosets evaluated lung lesions by rnaseq at days − postinfection. pathway analyses demonstrated that chemotaxis and cell migration, cell cycle progression, and cell proliferation and fibrogenesis were highly over-represented relative to uninfected controls. to a lesser degree, pathways associated with inflammation, vascularization, endothelial activation, proliferation of smooth muscle, and tissue repair were also overrepresented in infected animals. differences were most significant on days and postinfection during illness progression relative to day . recently, menachery and colleagues examined the interaction between mers-cov emc/ and the host ifnstimulated gene (isg) response by transcriptomics. isg responses in mers-cov infected calu cells, a lung adenocarcinoma cell line, had no discernible induction initially upon infection but were up-regulated by h postinfection. down-regulation of a subset of isgs resulted in altered histone modifications, a potential epigenetic contributor to early impairment of antiviral cellular defenses. in a separate analysis genetically distinct mers-cov strains, mers-cov sa and mers-cov eng , produced distinct gene expression profiles in calu- cells. these analyses may better inform early host-cell antiviral responses and the impact of viral evolution on these and other complex biological responses. proteomics analysis corroborated these transcriptional data with induction of isgs observed h postinfection. significantly reduced levels of stat and pkr compared with uninfected controls were also noted. differential host transcriptome responses to mers-cov sa and mers-cov eng highlight both the propensity of emerging viral pathogens to evolve rapidly and the importance of additional host response analyses for augmenting and clarifying such complex biological responses. kinome analyses of mers-cov infection. host responses to mers-cov infection through kinome analysis were recently assessed using huh- cells, an immortalized human hepatocyte cell line, that are highly permissive to mers-cov infection. temporal analysis of kinome responses by peptide arrays revealed selective modulation of extracellular signal-regulated kinases (erk)/mitogen-activated protein kinases (mapk) and phosphatidylinositol- -kinases (pi k)/akt (also known as protein kinase b)/mechanistic target of rapamycin (mtor) signaling responses. over-representation analysis (ora) revealed erk/mapk and pi k/akt/mtor signaling responses were consistently up-regulated during infection. multiple erk/mapk family members formed central components of functional networks and signaling pathways throughout infection. similar results were observed for intermediates of the pi k/akt/mtor signaling pathway at and h postinfection, suggesting that modulation of erk/ mapk and pi k/akt/mtor signaling may be important for productive mers-cov infection. downstream analysis of the phosphorylation patterns of pathway intermediates from the erk/mapk and pi k/akt/ mtor signaling supported observations from the kinome analysis. both investigations demonstrated that nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb)regulated family members were important mediators of mers-cov infection. il -and ifn-mediated signalings were also modulated during mers-cov infection, consistent with prior analyses. , these results were also in agreement with in vitro transcriptional analysis of mers-cov infection. prophylactic or therapeutic addition of fda-licensed kinase review inhibitors targeting activated kinases in mers-cov infection impaired viral replication. these hypothesis-generating data may inform directed investigations into mers-cov pathogenesis and, importantly, demonstrate the potential to identify novel host-centric therapeutic targets. ebolaviruses. the filoviridae family of viruses consists of three genera: ebolavirus, marburgvirus, and the newly identified cuevavirus. structurally, filoviruses have a pleomorphic enveloped, filamentous virion particle that encapsulates a negative-sense single-stranded rna genome. ebolaviruses were first described in following disease outbreaks in the democratic republic of congo and sudan and are composed of five viral species, including ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), taı̈forest virus (tafv), and reston virus (restv). sporadic outbreaks of ebov, sudv, bdbv, and tafv have occurred throughout central africa for more than three decades, resulting in thousands of infections. case fatality rates during these outbreaks have routinely exceeded %. isolated outbreaks of restv have occurred outside africa in nonhuman primate facilities in the united states, italy, and the phillipines, and infection results in high morbidity and mortality in nonhuman primates; however, restv has only been associated with asymptomatic infections in humans. although ebolaviruses have been historically associated with isolated outbreaks involving small cohorts of infected patients (< ), an outbreak of evd in west africa beginning in has resulted in , cases and , deaths ( % cfr) as of june (http://www.who.int/csr/ disease/ebola/en/). although virus transmission has greatly decreased in west africa, surveillance for sporadic infections continues. clinical findings in evd. ebov transmission occurs through exposure of infected body fluids or tissues to mucous membranes or nonintact skin. the mean incubation period is − days, ranging from to days. initial signs and symptoms are nonspecific including fever, myalgia, and malaise and cannot be reliably distinguished from other endemic illnesses in africa including malaria and enteric infections. whereas mild illness has been described, most patients develop severe disease within days of symptom onset. massive gastrointestinal fluid losses of up to − l per day due to vomiting and watery diarrhea may result in progressive dehydration and hypovolemic shock. even in the setting of adequate fluid and electrolyte replacement, sequential multiorgan failure may occur. ebov infects multiple organs and cell types throughout the body with the notable exception of lymphocytes that are indirectly depleted early during infection. organ injury due to direct viral or indirect host-mediated responses results in severe complications including meningoencephalitis, uveitis, respiratory failure, secretory diarrhea, disordered coagulation, renal failure, hepatic necrosis, and myositis. the clinical presentation, laboratory values, viral kinetics, and clinical management of evd patients in west africa, europe, and the united states during the − outbreak have been recently well-characterized. soluble immune mediators associated with ebov infections. there is a paucity of information regarding ebov pathogenesis in humans primarily due to the limited frequency of evd outbreaks prior to and limitations presented by sample acquisition from infected patients in the field as well as the overall size of patient cohorts. largely contradictory findings regarding the immune responses in those who survive or succumb to evd have further confounded the under-standing of ebov pathogenesis in human patients. for example, villinger et al. reported that serum cytokine concentrations (including ifnα, ifnγ, tnf-α, il- , and il- ) were elevated in patients with fatal infections in comparison to survivors. in contrast, additional studies have suggested that fatal infections were instead related to general immunosuppression including ifnγ, il- , and il- . − an investigation of sudv infection in humans by sanchez et al. demonstrated limited changes in the expression levels of cytokines, fas antigen, and fas ligand in pbmcs from infected patients relative to those found for uninfected patients. furthermore, an investigation of fatally infected evd patients by wauquier et al. has further confounded the role of host immune responses in fatal evd as hypersecretion of multiple cytokines and growth factors and decreased secretion of t lymphocyte-derived cytokines were associated with fatal disease. transcriptome analyses of ebola virus infection. to date, no investigations of host gene expression in ebov-infected patients have been reported, although limited data are available from animal models of infection or from in vitro investigations. in a study of pbmcs from ebov-infected crab-eating macaques, rubins et al. found few notable changes in the early stages of infection ( − days); however, broad changes were observed over days − post-infection. pro-inflammatory cytokines (il- β, il- , il- , and tnf-α) and chemokines (mip- α and mcp − ) were up-regulated at days − postinfection relative to healthy controls. multiple genes related to apoptosis including bcl- family members, multiple caspases, fas-associated death domain protein, and tnf superfamily member were also up-regulated at late time points. ifn-regulated genes were up-regulated by day postinfection and remained so through study day . yan et al. investigated pbmc gene expression in ebovinfected rhesus macaques with or without anticoagulant administration. untreated animals displayed up-regulation of immune response genes, b cell receptor signaling intermediates, nk cell mediated cytotoxicity, leukocyte activation, and lymphocyte activation compared with anticoagulant-treated animals during the early stages of infection. the expression levels of these gene clusters fell to pre-infection levels at the late-stage of infection. in contrast, genes related to defense responses, apoptosis, wounding, inflammation, coagulation, and leukocyte activation remained elevated during early-and latestage infection. following the isolation of restv from pigs, subsequent investigations have demonstrated that pigs were susceptible to both restv and ebov infection with preferential targeting of macrophages in the lungs. recently, nfon et al. demonstrated that ebov infection in pigs resulted in up-regulation of chemokine expression beginning on day postinfection as compared to mock-infected pigs. the most pronounced changes in gene expression were found on days and postinfection and included the up-regulation of a broad set of cytokines (il- , il- , il- , il- , il- , il- , il- , resistin), chemokines (ccl , ccl , ccl , ccl , amcf-ii, ccl l , ccl ), cell adhesion protein (selectin), antimicrobial protein, palate, lung, and nasal epithelium clone proteins, and pro-apoptotic molecules (multiple caspases, caspase recruitment domain-containing protein (card), apoptosisassociated tyrosine kinase (aatk), fas, fas-associated protein with death domain (fadd), tnf receptor-associated factor (traf ), tnfα-induced protein -interacting protein review (tnip )). in addition, expression of multiple genes related to microbial sensing (pattern recognition receptors) or antiviral responses (isgs) was up-regulated in the lungs of infected animals. although the localization of the cytokine response of pigs and humans or nhps differs during the course of ebov infection (localized responses in the lungs of pigs versus a predominantly systemic response in humans and nhps), the cytokine profiles of pigs, humans, and nhps were quite similar. for example, comparison of nhp and porcine responses during ebov infection demonstrated multiple gene expression similarities between the two species (i.e., il- , il- , caspase family members). it is also likely that direct comparison of both data sets would likely yield many common gene signatures that are conserved in their identity as well as their directionality (upregulation vs down-regulation). macrophages are an early target of ebov infection and support high-level viral replication. ebov attachment and entry into human macrophages in vitro induces pro-inflammatory mediators including il- , il- , and tnf-α as early as h postinfection. noncardiogenic pulmonary edema is a recognized complication of evd, and human autopsy data support that alveolar macrophages are a target of ebov infection. ebov infection of alveolar macrophages in vitro resulted in an early, transient increase in cytokine and chemokine expression, supporting that paracrine-soluble mediators of inflammation may contribute to vascular leakage in the lungs. gene expression responses of ebov-and marvinfected huh cells resulted in the global suppression of antiviral responses, including toll-like receptor (tlr), irf , and protein kinase r (pkr)-mediated pathways. however, signal transducers and activators of transcription (stat) phosphorylation in ebov-and marv-infected cells were differentially modulated. ebov-mediated ifn inhibition has been well characterized and is thought to be attributable to ebov proteins vp and vp . interestingly, restv infection, which does not induce clinical illness in humans, resulted in the activation of > % of the ifn-stimulated genes (isgs). kinome analysis of ebola virus. hepatocytes are an early target of ebov infection, directly contributing to diffuse hepatic necrosis observed in fatal cases. ebov infection of huh cells has been evaluated by kinome analyses, shedding light on liver pathogenesis in evd. ebov infection of huh- cells resulted in temporal modulation of the tgf-β signaling pathway as compared to mock-infected cells. pathway ora demonstrated that multiple tgf-β-mediated signaling pathways were up-regulated at and h post ebov infection. furthermore, these responses were associated with changes in the expression patterns of multiple cellular proteins associated with a mesenchyme-like transition. these included the upregulation of matrix metalloproteinase , n-cadherin, and fibronectin and down-regulation of e-cadhering and claudin . in this process cells lose polarity and cell-to-cell adhesion transforming into mesenchymal stem cells that contribute to wound healing or organ fibrosis; however, the role of these events in ebov infection remains to be elucidated. additional analysis demonstrated that inhibition of pi k/akt, erk/ mapk, or pkc pathways with kinase inhibitors reduced ebov replication when administered prophylactically or therapeutically. supporting this observation, a subset of kinase inhibitors administered to ebov-infected mice reduced lethality. defining mechanisms by which kinase inhibitors show benefit in these models will better clarify their role as potential therapeutics. monkeypox virus. mpxv, a member of the genus orthopoxvirus, causes zoonotic infections with a case fatality rate of ∼ %. mpxv, vaccinia virus (vacv), cowpox virus (cpxv), ectromelia virus, and variola virus (varv), the etiologic agent of human smallpox, comprise the orthopoxviridae family of viruses. mpxv was first isolated in from cynomolgus macaques in denmark; however, human mpxv infections were not recognized until following the isolation of the virus from a suspected case of smallpox infection in the democratic republic of congo. mpxv is composed of two distinct clades that are genetically, clinically, and geographically distinct. the congo basin mpxv (central african mpxv) clade is considered to have both higher lethality and morbidity than the west african mpxv clade as demonstrated from comparative infection models in various animal species (including nonhuman primates, mice, prairie dogs, and ground squirrels) and as well natural infection in humans. although human mpxv infections have been recorded in west africa, the majority of human mpxv infections have occurred in the congo basin region of central africa, largely in the democratic republic of congo. clinical findings in mpxv infections. clinical and epidemiological information regarding human mpxv disease has been derived from enhanced surveillance campaigns in the congo basin. from this work, it has been demonstrated that human mpxv infection and illness largely mirror those of discrete, ordinary smallpox. the incubation period for both viruses (varv and mpxv) is − days with an initial febrile prodromal period of − days. this prodromal period is normally accompanied by fever, headache, backache, malaise, and prostration. the rash period for both smallpox and mpxv (including lesion appearance and desquamation) normally occurs − days postinfection with highly similar appearance, distribution, and progression of lesions. , as with smallpox, mpxv-associated rash progresses through macular, papular, vesicular, and pustular phases. a second febrile period occurs when the lesions become pustular and is often associated with deteriorating conditions in the patient. lymphadenopathy (maxillay, cervical, or inguinal) is often associated with mpxv infections prior to, or concomitant with, rash development but is absent in varv infections. it has been postulated that this reflects the effective generation of host immune responses during mpxv infection as compared to varv; however, this has yet to be validated. , severe complications have been noted late in the course of mpxv infection, including pulmonary distress or bronchopneumonia, corneal scarring and permanent vision loss, and encephalitis. severe dehydration due to excessive vomiting or diarrhea may also occur. long-term sequelae in survivors are most commonly associated with pitted scarring. soluble immune mediators associated with mpxv infections. although mpxv infections in humans have been recorded for over four decades, there has been little information review regarding host immune responses during the course of natural infection. as disease presentation is highly similar during mpxv and varv infections, it has been postulated that immune responses would likely be highly conserved. recently, johnston et al. provided the first empirical evidence for a relationship between cytokine responses and disease severity during mpxv infection. serum cytokines were analyzed from patients with confirmed mpxv infections ranging from mild to severe as assessed by the who smallpox lesion scoring system. , serum concentrations of il- β, il- ra, il- r, il- , il- , il- , il- , il- , il- , il- , mcp- , and rantes were elevated in all disease groups (mild to severe) as compared to normal serum concentrations. il- concentrations were also elevated in all disease groups and were proportional to disease severity. however, patients with serious mpxv disease had significantly higher concentrations of il- compared to all other disease groups. mpxv infection resulted in elevated mip- α and mip- β; mild cases had significantly elevated levels above the moderate or severe disease groups. serum concentrations of il- r were elevated across all disease groups; however, patients with serious disease had significantly higher il- r serum levels than those with mild to severe mpxv disease. gm-csf levels were significantly elevated only in those with serious mpxv disease as compared to normal serum ranges. on the basis of these observations, mpxv infection resulted in prominent t helper (th ) and dampened th responses. transciptome analyses in mpxv infection. transcriptome analyses have largely been employed for the in vitro investigation of the molecular pathogenesis of mpxv infection. alkhalil et al. investigated the host transcriptome responses to mpxv infection during the first cycle of viral replication ( and h postinfection) in rhesus macaque kidney epithelial cells. interestingly, mpxv infection resulted in a strong downregulation of host transcriptional responses. of the transcripts that met the authors' criteria for significance, % of the transcripts were found to be down-regulated at both postinfection time points. comparative functional analysis from both time points suggested that the primary biological functions associated with these down-regulated transcriptional responses were largely related to cell morphology, cell development, metabolic responses, and post-translational modifications. canonical pathway analysis demonstrated a general conservation in the identities of over-represented pathways at both time points including multiple growth factor signaling pathways, p signaling, and cell cycle-related pathways. more recently, bourquain et al. investigated host transcriptome responses in mpxv-infected hela cells, a cervical epithelial cell line. at h post-mpxv infection, only . % of the transcripts analyzed were found to have > fold changes in gene expression. in contrast to alkhalil et al., the majority of these transcripts (∼ %) were found to be upregulated as compared to mock-infected controls. functional analysis of all transcripts with > -fold changes in gene expression demonstrated a strong over-representation of genes involved in the negative regulation of mapk signaling and the intracellular protein cascade. positive regulation of pathways related to toll-like receptor signaling, chemotaxis, and regulation of leukocyte migration was also predicted from the data. an investigation by rubins et al. compared the temporal host transcriptome response to mpxv in multiple human cells targeted by mpxv including primary macrophages, primary fibroblasts, and hela cells. the trasncriptome of mpxv-infected fibroblasts was found to have the most significant changes where mpxv infection resulted in the depletion of ∼ genes by a factor of ≥ . interestingly, mpxv infection resulted in the broad repression of many transcripts related to innate immune responses in all cell types tested. in contrast, inactivated mpxv resulted in strong upregulation of innate immune responses in all of the cell types. it was also noted that mpxv infection resulted in strong cytopathic effects across all of the cell types in contrast to an almost universal repression of innate immune responses. kinome analyses in mpxv infection. human mpxv infections and infection models of mpxv in various animal species have demonstrated that the congo basin mpxv clade is more virulent than the west african mpxv clade. however, there has been a paucity of information regarding the underlying molecular mechanisms mitigating these virulence differences. furthermore, previous investigations focusing on gene expression or proteomic changes during mpxv infection have focused solely on congo basin mpxv. to address this, host kinome analysis was performed on congo basin and west african mpxv-infected human monocytes, a host cell targeted by orthopoxviruses. as the genomes of both mpxv clades demonstrate considerable diversity in the regions coding host response modifier proteins, and in particular in genes associated with anti-apoptotic activities, it was postulated that the virulence differences of the two mpxv clades may be related to differential modulation of host cellular responses. hierarchical clustering of the kinome data sets suggested limited similarities at the level of host kinase modulation between the two mpxv clades. the congo basin mpxv kinome data set clustered most strongly with the kinome data set from cpxv-infected monocytes and moderately with the vacv-infected monocyte data set. both cpxv and vacv can cause serious disease in humans. the pathway ora of the kinome data demonstrated that congo basin mpxv infection resulted in strong down-regulation of a large proportion of host cell responses, most notably apoptosis, in comparison to west african mpxv. biological validation through fluorescenceactivated cell sorting (facs) and caspase activity analyses confirmed this phenomenon. from the perspective of individual phosphorylation events, the kinome data also suggested that akt phosphorylation at ser was increased in congo basin mpxv-infected cells as compared to west african mpxv-infected cells. pharmacologic inhibition of akt phosphorylation at ser resulted in a > -fold inhibition of congo basin mpxv virus yields, whereas those for west african mpxv were unaffected. prior investigations with cpxv and vacv demonstrated that pharmacological inhibition of akt resulted in decreased viral yields for both viruses. overall, this investigation provided significant insight into the host cellular response differences between the two mpxv clades. emerging and re-emerging pathogens are a continual threat to global health. in recent years, disease outbreaks associated with sars and the influenza pandemic have also demonstrated that these pathogens can have considerable effects on local, national, and international economies. as a consequence, regional outbreaks of emerging and re-emerging pathogens can have deleterious effects on global stability. thus, it is prudent that a concerted effort is employed to assimilate data that bridge both clinical and molecular information in investigations review of these pathogens. these efforts will not only provide considerable context in regard to the molecular events that potentiate clinical manifestations of pathogenesis but also better inform the design and implementation of novel therapeutics. to this end, global analyses of host molecular responses can provide considerable insight into the complex molecular events that underlie cellular responses. indeed, transcriptome analyses have provided important information regarding host transcriptional responses during emerging and re-emerging pathogen infection. these investigations often provide critical insight into the kinetics of host immune responses during the course of infection as well as mechanistic information regarding the cellular intermediates involved in these processes. however, the role of ptms in the regulation of these events cannot be captured by traditional transcriptome technologies. in particular, the role of kinase-mediated regulation of cell signaling pathways has remained poorly understood. given the central role of kinases in the regulation of cellular processes (e.g., homeostasis, metabolism, proliferation, and stress responses), it is of inherent importance that future investigations also address the role of the kinome in the cellular response to pathogen insult. furthermore, kinomics also provides a mechanism for the identification of novel therapeutic targets based on the direct assessment of the activation state of cell signaling pathways. for example, proinflammatory responses during early stages of infection, and in particular the dysregulation of specific cytokines or cell signaling events that contribute to these, may represent potential therapeutic targets in the early stages of highconsequence viral pathogen infection. however, the selection of immunomodulatory therapeutics that target these dysregulated host responses is complicated by the regulatory events (i.e., kinase-mediated cell signaling events) that occur upstream of changes in gene expression. in addition, mrna is subject to a variety of regulatory processes (including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/substrate subcellular sequestration, and/or protein activation/repression ptms). thus, from the standpoint of therapeutic discovery, the sole reliance on technologies for the global investigations of host responses that do not account for these regulatory processes or the role of ptms in the modulation of cellular responses could impede the identification of efficacious therapeutics. to this end, kinome analysis may also facilitate the identification of immunomodulatory therapeutics that have gained licensure through analysis of a quantifiable biological event (kinase-mediated phosphorylation) or for identifying novel host therapeutic targets for which therapeutics could be designed/developed. furthermore, kinase inhibitors may serve as primary or adjunctive therapies for emerging infectious diseases. in addition, preclinical data and the increasing number of kinase inhibitors that have gained regulatory approval for cancer and other maladies suggest this approach is feasible and efficacious. from the perspective of this review, kinome investigations have identified several therapeutic targets and licensed kinase inhibitors that have impaired viral replication in vitro and reduced the severity of disease in vivo (table ) . for example, it has been demonstrated that the erk/mapk and pi k/akt/mtor signaling pathways have a role in viral propagation during mers-cov infection. indeed, licensed kinase inhibitors that targeted these pathways (i.e., everolimus, selumetinib, and trametinib) resulted in decreased viral replication in vitro when added prior to, or following, infection. furthermore, the pharmacologic inhibition of pi k and pkc following ebov infection provided partial protection in a lethal model of evd in mice. it should be noted that although the modulation of an individual kinase may have suppressive effects on infection (i.e., viral replication), this might not provide the level of inhibition required to completely negate viral escape. in addition, given the ability of many cell signaling pathways to signal through both canonical and noncanonical mechanisms, inhibition at a single intermediary point within a pathway may not provide the overall level of inhibition required to negate a deleterious response (i.e., viral replication, changes in cellular phenotypes, etc.). thus, although previous investigations have demonstrated that individual kinases or cell signaling pathways may represent novel targets for anti-infective therapies, it is prudent that future investigations also examine combinations of inhibitors for efficacy and anti-infective activities. furthermore, the targeting of cell signaling pathways at or near the origin point for the cell signaling cascade should also be examined as these likely represent stronger inhibitory targets given the generally reduced branching of cell signaling networks at or near the cell receptor. in addition to host-directed therapeutic targeting, kinomics also confers the ability to identify novel inhibitors of pathogens through detailed characterization of the viral life cycle. hostmediated ptms, and in particular kinase-mediated phosphorylation, have been implicated in the viral life cycle and pathogenesis for several members of the order mononegavirales, including ebov. − thus, therapeutic targeting of kinases may represent a novel therapeutic strategy that can be employed to modulate host-centric or pathogen-centric molecular events during infection. for example, in silico prediction of viral protein phosphorylation sites provides a mechanism for the construction and, ultimately, the annotation of viral protein ptms that are critical to the viral life cycle. furthermore, the use of kinome peptide arrays has extended beyond the human kinome and now extends to a variety of animal species. − it has been suggested that the interspecies phenotypic variability may reflect differences in phosphorylation sites found within the proteome. thus, the development of species-specific kinome peptide arrays provides additional utility for kinome analysis as peptide arrays representing traditional laboratory animal species (mouse, guinea pig, nonhuman primate) can be employed to detail the species-specific host response. the results from such analyses, and the overlap between these and those described previously from the analysis of human infections, may inform review the selection of appropriate animal models that meet regulatory approval through the fda animal efficacy rule. taken together, it is of inherent importance that future investigations of emerging and re-emerging pathogens address the complex nature of biological responses. thus, molecular investigations of pathogenesis should be guided by available knowledge regarding the clinical and pathologic manifestations of disease. indeed, technologies that provide further granularity into the precise molecular events that potentiate cellular responses during the course of infection will assist investigations of emerging and re-emerging pathogens and the identification of novel therapeutic targets. to this end, kinomics-based analyses of host responses provide a mechanism to directly address the cellular events at the level of specific cell signaling phenomena that underlie the biological responses and, ultimately, the clinical presentation of disease for emerging infectious pathogens. global trends in emerging infectious diseases a new antibiotic and the evolution of resistance signaling − and beyond protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling the protein kinase complement of the human genome sample preparation and profiling: probing the kinome for biomarkers 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infection selectively modulates host cell signaling responses as compared to west african monkeypox virus activation of the pi k/akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication dynamic phosphorylation of vp is essential for ebola virus life cycle structural phosphoprotein m - of the human respiratory syncytial virus is an rna binding protein phosphorylation status of the phosphoprotein p of rinderpest virus modulates transcription and replication of the genome a comparison of the chicken and turkey proteomes and phosphoproteomes in the development of poultry-specific immuno-metabolism kinome peptide arrays characterization of the host response to pichinde virus infection in the syrian golden hamster by speciesspecific kinome analysis computational analysis of the predicted evolutionary conservation of human phosphorylation sites repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection identification of novel cellular targets for therapeutic intervention against ebola virus infection by sirna screening inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin productive replication of ebola virus is regulated by the c-abl tyrosine kinase pyridinyl imidazole inhibitors of p map kinase impair viral entry and reduce cytokine induction by zaire ebolavirus in human dendritic cells variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases the authors declare no competing financial interest. review key: cord- -yc wxgid authors: martínez, miguel j.; salim, abdulbaset m.; hurtado, juan c.; kilgore, paul e. title: ebola virus infection: overview and update on prevention and treatment date: - - journal: infect dis ther doi: . /s - - - sha: doc_id: cord_uid: yc wxgid in and , the largest ebola virus disease (evd) outbreak in history affected large populations across west africa. the goal of this report is to provide an update on the epidemic and review current progress in the development, evaluation and deployment of prevention and treatment strategies for evd. relevant information was identified through a comprehensive literature search using medline, pubmed and cinahl complete and using the search terms ebola, ebola virus disease, ebola hemorrhagic fever, west africa outbreak, ebola transmission, ebola symptoms and signs, ebola diagnosis, ebola treatment, vaccines for ebola and clinical trials on ebola. through july , a total of , evd cases and , deaths were reported from all affected countries. several therapeutic agents and novel vaccines for evd have been developed and are now undergoing evaluation. concurrent with active case investigation, contact tracing, surveillance and supportive care to patients and communities, there has been rapid progress in the development of new therapies and vaccines against evd. continued focus on strengthening clinical and public health infrastructure will have direct benefits in controlling the spread of evd and will provide a strong foundation for deployment of new drugs and vaccines to affected countries when they become available. the unprecedented west africa ebola outbreak, response measures, and ensuing drug and vaccine development suggest that new tools for ebola control may be available in the near future. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. . the scope and severity of the evd outbreak underscore the urgent need for development and evaluation of affordable therapeutic and prophylactic agents that can be made available for at-risk populations across africa. over the past months, the west africa evd outbreak has provided an important opportunity to consider use of and evaluate several therapeutic and prophylactic agents (e.g., vaccines) to determine their safety and efficacy [ , ] . for this review, we considered published and [ ] [ ] [ ] . in , a novel third genus of filovirus named cuevavirus was reported from post-mortem tissues of bats collected in in northern spain [ ] . cuevavirus has not been grown in cell culture, and its pathogenic potential for humans remains unknown. to date, a single species (lloviu cuevavirus) has been approved by the international committee on taxonomy of viruses (ictv) [ ] . the current west africa outbreak is caused by zaire ebolavirus, which shows % identity to ebov strains from the drc and gabon [ ] . the genome of ebov contains seven genes named nucleoprotein (np), virion protein (vp) , vp , vp , vp , glycoprotein (gp) and l protein [ ] . each one of these genes encodes a corresponding structural protein. part of the nucleocapsid and, together with vp , interferes with innate host immunity. the surface gp is responsible for the attachment to the cellular receptor and viral entry. l protein is the rna-dependent rna polymerase [ ] [ ] [ ] [ ] [ ] [ ] . early reports suggest that the ebov variant of the - west africa outbreak accumulated mutations that may have an impact on the performance of certain diagnostic tests or even on the efficacy of several experimental treatments. gire et al. analyzed the genetic sequence of ebov genomes from patients in the four most affected countries of the west african region [ ] . they found significant rates of genomic variation in ebov in the current outbreak when compared with the ebov genomic sequence in the ebola outbreak in the drc. although the impact of these mutations on the diagnostic tests and experimental therapeutics has not yet been proven, some mutations exist in viral genes that are targeted by primers of some reverse transcription-polymerase chain reaction (rt-pcr) protocols [ ] , as well as mutations in the binding sites of target proteins of some experimental treatments such as anti-gp monoclonal antibodies [ ] . in , hoenen and colleagues studied full-length sequences of two clusters of ebov imported from mali and found that the gene sequence of ebov has remained stable during the current ebola outbreak [ ] . ebola viruses have been responsible for outbreaks in six african countries [ ] . historically, the outbreaks have affected hundreds of individuals where effective control of outbreaks was achieved primarily through isolation of cases and contact tracing. however, from , evd outbreaks have been recognized almost every year with substantial variation in morbidity and case-fatality rates ranging from % to % [ ] . high case-fatality rates have been associated with the zaire and sudan subtypes [ ] . in the ongoing ebola outbreak, the overall case-fatality rate has been estimated to be approximately % for west africa and other affected countries [ ] . most recently, although the number of cases has declined substantially ( fig. ) , evd cases continue to be reported (please see the supplemental table for details) in guinea, liberia and sierra leone with who ebola situation reports noting weekly cases in july [ ] . in july , situation report statistics from the who suggest that the greatest current burden of evd is found in guinea (n = ) and sierra leone (n = ). liberia reported the lowest evd case number (n = ) in the week of june through july [ ] . the search for the natural reservoir host of ebov has been a matter of investigation during the last decades. there is mounting evidence that a number of mammal species may harbor and transmit the virus. several bat species (i.e., epomops franqueti, hypsignathus monstrosus and myonycteris torquata) have been found to carry filoviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ , ] . patients often present to health care providers within week of symptom onset [ , ] . in the early clinical phase of evd, patients manifest signs and symptoms that mimic common tropical illnesses (e.g., dengue, malaria, typhoid fever and other viral infections) [ , , ] . patients with evd. these investigators found hemorrhage, shortness of breath and myalgia were independently associated with death [ ] . clinical deterioration may progress rapidly resulting in death within to days. vulnerable populations include children under the age of years, pregnant women and the elderly [ ] . evd in these groups also include unspecific symptoms in the clinical presentation. qin et al. did not find differences related to mortality between patients less than years of age and others between to years old, but they found that patients aged \ years had a much lower case fatality rate than those aged [ years [ / ( . % ) and / ( . %), respectively, with p = . ] and that survivors attended ebola treatment centers earlier after the onset of symptoms [ ] . no evidence suggests that pregnant women are more susceptible to ebov infection than the general population. however, they might be at increased risk of severe illness and fetal loss. although no large series are available, the fetal outcome is generally fatal. immune suppression and a systemic inflammatory response due to the release of cytokines and other proinflammatory mediators lead to the impairment of vascular, coagulation and immune systems [ ] . this can result in multiorgan failure and shock resembling a septic shock syndrome. massive fluid losses due to intense vomiting and profuse diarrhea can result in dehydration and hypovolemic shock [ ] . severe lymphopenia as well as significant deterioration of renal and liver functions, which may be reflected in high blood urea nitrogen, serum creatinine and hepatic enzymes (i.e., aminotransferases and alkaline phosphatase), can occur [ , , ] . since ebov is a contagious pathogen, the who and centers for disease control (cdc) have issued recommendations for proper handling of biological specimens from suspected cases of evd [ , ] . extreme caution should take place at all stages (i.e., specimen acquisition, transport, processing and testing) of specimen processing, and appropriate biosafety laboratory procedures must be used when handling biological specimens from patients with suspected evd. tests such as loop-mediated isothermal amplification (lamp) assays [ , ] . prior to , antigen detection methods [e.g., enzyme-linked immunosorbent assay (elisa)] were the gold standard for ebov detection in some outbreaks [ ] . in the acute phase of evd, elisa has a relatively high sensitivity ( %), but ebov antigen levels decline as disease progresses, rendering lower sensitivity for antigen detection - weeks following symptom onset [ , ] . several other antigen detections tests are currently under evaluation and may be deployed in the near future to complement rt-pcr testing [ ] . elisa testing has been largely replaced by rt-pcr, which permits more rapid detection and can now be deployed in mobile (portable) testing platforms in outbreak settings [ ] . detection of igm antibodies against ebov is performed by elisa in the first week after the onset of symptoms with a peak of igm levels occurring in the nd week of illness [ , , ] . igm antibodies are cleared at variable rates from to months after illness onset [ ] . data showed that serology can be highly specific for the evd diagnosis but less sensitive in the intensive care unit setting. hence, antibody testing may be less clinically useful in the diagnosis and management of critically ill evd patients [ ] . although igg antibodies appear soon after the igm and may persist for years [ ], a substantial number of evd patients have died before they develop an igg antibody response [ ] . nucleic acid tests (nats), particularly rt-pcr, are regarded as the gold standard for evd diagnosis, in part because of their high sensitivity and specificity in detecting the ebola viral genome. this is generally accomplished by international mobile teams deployed in institutions such as the european mobile laboratory or cdc. rt-pcr is a rapid and highly sensitive nucleic acid amplification test to detect ebov nucleic acid [ ] . the sensitivity and specificity of rt-pcr are approximately % and %, respectively [ ] . within the first days of illness, molecular assays may not detect the viral genome, which may lead to false-negative results. therefore, rt-pcr should be repeated in subsequent samples [ , ] . to minimize false-negative results, proper sampling, collection, storage or transportation, and a proper rt-pcr technique have to be implemented to avoid cross-contamination [ , ] . quantitative rt-pcr has been developed and could possibly be used to monitor the viral load since data suggest high viremia might be associated with unfavorable outcomes and death [ , ] . for those patients receiving experimental treatments, ebov viral load monitoring could be useful to assess treatment response [ ] . the the provision of clinical supportive care is now a cornerstone of evd patient management, which includes rehydration, nutrition, analgesics and blood transfusion when appropriate, though no clear evidence proves their effectiveness [ ] . a key aspect of supportive care is the maintenance of intravascular volume with oral rehydration solution (ors) or intravenous fluids that provide appropriate electrolyte replacement. the use of antiemetics and antidiarrheal agents may also be important for patients with persistent vomiting and diarrhea [ , , ] favipirarvir is a nucleotide analog and viral rna polymerase inhibitor with a wide range of antiviral effects against numerous negative-or positive-strand rna viruses [ ] [ ] [ ] [ ] [ ] [ ] . initially, favipiravir was developed to treat influenza viruses, and a phase iii clinical trial was completed in which favipiravir was tested on several thousands of people and proven to be safe and effective [ ] . recently, favipiravir has also shown efficacy against ebov in vitro and in vivo in a mouse model [ ] been established, although early treatment in high-risk or potentially ebov-exposed individuals may be an option [ ] . oral administration of bcx may be feasible, although the pharmacokinetic data suggest that the intramuscular route may provide more favorable therapeutic levels [ ] . brincidofovir is a prodrug of cidofovir and a fairly recent oral nucleotide analog that prevents viral replication by inhibiting dna polymerase [ ] . brincidofovir has shown broad-spectrum antiviral activity against dna viruses such as herpes viruses and adenovirus and is currently in a phase iii clinical trial against cytomegalovirus and adenovirus [ , ] . although the exact mechanism of action for brincidofovir in evd is not yet well understood, brincidofovir may interfere with rna polymerase of ebov. the us fda has put brincidofovir on fast-track approval for treatment of evd based on in vitro data alone [ ] . a phase ii open-label multicenter study to assess the safety and efficacy of brincidofovir against ebov in humans has been withdrawn prior recruitment (clinicaltrials.gov identifier: nct ). as a result of the dramatic decline in the number of new cases in liberia in the month of january where the trial was first initiated, chimerix, inc., decided to discontinue the trial with no further discussions [ ] . there are a number of known agents and newly identified compounds that have shown anti-ebov activity. for example, compound is a benzodiazepine derivative that also prevents ebov entry into the host cells [ ] . preliminary analysis suggests that compound binds near a hydrophobic pocket near the ebov gp and gp interface. analysis of this compound suggests that it may have activity against several filoviruses including ebov [ ] . no animal or human trials have been reported to date. nsc is a small molecule, which has an antioxidant property, and was found to inhibit many viruses, including ebov, lassa virus, venezuelan equine encephalitis virus and rift valley fever virus [ ] . this compound, a reactive oxygen species, has shown in vitro activity against ebov as well as some evidence in the ebov mouse model for protection against lethal ebov infection at a treatment dose of mg/kg/injection (higher doses did not improve survival in the mouse model). further studies of this or related compounds in mouse and other animal models may be warranted to elucidate their role in the treatment of ebov and other filovirus infections. of additional interest are compounds that have been proven to be effective in preventing the entry of filoviruses, including ebov, into host cells [ ] . lj- binds to lipid membranes and prevents virus-cell fusion across a wide range of viruses. additional research will be needed to understand how such compounds can be optimally formulated to maximize both the safety and pharmacologic activity in vivo. fgi- , fgi- , and fgi- are a group of broad-spectrum antiviral agents that inhibit viral replication in a dose-dependent manner among multiple and genetically distinct viruses including ebov, bunyaviruses, dengue virus, hiv and hepatitis c virus [ ] . using a mouse model, aman et al. found that fgi- yields a protection after a challenge with a lethal dose of ebov. aman and colleagues showed that protection was also found when fgi- was administered in a prophylactic fashion. in related studies, fgi- and fgi- are also small molecules that inhibit filovirus infection and are found to protect ebov-or marburg-infected mice, although their mechanism of actions are unclear [ , ] . [ ] . for use of cp or cwb, the who has provided guidance to improve the safety of product development as well as safety for patients who receive these products [ ] [ ] [ ] . convalescent sera-based therapy may cause some toxicity related problems, such as transmission of undetected pathogen(s) and transfusion reactions. a recent case report from treating a spanish nurse who had contracted ebov during her care to a patient with evd in spain shed some light on the issue of using experimental therapeutics including cp [ ] . the infected nurse had received convalescent sera from two survivors of evd, high-dose favipiravir and other supportive care treatment. on the th day of clinical disease, the nurse developed clinical signs and symptoms suggestive of post-transfusion acute lung injury, which was managed conservatively without the need of supportive mechanical ventilation. although purified igg can lower these risks, lot-to-lot variation remains a potential problem. previous experience also highlights the risk of antibody-dependent enhancement of ebov infection [ ] . could overcome future zmapp shortages. using magnifection, . g of zmapp can be extracted and purified from kg n. benthamiana leaf biomass [ ] . while this product holds promise, a major hurdle in its future utility is to manufacture large quantities of each monoclonal antibody from plants in a way that ensures sustained high yield of monoclonal antibodies at reasonable cost [ ] . to overcome potential rate-limiting steps in large-scale production, zmapp can be manufactured using chinese hamster ovary (cho) cells. in despite the high safety profile compared with other newly discovered antivirals, these agents cannot be used alone because of their low efficacy, but they can be used in combination with other treatments available including supportive care measures. interferon: since its discovery in , interferon has not been widely used because of the associated adverse events [ ] . the potential use of interferons in the treatment of evd is rooted in the evidence that ebov interferes with functions of type i interferons [ ] [ ] [ ] . previous pre-clinical research showed that exogenous interferon-a or interferon-b could delay the occurrence of viremia or prolong survival time, but could not save animals from death [ , ] nhps (usually rhesus and cynomolgus macaque) can be infected with non-adapted strains and best mimic disease progression in humans, and therefore they are considered the ''reference'' animal model for vaccine studies [ ] . differences can also be found in the ebov antigens included in the vaccines. promising in providing more timely and accurate ebov detection, there will be a need for additional research to study optimal strategies for deploying these diagnostics to locations where testing is most needed. in the near future, there is a substantial likelihood that one or more drugs and vaccines will be approved for use in the treatment and prevention of evd. the 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infection in rodents successful treatment of advanced ebola virus infection with t- (favipiravir) in a small animal model post-exposure efficacy of oral t- (favipiravir) against inhalational ebola virus infection in a mouse model efficacy of favipiravir against ebola (jiki) world health organization. international clinical trials registry platform dose regimen of favipiravir for ebola virus disease french msf nurse 'cured' of ebola, health minister says protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx possible leap ahead in filovirus therapeutics progress in the development of new therapies for herpesvirus infections development of cmx (brincidofovir) for the treatment of serious diseases or conditions caused by dsdna viruses chimerix ends brincidofovir ebola trials to focus on adenovirus and cmv identification of a small-molecule entry inhibitor for filoviruses identification of an antioxidant small-molecule with broad-spectrum antiviral activity a broad-spectrum antiviral targeting entry of enveloped viruses development of a broad-spectrum antiviral with activity against ebola virus antiviral activity of a small-molecule inhibitor of filovirus infection fgi- : a broad-spectrum small molecule inhibitor of viral infection ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry clinical study to assess efficacy and safety of amiodarone in treating patients with ebola. virus disease (evd) in sierra leone nct ?&search=search. accessed treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and 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states rapid high-yield expression of full-size igg antibodies in plants coinfected with noncompeting viral vectors putative investigational therapeutics in the treatment of patients with known ebola infection accessed characterization of host immune responses in ebola virus infections evasion of the interferon-mediated antiviral response by filoviruses how ebola virus counters the interferon system evaluation of immune globulin and recombinant interferon-alpha b for treatment of experimental ebola virus infections interferon-beta therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever clinical features and pathobiology of ebolavirus infection treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys recombinant human activated protein c for the postexposure treatment of ebola hemorrhagic fever arca biopharma receives fda orphan drug designation for rnapc as a potential treatment for ebola ebola virus vaccines: an overview of current approaches evaluation in nonhuman primates of vaccines against ebola virus role of vp phosphorylation in the ebola virus replication cycle key: cord- -adfigzc authors: beniac, daniel r.; melito, pasquale l.; devarennes, shauna l.; hiebert, shannon l.; rabb, melissa j.; lamboo, lindsey l.; jones, steven m.; booth, timothy f. title: the organisation of ebola virus reveals a capacity for extensive, modular polyploidy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: adfigzc background: filoviruses, including ebola virus, are unusual in being filamentous animal viruses. structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level biological containment. the present study provides unique insights into the structure of this deadly pathogen. methodology and principal findings: we have investigated the structure of ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. here we report the three-dimensional structure and architecture of ebola virus and establish that multiple copies of the rna genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. we show that the helical ebola virus inner nucleocapsid containing rna and nucleoprotein is stabilized by an outer layer of vp -vp bridges. elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. the matrix protein vp forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. conclusions: the results of this study demonstrate a modular organization in ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope. viruses have evolved as genome packaging machines to efficiently transfer nucleic acids between susceptible host cells, ensuring replication. the majority of viruses have hollow, quasispherical shells rather than tubular structures, perhaps because this gives the most efficient packaging of nucleic acid with a fixed copy number of coat protein subunits. in non-enveloped viruses, the volume enclosed by the (usually) icosahedral structure is a constraint on the size of the genome, giving a limited capacity to encode capsid proteins, and usually restricts the genome copy number, or ploidy of the virion, to one [ ] . most membraneenveloped viruses are also quasi-spherical, but their symmetry is frequently less well-ordered, which is usually described as pleomorphic. this feature allows some flexibility in volume, which could accommodate variation in the size of the genome or its copy number. nevertheless, most viruses, irrespective of their architecture, appear to have evolved to encapsidate only a single copy of their genome within the protein or protein/lipid shell, or a dimeric copy in retroviruses. notable exceptions are the paramyxoviridae and the birnaviridae where particles may contain up to four copies of the rna genomes [ , ] . although some strains of influenza can produce elongated virions, there is a mechanism that selectively encapsidates only one set of genome segments in each virion [ ] . the filoviridae family, including the ebolavirus and marburgvirus genera, cause haemorrhagic fevers with high mortality in humans, and no effective treatments are currently approved [ ] , although candidate vaccines are promising [ ] . the . kb single-stranded negative-sense non-segmented rna genome of ebola virus (ebov) codes for at least eight proteins. the ribonucleoprotein complex is composed of the nucleoprotein (np), polymerase protein (l), vp , vp , and vp . the trimeric transmembrane glycoprotein (gp) forms surface spikes on the virion envelope and also has a soluble form, while the matrix protein, vp , is associated with the inner surface. [ , [ ] [ ] [ ] . the gp spike, a class i fusion protein, mediates cellular attachment and entry and is extensively glycosylated, especially in the glycan-rich mucin-like domain [ ] [ ] [ ] [ ] . three proteins, vp , vp and np are essential for nucleocapsid formation [ ] . although some of the major protein interactions that occur during ebov morphogenesis have been characterised [ , ] , the three-dimensional ( d) structure and molecular arrangements have not been previously determined. structural details are essential to understand how protection of the genome, cell binding, entry, and immune evasion are achieved in a filamentous animal virus, and to determine how this unique morphology plays a role in pathogenesis. research on filoviruses has been hampered by their status as biosafety level pathogens. previous investigations of filovirus structures within embedded, sectioned and metal-stained cells by electron tomography revealed few details of the high resolution oligomeric structure [ , ] . it has been demonstrated that aldehyde-fixation alone, and subsequent cryo-electron microscopic imaging in the frozen-hydrated state preserves structures, at least up to angstroms resolution [ ] , and in some cases, fixation improves the resolution achievable [ ] . in addition, it has also been shown that high-resolution x-ray structures can also be obtained in the presence of aldehyde fixatives [ ] . therefore, we analyzed purified and isolated ebov and ebola virus-like structures using cryo-electron microscopy (cryo-em), and cryoelectron tomography (cryo-et). in the current study, the zaire strain of ebov was purified and inactivated by paraformaldehyde fixation: excess fixative was then removed by dialysis to reduce beam damage for imaging in the frozen-hydrated state. the flashfreezing at liquid ethane temperatures used in cryo-electron microscopy preserves the structural and molecular detail, avoiding artifacts associated with conventional em methods, such as dehydration and/or sectioning or staining, that usually prevent detailed structural analysis. digital image processing reveals the d organization of ebov, including the structural arrangement of component molecules at resolutions of - Å . we identified filamentous ebov particles microns or longer, with a well ordered internal structure, and a helical nucleocapsid giving an internal ''herring-bone'' appearance using cryo-em and cryo-et (figures , , s , s ). the nucleocapsid, as observed within intact viral particles, has a uniform helical structure (figures , , , ) and is enveloped by a membrane coated by an external layer of gp spikes. from the same image data set, we combined extracted volumes from tomograms with -d single particle processing to determine the structure of the gp spikes ( figure ) to a resolution of Å as measured by the fourier shell correlation (fsc) . criterion. virions are rarely straight. variation in the overall length of virions is non-random, and they fit into ordered size classes ( figure ). analysis of distinct intact virions with a nucleocapsid from cryo-electron micrographs shows that the most common class length ( %) of virus particles is nm ( figure a , table s ). the other size classes are multiples of this length. enveloped filovirus particles have several empty and linked ebov structures were excluded from the histogram data. a single g -single/comma shaped ebov is shown (inset on the right, g = copy of genome). (b) low magnification cryo-images showing: g -single/comma shape, g -single/ linear, g -continuous (g = copies of genome). (c) high magnification of a g -(single genome) virion with a region filtered to emphasize the nucleocapsid. (d) low magnification image of a g -linked ebov, each genome copy is indicated and numbered, the red arrows show the transition points between nucleocapsids. the circular holes (filled with vitreous ice) appear as lighter regions and the support film (''quantifoil'') appears dark grey. a ''linker'' region is shown at higher magnification (inset). doi: . /journal.pone. .g different morphologies (shown in figures , s , s ). these configurations are ''single'' particles, containing a nucleocapsid of uniform length, (which we postulate to contain one copy of the genome), ''continuous'' particles, with nucleocapsids of a length of the single virion multiplied by an integer of or greater; and ''linked'' virions composed of a series single-genome nucleocapsids connected by short sections of empty envelope. negative staining can cause drying and staining artefacts which hamper accurate measurements and preclude d analysis. nevertheless, in previous studies using this technique, the average length of marburg virus figure . image processing of ebola virus. linear d averaging of ebov: the envelope and nucleocapsid are prominent features (a). the line trace is colour-coded as follows: red, spike; beige, lipid envelope; green, membrane-associated proteins; white, membrane-nucleocapsid gap; blue and purple, outer and inner nucleocapsid. (b) d class averages of envelope plus inner face. (c) vp vlps, showing d averages from the from side regions (first two) and end-on/central regions (last three). in (a-c) representative individual repeats have been highlighted in color using the same scheme as in (a). (d) schematic model of the nucleocapsid and envelope, highlighting the relative distribution of np to vp . (e,f) d reconstruction of the nucleocapsid with the same colour scheme as in (a). the location of the inner nucleocapsid, and the bridge are indicated. the reconstruction is presented at a volume threshold that would encompass a single copy of each of these proteins, and the viral rna. in (e) the vertical (protein-protein) and horizontal (protein-rna) contacts are indicated by yellow and white arrows, respectively. (g) various recombinant nucleocapsid-like structures, and authentic ebov, which have been studied by electron microscopy [ , , , ] . d schematics of these structures highlighting the rna and protein composition and the diameter of these structures, at the same scale for comparison to (e). doi: . /journal.pone. .g was measured as nm, and ebov as nm [ , ] : the latter is very similar to the mean length of nm measured in the current study by cryo-tem of frozen hydrated specimens. in addition, discrete sub-populations of virions of double or triple the average length were also previously reported in centrifuged virus preparations [ ] . we also observed ''empty'' filaments that lack nucleocapsids, with a random length and a smaller diameter ( figure s c ). these empty nucleocapsids are also visible in previously published thinsection micrographs of human ebov infected pathology specimens, e.g. [ ] , and are thus probably not an artifact of cell culture. it is also unlikely that the polyploidy we have observed in ebov is a peculiarity of the particular viral isolate or of the vero cell line that we used for this investigation. previous studies using negative stain em have shown that both marburg virus and several different ebov species can produce filamentous virions up to mm long when grown in several different cell lines [ , ] . blood specimens from guinea-pigs and monkeys that were inoculated with primary isolates of the marburg agent, when centrifuged and observed by negative stain, also showed filamentous virions of variable length, with the average reported as about mm in length but with a smaller proportion being over mm in length [ ] . in addition, viral filaments of lengths from . to . mm long, and occasionally as long as . mm, can also be seen in published ultrathin sections of ebov infected human and monkey tissues [ , ] . thin-section em always underestimates the lengths of virions, since filament profiles are frequently truncated by the section plane: in addition, tissue shrinkage of to % during dehydration and resin embedding is common. thus, it is probable that polyploid ebov with multiple nucleocapsids are also produced in naturally infected humans and animals, though it is not possible to make a direct comparison with cell cultured virus: it would be very difficult to obtain large enough quantities of concentrated virus from animals to carry out detailed cryo-tem analysis and measurements. the ebola nucleocapsid structure was solved to Å resolution (fsc . criteria) using linear regions of , images (taken at - mm defocus, figure ). since virus particles were not straight enough for conventional helical image processing, a combination of tomography, sub-tomogram averaging, single particle averaging, and the iterative helical real-space construction method were used [ , ] . the ebov nucleocapsid is a right-handed doublelayered helix with an outer diameter of nm and a hollow inner channel nm in diameter as determined by image analysis and tomography (figures , , , s , s , movie s ). the pitch is . nm, with . repeats per helical turn ( figure ). the inner nucleocapsid, composed of large subunits, is linked by vertical and horizontal contacts between the large subunits ( figure e ,f). the horizontal contacts occur between the large subunits at a diameter of . nm. the vertical contacts linking the coils have a higher density than the horizontal ones, so we interpret the horizontal contacts as involving viral rna (white arrow, figures e, e ; movie s ) and the vertical contacts as protein-protein interactions (yellow arrow, figure e ). the np subunits are also linked by an outer horizontal layer at a diameter of nm. this layer consists of a ring of bridges between adjacent large subunits ( figure ). the bridges joining the np subunits are composed of two lobes, one of which of which is slightly bigger than the other. previous studies that produced recombinant nucleocapsid-like structures showed that expressed vp and vp both independently associate with np, but that all three proteins together are necessary to produce , nm diameter helical nucleocapsid-like structures. when vp , vp , vp , and np were transfected together, approximately nm diameter helical nucleocapsid-like structure was also generated, whereas np alone generated helical np-rna complexes , - nm in diameter, which were nuclease sensitive [ , , ] . taken together these results suggest that vp and vp are the structural components of the bridge located on the periphery of the nucleocapsid ( figure g ). it is not possible to accurately delineate vp and vp within the bridge at this resolution, however the density of the bridge is consistent with a predicted total mass of kd, thus each bridge is composed of one molecule of vp and one molecule of vp . it is likely that the larger lobe is vp and that vp therefore resides within the smaller lobe ( figure f ). thus, each bridge is composed of a vp -vp heterodimer that holds adjacent np molecules together horizontally. this structure explains how vp and vp are able to independently interact with np, and why all three are required for the formation of a double-layered nucleocapsid. in addition, it implies that vp and vp can interact with each other as well as each interacting with a different site on the np molecule in order to make an oligomeric structure. the recombinant nucleocapsid data indicates that both vp -vp -vp -np and vp -vp -np produce approximately nm diameter helical structures that are indistinguishable from nucleocapsids produced by ebov. this indicates that vp does not increase the diameter of the nucleocapsid. we propose that vp lies in the interior of the nucleocapsid and is not part of the bridge on the periphery of the nucleocapsid. this localization is consistent with previous work showing that vp is a component of the nucleocapsid, and associates with np, but is non-essential for nucleocapsid formation [ , , ] . our model, in which the inner layer at . nm diameter is rna-np, and the outer bridge centered at nm is composed of vp -vp heterodimers, with vp bound to np, is thus consistent with these previous observations [ , , ] , and suggests that the outer vp -vp heterodimer bridge functions in the stabilization and/or protection of the nucleocapsid. we have modeled the arrangement of the rna within the ebov nucleocapsid (table s ). since no atomic resolution data on the ribonucleoprotein structure of filoviruses have yet been reported, the ribonucleoprotein ring-structures of other members of the mononegovirales are useful for comparison. although the mass of the nucleoproteins, and the number of nucleotides per subunit differs amongst these virus families, a model of the . kb ebov genome with the rna following a circular fixed radius as in respiratory syncytial virus (rsv) [ ] would make a nucleocapsid containing one copy of the ebov genome about nm long (table s ). this fits closely with the nm length class ( % of the particles observed in this study) containing a single copy of the ebov genome threaded through the np at a fixed radius. allowing nm of space for the membrane envelope to curve around at each end of the virus particle gives an estimated length for a single-genome virus particle of nm, which is very close to that observed ( nm). the majority of the virus particles fall into size categories that are a multiple of the putative single genome length (g ), giving size classes of . . mm (g : . % of the particles having genomes); . . mm (g : % of particles having genomes) and so on ( figure a and table s ) . the length of the longest particle measured was consistent with having genome copies. our model predicts a nucleotide to np ratio of . (table s ) , which is within the range of to nucleotides per np molecule as measured by biochemical studies of marburg virus [ ] . in partially full virions, the membrane envelope is constricted at the transition point where the nucleocapsid ends and the empty membrane tube begins (figures , s c ). empty virions ( figure s c ) have a similar structure to vp -gp virus-like particles ( figure a ). both full and empty virions have a continuous layer of spikes projecting from the surface (figures s , s , s , movie s ), giving an overall diameter of nm for full particles. the majority of virions are linear (figures , s a, s ), others have a ''comma-shaped'' appearance, with a globular head containing portions of the nucleocapsid that are curled-up or bent at one end ( figures a,b and s b). it is clear that ''toroidal'' virions previously identified by negative staining [ , ] are a variation of comma-shaped virions. internal vesicles of - nm in size are also sometimes observed at the ends of virions ( figure s d ). these vesicles appear to be formed during the process of envelopment of the nucleocapsid, since they were not observed in preparations of vp or vp -gp vlps. vp vlps had wavy envelopes with an irregular diameter ranging between nm and nm (n = ) compared to vp -gp vlps which were more ordered with a diameter between nm and nm (n = ), (figure ). thus the presence of gp, and certain contacts between the gp and vp , play a part in stabilizing the tubular membrane envelope structure, and our observed structure agrees with previous reports that gp enhances vp vlp budding [ , ] . the previously reported ''branched'', filamentous forms [ ] were rarely observed: these consist of empty tubes (data not shown). it is possible that centrifugal virus purification disrupted most branched structures that were seen previously with negative staining of cell culture supernatant. the vp matrix protein shows a regular nm lattice spacing ( figure b , c). both the nucleocapsid and the vp layers are ordered, however the contacts between them appear to be nonsymmetrical ( figure d ), implying some flexibility in their intermolecular contacts. it has been shown that vp interacts with the vp and can be packaged into vp vlps [ ] . the localization of the vp -vp bridges on the periphery of the nucleocapsid, may allow interactions of one or more of the nucleocapsid proteins with vp , possibly through projecting low-density protein loops. there is a - nm gap of low density between the nucleocapsid and the vp layer, but tomography also shows discrete areas of connectivity between the nucleocapsid and matrix protein layers, which may be connections between the envelope and the nucleocapsid ( figure i ). these results are substantiated by the analysis of sub-tomograms where helical symmetry is clearly evident but was not imposed (figures , s ) . the use of sub-tomogram analysis improves the resolution of the data by averaging sub-tomograms together which are at different angular orientations. the helical nucleocapsid is clearly identified in the structure, including the gap between the envelope-vp , and the nucleocapsid-vp . the right-handed pitch of the helix is clearly discernable (figure e ), as well as the putative location of several vp proteins, although the resolution of the tomographic data by itself is slightly lower than with single particle analysis. while showing the overall organization of ebov, tomography also allows estimation of the stoichiometry of the major structural proteins (figure , movie s ). both ebov and vp -gp vlps have an irregular distribution of the gp spikes on the surface (figures , s ) demonstrating the lack of any ordered lattice-like arrangement with the matrix protein vp . the spikes are clustered, and the average centre-to-centre spacing is . nm ( figure s , and movie s ) . we calculate that a virion of nm in length would have about copies of the gp spike, and copies of vp . the wide spacing of the ebov gp in the viral envelope allows plenty of space for free access and binding of any neutralizing antibodies directed at both the club-shaped head and stem region without stearic hindrance (figures j, s ) . the nucleocapsid structure implies equimolar ratios of np, vp , vp , and vp . a genome of . kbp with bases per np with a single genome copy gives molecules of np, vp , vp , and vp per virion. previous analyses of coomassie blue stained gels of purified ebov predicted , , , and protein molecules of np, vp , vp , and vp , per virion respectively [ ] , which is in the same stoichiometric range as predicted by our model for the nucleocapsid, taking into account the variability of individual protein band staining by coomassie blue, which is affected by factors such as distance of migration [ ] , basic amino acid content [ ] , and the extent of glycosylation [ ] . since np is glycosylated, we anticipate underestimation of the np content of virions [ , ] . the gp spike is necessary for cellular attachment and fusion of ebov. a definitive cell surface ligand for the receptor binding domain has not been identified, and a number of different cell surface proteins are able to enhance infection [ , ] . cleavage of gp results in two domains: gp containing the receptor binding domain and gp that contains the fusion and transmembrane domains. the structure of a smaller engineered fragment of gp -gp has recently been determined by x-ray crystallography ( csy pdb [ ] ). we determined the structure of the entire ebov gp trimeric spike at a resolution of Å (fsc . criteria), by combining sub-tomograms from the spikes of vp -gp vlps ( images) with ebov images for the side perspective data ( images) using projection matching as previously described [ ] ( figure and movie s ). in our reconstruction, the spike is in situ in the viral membrane, thus the transmembrane region and base of the spike, adjacent to the membrane, are less well defined than the distal region of the spike, due to the smaller differences in contrast between lipid and protein versus water and protein, as well as fresnel fringes at the edge of the viral membrane. the spike extends nm from the surface of the envelope, with a clubshaped head . nm in height, and a . nm long stalk. docking of the previously determined x-ray structure into our cryo-em map shows a good fit, with a correlation coefficient of . using the docking and correlation program situs [ ] . the difference map calculated between our cryo-em map and the gp -gp structure identified volumes corresponding to the domains deleted to generate the csy gp -gp structure ( figure ). we show that the mucin-like domains (connected at v and e -shown in green in figure ) completely fill the previously described bowllike chalice which contains the putative receptor binding sites [ ] . the proximity of glycosylation sites in the gp -gp structure suggests that the distal density of the spike contains the glycans that were deleted in order to construct the gp -gp structure. in addition, each mucin-like domain has an ''arm-like'' projection, which extends radially at the distal end of the spike, to a maximum diameter of nm. the localization of the mucin-like domain is consistent with previous studies showing that endosomal proteolysis plays a role in enhancing infectivity as well as binding of the ebola gp to the plasma membrane [ , ] . the other two major deletions in the csy structure (n -r and a -y ) are situated at the midpoint of the structure just above the stalk (shown in pink in figure ). inclusion of the kz fab in the docked structure demonstrates that this neutralizing epitope (from a human survivor) is localized on the side of the stem region of ebov gp trimer at the base of the clubshaped head, and that the fab domain lies close to the lipid envelope when bound, and approximately tangential to the viral envelope. the densities putatively corresponding to n -r and a -y are close to the kz neutralizing site, but do not obstruct antibody binding. the mucin-like domains are out of the way and cannot interfere stearically with kz fab binding. this is consistent with previous results indicating that kz binding does not require cathepsin cleavage [ ] . we have thus delineated the low resolution structure of the glycocalyx or ''glycan cap'' that covers the distal end of the uncleaved ebov gp spike, which is consistent with a proposed role in immune evasion [ ] . our data will enable docking of future structures to investigate receptor binding, antigenicity, and fusion mechanisms. we have shown that ebov particles are capable of a high degree of polyploidy, made possible by the extreme length polymorphism of budding virus particles. polyploidy in filoviruses may be more extensive than in any other virus family, with % of virions having more than one genome copy, and some having up to copies. polyploidy has been shown to increase infectivity rates in paramyxovirus and birnavirus [ , ] . attempts to investigate infectivity rates of ebov by centrifugal fractionation of the different sized particles are stymied by the extreme filamentous morphology, as well as that fact that ebov of different lengths have the same buoyant density (personal observations). a complex double layered helical nucleocapsid appears to be unique to filoviruses. in the case of rhabdoviruses, the bullet-shaped nucleocapsid precludes them from being linked sequentially. although some influenza strains can produce filamentous virions, this morphology appears to be driven by the matrix protein only [ ] . a filamentous morphology may have evolutionary implications by allowing genome length flexibility. it could also enhance the ability for viral dissemination in infected tissues, for example by diapedesis of budding filamentous virions through epithelial layers. zaire ebolavirus was propagated in vero e cells and purified as previously described [ ] . ebola enriched samples were checked by sds-page and western blotting, and rendered non-infectious by fixation with % paraformaldehyde. excess fixative was removed by placing the fixed samples in a slide-a-lyzer g cassette with a . ml capacity, and a , mwco (thermo scientific pierce protein research products, rockford, illinois, usa), followed by dialysis against pbs. virus-like particles were produced as previously described [ ] . all work with infectious ebola virus (virus culture and purification) was performed in the biosafety level laboratories at the national microbiology laboratory of the public health agency of canada, winnipeg, manitoba. samples for cryo-electron microscopy (cryo-em), and cryoelectron tomography (cryo-et) were mixed with bsa coated accessories, wageningen, the netherlands) at a ratio of : (virus:gold) for cryo-et, and ( : ) for cryo-em. specimens ( ml) were then applied to glow-discharged quantifoil grids with mm holes spaced at mm intervals (quantifoil microtools gmbh, jena, germany). grids were subsequently plunge cooled in liquid ethane using a vitrobot mark iv (fei company, hillsboro, oregon, usa). specimens were transferred to a tecnai g transmission electron microscope (fei) operated at kv, equipped with a gatan ct tr single tilt rotation lowtemperature specimen holder. for cryo-em imaging was conducted at temperatures of , uc. images were recorded using an eagle k ccd camera (fei company, hillsboro, oregon, usa). for single particle image analysis, images were taken at , or , magnification at - mm defocus, with a dose of electrons/Å . this corresponded to a pixel size at the ccd detector of . Å /pixel and . Å /pixel, respectively. for virus length measurements low magnification cryo-em images were taken at , , , and , . for cryo-et single axis tomograms were taken at , , , or , magnification, at m or m defocus, with angle steps of u u. data were collected within tilt ranges of u, or u, with a total dose/tomographic data set of - electrons/Å . for cryo-em, data collection was done using the low-dose unit and software coupled with the tem imaging & analysis (tia) software (fei company, hillsboro, oregon, usa). automated eucentricity determination, and focusing were performed using the xplore d data acquisition software (fei company, hillsboro, oregon, usa). for cryo-et, data collection was done using the xplore d data acquisition software, the low-dose unit, and the tia software (fei company, hillsboro, oregon, usa). the exact magnification in the microscope at the ccd detector was determined using a calibration grid (pelco international, redding, ca). ebola virus length measurements (n = ) were made using the image j software package [ ] using the free hand line tool, and the analyse/measure function. for this analysis only viruses containing a continuous nucleocapsid were measured. viruses with linked nucleocapsids and empty viruses were omitted. the measurements that were made in image j were then collated, analysed, and plotted, using microsoft excel. tomographic image analysis of cryo-et data was carried out with the inspect d xpress software package (fei company, hillsboro, oregon, usa). the tomographic images were aligned to each other by a two-step process. the first step involved alignment of adjacent images by cross correlation. this process was repeated several times until the shift between adjacent images was below one pixel in either the x or y plane. the second step involved the alignment of the entire image stack using nm colloidal gold particles as fiducial markers. in this instance the term ''image stack'' refers to all the images collected in a single tomographic tilt. this procedure involves the selection of ten to twenty of the nm gold particles and the subsequent identification and tracking of these particles in all of the images in the image stack. the locations of these particles are then used in conjecture with the tilt angles of each image to globally align all of the images to each other. the last step in this process was to calculate the three dimensional reconstruction of the tomogram from the aligned images. in this study we used the simultaneous iterative reconstruction technique (sirt) algorithm with iterations to calculate the final three-dimensional reconstruction (tomogram). sub-tomogram image analysis of cryo-et data was carried out with the automated recognition of geometries, objects, and segmentations (argos) software package (fei company, hillsboro, oregon, usa). for this analysis an pixel subtomogram was extracted from a tomogram using the chimera [ ] software package. in this analysis the tomogram used contained a linear region of the ebola virus ( figure s ), and the pixel sub-tomogram contained a single linear segment. this template was then used by the argos software to conduct an exhaustive search of the original tomogram for similar structures. this analysis involved a six dimensional search matrix (three positional variants, and three rotational variants). the entire search process was sped up by the argos software by utilizing parallel processing on the computer's graphics processing unit (gpu). once individual sub-tomograms were selected based on correlation, they were inspected and compared to the initial template sub-tomogram. the extracted and aligned sub-tomograms were subsequently averaged with a filter that minimized the missing wedge artifact. this average structure then was used as the reference and the entire procedure was repeated several times. single particle image analysis: software and hardware single particle cryo-em image processing was carried out using the eman/eman and spider/web image processing program packages [ , ] . particle selection (eman) and contrast transfer function correction (eman ) were conducted on an apple inc. mac pro computer ( -core, intel xeon nehalem processors . ghz, gb ram, mac os x . . ). all subsequent calculations were performed on a dell poweredge r -way -bit xeon x processors, six core . ghz cpus with gb ram running linux (centos . ). images were corrected for contrast transfer function (ctf) using the ''e ctf.py'' function in the eman software package , which estimates defocus and corrects for ctf by phase-flipping. images of the spike (n = side perspective; n = end-on perspective) and nucleocapsid (n = , ) were selected for image analysis. the resolution of the cryo-em reconstruction was estimated by fourier shell correlation using the fsc . criteria. in all subsequent sections image analysis procedures were conducted using the spider software package unless otherwise stated. analysis of initial images of the ''straight'' linear segments of the ebola virus using fourier transformation indicated that there was sufficient bending of the helical nucleocapsid to make standard helical analysis problematic. therefore, an initial reference free single particle d analysis was conducted in eman using the ''startnrclasses'' program to identify any potentially recurring motifs within linear regions of the ebola virus. in order to further investigate the nucleocapsid repeat identified in the d analysis the iterative helical real space reconstruction method (ihrsr) was implemented [ , ] . this procedure requires an initial d helical reference structure which is used for image alignment. in this investigation a linear region of the ebola virus which was extracted from a tomogram was used to generate this helical reference structure. this initial d structure was first pre-treated with a gaussian mask to select only the nucleocapsid-containing region of the virus tomogram. an auto correlation function was then performed in which the volume was rotated around the helical axis, and translated along the axis of the nucleocapsid. at each rotational and translational position and autocorrelation value was calculated (between the shifted and unshifted volume). the net result of this process was the determination of the helical symmetry present in the tomogram. in order to analyse the data generated by this procedure the microsoft excel spread sheet program was used. the correlation plots generated by this process solved the handedness, pitch, and number of repeats per turn for the nucleocapsid. these symmetry parameters were then applied to the tomogram to generate the initial d model. the ihrsr method was then applied to the , single particle images of the nucleocapsid as previously described [ , ] . for the spike dataset two image populations were combined composed of side, and end-on perspectives. for the side view perspective images were subfield directly off of the ebola virus cryo-em images. for the end-on perspectives sub-tomographic volumes were extracted from the tomographic reconstructions of the ebola vlp. the d volumes were then added in the ''z'' plane to generate d projection averages which were then used for the subsequent single particle image analysis. the data were the processed using eman to generate an initial d reconstruction, which was then refined in spider using the projection matching technique as previously described [ , ] . the docking of the csy.pdb [ ] structure to the cryo-em structure of the spike was accomplished using the situs [ ] software package with the exception that only the gp and gp components of the csy structure were used for the docking process. the ''floodfill'' program in situs was used to segment the spike component of the d cryo-em reconstruction from the envelope component of the reconstruction. the segmented volume was then used for the docking procedure using the 'colores' function in situs. once docked the entire csy.pdb structure which included the fab of the kz neutralizing antibody was superposed over the docked gp /gp component of the structure. the d cryo-em reconstructions, cryo-et reconstructions, d models of the ebola virus, and the atomic resolution structure csy.pdb were visualized using ucsf chimera software package (computer graphics laboratory, university of california, san francisco, supported by nih p rr- ) [ ] . the d images and movies presented in this manuscript were generated directly by ucsf chimera software package. , region three is shown, with the locations of correlation maxima shown with an x. the angular distance between each maximum was calculated from several plots. a total of measurements gave an average angular distance of . u+/ . u between helical repeats, resulting in . repeats per turn. using the . nm pitch (fig. s ) the step in z per helical repeat was calculated as . nm. these helical symmetry values were then imposed on the nucleocapsid tomogram and this structure was used as the initial reference volume for refinement using the iterative helical real space reconstruction method. (tif) figure s surface spike distribution in the ebola vlp. longitudinal z-slices through the top and middle of the particle are shown, as well as the end-on view (a). the tomogram is shown as a shaded surface at a density threshold that indicates the spikes (b). the volume from one side of the tomogram has been extracted, and a red-blue color scheme shows the depth at which the spikes are located. this region of the envelope has a surface area of , nm . selected spikes have been identified by red circles, the single particle reconstruction of the spike is shown at the same scale to the right in a red square for comparison. the same region in (b) is shown in (c) with a solid orange cylinder to provide a visual cue for the viral envelope. eighty-six individual spikes were counted (white spheres) and have a patchy distribution (d), each spike would occupy an average area of nm , giving an average spacing between spikes of . nm. the reconstruction of the spike (blue) with the docked kz fab (purple) has been included to show that there is ample room for antibody attachment. (tif) figure s extraction of ebola nucleocapsid structure for sub-tomographic analysis. the tomogram of a linear region of the ebola virus was used as the first reference for subtomogram analysis (a). when viewed along the helical axis (y) or from the end perspectives (x,z) the basic components are visible. the tomographic volume was also cylindrically masked along the x-axis, selecting only the density containing the nucleocapsid, to highlight the components of the nucleocapsid in the tomogram (b). two-dimensional single particle image analysis was carried out with cryo-images (c) (not tomographic data sets), for comparison to the d tomographic data. the average shown in this panel was generated by reference free classification, using the ''startnrclasses'' program in eman [ ] . the . nm helical pitch can be easily seen in the d average, but is also visible in the projections of the tomographic volume in (a, b) . (tif) figure s representative low-magnification images of ebola virus. frozen hydrated virus is clearly visible with sections of the filamentous virus over both the support film and across the holes in the quantifoil film. individual g (single genome copy) virus is circled in red, several sections containing a nucleocapsid are indicated by a blue arrowhead, and regions without a nucleocapsid are indicated by a magenta arrowhead. globular heads are identified by yellow arrowheads. in this image the circles (light grey, m diameter) are filled with frozen hydrated virus in a thin aqueous layer, and the quantifoil support film appears as darker grey. table s length analysis of ''continuous'' ebola virus particles. the length of ebov particles were measured using imagej [ ] . the values in the ''model length'' column are based on multiples of the g mean length. the values in the in the ''mean length'' column were calculated directly from the data. only full particles containing a continuously packaged nucleocapsid were measured, all others (linked-nucleocapsid and empty particles) were omitted from this analysis. the terms g -g indicate the number of genomes/viral particle (i.e. g = genomes). all measurements are in mm. modeling of rna in the ebola nucleocapsid. two previously determined atomic resolution structures of negative stranded rna viruses (vsv ( gic.pdb) [ ] , and rsv ( wj .pdb) [ ] ) were used to estimate the ebov nucleocapsid length and number of nucleotides per nucleoprotein. images of vsv (a) and rsv (b) are shown as a molecular surface with the protein in orange and the rna as a green ribbon. from left to right, they show a surface view from the side, a side-on cross section, an end-on view, the rna density alone in projection, and a rotational average of the projection. the vsv-based estimate, with a saw-tooth pattern of rna in the helix, gave a nucleocapsid which . nm long, too short for the measured length of the g ebov ( nm). the rsv-based model, with a relatively straight/circular pattern of rna in the helix predicted a nucleocapsid . nm long, which closely fits the measured length of g virions, after allowing , nm space at each end to accommodate the curve of the envelope containing gp spikes and matrix proteins. the rsv-like model gives nucleotides per nucleocapsid protein which is similar to previous biochemical estimates of - for marburg virus [ ] , suggesting that the rna in the ebov nucleocapsid is arranged in a smooth helical pattern at a diameter of , nm. (tif) movie s -d reconstruction of ebola virus nucleocapsid. this movie shows the three-dimensional structure of the of the ebola virus nucleocapsid as shaded surface representation. the surface is set at a density threshold which would include one copy of np, vp ,vp , vp , and the rna. the nucleocapsid rotates, and then is sliced through the z-axis to show the internal components of the structure. movie s ebola virus spike distribution. this movie shows one surface of a cryo-electron tomogram of an ebola viruslike particle, generated by expressing the vp and gp proteins. the structure rotates showing the distribution of spikes. the locations of individual spikes are identified by white spheres, and the reconstruction is replaced by a cylinder to show the patchy distribution of spikes on the surface of the virus-like particle. movie s -d reconstruction of ebola virus spike. this movie shows the three-dimensional structure of the of the ebola virus gp spike. the structure rotates showing views from different angles, and indicates the location of the docked csy.pdb [ ] structure with the gp and gp domains and kz antibody (purple). (mov) physical principles in the construction of regular viruses studies on the pleomorphism of hvj virons infectious bursal disease virus is an icosahedral polyploid dsrna virus architecture of ribonucleoprotein complexes in influenza a virus particles ebola virus: new insights into disease aetiopathology and possible therapeutic interventions live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses ebola virus: from discovery to vaccine the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases ebola virus glycoprotein : identification of residues important for binding and postbinding events conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor comprehensive analysis of ebola virus gp in viral entry identification of two amino acid residues on ebola virus glycoprotein critical for cell entry the assembly of ebola virus nucleocapsid requires virion-associated proteins and and posttranslational modification of nucleoprotein functional mapping of the nucleoprotein of ebola virus nucleocapsid-like structures of ebola virus reconstructed using electron tomography electron tomography reveals the steps in filovirus budding grafix: stabilization of fragile macromolecular complexes for single particle cryo-em grafix: sample preparation for single-particle electron cryomicroscopy post-crystallization treatments for improving diffraction quality of protein crystals differentiation of filoviruses by electron microscopy ebola and marburg viruses: i. some ultrastructural differences between strains when grown in vero cells ultrastructure of ebola virus particles in human liver on the etiology of an unknown human infection originating from monkeys apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses a robust algorithm for the reconstruction of helical filaments using single-particle methods the iterative helical real space reconstruction method: surmounting the problems posed by real polymers assembly and budding of ebolavirus characterization of the ebola virus nucleoprotein-rna complex filoviridae: marburg and ebola viruses the ebola virus ribonucleoprotein complex: a novel vp -l interaction identified crystal structure of a nucleocapsid-like nucleoprotein-rna complex of respiratory syncytial virus morphology of marburg virus np-rna ebola and marburg viruses: ii. their development within vero cells and the extra-cellular formation of branched and torus forms analysis of ebola virus and vlp release using an immunocapture assay contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles ebola virus vp -vp interaction is sufficient for packaging e- e minigenome rna into viruslike particles descriptive analysis of ebola virus proteins quantitative densitometry of - g protein in acrylamide gel slabs with coomassie blue why does coomassie brilliant blue r interact differently with different proteins? a partial answer interference of the carbohydrate moiety in coomassie brilliant blue r- protein staining structure of the ebola virus glycoprotein bound to an antibody from a human survivor biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity architecture of the sars coronavirus prefusion spike using situs for flexible and rigid-body fitting of multiresolution single-molecule data endosomal proteolysis of the ebola virus glycoprotein is necessary for infection proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms structural organization of a filamentous influenza a virus replication-deficient ebolavirus as a vaccine candidate the creation of stable cell lines expressing ebola virus glycoproteins and the matrix protein vp and generating ebola virus-like particles utilizing an ecdysone inducible mammalian expression system ucsf chimera-a visualization system for exploratory research and analysis eman: semiautomated software for high-resolution single-particle reconstructions spider and web: processing and visualization of images in d electron microscopy and related fields conformational reorganization of the sars coronavirus spike following receptor binding: implications for membrane fusion situs: a package for docking crystal structures into low-resolution maps from electron microscopy structure of the vesicular stomatitis virus nucleoprotein-rna complex we thank b. szklarczuk and s. silcox for compiling the movies. key: cord- -p v p authors: ekins, sean; freundlich, joel s.; coffee, megan title: a common feature pharmacophore for fda-approved drugs inhibiting the ebola virus date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: p v p we are currently faced with a global infectious disease crisis which has been anticipated for decades. while many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the ebola or similar filoviruses that cause haemorrhagic fever. two recent high throughput screens published in did however identify several hits that progressed to animal studies that are fda approved drugs used for other indications. the current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore. this ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored. a recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein (vp ). when receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on fda approved drugs. these previously identified fda approved drugs with activity against ebola were therefore docked into this protein. the antimalarials chloroquine and amodiaquine docked favorably in vp . we propose that these drugs identified to date as inhibitors of the ebola virus may be targeting vp . these computational models may provide preliminary insights into the molecular features that are responsible for their activity against ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested. the current ebola virus (ebov) crisis has demonstrated that globally we are not prepared to respond with therapeutics to treat existing infections or act as prophylactics as there is no food and drug administration (fda) or european medicines agency (emea) approved therapeutic. more importantly this suggests we should have been prepared for a pathogen which has been known about for nearly forty years. the current ebov outbreak is already proving remarkably costly in terms of the mortality and financial ramifications , . the best approaches to ebov so far have relied on public health measures for containment which have been used in past outbreaks . these lessons with ebov will undoubtedly be important for the next virus outbreak but they also raise many questions which point to how little we know about these viruses in general, as well as how best to share knowledge openly . there have been a relatively small number of studies that have attempted to identify compounds active against ebov. two recent studies utilized high-throughput screens of a subset of fda approved drugs against different ebov strains (zaire and sudan) in vitro and in vivo. these independent reports suggested the promise of the antimalarials amodiaquine and chloroquine in one study , while the selective estrogen receptor modulators (serms) clomiphene and toremifene were active in another . chloroquine to date has not progressed beyond the mouse ebov model used in these studies. we hypothesized that we could use these four molecules to computationally define the features that are important for activity. the previous studies were not exhaustive screens of all fda drugs and so we have taken this opportunity to suggest additional compounds. looked at from another perspective "non-antiviral" drugs may be worth following up even though their molecular mechanism is unknown. these compounds may themselves have broad antiviral activity as reports describe modest inhibitory activity against other viruses - . several studies have identified non-fda approved drugs including an in silico docking approach to identify molecules targeting the viral nedd -ppxy interface . these molecules were similar to the fda benzimidazole and aminoquinoline , compounds that were active against ebov. another good example is the recent in silico docking of . million drug-like compounds docked in the viral protein vp protein . this identified multiple pyrrolidinones which inhibit its polymerase cofactor activity . the pyrrolidinones bind to an alpha helix which is proposed as important for viral function . with the limited knowledge of small molecules and potential targets we have studied whether the fda-approved drugs that are active in vitro and in vivo versus ebov could be targeting vp . common features pharmacophore for ebov actives two papers from described compounds active as inhibitors of different ebov strains in vitro and in vivo, namely amodiaquine and chloroquine in one study , clomiphene and toremifene in another . these active molecules were used as they have both in vitro and in vivo activity to build a common features pharmacophore with discovery studio . (biovia, san diego, ca) from d conformations of the molecules generated with the caesar algorithm. this identified key features. the pharmacophore was then used to search various databases (for which up to molecule conformations with the fast conformer generation method with the maximum energy threshold of kcal/mol, were created). the pharmacophore was then used to search the microsource spectrum database (http://www.msdiscovery.com/spectrum.html) as well as the cdd fda drugs dataset (https://www.collaborativedrug.com/ pages/public_access). in both cases over hits were retrieved initially. the van der waals surface of amodiaquine (which was more potent than chloroquine ) was added to limit the number of hits retrieved - . receptor-ligand pharmacophores for the vp protein were generated from crystal structures ( ibb, ibc, ibd, ibe, ibf, ibg, ibi, ibj, ibk) in the protein data bank pdb. pharmacophores were constructed using the receptor-ligand pharmacophore generation protocol in discovery studio version . (biovia, san diego, ca) with a maximum number of pharmacophores ( ), minimum features ( ), and maximum number of features ( ) as are described elsewhere . in silico docking of molecules in vp structure pdb ibi was used for docking using libdock in discovery studio (biovia, san diego ca) . the proposed binding site was centered on the ligand and a site sphere created (coordinates . , . , . ) with . Å diameter. the protocol included hotspots and docking tolerance ( . ). the fast conformation method was also used along with steepest descent minimization with charmm. further parameters followed the default settings. the ligand vpl was removed from the binding site and re-docked. the four fda approved drugs with activity against ebola were docked in the structure from an sdf file. molecules were visualized alongside the original ligand vpl and the d interaction plots generated. common features pharmacophore for ebov actives the pharmacophore was generated using the in vivo and in vitro active amodiaquine, chloroquine, clomiphene and toremifene we have responded to the reviewers suggestions. we have made a small labeling addition to figure and added a new figure s which is an expanded view of figure b . (supplemental table ) as these represent the most relevant fda approved drugs to date. this pharmacophore consists of hydrophobic features and a hydrogen bond acceptor feature ( figure ). the pharmacophore with van der waals surface was also used to search fda drug various libraries (supplemental table and supplemental table ). the most interesting observations from this virtual screen are that various estradiol analogs score well (e.g. estradiol valerate fit value . ). previously estradiol was suggested to be active in the ebov pseudotype assay in vitro . in addition, dibucaine was also retrieved (fit value . ) which was also active in the ebov pseudotype assay . amodiaquine, chloroquine, clomiphene and toremifene can be used as positive controls for future screens. because the original complete sets of fda approved compounds screened are not publically accessible it is difficult to compare hit rates versus all compounds tested to date. the nine receptor-ligand pharmacophores created all consisted of three to four hydrophobic features and one to two hydrogen bonding features ( table ). eight of these pharmacophores also had a negative ionizable feature. these suggest that the receptor-ligand based approach results in a general similarity across the nine structures, likely indicating the similar binding mode and importance of features for interfering with this generally hydrophobic pocket for protein-protein interactions. in silico docking of molecules in vp structure redocking the ibi ligand in the protein resulted in an rmsd of . Å, which generally indicates the difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket ( figure s ). this molecule was ranked the th pose and had a libdock score of . ( figure s higher scores are better). the four fda approved drugs were docked into the vp structure ibi. all compounds docked similarly and overlapped with the co-crystal ligand ( figure ). amodiaquine and chloroquine had higher libdock scores (> ) than the ibi ligand, while clomiphene and toremifene had libdock scores less than . all four fda approved drugs bound similarly to the pyrrolidinone ligands in the pocket formed by residues from the α-helical and β-sheet subdomains . we have highlighted proposed energetically favorable interactions of the antimalarial candidate binders with ile , lys and gln , which scored favorably. previously published studies suggested mutation of ile , lys resulted in near-complete loss of binding activity . our previous experience with common feature and quantitative pharmacophore models has demonstrated their value in predicting novel actives from collections of fda approved drugs - . candidate predicted actives may be assessed by their fit value to the pharmacophore model. this score can be used to prioritize compounds for eventual testing. in the current study it was hypothesized that two different classes of compounds showing activity against ebov in vitro and in vivo may share a common pharmacophore. construction of this pharmacophore ( figure ) indicated four hydrophobic features and a hydrogen bond acceptor feature. this pharmacophore (with an added van der waals surface to limit the number of hits retrieved) was then used to screen and score other fda drugs from score particularly well in terms of docking to vp . if this is the case it could provide a means to follow up with other small molecule analogs and/or additional fda approved drugs that could target this protein-protein interaction. as with our other tuberculosis-focused research , , and computational approaches to repositioning compounds we embrace the essentiality for computational predictions to be interrogated through rigorous experimental studies. for example at least two in silico docking studies screened commercially available compounds , . we propose that docking fda approved drugs could also be a viable first step to identifying potential compounds that could be used. we are actively seeking collaborators with experience with ebov assays to enable further translational studies. we believe this computationally inspired approach may be applicable for other known infectious pathogens that do not have current treatments such as other viruses related to ebola. ultimately we need to be able to leverage such approaches to provide antivirals for future pathogens. f research: dataset . pharmacophores, receptor ligand models and docking data for fda-approved drugs inhibiting the ebola virus, . /f research. .d . the ligand-based pharmacophore was previously made available: http://figshare.com/articles/ebola_active_cpds_pharmacophore/ . the following pdb structures were used in this study ( ibb, ibc, ibd, ibe, ibf, ibg, ibi, ibj, ibk). for models and advice please contact sean ekins (ekinssean@ yahoo.com). author contributions s.e. and m.c. came up with the general idea for the study based on the published in vitro and in vivo data. all authors contributed to the collaborative writing of this project. s.e. works for collaborations in chemistry, and consults for collaborative drug discovery inc. the author(s) declared that no grants were involved in supporting this work. dr. christopher d. southan, dr. peter madrid and dr. nadia litterman are acknowledged for discussions on ebola. biovia are kindly acknowledged for providing discovery studio. an earlier preliminary version of this pharmacophore was described previously: http://figshare.com/articles/a_pharmacophore_of_ebola_active_ compounds/ . a small database and identified and structures for future evaluation in vitro testing (supplemental table and supplemental table ). out of these compounds estradiol and dibucaine had been previously described as active in in vitro ebov assays. this suggested the pharmacophore could retrieve some structurally diverse classes of known hits . recently identified co-crystal structures of the ebov vp protein were used to derive receptor-ligand pharmacophores. these nine receptor-ligand pharmacophores suggested the importance of hydrophobic, hydrogen bonding and negative ionizable interactions to interfere with this protein-protein interaction ( table ) . eight out of nine of the pharmacophores had one or more hydrogen bond acceptor feature. these pharmacophores are grossly similar to our ligand based pharmacophore (derived from four fda approved drugs that inhibit ebov), as both types of model had multiple hydrophobic features and at least one hydrogen bond acceptor. when we docked the antimalarials and serms into a representative vp structure these compounds were found to overlap with the x-ray ligand to differing extents. amodiaquine and chloroquine had libdock scores greater than and higher than that for the redocked x-ray ligand. this indicated that vp may be a potential target for these two distinct classes of compounds. however, it is important to point out that we have not compared docking to other proteins in ebov and it could also be possible that these molecules are active elsewhere as well as via other mechanisms than by specific binding to proteins , . further, vp may be a preferred target for the antimalarials while the serms are not predicted to bind as well as the x-ray ligand. the use of other docking and scoring methods may produce differences in the pose and predicted binding affinity, which could be of interest for further studies. a combination of the promising efficacy of chloroquine (ec μm ) and amodiaquine (ec . μm ) versus ebov, their availability and likely low cost should prioritize their further laboratory exploration. mechanistic studies against vp and possibly other proteins should also be pursued and may be enlightened by the observation that both of these compounds also have reported activity against other viruses. for example, chloroquine is active against human coronavirus oc (in vitro and in infected mice) as well as sars (in vitro) , , , while amodiaquine also inhibits dengue virus replication and infectivity in vitro . in summary, this study has built on the previous publications that identified four fda approved compounds active against different strains of ebov , . our pharmacophore model for serms and aminoquinolines suggests that these compounds share multiple chemical features based on their overlap to the four hydrophobic features and a hydrogen bond acceptor ( figure e ) and they may have a common mechanism or target. we suggest that vp may be the likely target based on the overlap of receptor-based pharmacophores and docking into the crystal structure. amodiaquine and chloroquine figure s . redocking vpl in ibi. the ibi ligand was removed from the structure and redocked. the closest pose (grey) was ranked with rmsd . a and libdock score . when compared to the actual ligand in ibi (yellow). table . fda drugs and common features pharmacophore. the dataset of molecules was downloaded from the cdd public access (https://www.collaborativedrug.com/pages/public_access) as an sdf and then a d database was created in discovery studio using fast conformer generation with up to conformations. the database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene. the search d database protocol was used with the fast search method. in some cases the indication for the molecules is not described (nd). table . microsource spectrum and common features pharmacophore. the dataset of molecules was provided by microsource (http://www.msdiscovery.com/spectrum.html) as an sdf and then a d database was created in discovery studio using fast conformer generation with up to conformations. the database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene. the search d database protocol was used with the fast search method. have presented a crisp and lucid manuscript on a very relevant topic. they have suggested a et al. methodology to extract common features from four approved compounds that have recently been found to work against the ebola virus (amodiaquine, chloroquine, clomiphene and toremifene), and define a pharmacophore, which has been used to search databases, and identify further compounds for and in vitro in vivo testing. the methodology described here provides an excellent method to quickly screen known in silico compounds for possible therapies against ebola in particular, and other viruses in general. the result that serms show lower scores for binding to vp is rationalized by the finding that 'clomiphene and toremifene inhibit ebov vlp entry with some specificity to gp' , and therefore does not probably inhibit vp . 'chloroquine to date has not progressed beyond the mouse ebov model used in these studies.' this statement is not clear, does it mean that the others have progressed beyond the mouse ebov model? the first few compounds in supplemental table should be part of the main manuscript as a table. color coding of pharmacophore features should be in fig too (it comes earlier than table , where it is described). the structures look better with a white background. a Å rmsd for redocking a given ligand is quite high . the authors should consider the use of other docking methods, as a comparison. a table of libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score). the major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures. further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future. response: because this data is available easily on the website, i do not see any benefits of taking these compounds out of this supplemental table and putting them into the body of the manuscript. it might also add more confusion cutting the table up. color coding of pharmacophore features should be in fig too (it comes earlier than table , where it is described). response: thank you -this has now been added. response: i think this is a personal preference, the structures are clear in our opinion with a black background. i have not had this suggestion previously with other publications regarding the background color. the study was not intended as an exhaustive docking comparison, there are plenty of these in the literature as noted by the reviewer. i agreed the redocking rmsd was high, but i also provided some justification for the result (difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket). if others want to use different methods and perform a comparison for this target i would be supportive. a table of libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score). the libdock scores for the best poses are in the ' ibi libdock docking data best poses" file. a higher score is better and this has been added to the results section the major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures. further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future. all of the models were generated with the proprietary software discovery studio, and response: all files have been provided. the comment about such software is true, while i support using open software, i have yet to find an open source pharmacophore tool as good as that in discovery studio to date. it is also more convenient to use this software generating pharmacophores, receptor-ligand pharmacophores and docking in the same place. the types of analysis i have described could be repeated with any software, open source or proprietary. my hope is that by making this work openly accessible others will be inspired to pursue computational approaches with ebov. perhaps the community could propose the use of standards for open pharmacophore files as well. by publishing in this journal we are making all our data open even though they are in proprietary formats, i do not think this should preclude publication. no competing interests were disclosed. the current ebola crisis in west africa has shattered all expectations by continuing to grow months following the initial case. this has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community. the authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model. the key features of such a model are used to scan databases of small molecules. they have come up with a list of putative inhibitors. their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model vp -inhibitors that they have built. in parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein . based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein. in this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results. as they mention in the text (page ), the redocking of the ligand in ibi to the protein does not show the crystal structure binding mode accurately. these different settings could help in a better prediction of the ligand orientations. concerning the docking and proposed mechanism, i wonder how different the other solved nucleocapsid proteins are from a structural and sequence point of view, in order to make the authors point that vp is indeed the target. would it be possible to explore for surface patches with similar physico-chemical features? in the case of vp , a potential binding pocket for small-molecule inhibitors has been suggested by . how good or bad the overlapping with the built pharmachophore is hartlieb ( ) et al. for this case? to complete the structural understanding of the action of these compounds, a figure displaying their location on the protein surface as well as the binding site for rna would clarify their role in the inhibition of protein-protein interactions. in summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as ebola virus. although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value. no competing interests were disclosed. the current ebola crisis in west africa has shattered all expectations by continuing to grow months following the initial case. this has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community. the authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model. the key features of such a model are used to scan databases of small molecules. they have come up with a list of putative inhibitors. their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model vp -inhibitors that they have built. response: to clarify, we have focused on vp not vp . i am not aware of an x-ray structure with ligand bound for vp . the same type of approach could certainly be pursued with other ebov targets. we produced a common features pharmacophore for the compounds, and after looking at the vp receptor-ligand pharmacophores proposed that there may be some overlap, and then this led to docking the compounds in the x-ray structures. our intent was not to design molecules but to use the available methods to perhaps infer a potential target/mechanism and then perhaps researchers would want to test the compounds. we do not have access to experimentally test these predictions, but this manuscript may lead to others doing this work perhaps. whether one wants to use the libdock score for (absolute) prediction of binding affinity interactions is debatable, rather this approach might help to limit or prioritize which compounds to test. in parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein . based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein. response: vp may be a target for these compounds although we do not discount other targets or non-target related mechanisms. in this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results. as they mention in the text (page ), the redocking of the ligand in ibi to the protein does not show the crystal structure binding mode accurately. these different settings could help in a better prediction of the ligand orientations. response: as explained earlier, our study is not intended to be an exhaustive evaluation of docking tools, we have used different computational approaches to suggest that the fda drugs may have a common pharmacophore, which seems to be similar to that of the ligands co-crystallized with vp- . finally docking suggests they may fit into the pocket that the co-crystal ligands bind to. the work proposes that the compounds could fit in the binding site, but it is unclear what additional value more docking would add unless we were going to try to predict and then generate the x-ray structure of these fda drugs. certainly if experts in docking or crystallography want to pursue this target they can. ebola by the numbers: the size, spread and cost of an outbreak , and define a pharmacophore, which has been used to search databases, and identify further compounds for in vitro and in vivo testing. the in silico methodology described here provides an excellent method to quickly screen known compounds for possible therapies against ebola in particular, and other viruses in general.thank you for your constructive comments. some minor comments.the result that serms show lower scores for binding to vp is rationalized by the finding that 'clomiphene and toremifene inhibit ebov vlp entry with some specificity to gp' , and therefore does not probably inhibit vp .response: while the lower docking scores are noted i do not think this necessarily excludes them from inhibiting, as we know docking scores may not be that accurate and in this case, docking was used to answer the question could they fit. it's pretty clear that a wide variety of drugs could fit based on the binding site size and accessibility. response: from discussions with the author on the paper that described chloroquine as active versus ebov and in mouse, this work has not gone beyond the mouse model in vitro of ebov. response: this is indeed a very good point. we are not experts on these proteins. i think the proposed work could be done, the difficulty may be that there is no crystal structure (that i can see) with a ligand bound that would be a useful guide to binding in this pocket and would be essential for a receptor-ligand pharmacophore to be built. in summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as ebola virus. although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value. response: thank you for your suggestions, we agree and would encourage other scientists to test whether these compounds are targeting vp or vp as you propose, or having an alternative mechanism. i think we would also be happy to see any of these molecules progress into other animal models of ebov.no competing interests were disclosed. competing interests: key: cord- -bw tziup authors: perez-zsolt, daniel; martinez-picado, javier; izquierdo-useros, nuria title: when dendritic cells go viral: the role of siglec- in host defense and dissemination of enveloped viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bw tziup dendritic cells (dcs) are among the first cells that recognize incoming viruses at the mucosal portals of entry. initial interaction between dcs and viruses facilitates cell activation and migration to secondary lymphoid tissues, where these antigen presenting cells (apcs) prime specific adaptive immune responses. some viruses, however, have evolved strategies to subvert the migratory capacity of dcs as a way to disseminate infection systemically. here we focus on the role of siglec- , a sialic acid-binding type i lectin receptor potently upregulated by type i interferons on dcs, that acts as a double edge sword, containing viral replication through the induction of antiviral immunity, but also favoring viral spread within tissues. such is the case for distant enveloped viruses like human immunodeficiency virus (hiv)- or ebola virus (ebov), which incorporate sialic acid-containing gangliosides on their viral membrane and are effectively recognized by siglec- . here we review how siglec- is highly induced on the surface of human dcs upon viral infection, the way this impacts different antigen presentation pathways, and how enveloped viruses have evolved to exploit these apc functions as a potent dissemination strategy in different anatomical compartments. dendritic cells (dcs) are the most potent antigen presenting cells (apcs) found in humans [ , ] and their immune function is key to initiate immunity against invading viruses [ ] [ ] [ ] . these cellular sentinels patrol distinct mucosae and upon infection, viral sensing triggers rapid innate immune responses that might initially contain viral spread. dc activation also elicits cellular migration towards secondary lymphoid tissues, where dcs acquire a fully mature phenotype and become competent for antigen presentation, activation of naïve t cells, and expansion of antigen-specific adaptive t cell responses [ , ] . despite the immune activity exerted by dcs after viral infection, it has been known for decades that viruses evolved different strategies to escape dc antiviral activity [ ] [ ] [ ] . furthermore, certain viruses exploit the immune function of dcs as a way to colonize distant tissues and effectively disseminate systemically [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the discovery of the role of the receptor siglec- /cd , a sialic acid-binding ig-like lectin- expressed by dcs, has greatly contributed to our understanding of how viruses subvert dc activity. the siglec- receptor acts as an immuno-surveillance molecule [ ] but can also be effectively hijacked by distinct enveloped viruses, which either infect dcs directly or are effectively transferred to bystander target cells that become productively infected [ ] [ ] [ ] . hence, siglec- function on dcs clearly illustrates how these apcs can trigger antiviral immunity but also enhance viral spread via this receptor. siglec- is a type i transmembrane lectin with an amino-terminal v-set domain that interacts with sialylated ligands, preferentially n-acetylneuraminic acid (neu ac) in an α - linkage [ , ] . several enveloped viruses including human immunodeficiency virus (hiv)- [ ] [ ] [ ] and ebola virus (ebov) [ , [ ] [ ] [ ] incorporate such sialylated ligands within their membranes as an integral part of the gangliosides that are dragged from the plasma membrane when viruses bud from infected cells. although siglec- affinity for sialylated ligands is in the micromolar range, high-avidity binding can be achieved upon clustering of thousands of gangliosides in the viral membrane with their receptors on the cellular membrane [ , ] . moreover, as siglec- contains ig-like c -type extracellular domains that separate the ligand-binding site from the cell surface, it is available for interaction with external ligands and not bound in cis to cell-surface molecules, which is what usually happens with shorter siglecs that are also expressed by dcs [ , , ] . in vivo, the role of siglec- during viral infection has been mostly studied in murine models, focusing on resident tissue macrophages that express this lectin and play key immunomodulatory functions. siglec- -expressing macrophages are located in the subcapsular sinus of the lymph nodes, and they protect mice against vesicular stomatitis virus (vsv) infection by containing incoming viruses. viral sensing triggers cytokine release and promotes antigen presentation to b cells [ , ] . however, studies using different retroviruses to infect mice have shown that the protective function of these macrophages can be hijacked for efficient viral infection and dissemination within tissues. indeed, robust infection of a particular retrovirus in lymphoid tissues and spleen requires siglec- -expressing macrophages [ ] . the pathogenicity of the infecting retrovirus is key to tip the balance of these siglec- -expressing macrophages in favor of the protective immune function. the antiviral response dominates when the replicating virus has an expanded tropism [ ] , as it also happens in the case of the amphotropic vsv infection [ , ] . under these pathogenic conditions, viral capture via siglec- macrophages is necessary to elicit an effective antiviral cd + t cell response via antigen cross-presentation by dcs [ ] . overall, these murine studies explain how siglec- can contain viral replication and induce antiviral immunity against highly pathogenic viruses, but also favor viral spread within tissues when retroviruses have a limited tropism. yet, how these findings correlate with the pathogenesis of different siglec- -interacting human viruses, such as hiv- or ebov, remains largely unexplored. hiv- is the causative agent of acquired immunodeficiency syndrome (aids), a pandemic that has affected more than million people worldwide [ ] , while ebov is responsible for the intermittent outbreaks that produce a filovirus-associated disease (fvd) with high fatality rates [ ] . in this review, we discuss how siglec- is induced on human dcs upon viral infection, to what degree that impacts different viral antigen presentation routes, and in which ways distant enveloped viruses have evolved to exploit siglec- function as a dissemination strategy in distinct anatomical compartments. siglec- is a receptor codified by an interferon-stimulated gene and is therefore potently upregulated on distinct human dcs, monocytes, and macrophages when these cells sense type i interferons (ifns) such as ifnα [ ] [ ] [ ] . thus, infection with viruses such as hiv- or ebov tightly upregulates siglec- expression on apcs, as they directly trigger or indirectly promote the release of type i ifns via immune activating factors (figure ). hiv- induces secretion of type i interferons (ifns) by plasmacytoid dcs (pdcs) through toll-like receptor (tlr) - and - sensing, which upregulates siglec- on dcs in a paracrine manner. in addition, lipopolysaccharide (lps) from bacterial translocation upregulates siglec- on dcs via tlr sensing and autocrine type i ifn release. (b) during ebov infection, type i ifns might also play a central role in enhancing siglec- expression on dcs, although this needs further investigation. pdcs may produce type i ifns in response to ebov infection in vivo, while bacterial translocation was suspected during a case of gram-negative septicemia in an ebov-infected patient. in parallel, viral components such as secreted ebov glycoprotein may induce activation of myeloid cells through tlr signaling, providing an alternative stimulus of autocrine type i ifns during ebov infection. while solid arrows indicate established mechanisms, dotted arrows suggest processes that require further investigation. ifnar: ifnα/β receptor; sgp: secreted glycoprotein. in the case of hiv- infection, ifnα levels are potently boosted during acute infection, and sustained-although to a lower extent-throughout the chronic stage, which is characterized by a persistent immune activation [ ] [ ] [ ] . several dc types have been identified as the sources of ifnα production during the course of hiv- infection, and therefore contribute to siglec- induction. plasmacytoid dcs (pdcs) are considered the most potent type i ifn producers in blood [ ] , and their capacity to secrete ifnα in response to hiv- sensing has been demonstrated in vitro [ ] [ ] [ ] [ ] [ ] and in vivo [ ] [ ] [ ] , both during the acute and chronic phases of the disease [ , , ] . of note, pdc activation in response to hiv- sensing induces ifnα secretion through toll-like receptor (tlr) - and - signaling [ , ] and maturation of bystander myeloid dcs [ ] , and this ifnα response is more potent on pdcs derived from females [ ] . in turn, secretion of this cytokine can directly upregulate siglec- expression on dcs [ ] ( figure a ). in addition to pdcs, myeloid dcs also secrete type i ifns, although this release is mostly and indirectly triggered by immune activating signals present during the course of hiv- infection, which can induce the expression of ifn-stimulated genes on dcs in an autocrine manner [ ] [ ] [ ] [ ] . one of those factors is bacterial lipopolysaccharide (lps), which is increased in the plasma of hiv- -infected individuals due to the bacterial translocation that takes place in the gut-associated lymphoid tissue as a consequence of the gut epithelial barrier disruption occurring early after hiv- infection [ , , ] ( figure a ). lps induces siglec- expression on dcs [ ] . moreover, plasma from hiv- -infected individuals also stimulates siglec- expression on dcs signaling via type i ifn receptor [ ] . this explains why on circulating monocytes of hiv- infected individuals, siglec- expression correlates in vivo with the levels of plasma viremia, and why these levels only diminish after introduction of combined antiretroviral treatment [ ] . moreover, both types of siglec- -inducing factors are also present throughout the course of ebov infections ( figure b ). secretion of ifnα has been detected in humans and nonhuman primate models [ , ] , especially in lethal cases [ ] , while asymptomatic ebov infections are characterized by the absence of this cytokine [ ] . although in vitro pdcs exposed to ebov do not secrete ifnα [ ] , activated pdcs have been found in ebov-infected nonhuman primates, suggesting that these cells might produce ifnα in vivo [ ] ( figure b ). aside from pdcs, myeloid cells could contribute to ifnα secretion during ebov infection, as ebov-like particles induce ifnα production by murine bone marrow-derived dcs through tlr signaling [ ] . ebov glycoprotein interaction with human monocyte-derived macrophages induced tlr -dependent ifnα secretion by these cells [ ] . moreover, a cleaved and secreted form of ebov glycoprotein signals through tlr [ ] , although ifnα secretion in response to these glycoproteins remains unexplored ( figure b ). noteworthy, lps was also found in a case of ebov infection complicated with septicemia, possibly due to bacterial translocation [ ] , which might account for indirect ifnα secretion during ebov infection as described for hiv- ( figure b) . overall, the presence of type i ifns throughout the course of these viral infections is well-established, although both hiv- and ebov have evolved particular molecular mechanisms via viral antagonistic proteins that aid to evade cellular immune sensing [ ] [ ] [ ] [ ] . intriguingly, the protective role of type i ifn responses is controversial, since the apparent antiviral function during the earliest stages of infection may, in turn, fuel pathogenesis during the later stages of viral disease. that seems to be the case not only for hiv- [ ] , but also for ebov [ ] , where clinical data collected during human outbreaks have indicated that elevated levels of circulating ifnα, as well as upregulation of type i ifn-inducible genes, correlates with fatal disease outcome [ , [ ] [ ] [ ] . thus, hiv- and ebov infections trigger an immune activation state that upregulates siglec- expression on dcs, a situation that might favor early viral dissemination events in an otherwise antiviral environment [ ] . viral sensing enhances siglec- expression on apcs, and this facilitates hiv- infection of dcs and other myeloid cells (figure a , top), such as macrophages [ ] . although dcs are generally resistant to hiv- infection, a recent study showed that siglec- mediated hiv- productive infection of a population of human dc precursors known as pre-dcs [ ] . even though all dcs express the hiv- -interacting cellular receptor cd and the viral coreceptors [ , ] , being therefore susceptible to viral infection in vitro [ ] [ ] [ ] , infectivity was less prominent than in activated cd + t cells [ ] [ ] [ ] [ ] . importantly, the activation process that enhances siglec- expression on dcs further restricts viral infection, as mature dcs are -fold to -fold less susceptible to hiv- than immature dcs [ , , [ ] [ ] [ ] . infection of dcs with hiv- also appears to be uncommon in vivo, although it has been reported for both cutaneous and mucosal dcs, including vaginal epithelial dcs [ , , ]. yet, the role of siglec- in promoting hiv- infection of these dcs remains largely unexplored. in most dc subtypes, however, the restriction factor samhd inhibits reverse transcription, thus precluding immune sensing and the synthesis of viral antigens. bottom: conversely, hiv- encodes vpx, which counteracts samhd activity allowing reverse transcription of the viral genome, that can be sensed via cgas and trigger cytokine release. newly synthesized proteins lead to the production of viral particles, but also to proteosomal cleavage the de-phosphorylated form of the host factor sterile alpha motif histidine-aspartate domain-containing protein (samhd ) is the most potent restriction factor that limits hiv- infection in dcs [ ] (figure a, top) . however, if this lack of infectivity is actually beneficial for an immune control remains controversial, as the absence of viral replication impairs viral sensing and limits presentation of hiv- -specific antigens to prime adaptive immune responses [ ] [ ] [ ] . in contrast to hiv- , hiv- naturally replicates on dcs [ ] , which depends on the counteraction of samhd by the viral antagonist vpx [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure a, bottom) . hiv- genome replication in infected dcs is detected by the innate cytosolic dna sensor cyclic guanosine-adenosine monophosphate synthase (cgas), which triggers immune responses upon dna sensing [ , ] (figure a, bottom) . in contrast, hiv- restriction by samhd prevents viral dna (vdna) retrotranscription in the cytoplasm, impairing the induction of antiviral type i ifn responses [ ] (figure a, top) . thus, viral replication on dcs can provide an additional source of viral components to be detected via immune sensors or presented to t cells [ , ] (figure a, bottom) , but also compromise cell viability and release inflammatory factors that fuel viral pathogenesis, as previously described for sustained or chronic type i ifn responses. while hiv- replication on dcs remains hard to identify in vivo, it has been known for more than a decade that dcs are among the first target cells encountering ebov [ ] . dcs are highly susceptible to ebov infection [ , ] , and this is a complex process that involves several host factors whose function is still being identified [ ] . indeed, siglec- expressed on activated dcs has recently emerged as a new host factor implicated in ebov attachment, a mechanism that facilitates subsequent cytoplasmic viral entry [ ] ( figure b ). initial ebov attachment to the dc surface is mediated by several receptors that recognize different elements on the viral membrane and often have a redundant activity [ ] . c-type lectin receptors (clrs) such as the dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign) and the liver/lymph node sinusoidal endothelial c-type lectin (lsectin) mediate viral attachment through binding to viral glycoproteins [ , ] , while receptors of the tim/tam families (comprising the t cell immunoglobulin and mucin domain receptor along with tyro-axl-mer receptors) recognize phosphatidylserine lipids present on the viral envelope [ ] (figure b ). ebov incorporates sialylated gangliosides on their membrane [ ] , and we have recently shown that these viruses are effectively recognized by the siglec- receptor [ ] (figure b ). siglec- recognition of sialylated gangliosides on ebov modulates the binding, uptake, and trafficking of filoviral particles into a sac-like virus-containing compartment (vcc) continuous with the plasma membrane ( figure b ). viruses stored in this compartment can be redirected into the classical endosomal pathway and facilitate viral entry into the cytoplasm [ ] . indeed, ebov macropinocytosis allows trafficking into late endosomes, where cleavage of viral glycoproteins by cathepsin b (ctsb) facilitates the interaction with the endosomal receptor niemann-pick c [ ] [ ] [ ] (ncp ) that triggers cytoplasmic viral entry [ ] [ ] [ ] (figure b ). thus, siglec- -mediated attachment facilitates viral access to the cell cytoplasm [ ] . while siglec- contributes to ebov entry into dcs, filoviral replication within these cells compromises immune function and prevents adaptive immune responses by limiting cytokine secretion, downregulating the expression of major histocompatibility complex (mhc) and costimulatory molecules and also by reducing the ability of dcs to stimulate t cell proliferation [ , [ ] [ ] [ ] . these results suggest that ebov suppression of dc function prevents initiation of adaptive immune responses and facilitates uncontrolled systemic virus replication [ , ] through a mechanism that is enhanced by siglec- activity [ ] . however, clinical data gathered during the west african - outbreak showed strong and sustained t cell activation [ ] , challenging the in vivo relevance of this viral dc-escape mechanism [ ] . overall, both hiv- and ebov can exploit siglec- activity to boost dc infectivity, although ebov replication is more prominent in these cells. yet, viral infection poses a difficult balance for apcs. on the one hand, infectivity triggers antiviral immunity via viral sensing and antigen presentation, but on the other hand, it also promotes cell death and suppression of immune responses through the activity of particular viral antagonist proteins. recently, it has been suggested that this apparent paradox is overcome by a division of labor between distinct dc subsets [ ] . there is therefore a dissociation between viral infection and antigen presentation, which occurs in distinct dc subpopulations. by these means, susceptible infected dcs transfer viral antigens to resistant dcs, which remain competent to launch adaptive immune responses against viral infections [ ] . while several pathways allow for antigen transfer between dcs, secretion of extracellular vesicles bearing particular antigens is among the most effective ones. although the functional paradigm of dc biology states that the particular apc that interacts with incoming viruses in the mucosa would be the one processing these viruses and then traveling to the lymphoid tissue, these cells may not always be the only ones presenting the captured antigens. rather, these pathogen-interacting dcs may transfer captured antigens to other apcs by several mechanisms, including secretion of extracellular vesicles bearing antigen-loaded fragments, which can even be already processed and presented in mhc molecules (figure , top) . by these means, the number of dcs bearing viral-specific antigens can be increased very quickly upon infection, thus amplifying the initiation of primary adaptive immune responses [ ] [ ] [ ] . importantly, to induce naïve t cell stimulation in vitro, these extracellular vesicles require a competent activated dc to deliver the co-stimulatory signals to t cells [ ] (figure , top) . thus, antigen-containing extracellular vesicles do not overcome the need for a competent apc to activate naïve t cells. among the distinct cellular receptors expressed by dcs, siglec- is key to capture secreted extracellular vesicles through the same mechanism hijacked by enveloped viruses [ ] (figure ). siglec- interacts with extracellular vesicles via recognition of sialylated gangliosides packaged on the vesicle membrane [ ] , which assemble and bud from cellular membranes as viruses do [ , ] . this result has been confirmed not only in vitro [ , ] with extracellular vesicles derived from cell lines or primary cells but also in murine models where siglec- expressed on lymphoid tissues was required to trap extracellular vesicles in vivo [ ] . upon capture of extracellular vesicles on activated dcs via siglec- , these vesicles are trafficked along with the receptor towards a sac-like compartment invagination that is continuous with the plasma membrane and allows for extracellular vesicle retention [ , ] (figure , top) . the siglec- positive compartment formed within activated dcs may serve as an antigen depot, controlling and sustaining adaptive immunity even if the source of antigen is not directly in contact with the apc, that still can trigger antigen-specific immune responses. these antigens could maintain immunity for prolonged periods, as it happens when dcs control endosomal acidification to preserve antigen cross-presentation over time [ ] . although mature or activated dcs markedly downregulate their macropinocytic capacity, these cells are still able to capture, process, and present antigens internalized via endocytic receptors [ ] , and that may also be the case for siglec- via extracellular vesicle trapping. moreover, as dcs continue to capture and present antigens after maturation in vivo [ ] , they could also initiate responses to newly encountered antigens during the course of viral infections, a process that would be boosted by siglec- expression. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via transinfection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation (figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] ( figure ) . although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical the fate of trapped extracellular vesicles on dcs is diverse, as they provide a source not only for antigen cross-presentation to cd + t cells, but also to stimulate antigen-specific naïve cd + t cell responses in vivo [ , ] . cd + t cell stimulation can take place either by reprocessing the antigens contained in the captured extracellular vesicles or by the direct presentation of previously processed functional epitope-mhc complexes exposed in the vesicle surface [ , ] . direct extracellular vesicle antigen presentation in the absence of lytic degradation within dcs was initially described using dc populations devoid of particular mhc-ii molecules, that were still able to activate cd + t cells because the necessary mhc-ii molecules were already presenting the antigen on the extracellular vesicles trapped by those dcs [ ] . thus, extracellular vesicles displaying previously processed functional epitope-mhc complexes on their surface can be recognized, retained, and directly transferred from dcs to antigen-specific cd + t cells [ ] (figure , top) . in turn, siglec- upregulation on activated dcs, which are competent apcs, could boost extracellular vesicle uptake and magnify antiviral immune responses. intriguingly, hiv- and other retroviruses exploit this antigen dissemination pathway usually engaged by extracellular vesicles to reach cd + t cells [ ] , which are the main cellular targets of this particular retrovirus (figure , bottom left) . siglec- directs captured hiv- particles to the same vcc where ebola viral particles are retained [ ] , that is in addition the same compartment where extracellular vesicles are trapped in activated dcs [ , ] (figure , bottom left) . however, in the case of hiv- recognition, viral entry via siglec- does not lead to the productive infection of dcs as it happens with ebov, but favors the transfer of trapped viruses to bystander cd + t cells. thus, trapped viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes [ , , ] ( figure ). this mechanism of viral transmission is known as trans-infection [ ] and is mediated by siglec- on activated monocyte-derived dcs, monocytes, blood conventional dcs, pre-dcs, and primary myeloid cells isolated from lymphoid and cervical tissues [ ] [ ] [ ] , , ] . filoviral trans-infection from dcs to cd + t cells is improbable as lymphocytes are largely resistant to ebov infection [ ] . nonetheless, filoviruses display a broad cell tropism, infecting hepatocytes, adrenal cortical cells, and endothelial cells, among other cellular targets [ ] [ ] [ ] [ ] . thus, aside from lymphocytes, other cellular targets could be transinfected (figure , bottom right), as it was previously shown for a human cell line binding ebov that transinfected hela cells [ ] . however, further research will be required to determine in which anatomical context dcs trapping ebov via siglec- could transfer that infectivity to susceptible cellular targets in vivo. overall, siglec- retention of distinct viruses on extracellular vesicle-containing compartments highlight how these cells might act as "trojan horses", capturing filoviruses or retroviruses in the peripheral mucosae and carrying them to secondary lymphoid tissues, where viruses can be effectively transmitted to target cells and contribute to the systemic spread of infection [ ] [ ] [ ] , ] . recently, we have identified the presence of siglec- -expressing cells in cervical mucosa. these dcs are capable of capturing hiv- and mediate viral transmission to target cd + cells via trans-infection, even in a basal state where no apparent activation is detected [ ] (figure ) . indeed, dcs directly isolated from human ectocervix displayed a basal siglec- expression that was sufficient to mediate viral transfer. however, at the endocervix, where the expression of siglec- is lower than at the ectocervix, this capacity was enhanced upon ifnα stimulation ( figure ) . hiv- is mostly acquired by sexual transmission, and in the cervical mucosa, there are two major sources of antiviral type-i ifn responses after retroviral infection: resident myeloid cells [ ] and pdcs, which are the most potent producers of ifnα [ ] and are soon recruited to the cervix [ ] (figure ). although increased antiviral ifnα secretion could limit initial viral infection, it could promote viral capture on cervical myeloid cells via siglec- induction as well. of note, in the cervical biopsy of a viremic hiv- + patient, siglec- + cells harbored hiv- -containing compartments, demonstrating that in vivo, these cells can trap viruses [ ] . interestingly, similar vcc-like structures have been detected in urethral macrophages of hiv- -infected individuals under suppressive combination antiretroviral therapy [ ] , but if siglec- is implicated in the formation of these particular structures remains to be determined. siglec- allows transferring viruses to bystander cd + t cells in the mucosa, but also the systemic viral dissemination upon dc migration to lymphoid tissues ( figure ) . indeed, dcs bearing retroviruses are found in the draining lymph nodes of distinct animal models as soon as h after vaginal challenge [ , [ ] [ ] [ ] and these findings originally led to formulation of the trojan horse hypothesis, which states that dcs can serve as vehicles transporting the virus from the entry sites to distant tissues [ , ] . . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such . proposed mechanism of hiv- dissemination from the female reproductive tract. in the vaginal/ectocervical mucosa, basal siglec- + dcs may mediate local hiv- trans-infection to target cd + t cells. at the endocervical mucosa, lower siglec- expression is boosted in response to ifnα released by recruited pdcs sensing hiv- . siglec- + dcs may also contribute to systemic hiv- spread due to their ability to migrate to secondary lymphoid tissues, where cd + t cells accumulate. while a similar mechanism could be exploited by ebov, further work needs to address this possibility. sexual transmission is not considered a major route of ebov infection. however, a case of sexually transmitted ebov has been well documented [ ] . moreover, a recent mathematical model is consistent with a significant contribution of sexual ebov transmission during the - outbreak in west africa [ ] . importantly, infectious viral particles are found in semen of ebov convalescent individuals several months following symptoms onset [ ] [ ] [ ] [ ] , and seminal fluid amyloids may enhance ebov infection [ ] . as the cytokine tgf-β is abundant in semen, and it also upregulates siglec- expression on dcs [ ] , the role of this receptor should be further assessed in the context of ebov sexual transmission. moreover, dcs are early and sustained targets of ebov that can disseminate infection from the portals of viral entry to the regional lymph nodes, spleen, and liver [ ] . since siglec- is expressed in all these ebov replicating-tissues [ , ] , this receptor could also boost systemic viral spread as previously suggested for hiv- [ , , , , ] . collectively, these data indicate that siglec- on dcs may not only participate in viral transmission at the mucosa, but also promote systemic viral dissemination to secondary lymphoid tissues. we hypothesize that the outcome of early interactions between dcs and enveloped viruses may be key to mount effective antiviral responses, but these early encounters also foster viral dissemination to distant tissues. the emerging roles of siglec- receptor on dcs clearly exemplify how the immune-mediated activity of this lectin can be effectively hijacked by unrelated viruses such as hiv- or ebov. future work should address if other enveloped viruses causing relevant human infectious diseases may contain sialylated gangliosides on their membranes and be recognized by siglec- , as it has been already shown for the henipavirus [ ] . moreover, the precise contribution of siglec- to viral immune containment or to pathogenic viral dissemination should be carefully evaluated, as understanding both mechanisms may provide novel avenues to combat infectious agents. the identification of individuals which naturally lack siglec- expression due to the presence of an early stop codon in the siglec gene in homozygosis [ ] indicates that the role of this protein is not essential and that it may be therefore a safe therapeutic target. developing such pharmacological agents to block siglec- interaction with viruses could pave the way to ameliorate viral systemic dissemination. moreover, exploiting the immune-surveillance function of siglec- receptor to induce antiviral immune responses may also prove valuable. in turn, dissecting how distinct viruses exploit common molecular pathways will advance future antiviral strategies to generate broad spectrum 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efficient interaction of hiv- with purified dendritic cells via multiple chemokine coreceptors diversity of receptors binding hiv on dendritic cell subsets susceptibility of human peripheral blood dendritic cells to infection by human immunodeficiency virus replication of human immunodeficieacy virus type in primary dendritic cell cultures differential susceptibility to human immunodeficiency virus type infection of myeloid and plasmacytoid dendritic cells immature dendritic cells selectively replicate macrophagetropic (m-tropic) human immunodeficiency virus type , while mature cells efficiently transmit both m-and t-tropic virus to t cells virus replication begins in dendritic cells during the transmission of hiv- from mature dendritic cells to t cells during hiv- infection most blood dendritic cells are not productively infected and can induce allogeneic cd + t cells clonal expansion low levels of hiv- infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory cd + t cells the maturation of dendritic cells results in postintegration inhibition of hiv- replication human immunodeficiency virus fusion to dendritic cells declines as cells mature dendritic-cell interactions with hiv: infection and viral dissemination blockade of attachment and fusion receptors inhibits hiv- infection of human cervical tissue hiv- replicates and persists in vaginal epithelial dendritic cells samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx a cryptic sensor for hiv- activates antiviral innate immunity in dendritic cells the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses vpx relieves inhibition of hiv- infection of macrophages mediated by the samhd protein mhc-i-restricted presentation of hiv- virion antigens without viral replication ebola and marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation mechanisms of filovirus entry characterization of the filovirus-resistant cell line sh-sy y reveals redundant role of cell surface entry factors c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus phosphatidylserine receptors: enhancers of enveloped virus entry and infection endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry: a curious and complex series of events host factors in ebola infection filovirus entry: a novelty in the viral fusion world impairment of dendritic cells and adaptive immunity by ebola and lassa viruses ebola virus: the 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dendritic cell-mediated capture and transfer of hiv- and henipavirus identification of siglec- null individuals infected with hiv- this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -jq xumrh authors: postnikova, elena; cong, yu; dewald, lisa evans; dyall, julie; yu, shuiqing; hart, brit j.; zhou, huanying; gross, robin; logue, james; cai, yingyun; deiuliis, nicole; michelotti, julia; honko, anna n.; bennett, richard s.; holbrook, michael r.; olinger, gene g.; hensley, lisa e.; jahrling, peter b. title: testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jq xumrh identifying effective antivirals for treating ebola virus disease (evd) and minimizing transmission of such disease is critical. a variety of cell-based assays have been developed for evaluating compounds for activity against ebola virus. however, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-ebola virus activity using established cell lines and human primary cells. the effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for % and/ or % inhibition (ec( ), ec( )) was evaluated using the fda-approved compound, toremifene citrate. in these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the ec( ). these results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals. ebola virus (ebov) infection in humans and nonhuman primates is often associated with high morbidity and mortality rates, as well as severe hemorrhagic fever [ ] [ ] [ ] [ ] . ebov is a biosafety level- pathogen transmitted by contact with bodily fluids, fomites, or droplets from plos infected patients. ebov is considered a significant threat to public health and global security due to its potential to be used as a bioweapon [ ] [ ] [ ] [ ] . currently, no fda-approved vaccine or therapeutic agents are available, and supportive care remains the standard for ebola virus disease (evd) treatment. therefore, accelerated efforts in the development of therapeutics is a key objective in the ebov research community, especially since the - evd epidemic in western africa. drug discovery and development requires considerable time and resources to identify an effective drug that will progress to clinical trials [ , ] . as a result, research investigating the repurposing of drugs for additional indications have become increasingly more prevalent to accelerate the identification of therapeutic drugs for evd. the off-label use of fda-approved drugs is particularly advantageous as safety concerns and ethical problems have already been addressed [ ] [ ] [ ] [ ] . to effectively identify potential compounds of interest from large libraries of chemical compounds, share more reliable and reproducible data between laboratories, and provide data to the international community, appropriate methods or models need to be established. furthermore, these models should be evaluated to determine how predictive they are for identifying compounds most likely to be efficacious in humans. for evd, indications of efficacy could include successful treatment and survival of patients, alleviation of disease severity, or mitigation of clinical symptoms associated with ebov infection. a variety of methods are available to measure antiviral activity in vitro. however, the development of a screening assay to detect compounds with anti-ebov activity was previously limited due to the difficulty of developing a suitable high-throughput screening system for a biosafety level- viral pathogen. classical methods evaluate drug efficacy include the reduction of virus yield [ ] or a decrease in viral rna transcription as determined by real-time polymerase chain reaction (pcr) [ ] [ ] [ ] . recent therapeutic screening methods have transitioned from the classical methods of measuring viral inhibition to assays with the ability to be automated, resulting in higher-throughput. assay chemistries have been developed to enable the homogeneous measurement of a variety of different endpoints such as cytopathic effect, viral protein or reporter gene expression, which can serve as markers of viral replication [ ] . the growing interest in identifying drugs with activity against ebov has resulted in a variety of assays and readouts for activity as well as cytotoxicity. the use of cell-based assays for highthroughput screening of compound libraries has increased steadily over recent years [ ] [ ] [ ] [ ] . as cell-based assays monitor specific viral proteins and provide the means to screen for potent viral inhibitors intracellularly, these assays identify drug candidates with desired pharmacological properties in the primary drug-discovery pipeline. in this study, the susceptibility to ebov using both immortalized cell lines and primary monocyte-derived macrophages (mdms) was investigated under a variety of conditions such as different multiplicities of infection (mois), times of exposure, and the cell passage numbers. a cell-based assay with ebov vp -specific antibody was used to detect infected cells. fluorescence or chemiluminescence readout was used for determining signal-to-noise (s/n) ratio, and a high-content imaging system was applied to determine the percentage of ebov-positive cells. toremifene citrate, which the world health organization considered evaluating in a clinical trial for treatment of evd, was chosen as a positive control to measure ebov inhibition under each condition [ ] [ ] [ ] [ ] . the conditions under which drugs are tested can influence their apparent potency. while testing drugs for the world health organization (who) community, we received many requests to repeat experiments under specific conditions to confirm activity identified by another laboratory. the resulting data sets indicated just how variable the ec value can be under varying assay conditions. the data presented here provides insight on how different assay parameters can impact the in vitro efficacy of potential anti-ebov antivirals using toremifene citrate as a model compound. vero e (african green monkey kidney; atcc ) cells were obtained from the american type culture collection (manassas, va). vero c (e ) cells (african green monkey kidney, working cell bank nr- ) were obtained through bei resources (national institute of allergy and infectious diseases [niaid] , national institutes of health [nih], manassas, va). huh cells (human hepatocellular carcinoma) were obtained from dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt). all cell lines were maintained at the integrated research facility (irf) following cell source instructions. a primary vero e and huh cells culture were grown to % confluency in a t- (fisher scientific) or triple layer tissue culture flask (nunc) containing dulbecco's modification of eagle medium (dmem) (gibco) supplemented with % heat-inactivated fetal bovine serum (fbs) (sigma). cells were dispersed by trypsin (gibco) treatment and then reseeded into secondary cultures. the process of removing cells from the primary culture, diluting, and then transferring them to secondary cultures constitutes a passage. both cell lines were provided at passages - , at which point a new culture was introduced and the previous passage series was ended. additionally, cell cultures were required to be a least % viable in order to achieve acceptance criteria and to be plated for use in a screening assay. the generation of mdms has been described in previous studies [ , ] . briefly, pbmcs were isolated from human whole blood by density-gradient centrifugation over histopaque ( . g/ml, sigma-aldrich, st. louis. mo). monocytes were purified using human cd -specific microbeads (miltenyi biotec, san diego, ca, - - ) following manufacturer's instructions. cd + monocytes were differentiated into mdms by culturing for - days with recombinant human macrophage colony-stimulating factor (bio-techne, minneapolis, mn, -mc- ) and conditioned medium from kpb-m cells (kind gift from dr. atsunobu hiraoka, scgf research laboratory, kyoto, jp). media were replaced every - days during the incubation for a total of - days. the cells were harvested and plated on desired -well plates day prior to the drug screen assay. the differentiated mdms were characterized by flow cytometry before assay initiation. toremifene citrate (oral solution) tested in this study was purchased from sigma-aldrich (cas - - ; t - mg). the makona isolate of ebov (h. sapiens-tc/gin/ /wpg-c ) (ebov/mak, genbank accession no. kp ), a kind gift from dr. gary p. kobinger (public health agency of canada, winnipeg, ca), was used in these studies. to generate virus stocks, ebov/mak was inoculated at an moi of . in vero c cells (bei resources, manassas, va, catalog nr- ). when the cytopathic effect was visible at day - after infection ( - % of monolayer showing cpe), cell culture supernatants were harvested and clarified by centrifugation. the ebov/mak titer was determined by plaque assay in vero e cells. virus titers were measured using -fold serial dilutions of culture supernatant in triplicate infections of vero e cell monolayers in -well plates. after incubation at ˚c for h (plates were rocked every minutes), ml of medium containing x mem (gibco), . % avicel (fmc biopolymer), and x antibiotic-antimycotic (gibco) were added to each well ( ml/well). after days post-incubation, virus plaques were stained with . % crystal violet (ricca chemical) in % neutral buffered formalin (thermo scientific) and infectivity titers were measured in plaque forming units per ml (pfu/ml). all procedures using live ebov were performed under biosafety level- (bsl- ) conditions. vero e and huh cells were seeded overnight at to × cells per well and mdm cells were plated at × cells per well in μl of dulbecco's modified eagles's medium with % fetal bovine serum in black opaque (thermo fisher scientific, waltham, ma, corning , - - ) or clear bottom -well greiner microplates (greiner bio-one, monroe, nc, ). ebov/mak isolate was diluted in culture media to the specified mois (the titers used to determine moi hereby were generated on vero e cells) in -well plates. the cells were then infected by transferring μl of ebov/mak isolate from the virus dilution plates to cell plates using the -well liquidator (rainin instrument, oakland, ca). the cells were incubated at ˚c and % co for the indicated periods of time. the plates were fixed by adding μl of % neutral-buffered formalin (final concentration %) at , , or h postinoculation (hpi). after fixing for h, the plates were transferred to a bsl- lab for antibody staining as described previously [ ] . briefly, ebov was detected by exposure of the infected, fixed, and permeabilized cells to a monoclonal mouse antibody specific to the ebov vp matrix protein (b-md -bd -ae , made by us army medical research institute of infectious diseases, frederick md under centers for disease control and prevention contract) [ ] , followed by staining with an alexafluor goat anti-mouse igg (heavy + light chains) antibody (life technologies, carlsbad, ca) at ˚c for h. fluorescence was quantified using a tecan plate reader (infinite m , tecan us, morrisville, nc) or an high-content imaging (hci) system (operetta, perkinelmer, waltham, ma). hci images were collected at x magnification using to fields of view in each well to quantify the percent of ebov-positive cells. the viability of the cell layer was monitored by staining cell nuclei with the hoechst dye (molecular probes) at ˚c for h. columbus . . software (perkinelmer) was used to analyze the hci data. the chemiluminescent enzyme-linked immunosorbent assay (celia) was performed by detecting ebov with the anti-ebov vp antibody followed by staining with the horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody (seracare, milford, ma, cat. # - ). chemiluminescence was quantified using pico chemiluminescent substrate (thermo fisher scientific inc., rockford, il) and a plate reader (infinite m tecan). vero e , huh and mdm cells were seeded as described above in μl media overnight at ˚c with % co . compounds in dimethyl sulfoxide were prediluted to reduce dimethyl sulfoxide concentration to . % or lower. compounds were prediluted in dilution blocks before performing a final : dilution by transferring μl of each compound to cell plates containing μl of cell culture media. this dilution achieved a desired compound concentration in μl of cell culture media. the process of performing the drug screen assay is shown in fig . three plates were set up per experiment, two plates (clear bottom -well greiner microplates) for detecting inhibition of ebov, and one mock plate (black opaque plate) for determining drug cytotoxicity. after h of predilution and transport to the bsl laboratory, μl of virus (or mock control) at the desired moi was added to cells. at , or hpi, assay plates were fixed at final concentration of % nbf for h before transferring to a bsl- lab for staining. infected cells were detected as described above. to further confirm the accuracy of assays with high background, chemiluminescence assay was performed afterwards. cytotoxicity in mock infected cell plates was measured or h after treatment with compounds using the celltiter glo luminescent cell viability assay kit according to the manufacturer's instructions (promega, madison, wi). luminescence was read on the infinite m tecan plate reader (fig ) . non-linear regression analysis and curve fitting parameter were performed to calculate ec s, ec s and % cytotoxic concentration (cc s) (graphpad software, la jolla ca) [ ] using dose-response curves for the compounds (toremifene citrate). error bars of dose-response curves represent the standard deviation of three replicates. equations for the ratio of s/n, percentage of ebov-positive infected cell, and z' factor were defined previously [ , ] , and ec s were used as parameters for assay validation. the quality control of cell-based assay is at specific assay endpoints, cells are fixed and transferred to the bsl- . immunostaining was performed with a ebov-specific antibody against vp and a fluorescent or chemiluminescent secondary antibody using a plate washer/dispenser. fluorescence is quantified on a plate reader. the hci system (operetta) is used to detect ebov-positive cells and count cells with a nuclei stain (hoechst ). in parallel, cytotoxicity assays (celltiter glo) with mock infected cells are performed at bsl- . luminescence is read on the infinite m tecan plate reader. data are analyzed using graphpad prism and/or columbus software (operetta). https://doi.org/ . /journal.pone. .g factors that influence ebola antiviral activities in cell-based assays plos one | https://doi.org/ . /journal.pone. march , represented by z' factor which is defined as equation z' = [ -(( à sd pos )+( à sd neg ))/(imean pos -imean neg )]. in our assay, the z' criteria is as follows: z' = . - . corresponds to an excellentassay; z' = - . corresponds to a suboptimal assay; z' < corresponds to a unsuccessful assay [ ] . the susceptibility to ebov infection was evaluated in multiple cell types in cell-based assays measuring anti-ebov activity. vero e , huh and mdm cells were infected with ebov/ mak isolate at different mois, and the assay was terminated after , , or hpi. cells were stained with a fluorescent antibody, and hoechst dye was used to visualize the cell nuclei. infectivity was measured using the high-content imaging (hci) system as percentage of vp positive cells. the growth of ebov/mak isolate in three cell types was compared over time. in vero e cells, ebov spread slightly slower, and the number of positive cells were overall lower compared to huh cells (fig a- d ). mdms were the most susceptible to ebov among the cell types. at hpi, mdms already exhibited a typical dose response relative to virus input, while only minimal ebov replication was observed in vero e and huh cells at hpi at all mois tested. virus spread effectively at hpi with higher ( to . ) moi of vero e , huh cells and most of mois in mdms (fig a- f ). the infection in mdms was saturated at hpi at almost all mois (fig e and f ). the nuclear stain for all cell types showed that the cell layer deteriorated with increased virus inoculum and duration of infection as was clearly evident at the hpi time point and at higher virus input (mois of . ) (fig b, d , f and g). the cell layer frequently became fragile, and at later time points, the cell layer would lift off the well surface possibly because of increased exposure to virus, higher moi, or manipulation of plates during the fixing/staining procedure (fig g) . the hci system proved to be invaluable for determining optimal assay conditions (end point and virus input) and in identifying potential issues such as cell layer integrity. different virus input and endpoints were assessed to identify an acceptable range for testing drugs in vero e and huh cells (tables and ). the same experimental plates from fig a- d were used for this analysis. the fluorescence s/n ratio was calculated using a tecan plate reader, and the percentage of ebov-positive cells was determined using hci. for reproducible data, we determined that the percentage of ebov-positive cells should be kept within a range of to % and the s/n ratio at to . at lower s/n ratios (< ) or lower percentage of infected cells (< %) distinguishing activity of a drug from non-activity becomes increasingly difficult. on the other hand, at higher s/n ratios (> ), when cell infection rate trends towards - % and cells overgrow at later time points, the risk of a disrupted cell layer is greater. cell layer disruption increases the variability of the assay. therefore, the optimization of assay conditions (i.e., duration of infection, virus input) was carefully calibrated resulting in a compromise between signal strength and cell viability. based on the data obtained from fig and tables and , two optimized conditions for the fluorescence assays were selected. an moi of with hpi as endpoint and an moi of . with an endpoint of hpi produced a robust signal for all cell types, vero e , huh and mdms (fig a- f , tables and ). vero e cells were infected with ebov at around . %, and mdms and huh cells usually had a stronger signal with around % or higher of ebov-positive cells (fig c- f ). the two conditions had a similar signal or ebov infection during the assay development phase, vero e cells appeared less reliable in producing a consistent viral spread, and this lack of reliability appeared to be due to cell culture passage history. to address this matter, vero e and huh cells were analyzed at different cell passage numbers by determining percentage of ebov-positive cells (fig a and b ). both cell types were infected with a moi of for h. the viral spread of huh cells were consistent over cell culture passages - , indicating that the passage number does not impact ebov spread c the percentage of ebov-positive cells was determined with a vp /alexa- antibody stain to detect ebov and a nuclear stain to quantify the number of cells in a high-content imaging system. d s/n is the ratio of the fluorescent signal (mean from infected wells) to the noise (mean from mock-infected wells) quantified using a plate reader. abbreviations: ebov + , ebola virus-positive; hpi, hours post-inoculation; moi, multiplicity of infection; s/n, signal-to-noise ratio. in huh cells in the range tested. in contrast, ebov spread on vero e cells varied considerably between cell passages - with peak infection (replication efficacy) at cell culture passages - ( fig a) and equally lower for early or late passages. significant differences were observed at passages , , , , and when compared to the passage or using an ordinary one-way anova following dunnett's multiple comparison in graphpad prism . . occasionally, exceptions to this trend were observed (fig ) . the infectivity of ebov in vero e and huh cells were compared at an early (p ) and a late passage number (p ) at a range of mois ( . - . ) for h. in this case, vero e cells tested at the later passage showed a lower infection rate (< %) as expected, while the early passage demonstrated an unusually high infection rate (up to %). infectivity in huh cells remained consistent regardless of passage number, vero e cells were inconsistent overall despite the trend originally observed. both the virus input and duration of the experiment can have an impact on drug activity. to address this, we evaluated the in vitro efficacy of toremifene citrate, an fda-approved drug with proven anti-ebov activity [ ] , under different parameters using fluorescence as the read out. huh cells were infected with a constant moi of and treated with toremifene citrate for , , or h (fig a) . later time points resulted in a decrease in activity with the ec at hpi . -fold higher than at hpi. when the time point remained constant at hpi the activity of toremifene citrate increased as the virus input decreased (mois of . , . , , or ) (fig b) . the ec at a moi of . was . -fold lower than at a moi of . in addition to time and virus input, the cell type used in the assay can result in differences in the calculated ec . anti-ebov activity of toremifene citrate was measured using variable assay endpoints ( , and hpi), mois ( . , . , . , and ), and cell lines (vero e and huh cells, fig ) . ec values for toremifene citrate increased with exposure time and virus input in both cell types. overall, activity in vero e cells was higher with maximum activity (ec = . μm) at hpi and an moi of . . in huh cells, maximum activity (ec = . μm) was detected at hpi and an moi of . . ec values for toremifene citrate increased with exposure time and virus input in both cell types indicating that more drug is required to produce the same anti-ebov effect. factors that influence ebola antiviral activities in cell-based assays to increase sensitivity of the drug screen assays, a chemiluminescent enzyme-linked immunosorbent assay (celia) was developed for evaluating compounds for anti-ebov activity. the celia combines the advantage of specificity of an immunoassay with the high sensitivity of a chemiluminescent enzyme detection assay and is a simple and low cost screening assay [ , ] . the celia was compared to the fluorescent assay (detected by regular plate reader or the hci system) by testing the efficacy of toremifene citrate against ebov infection in huh cells with a range of mois ( . , . , , and ) at three different time points ( , and hpi) . assay parameters, signal-to-noise (s/n), and z' factor, were compared between the assays (table ) . at the earliest time point ( hpi), the celia had higher sensitivity than the fluorescent detection assay as the z' were in the acceptable range (> . ) even at the lowest moi of . . in contrast, the fluorescent assay required higher mois for reliable data sets (z'> . ). at the hpi time point, both detection methods generated high quality data sets with the celia providing improved z' factors (> . ). at hpi, the celia and the fluorescent assay also produced excellent data using hci for detection, while the fluorescent data detection by regular plate reader showed higher variability, maybe due to cytopathic effects. the ec s and ec s factors that influence ebola antiviral activities in cell-based assays were determined on those data sets with acceptable z' (> . ) and overall there was good correlation between the different detection methods (table ). all detection assays demonstrated that higher moi and longer times of exposure led to an increase of the ec values for toremifene citrate. in contrast, the ec values showed considerably lower fluctuations under the different conditions tested. the established celia was used to compare anti-ebov activity of toremifene citrate in different cell types, vero e , huh and mdms using an moi of . and a time point of h (fig ) . huh cells and mdms had s/n ratios in the range of s with z' factors generally above . (fig a) . the s/n for vero e cells ranged from > to over , leading to more variability as reflected in the wider range of z' factors ( . - . ) (fig a) . the activity (ec ) of toremifene was compared, and considerable differences were detected for the cell types ( fig b) . the efficacy of toremifene citrate was -fold higher in huh cells than in mdms, and -fold higher than in vero e cells (fig b) . in summary, the chemiluminescent drug screen assay for evaluating anti-ebov activity of compounds is a very robust, reliable and reproducible method. the data presented in this study exemplify the importance of having guidelines for testing drugs in vitro for antiviral activity and generating reproducible data sets that can be shared and confirmed by outside laboratories. several parameters should be considered when testing factors that influence ebola antiviral activities in cell-based assays drugs in vitro for antiviral efficacy. cell type, assay endpoint, and the virus input are among the most important factors [ ] . the vero e cell line, derived by immortalization of african green monkey kidney cells, is the most commonly used cell line for testing antivirals against filoviruses. ebov propagates very well in this cell line, and many laboratories, including ours, generate their virus stocks and determine virus titers in vero e cells. however, the argument to use a cell line with more relevance to human disease has come up repeatedly. hence, the human liver cancer cell line, huh and human mdms were chosen for these studies. macrophages are relevant to human disease, and they are considered to play an important role in virus dissemination in pathogenesis of evd [ ] . therefore, a drug that is highly effective at inhibiting ebov infection in mdms may better translate to in vivo potency than vero e cells. the susceptibility of three cell types was compared over a range of virus input and over time. all cell types were permissive to ebov infection, but to different degrees. mdms demonstrated optimal replication starting as early as hpi at an of moi . . in contrast, ebov grew slower in vero e and huh cells. the optimal conditions for evaluation of drug efficacy using the fluorescent assay were at an moi of . for the h time point or at an moi of for the h time point for all cell types to ensure a strong virus signal and avoid destruction of cell layer (fig g) . however, an moi of is not suitable for detecting inhibitors of later steps in the virus life-cycle. the ability to detect inhibitors of virus assembly or egress will be reduced with increasing moi, as higher proportions of the cells will be infected even in the factors that influence ebola antiviral activities in cell-based assays though vero e cells are one of the most broadly used cell lines for testing compounds in in vitro assays, our data show that the performance of these cells is not ideal especially when evaluating drugs for anti-ebov activity. although it is never a good idea to passage cells for too long [ , ] , we found that at lower or higher passages the vero e cells can show a decreased percentage of ebov-positive cells. huh cells showed more efficient and more reliable virus replication irrespective of passage number. immortalized cancer cell lines are in general an easy choice for in vitro drug testing because they are easy to handle, propagate, expand, and plate on a week-by-week basis with relative consistency. in contrast, primary cell types such as human primary macrophages do not proliferate in cell culture and are ideally generated fresh from human blood upon use. for large scale testing, procuring the blood volumes needed for generating mdms from a cumbersome isolation technique could be a challenge. mdms demonstrated very efficient spread of virus through cell culture over time. donor-todonor variability precludes consistency, and in general, macrophages from different donors are not pooled. despite of the constraints, testing a selection of drugs that are closer to human clinical studies in human macrophages makes sense to get a better idea on efficacy in a more relevant target cell. fluorescence was detected by two different read outs and each technique has its advantages. the plate reader will measure the average fluorescence across the whole well. readings are quick and conducive to handling large numbers of plates during screening. however, the quality of the cell layer cannot be monitored. hci provides images of or more fields of each well, and a nuclear stain can be added in parallel to indicate the viability of the cell layer. in the assay development phase, this tool is especially useful in identifying conditions that will ensure a healthy cell layer or identify issues such as plate corner or edge effects for assay quality control. one drawback is that not the whole well is imaged, but only a certain number of fields inside the well. it is important to pick the fields wisely to avoid bias or skewing of data. scanning at least fields per well is recommended for statistical purposes, but that will increase reading time per plate. the time to read and analyze the hci data can take considerably longer than the data acquired with the regular plate reader. hci measures different parameters such as percentage of ebov-positive cells and mean signal intensity per cell. both the regular plate reader and hci have unique applications in a drug testing program. while a plate reader is good for a quick readout of large number of plates, the hci system is used to cross check on quality of cells and to clarify issues of noise and signal variability. in addition to the fluorescent assay, we also implemented a celia using an hrp-labeled antibody, which amplified the signal and increased the sensitivity of virus detection. as expected, the celia showed an improvement in the quality of data sets compared to the fluorescent assay. s/n ratio and z' factor were in an acceptable range as early as hpi with the lowest virus input detected at an moi of . . an ec could also be determined at this time point. at or hpi, the two fluorescent read outs were similar to the chemiluminescent read out. toremifene citrate is a selective estrogen receptor modulator reported to be active in vivo against ebov in mice [ , ] . this is an fda-approved drug for treating breast cancer [ ] . mechanism of action studies showed that toremifene citrate affects ebov virus entry at the stage of virus-fusion with the endosomal membrane [ ] . toremifene citrate was chosen by the who as a potential candidate for clinical evaluation in evd patients. we evaluated the performance of toremifene citrate on anti-ebov activity using different conditions. a trend towards higher ec s with increasing input virus and longer assay time was observed. the data indicate that if the amount of virus present in cells or tissues is high enough, the drug will be less active. also with longer time for the virus to replicate, drugs may be unable to stem high viral replication. in contrast, the ec values were less affected by high moi or longer time points, which may, therefore, be a more reliable parameter for comparing data sets between laboratories. serum in the media and pretreatment of cells with the compound can also have a considerable effect on antiviral activity. toremifene was compared directly to brincidofovir under a panel of different conditions [ ] showing that activity of brincidofovir was dependent on media conditions with low serum (< %) and h pretreatment before adding virus. in contrast, the serum concentration had no effect on the activity of toremifene citrate, and pretreating cells for h (instead of h) decreased the activity of this drug. many reports on the repurposing of fda-approved drugs discuss and compare a drug's in vitro ec (determined in cell cultures) with its maximum concentration in human plasma (determined in clinical trials) when evaluating the potential of the drug to have in vivo activity. however, our data show that the ec of a drug can range over more than a log based on testing conditions (e.g, moi, time of endpoint, serum). johanson et. al. [ ] reported ec s of . and . μm for toremifene with two different ebov strains ebov/kik and ebov/may, respectively, at hpi with . moi by celia using the c antibody. these data are comparable to the ec of . ± . μm for ebov/mak determined in our assay under similar conditions (table ; moi . / hpi). understanding how assays are performed and the potential variables is important, and caution should be used when using in vitro data for making decisions to advance a drug to in vivo studies. addition, we acknowledge laura bollinger and jiro wada at the irf for technical writing services and figure preparation, respectively, for this manuscript. multiple ebola virus transmission events and rapid decline of central african wildlife ebola virus: from discovery to vaccine exotic emerging viral diseases: progress and challenges phase clinical trial of apical membrane antigen : an asexual blood-stage vaccine for plasmodium falciparum malaria the soviet union's anti-agricultural biological weapons 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toxin, and plating density in cell-based cytotoxicity assays cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death inhibition of ebola virus entry by a c-peptide targeted to endosomes the lipid moiety of brincidofovir is required for in vitro antiviral activity against ebola virus we thank irf cell culture staff in preparing the cells used in this study. we thank dr. atsunobu hiraoka (scgf research laboratory, kyoto, jp) for the kind gift of conditioned medium from kpb-m cells. we thank dr. gary kobinger (public health agency of canada, winnipeg, ca) for the makona isolate of ebola virus (genbank accession no. kp ). we thank dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt) for huh (human hepatocellular carcinoma) cells. we thank dr. sheli radoshitzky (usamriid, frederick md) for her gift of vp ebov vp matrix protein (b-md -bd -ae . in key: cord- - hlj r authors: brauburger, kristina; hume, adam j.; mühlberger, elke; olejnik, judith title: forty-five years of marburg virus research date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hlj r in , the first reported filovirus hemorrhagic fever outbreak took place in germany and the former yugoslavia. the causative agent that was identified during this outbreak, marburg virus, is one of the most deadly human pathogens. this article provides a comprehensive overview of our current knowledge about marburg virus disease ranging from ecology to pathogenesis and molecular biology. marburg virus (marv) first appeared in august , when laboratory workers in marburg and frankfurt, germany and belgrade, yugoslavia (now serbia) were infected with a previously unknown infectious agent. the patients ( primary, six secondary infections) developed severe disease that progressed to a fatal outcome in seven of the cases. an additional case showing symptoms of disease was diagnosed retrospectively (reviewed in [ ] ). the source of infection was traced back to african green monkeys (chlorocebus aethiops) that had been imported from uganda and were shipped to all three locations. the primary infections ironically occurred when the monkeys were necropsied for the purpose of obtaining kidney cells to culture poliomyelitis vaccine strains. in the remarkable period of less than three months the etiologic agent was isolated, characterized, and identified by the joint effort open access of scientists in marburg and hamburg [ ] and was later confirmed by kunz and colleagues [ ] and kissling and colleagues [ ] . the pathogen was named marburg virus after the city with the most cases and represented the first isolation of a filovirus. erroneously, a study published in 'the lancet' claiming that the mysterious disease was caused by rickettsia or chlamydia has frequently been cited as the first report on the causative agent of marburg virus disease (mvd) [ ] . it was not until that the now better-known member of the family, ebola virus (ebov), first emerged in africa [ , ] . shortly thereafter marburgviruses and ebolaviruses were classified together in a newly established family termed filoviridae, so-named after their distinctive thread-like structure (filum being latin for thread). marv had not been heard of for eight years, when a young australian who had traveled throughout zimbabwe was admitted to a hospital in johannesburg, south africa with symptoms reminiscent of those observed during the outbreak in europe [ ] . when he died and the infection spread to his travel companion and later also to a nurse, lassa fever was initially suspected resulting in strict barrier nursing techniques and isolation of the patients and their primary contacts. this lead to a quick containment of the outbreak, and while the secondary cases recovered, marv was identified as the causative agent of the disease. in the following years from through , only sporadic outbreaks that affected small numbers of individuals were caused by marv on the african continent (table , figure a ). as the case fatality rates associated with mvd were also lower than those seen in the devastating outbreaks associated with ebov disease that reached up to %, marv was long thought to be less threatening (table ) . however, this view had to be revised as marv reemerged in two large outbreaks occurring in the democratic republic of the congo (drc) in - [ ] and then, for the first time also in western africa, in angola, in - [ ] . the total number of cases and the high fatality rates ( % in drc and % in angola) revealed that marv was as big of a threat for public health as ebov [ , ] . the variation observed in disease severity and case fatality rates between these outbreaks versus the initial one in may depend on many complicating/mitigating factors. these include quality and availability of medical care, infectious dose and route of infection, differences in host population susceptibility (depending on immune and nutritional status) and genetics, inherent differences in viral variant virulence, and the prevalence of co-infections (particularly malaria and aids in patients from sub-saharan africa) [ ] . the assumption that marv angola might be inherently more virulent than other marv variants has been proposed mainly based on infection studies with nonhuman primates (nhp) [ ] [ ] [ ] but is a matter of debate [ ] . the genomes of the angolan isolates differ about % at nucleotide level from the majority of the east african marv isolates, including the ones from [ ] . there is no evidence so far that the observed genetic differences result in higher virulence in humans. the drc outbreak was unique, as there were at least nine different virus variants circulating in the tested patients indicative of several different spillover events from the natural reservoir to the human population [ ] . in contrast, sequence data from the angolan outbreak suggested a single introduction of marv to an unidentified index patient and subsequent spread via person-to-person contact. the viral genomes showed a remarkably high genetic stability within this outbreak. identical marv genomes were isolated from patients even after two to three human to human transmissions [ ] . mvd is considered a zoonotic disease that is thought to persist in a healthy reservoir host in the endemic areas in africa. humans and nhps are spillover hosts and show a high rate of fatal disease outcomes. several large-scale attempts to identify the natural host of filovirus infection throughout sub-saharan africa had been undertaken in the years since filoviruses first emerged with frustratingly little success [ ] [ ] [ ] [ ] . consistent with ecologic niche modeling of outbreaks and epidemiological patterns, isolated cases have suggested that ebov is endemic in the rain forests of central and western africa while marv is more prevalent in open, dry areas of eastern, south-central africa [ , ] . almost all of the primary infections of natural mvd outbreaks so far have been linked to human entry into caves inhabited by bats (e.g., cave visitors, mine workers) (table ) . thus, bats have long been suspected to play an important role in the transmission cycle of the disease [ , , ] . in , evidence was detected for marv infection of the common egyptian fruit bat (rousettus aegyptiacus) [ , ] (figure ), and marv was isolated from healthy infected r. aegyptiacus bats caught in the same year in uganda [ ] . the bats were collected in kitaka cave around the same time as human infections occurred that had been linked to the cave (table and see above, . epidemiology). genomic analysis of the few isolates of marv acquired from bats showed that the sequences matched closely to the marv genomes isolated from patient samples (figure b) . this was also the case for partial marv sequences isolated from bats inhabiting the goroumbwa mine in the drc that was suspected to be the major location for several independent spillover events to gold miners between and . the bat marv sequences were closely related to the distinct isolates that had been reported during these outbreaks in humans [ ] . a study analyzing marv prevalence in bat populations in gabon found marv-specific nucleic acids in r. aegyptiacus bats in several local caves [ ] . together with previous data showing a high prevalence of marv-specific antibodies in gabonese bat populations [ ] and with an observed relation of the isolated sequences with previously reported gabonese bat isolates [ ] this study viruses , suggests that marv is enzootic in gabon and raises the concern of further spread of marv into other countries. therefore, close ecological as well as serological surveillance of the bat populations in sub-saharan africa could help to predict and prevent further mvd outbreaks especially in areas where bats are still used as a food source. it is not currently clear whether r. aegyptiacus bats are the exclusive reservoir for marv or if other bat species reported to be positive for viral antibodies and rna are also natural reservoirs or merely intermediate hosts [ ] . the genus marburgvirus includes a single species, marburg marburgvirus (formerly referred to as lake victoria marburgvirus) [ , ] . phylogenetic analysis based on genomic sequence data suggests that the known members of this species can be assigned to at least five different lineages of which four are very closely related (nucleotide sequences differ up to %) while the fifth is divergent (a nucleotide difference of %) ( figure b) [ , , ] . as the genomic divergence between all isolates is below %-the cutoff for the classification of the five different ebolaviruses into five different species-the five marburgvirus lineages were recently reclassified as two viruses. ravn virus (ravv) is represented by the ravn isolates from , one isolate from the drc outbreak in - , and one human and several bat isolates from infections that took place in uganda in . marburg virus (marv) is represented by all other sequenced isolates ( figure b , table ) [ ] . for the sake of simplicity, in this review the abbreviation "marv" is used for all marburgviruses and the abbreviation "ebov" for all ebolaviruses. initial mvd patients are believed to contract the virus via exposure to an infected animal: either a reservoir host (several bat species) or a spill-over host such as nhps as described in the first mvd outbreak (see above, . epidemiology) [ , ] . following transmission to humans, spread of the virus between individuals is the result of direct contact with blood or other body fluids (saliva, sweat, stool, urine, tears, and breast milk) from infected patients. typical risks of exposure include administration of medical care to infected individuals as well as handling of corpses without use of proper protection [ ] . of particular note, virus has been found in tears, semen, and in a liver biopsy weeks to months following the onset of symptoms highlighting the importance of monitoring convalescent patients [ , [ ] [ ] [ ] . much of what we know about typical mvd symptoms comes primarily from clinical data obtained during the three largest recorded mvd outbreaks: the outbreak in germany and yugoslavia, the - outbreak in the drc, and the - outbreak in angola. although the case fatality rates were significantly higher in the latter outbreaks, most of the clinical symptoms observed were similar. based on the most reliable documented cases of exposure and subsequent illness, mvd has an incubation period ranging from to days (typically to days), which is likely modulated by factors such as infectious dose and possibly by route of infection. the course of mvd has conventionally been broken down into three phases [ ] : an initial generalization phase, an early organ phase, and either a late organ phase or convalescence phase depending upon disease outcome [ ] . a summary of mvd symptoms is reviewed below [ , [ ] [ ] [ ] [ ] . the onset of illness begins with generic flu-like symptoms; a characteristic high fever (typically - o c), severe headache, chills, myalgia, prostration, and malaise. for many patients ( - %) this is followed by rapid debilitation characterized by gastrointestinal symptoms including anorexia, abdominal pain, severe nausea, vomiting, and watery diarrhea. starting on day four to five patients commonly develop enanthem, dysphasia, and pharyngitis. additionally, a characteristic maculopapular rash is typically the first distinctive feature indicating a filovirus infection versus influenza or malaria. other common symptoms include lymphadenopathy, leukopenia, and thrombocytopenia. many of the initial symptoms may persist in the early organ phase, and patients may sustain a high fever. they may additionally display neurological symptoms including encephalitis, confusion, delirium, irritability, and aggression. patients can also develop dyspnea and abnormal vascular permeability, particularly conjunctival injection and edema. during the latter part of this phase more than % of patients present with some form of clear hemorrhagic manifestation such as petechiae, mucosal bleeding, melena, bloody diarrhea, hematemesis, and ecchymoses. due to the unusualness of hemorrhagic symptoms, diseases caused by filoviruses have sometimes been referred to as hemorrhagic fevers (marburg hemorrhagic fever (mhf) and ebola hemorrhagic fever (ehf)), although these terms are currently disfavored since not all patients display hemorrhagic symptoms. at this stage, multiple organs are affected including the pancreas, kidney, and liver. elevated serum activity of a number of liver enzymes including sgot and sgpt have been observed in most patients sampled. the late stages of mvd result in one of two potential outcomes: patients either succumb to the disease or enter a prolonged phase of recuperation. typical preagonal symptoms include restlessness, obtundation, confusion, dementia, convulsions, reduced circulation due to severe dehydration, metabolic disturbances, severe diffuse coagulopathy, multiorgan failure, shock, and coma. fatalities typically occur - days following the onset of symptoms, with death usually the resulting of shock and multiorgan failure. non-fatal cases are typified by an extensive convalescent period during which myalgia, exhaustion, sweating, peeling of the skin at the sites of rash, partial amnesia, and secondary infections are all common. prevention of newly emerging marv infections and effective containment during ongoing outbreaks is both essential and challenging, as there is currently no licensed vaccine or treatment available for general use. following the outbreak of mvd in europe and cases of infection with ebolavirus reston in imported crab-eating macaques (macaca fascicularis) in the usa in / and as well as in italy (reviewed in [ ] ), strict quarantine procedures have been put in place that have so far prevented infections acquired by imported nhps into non-endemic countries [ , ] . to avoid the spread of filoviruses by tourists, python cave was closed to the public following the diagnosis of the dutch patient in . the prevention and control of outbreaks and infections in endemic countries is much more challenging. in the past, joint efforts of teams from the who, doctors without borders, the red cross, the cdc and others in collaboration with the local ministries of health have been undertaken to cease the spread of mvd. the main focus of outbreak control is the prevention of secondary transmission and further primary infections. the first measures in response to a mvd outbreak include setting up isolation wards in hospitals to assure rapid isolation of marv-infected patients and prevent person-to-person transmission (figure b ). proper and fast laboratory diagnosis of suspected cases is key to eliminate further spread. nosocomial infections were commonly seen in earlier outbreaks [ , , ] . however, reinforcement of barrier nursing techniques and education of health care workers have limited these infections in recent outbreaks ( figure a ). epidemiological surveillance has been crucial in the identification of index cases as well as the predominant modes of transmission. in endemic areas, secondary infections mainly occurred while taking care of ill patients and family members or during traditional burial practices involving close contact to corpses [ ] . therefore, the execution of safe burial and disinfection techniques and information campaigns to educate the local population are essential in order to contain the spread of infections in endemic areas ( figure a ) [ , , ] . biosafety and epidemiological efforts alone were not sufficient for efficient outbreak control during large outbreaks, emphasizing the need for additional psychosocial support of the affected communities [ ] . the fast progression and high lethality rates associated with mvd even-and especially-after hospital admission resulted in a high level of fear and suspicion by the resident population. the fact that health care workers wearing recommended personal protective equipment (ppe) were fully masked and not identifiable further increased anxiety (figure b ). this resulted in the hiding of infected family members and verbal and in some cases physical aggression towards members of aid organizations [ ] . communicating necessary protective measures while respectfully considering the affected families' and communities' traditions and culture during ongoing outbreaks is therefore essential for successful outbreak management. the recent identification of bats as the potential reservoirs for marv as well as ebov [ , , , , ] will help to increase not only the public awareness, but also the effectiveness of the preventive measures taken in endemic areas to minimize contact with infected animals (i.e. closing of bat inhabited caves for the public, serosurveillance of bat populations) [ , ] . this is a challenging task, emphasized by the fact that during the last cluster of marv infections linked to a gold mine in uganda, the miner hired to enforce the restricted access to the mine got infected. the mine had been closed in response to the ongoing outbreak and even though he was aware of the risk, he had entered the mine without the suggested ppe [ ] . later, the bat population of this mine was eliminated by the owner by means of fumigation [ ] . as bats of most species are endangered, this does not seem a viable option and educational campaigns aimed at villagers living close to bat-inhabited caves as well as tourist groups and tour operators might prove more sustainable in the future. in , during the first reported filovirus disease outbreak in europe, the identification of the previously unknown causative agent of the deadly disease was performed by electron microscopy (em) (figure ). the unusual filamentous structure of the particles led to some confusion and it was even suggested that the causative agent of the disease might be related to the spiral-shaped leptospira, a genus of the spirochaetes bacteria [ ] . others concluded that the observed particles were viruses morphologically related to rhabdoviruses and named the newly discovered pathogen marburg virus [ , ] . marburg virions are pleomorphic particles, which appear as rod-or ring-like, crook-or sixshaped, or branched structures. cryo-em analysis of purified virions showed that about % of viral particles released from infected vero cells were filamentous, % were six-shaped, and % were round [ ] . the same study revealed a mean particle length of nm and a mean diameter of nm. previous conventional em studies showed that the marv particles were uniformly nm in diameter, whereas the length varied widely with virions measuring up to , nm. the average particle length was nm [ ] [ ] [ ] . the reported differences in particle size might be due to experimental differences between cryo-em and conventional em [ ] . notably, marv particles are considerably shorter than ebov virions, although marv genomes are slightly longer than ebov genomes [ , ] . marv particles are surrounded by a host-derived membrane that is coated with spikes of - nm in length, which are formed by trimers of the viral glycoprotein (gp) ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] . the central core of the viral particles is the ribonucleoprotein complex (nucleocapsid) formed by the viral rna genome and tightly associated nucleocapsid proteins ( figure ). the nucleocapsids are highly organized tubular structures with an outer diameter of - nm and an electron-dense central axis of - nm. the central axis is surrounded by a helical capsid with cross-striations at a nm interval [ ] [ ] [ ] . a recently published detailed cryo-electron tomography analysis of marv virions has shed some light on the structural organization of the nucleocapsids. reconstructions of virion-associated nucleocapsids using subtomogram-averaging analysis revealed that the marv nucleocapsids form a left-handed helix with a pitch of . nm and a flexible average symmetry of . protrusions per turn with two inner lobes of density per protrusion. the inner lobes represent the nucleoprotein (np), suggesting that the marv nucleocapsid contains an average number of . np molecules per turn with each np molecule packaging six rna bases [ ] . marv nucleocapsids show directionality, having a pointed and a barbed tip [ ] . the nonsegmented negative-sense (nns) rna genomes of the various marv isolates range in size from , to , nts and contain seven monocistronic genes in a linear order ( figure ) [ , ] . each gene is composed of a highly conserved transcription start and stop signal, an unusually long ' and ' untranslated region, and the open reading frame (orf). the genes are either separated by short intergenic regions that range from to nts, or the transcription stop signal of the upstream gene and the transcription start signal of the downstream gene overlap, sharing five highly conserved nts ( figure ). the structure of this gene overlap is found among all filoviruses and is unique among members of the order mononegavirales (for review see [ ] ). the ' and ' genome ends are extracistronic regulatory regions that contain cis-acting signals essential for transcription and replication, including transcription and replication promoters. there are generally two types of genomic replication promoters for nns rna viruses: a bipartite promoter found in members of the paramyxovirus subfamily paramyxovirinae and one continuous more compact replication promoter for rhabdo-and pneumoviruses [ ] . the bipartite promoter structure of the paramyxovirinae subfamily is associated with the "rule of six", i.e., the total genome length must be a multiple of six [ ] . given that filoviruses do not obey the rule of six, it was surprising that mapping of the marv genomic replication promoter revealed a bipartite structure. the ' genome end, the leader, comprises nts and contains the first promoter element of the bipartite genomic replication promoter. the second promoter element consists of a (unnnnn) motif with three conserved uridine residues separated from each other by five non-conserved nucleotides. the un ( ) hexamers are located within the ' untranslated region of the first marv gene, the np gene, and are separated from the first promoter region by the nts long transcription start signal. substitutions in the np transcription start signal do not affect replication activity but do interfere with transcription initiation [ ] . the ' extracistronic region, the trailer, spans the last nts of the marv genome and contains the complement of the antigenomic replication promoter (see below, . . transcription and replication). the structure of the marv antigenomic promoter has not yet been determined. however, due to the presence of un ( ) hexamers it is likely that it is of bipartite nature, similar to the genomic promoter. a common feature of the leader and trailer regions of all nns rna viruses is a high degree of complementarity of the - most terminal ' and ' nucleotides [ ] . although filoviruses share this feature, both the leader and the trailer also have the capability to form an internal secondary structure, which is not the case for the leaders and trailers of other nns rna viruses [ , [ ] [ ] [ ] . the marv genome encodes seven structural proteins listed in table . marv has a single surface protein, gp, which is encoded by the fourth gene and mediates attachment to target cells and virus entry [ ] . gp is a type i transmembrane protein which is inserted into the viral envelope in the form of homotrimeric spikes [ ] . in contrast to ebolaviruses, which use transcriptional editing to express the membrane-bound gp and at least two nonstructural glycoproteins [ ] [ ] [ ] [ ] , the marv gp gene contains a single open reading frame (orf) encoding the full-length gp. during its transport from the endoplasmic reticulum (er) to the plasma membrane via the secretory pathway, the precursor gp is the target of various posttranslational modifications including glycosylation [ , ] , acylation [ ] , and phosphorylation [ ] . gp is heavily glycosylated by complex and high mannose-type n-linked glycans as well as by mucin-type o-linked glycans, with the carbohydrates contributing about % of the apparent molecular weight of the protein [ , , ] . similar to ebov gp, the o-linked glycans and many of the n-linked oligosaccharides are clustered in a mucin-like domain [ ] . after synthesis in the er, the precursor gp is cleaved at amino acid by furin or a furin-like protease in the trans golgi network, resulting in two disulfide-linked subunits, gp ( kd) and gp ( kd) [ ] . while the ectodomain, which is mainly formed by gp , mediates binding to entry factors and receptors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the transmembrane subunit gp contains the fusion peptide and is presumed to mediate fusion of the viral and the cellular membrane based on similarity to ebov gp both at the amino acid and structural level [ , ] . the amino acid long transmembrane domain of gp is required for the incorporation of gp into virions [ ] . in addition, the cytoplasmic tail of gp is involved in enhancing the efficiency of viral entry by maintaining the structure of the ectodomain [ ] . the receptor binding domain of marv gp was mapped to the aminoterminal region of gp spanning amino acids to [ ] , whereas the highly glycosylated mucin-like domain is not essential for virus entry [ ] . an important step in marv entry is the proteolytic activation of gp by endosomal proteases, facilitating binding of the receptor binding region to the endosomal entry factor niemann-pick c protein (see below, . . entry) [ , ] . besides its function in entry and budding, gp may also play a role in immune evasion. the ifn-inducible antiviral protein tetherin was shown to block the release of vp -induced virus-like marv and ebov particles, suggesting that tetherin might act as a restriction factor for filovirus release [ , ] . however, co-expression of gp was sufficient to counteract the antiviral activity of tetherin by a yet unknown mechanism [ , ] . it is possible that gp not only subverts innate immune responses but also suppresses the adaptive immune response. filoviral gp subunits, including marv gp , contain a domain resembling an immunosuppressive motif found in retroviral envelope proteins [ ] . a -mer peptide corresponding to the putative immunosuppressive domain of marv gp was shown to induce lymphocyte death and suppression of cytokine responses [ ] . it is not yet known if this motif plays a role in the induction of lymphocyte apoptosis observed in marv infection. finally, it has been suggested that shedding of the ectodomain of membrane-bound ebov gp by tumor necrosis factor α-converting enzyme (tace) may play a role in blocking the activity of neutralizing antibodies during infection [ ] . it has been reported for marv that considerable amounts of gp shed from infected cells, although it is not clear if marv gp is a target for tace cleavage [ , ] . the matrix protein vp is encoded by the third marv gene and is the counterpart of the m proteins of other nns rna viruses. vp plays a major role in the formation of virions by redistributing nucleocapsids from the perinuclear region to the plasma membrane, recruiting gp to the sites of budding, and mediating particle release [ ] [ ] [ ] . overexpression of vp led to reduced reporter gene expression of marv minigenomes, suggesting a regulatory role of vp in transcription and/or replication [ ] . as a peripheral membrane protein, vp coats the inner side of the virion's membrane ( figure ) [ ] . cryo-em tomography studies suggest that vp associates with the nucleocapsid through flexible interactions [ ] . it can be easily removed from the nucleocapsid by salt dissociation, indicating that it is only loosely connected to the nucleocapsid [ ] . after synthesis in the cytoplasm of the infected cell, vp associates rapidly with cellular membranes and accumulates in membranous structures of the late endosomal compartment, the multivesicular bodies. a minor portion of vp is also found in association with viral nucleocapsids and in inclusions. additionally, vp appears in patches beneath the plasma membrane where it is transported via the retrograde late endosomal pathway [ , , ] . similar to ebov vp , marv vp is the major factor in particle formation and budding. expression of vp in the absence of other viral proteins leads to the formation and release of filamentous virus-like particles (vlps) resembling authentic virions. this process is enhanced in the presence of gp [ , [ ] [ ] [ ] . the role of vp during budding is described in more detail below (see . . budding). compared to ebov vp , little is known about the structure of marv vp . the n-terminal domain of marv vp folds into ring-like structures, which have the tendency to polymerize into rod-like structures. while ebov vp has been shown to form hexamers and octamers, the stoichiometry of marv vp oligomers is not known [ ] . marv vp is phosphorylated at several tyrosine residues located in the n-terminal region of the protein. a non-phosphorylatable mutant of vp is impaired in its ability to recruit nucleocapsids to the sites of budding, but is still able to efficiently induce particle release [ ] . vp also possesses a pppy late domain motif in its amino terminus which is important for its interaction with components of the endosomal sorting complex required for transport (escrt) machinery in order to mediate budding, including tumor susceptibility gene (tsg ) and the membrane-bound e ubiquitin ligase nedd . [ , , ] . besides the pppy motif, other motifs and single amino acids have been found to be important for particle release [ , ] . besides its role as a classical matrix protein, marv vp also acts as a virulence factor by counteracting the innate immune response and determining the host tropism for marv [ , ] . marv vp blocks the phosphorylation of janus kinases, which play an important role in multiple signaling pathways by phosphorylating and activating stat proteins ( figure ). when marv-infected cells were treated with various stimuli, including ifnα, ifnγ, and il , it was shown that the stat proteins were neither phosphorylated nor translocated into the nucleus [ , ] . it was then shown that in marv-infected cells treated with exogenous stimuli, janus kinases were also not phosphorylated and vp was identified as the viral protein inhibiting ifn signaling. it is believed that jak is the target for vp , however, the mechanism of vp -induced inhibition is not completely understood [ ] . intriguingly, ebov is also able to block ifn signaling by employing a completely different mechanism. ebov vp blocks the nuclear translocation of phosphorylated stat proteins by binding to stat and importins involved in the nuclear transport of specific stat proteins ( figure ) ( [ ] , reviewed in [ ] ). when marv was adapted to non-or less permissive animals, such as mouse and guinea pig, the adapted viruses showed mutations in vp . two of the amino acid changes in the mouse-adapted marv vp have been shown to be essential for the inhibition of ifn signaling in mouse cells, underlining the importance of ifn suppression for the virulence and host specificity of marv [ , , ] . the protein product of the sixth gene, vp , is unique to the filovirus family. vp is generally addressed as a second, minor matrix protein. however, cryo-electron tomography analysis of viral particles showed that vp is located in close proximity to the nucleocapsid proteins, suggesting that it might be part of the nucleocapsid complex [ ] . vp can easily be released from virion-associated nucleocapsids by treatment with increasing salt concentrations, indicating that it is only loosely connected to the nucleocapsid [ ] . intracellular localization studies of vp showed that a minor part of the protein (approx. %) is weakly bound to cellular membranes, including filopodia enriched with vp . vp is also distributed diffusely in the cytoplasm, relocalizes to nucleocapsid-containing inclusions, and is found in association with free nucleocapsids. co-expression of np and vp is sufficient to direct vp to np inclusions in the cytoplasm [ ] . functional studies on marv vp suggest that the protein is important for the release of viral particles in the context of infection, although it influences neither the morphology of vp -derived vlps nor the efficiency of vlp release. in addition, rnai-mediated knockdown of vp in marv-infected cells had no impact on viral genome replication, indicating that vp is involved in a step after replication and before budding [ ] . according to the model that has been proposed based on these data, vp is involved in the maturation of transport-competent nucleocapsids and/or mediates the interaction between nucleocapsids and budding sites at the plasma membrane [ ] . there is also evidence that marv vp affects transcription and replication in a transcription and replication competent vlp system [ ] . structural information about marv vp is very limited. it has been shown that it forms oligomers, preferentially tetramers [ ] . structure prediction studies have proposed an ancestral link between vp and the armadillo repeat family [ ] . the marv nucleocapsid complex consists of the genomic rna and four tightly associated proteins, np, vp , vp , and l ( figure ). encapsidation of the viral rna by the nucleocapsid proteins protects it from both rnase degradation and detection by cellular pattern recognition receptors. similar to the genomic rna, the antigenomic rna, a replicative intermediate, is also encapsidated by the nucleocapsid proteins (see below, . . transcription and replication). in contrast, the viral mrnas are not encapsidated [ ] . the nucleocapsid rather than naked rna serves as the template for viral transcription and replication. in a marv minigenome system, np, vp , and l are essential for transcription and replication [ , ] . the role of vp in marv transcription and replication is not well understood and the steps in genome amplification that require, or do not require, vp are not yet defined. the nucleoprotein np enwraps the genomic and antigenomic rnas. replication and transcription activity in a marv minigenome system depends on the presence of np [ ] . when np is expressed in the absence of other nucleocapsid proteins, it self-assembles into highly organized helical tubular structures that resemble the nucleocapsids in infected cells, indicating that it is the driving force for nucleocapsid formation [ , , ] . recently, it has been shown that the conserved n-terminal residues of marv np are sufficient to form the helical structure of the nucleocapsid core [ ] . indeed, np serves as a viral hub protein. it forms interactions with most of the other viral proteins, leading to the subcellular redistribution of these proteins. the strong binding of np to the nucleocapsid proteins vp and vp redirects both proteins into np-derived inclusions [ ] . a bipartite coiled-coil motif in the central part of np has been shown to play an important role for self-assembly and np-vp interaction [ ] . as mentioned above, there is also a weak interaction between np and vp , leading to the partial relocalization of vp into np-containing inclusions [ , ] . in addition, np interacts with vp , which is important for the transport of newly synthesized nucleocapsids to the plasma membrane [ , , , ] . interestingly, np contains a c-terminal late domain motif, psap, which has been shown to be required for budding. np recruits tsg , a component of the escrt i complex, through its late domain motif, leading to enhanced vp -induced budding [ ] . np is heavily phosphorylated at serine and threonine residues clustered in seven regions in the c-terminal part of the protein. only the phosphorylated form of np is incorporated into virions [ , ] . recent studies suggest that the phosphorylation level in region ii modulates transcription and/or replication activity [ ] . vp is a polymerase cofactor and essential for transcription and replication. together with the catalytic subunit l, vp forms the rna-dependent rna polymerase complex [ , ] . vp is tightly associated with np and serves as a bridging protein between the nucleocapsid complex and l. without vp , l is not associated with the nucleocapsids which serve as the templates for viral transcription and replication [ , ] . vp forms homo-oligomers mediated by a coiled-coil motif located in the n-terminal part of the protein. homo-oligomerization of vp is essential for its interaction with l but not needed for redistribution of vp into np-derived inclusions [ ] . vp shares many features with the phospho (p) proteins of other nns rna viruses, including its position as the second gene in the viral genome and its role in transcription and replication. however, in contrast to the p proteins, vp is either not or only very weakly phosphorylated [ ] . besides its function in transcription and replication, marv vp acts as an ifn antagonist. while the impact of ebov vp on the host's antiviral response has been intensively investigated (reviewed in [ ] ), much less information is available about similar functions of marv vp . when we tested marv vp for its ability to block ifn induction in a reporter gene assay, it blocked reporter gene expression as efficiently as ebov vp (unpublished data). in addition, bosio and colleagues [ ] reported that expression of marv vp in the absence of other viral proteins was sufficient to completely block the induction of ifnα in stimulated human dendritic cells. besides its ability to inhibit the induction of type i ifn, ebov vp has been shown to block the activation of the antiviral protein pkr and to interfere with rna silencing pathways. importantly, ebov vp is a dsrna binding protein. the c-terminus of ebov vp contains a domain with patches of basic amino acids which is important for dsrna binding and the protein's inhibitory functions (for review see [ ] ). this c-terminal region, the so-called ifn inhibitory domain, is conserved in marv vp [ ] , suggesting that marv vp possesses similar inhibitory functions. marv and ebov vp proteins show many structural similarities. both marv and ebov vp proteins are tightly associated with the nucleocapsid via their binding to np ( figure ) [ , ] . both are highly phosphorylated at n-terminally located serine and threonine residues, and phosphorylation is crucial for their interaction with np [ , ] . both contain an unusual c h zn binding domain, which is essential for the function of ebov vp as transcription initiation factor, but whose functional relevance for marv vp is not known [ ] . it has also been shown that ebov vp forms hexamers [ , ] , binds single-stranded rna [ ] , and interacts with l [ ] . however, to date, similar data for marv vp are not available. the role of marv vp in viral transcription and replication is not well understood. in contrast to ebov vp , which plays an important role in regulating transcription initiation [ , , [ ] [ ] [ ] , marv vp is not essential for transcription or replication activity in a marv minigenome system [ , ] . nevertheless, it seems to play an important role in viral amplification, since rescue of a full-length marv clone is only successful in the presence of vp [ ] . in addition, down-regulation of vp by rna interference in marv-infected cells led to the reduction of both viral protein synthesis and virion production [ ] . among the nns rna viruses, only the members of the subfamily pneumovirinae possess a protein similar to vp , m - , which functions as a transcription processivity factor [ ] . the major component of the marv polymerase complex, l, has an estimated molecular weight of kd [ ] . it is essential for transcription and replication and together with vp forms the rna-dependent rna polymerase complex (see above, . . viral proteins, vp ). l contains the enzymatic functions of the polymerase. the binding site for vp has been mapped to the n-terminal amino acid residues of l [ , ] . the l proteins of the nns rna viruses are highly conserved multifunctional proteins, which are organized in functional domains [ ] . based on this conservation with other nns rna polymerases, marv l is believed to carry out rna synthesis, capping, and polyadenylation of viral mrnas although these functions have not been shown experimentally. to date most of the studies characterizing the marv replication cycle have utilized recombinant systems, allowing for these experiments to be performed in a biosafety level (bsl- ) context, unfettered by the restrictions of a bsl- setting. surrogate systems mimicking specific steps in the marv replication cycle include marv gp-pseudotyped retroviruses or recombinant vesiculoviruses expressing gp to study entry, vlps to study budding, and minigenome systems to study replication and transcription. while such experiments allow for the more facile examination of the marv replication cycle, the findings must be recapitulated with infectious marv since all surrogate systems lack elements of the infectious virus such as the distinct morphological features and virion protein composition of marv. marburg virus entry consists of three distinct phases: cellular attachment, endocytosis, and fusion ( figure ) . based on the sequence similarity between ebov and marv gps many investigators have presumed identical functions and characteristics between the filovirus glycoproteins. this is presumptuous given the differences in glycosylation and sialic acid linkages [ ] and dependence upon endosomal proteases (see below, . . . endocytosis). despite the existence of a number of detailed studies and structural analyses of ebov gp [ ] [ ] [ ] [ ] , relatively few mechanistic studies of marv gp have been performed [ ] , although one recent post-fusion structure of marv gp has been reported [ ] . the structure of marv gp is nearly identical to that of ebov gp , indicating that the mechanisms of fusion between the two viruses is likely conserved [ ] . ( ), gp is cleaved by endosomal proteases ( ) facilitating binding to npc , the entry receptor ( ). fusion is mediated in a ph-dependent manner by gp . following release of viral nucleocapsid into the cytosol ( ), transcription of the viral genome takes place ( ) . mrna is subsequently translated by the host cell machinery ( ) . synthesis of gp takes place at the er and undergoes multiple posttranslational modifications on its way through the classical secretory pathway ( ) . positive sense antigenomes are synthesized from the incoming viral genomes ( ) . these intermediate products then serve as templates to replicate new negative sense genomes ( ) . after cleavage in the golgi, gp is transported to multivesicular bodies (mvb) and to the cell membrane where budding takes place ( ) . nucleocapsids and vp are also recruited to sites of viral budding ( ) , which is driven by vp ( ). marv gp mediates both cell attachment and fusion of the virus. there is convincing evidence that initial virus attachment at the cell surface can occur via the binding of gp carbohydrates to various cellular c-type lectins, including the hepatocyte-specific asgp-r [ ] , dc-sign and dc-signr (also known as l-sign) [ , , ] , hmgl [ , ] , and lsectin [ , ] . other cell surface proteins have also been implicated in facilitating marv entry including the tam receptor protein kinases ax , dtk, and mer [ ] , and tim- [ ] . however, although these proteins may play a role in attachment or entry of certain cell types, the ability of marv to infect cells lacking these receptors [ , , ] indicates that there might be redundancy in cellular molecules required for marv attachment to cells. a number of key residues of ebov gp that are involved in virion incorporation and gp-mediated entry have been identified [ ] and found to play a similar role in marv gp [ ] , indicating that the viruses might utilize similar mechanisms to enter the cell. following attachment, marburg virions undergo endocytosis mediated through a mechanism that currently remains undetermined. (figure ) [ , ] initial studies investigating caveolin-mediated endocytosis showed that depletion of host cell cholesterol reduced viral infectivity but presented no direct evidence of caveolae involvement [ ] . in addition, studies examining the role of caveolae in ebov endocytosis are conflicting [ , ] . a major role for clathrin in marv entry has also been proposed based upon the ability of chlorpromazine (an inhibitor of both clathrin-mediated endocytosis and macropinocytosis) as well as rnai-knockdown of clathrin heavy chain to inhibit marv gp-pseudotyped hiv- entry [ ] . a caveat to these analyses of marv endocytosis is that they were performed only in the context of marv gp-pseudotyped retroviruses which lack the characteristic filamentous morphology and size of marburg virions. while other reports have verified that cholesterol is important for live marv particle uptake [ ] , canonical caveolae-and clathrin-mediated mechanisms are unlikely to be the primary mechanism of marv entry due to steric issues. the typical marv particle size (average nm) is much larger than canonical caveolae ( - nm) or clathrin-coated pits (up to nm) whereas pseudotyped murine leukemia virus (mlv) ( × nm) and vesicular stomatitis virus (vsv) ( × nm) are not [ ] . these findings indicate that involvement of the caveolae-and clathrin-mediated endocytic pathways for virus entry may therefore be the result of the artificial nature of the pseudotyped virions and highlights the need to confirm such experiments with live marv. macropinocytosis has been identified as a major entry pathway of ebov by research using the morphologically more relevant vlps and live ebov [ ] [ ] [ ] [ ] . although none of these analyses examined the role of this pathway during marv entry, it remains an intriguing possibility given the cholesterol-dependence and large size of macropinocytotic vesicles (up to - µm) [ ] . another important process in marv entry is believed to occur while virions are being trafficked within endocytic vesicles; the proteolytic cleavage of gp . endosomal cleavage of gp has been shown to be critical for the efficient entry of marv [ , ] . the current model for marv entry involves the cleavage of gp by host endosomal cysteine proteases. this removal of a large portion of gp (including the mucin-like domain) is believed to expose the putative receptor-binding domain based on studies conducted with ebov gp [ , ] . studies examining the roles of endosomal proteases on the entry of marv and ebov have produced mixed results. experiments analyzing recombinant vsv expressing ebov gp indicate a primary role for cathepsin b (catb) and minor role for cathepsin l (catl) [ ] . entry of recombinant vsv particles containing marv gp was inhibited when cells were treated with an inhibitor of both catb and catl [ ] . these reports are confounded by a report conducted with infectious marburg and ebola viruses in which catb and catl inhibitors greatly reduced ebov infection but showed mixed results with marv [ ] . yet two other, more recent analyses determined that catb was not required for marv entry (although over-expression did enhance infectivity) and that catl was required for entry into mouse embryonic fibroblasts but not vero cells, t cells, or human macrophages [ , ] . these data as well as the ability of other proteases to greatly diminish marv infectivity [ , ] , indicate that although catb and catl likely play a role in cleavage and activation of gp in certain cell types, other endosomal proteases may also be able to facilitate gp activation via cleavage. recently, two independent studies elegantly showed the requirement of the endosomal cholesterol transporter niemann-pick c (npc ) for the entry of both marv gp-pseudotyped viruses (vsv and mlv) as well as infectious marv [ , ] . it was also shown that npc catalytic activity is not required for ebov infection indicating that specific binding to npc rather than its role in cholesterol transport is required, although this was not tested for marv [ ] . in one of the studies identifying npc as the marv entry receptor, it was also determined that members of the homotypic fusion and vacuole protein-sorting (hops) complex were important for ebov entry, although they appeared to play a less important role in marv entry [ ] . the current model of ebov and marv fusion is that gp cleavage by endosomal proteases removes heavily glycosylated domains, exposing the receptor binding domain on gp and enabling binding to npc [ ] . the membrane-bound fusogenic gp undergoes a low ph-dependent rearrangement to an extended conformation resulting in the fusion of virion and endo-lysosomal membranes [ ] . in support of the ph-dependence of gp-mediated fusion, pre-treatment of cells with ammonium chloride prevented entry of a marv gp-pseudotyped virus [ ] . a recent report with live marv showed that ammonium chloride inhibited entry and replication, but that bafilomycin a , which specifically inhibits vacuolar-type h(+) atpase and prevents re-acidification of vesicles of the central vacuolar system, surprisingly had no effect [ ] . following viral fusion with the endosomal membrane, the nucleocapsid is released into the cytoplasm (figure ). after the nucleocapsid is released into the cytoplasm of the infected cell, transcription and replication of the viral rna genome takes place (figure ). the first morphological sign of viral replication observed by em analysis is the appearance of granular material containing rna and viral proteins in the cytoplasm of the infected cells at h post infection. later on, tubular structures can be detected in the granular material representing the newly synthesized nucleocapsids embedded in the viral inclusions [ ] . while experimental data on the sites of marv replication and transcription are not available, recent studies on ebov have shown that viral replication takes place in the inclusions, while transcription was observed prior to inclusion formation [ ] . the encapsidated negative-sense rna genome is transcribed resulting in seven monocistronic mrnas by the viral polymerase. they are co-transcriptionally capped and polyadenylated and subsequently translated by the cellular machinery (figure ) . the genomic rna also serves as the template for the production of positive-sense antigenomes, which are complementary copies of the genomes. the antigenomes are encapsidated by the nucleocapsid proteins and are in turn used as templates for genome synthesis (figure ) (for review see [ ] ). as mentioned above, np, vp , l, and probably vp are needed for viral transcription and replication. analogous to ebov, it is conceivable that vp and vp inhibit transcription and replication [ , , ] . it is hypothesized that negative regulators of replication convert the active polymerase complex into an inactive state, resulting in mature and transport-competent nucleocapsids. following assembly, newly synthesized nucleocapsids are recruited to the sites of virus budding (figure ) . release of viral particles is mainly mediated by vp via recruitment of nucleocapsids from the inclusions to the plasma membrane, recruiting gp to the sites of budding, and inducing the formation and release of filamentous vlps. vp -induced budding is enhanced by np, gp, and vp [ , , ] . as is the case with many other viruses, marv exploits the vesicular transport machinery of the infected cell for viral egress, including the copii vesicular transport system and the escrt machinery. the copii vesicular transport system is used by vp for its intracellular trafficking to the multivesicular bodies, where marv budding takes place [ , ] . cellular proteins that promote particle release and are linked to the escrt machinery include tsg , vps a/b, and nedd . [ , , , ] . marv budding not only takes place at internal membranes but also at the plasma membrane [ , , ] . in cell culture, marv particles are preferentially released at filopodia, filamentous cellular protrusions [ , , ] . filopodia are used by cells to explore the extracellular environment, which includes neighboring cells, and it is believed that viral particles can bud directly into adjacent cells via filopodia-mediated cell-to-cell contact [ , ] . budding at filopodia depends on actin and is not sensitive to the depolymerization of microtubules [ ] . marv budding was observed at the basolateral membrane of polarized epithelial cells and hepatocytes [ , ] , whereas viral particles were predominantly released from the apical membrane of infected endothelial cells [ ] , suggesting that cell-type specific components determine the sites of virus release. electron tomography studies of marv-infected cells led to the following model for marv particle release: the budding process is initiated when intracellular nucleocapsids associate laterally with the plasma membrane. starting from one end, the nucleocapsids are then subsequently wrapped by the plasma membrane until the viral particles protrude vertically from the cell surface. the release of infectious filamentous marv from cultured cells peaked at - days post infection, when the cells were still intact. at d post infection, when most of the cells were vesiculated, the released virions were round or bent and infectivity was decreased [ ] . determination of the nucleocapsid orientation at the sites of budding by -d reconstructions revealed that the pointed tip of the budding nucleocapsids is oriented towards the membrane, indicating that marv budding is directional [ ] . marv infections usually occur by direct contact with infected body fluids or direct personal contact with infected animals or humans. the viruses enter the body through small skin lesions or mucosal membranes (reviewed in [ ] ). cells of the mononuclear phagocyte system, including monocytes, macrophages and dendritic cells, are early target cells of marv, as shown in different experimental animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . marv replication was observed as early as hours post infection in macrophages of infected guinea pigs [ ] , and infected monocytes have been found in cynomolgus macaques at days post infection [ ] . monocytes and macrophages were also identified as early target cells in human patients [ ] . this has been confirmed by cell culture experiments showing that primary human monocytes and macrophages are highly susceptible to marv infection and produce infectious virus [ ] [ ] [ ] . in addition, primary human monocyte-derived dendritic cells (mdcs) and endothelial cells support marv replication [ , , ] . early sites of virus replication are the lymph nodes, liver, and spleen where the most severe necrotic lesions are observed [ , , , , , , ] . these organs contain high numbers of monocytes and macrophages. migration of infected monocytes and macrophages into surrounding tissues or transport of free virus via the lymph-or bloodstream is believed to facilitate the dissemination to multiple organs, resulting in a systemic infection [ , ] . cell-free virus has been observed in the tissue and organs of infected animals, and high levels of virus have been detected in the blood [ ] [ ] [ ] , , , , ] . besides monocytes, macrophages, and dendritic cells, a wide range of cell types including hepatocytes, adrenal cortical and medullary cells and fibroblasts are permissive to marv infection [ , , [ ] [ ] [ ] [ ] , , ] . endothelial cells are late target cells during marv infection in multiple tissues. whether or not replication of marv in endothelial cells is associated with the observed vascular impairment during mvd is a matter of debate [ , ] . only low numbers of infected endothelial cells are observed in nhp infection and therefore changes in the endothelium are likely caused by paracrine effects of cytokines [ ] . in late stages of infection marv particles can be isolated from nearly every organ [ , , , , ] . despite high viral load and necrotic lesions, only minor inflammation is observed in infected tissues and organs, indicating a dysregulated immune response [ , , , ] . strong liver pathology is observed, including increased serum activity of liver enzymes. this might influence synthesis of clotting factors and contribute to the observed coagulation defects in mvd [ , , , ] . these factors together with systemic virus replication and associated pathology probably trigger the multiorgan failure associated with fatal cases. although lymphocytes are not susceptible to marv infection [ , , , ] , massive bystander lymphocyte apoptosis is a hallmark of mvd [ , , , , , ] . however, the molecular mechanisms for lymphocyte depletion and the role it may play in the pathogenesis of mvd are far from being understood. cytokine secretion may play a role in the induction of lymphocyte apoptosis, since marv-infected cells secrete cytokines known to induce apoptosis, including tnfα [ , , , ] . increased levels of tnfα have been observed in infected rhesus macaques [ ] and mice [ ] , although no increase was observed in infected cynomolgus macaques [ ] . elevated tnfα levels may also play a role in the formation of endothelial gaps in the context of marv infection [ , ] . in addition, increased survival of marv-infected guinea pigs treated with anti-tnfα antibodies suggests that tnfα indeed plays an important role in mvd pathogenesis [ ] . increased serum levels of additional proinflammatory cytokines and chemokines have been observed in infected nhps and in mice, but the reported data are not completely consistent [ , , , , ] . cytokine and chemokine secretion has also been observed in infected primary human monocytes and macrophages [ , ] . however, data about the cytokine levels in the serum of marv-infected patients are not available, but high levels of cytokines have been observed in ebov-infected patients [ ] [ ] [ ] [ ] . upregulation of the proinflammatory cytokines il (mediator of fever and acute inflammatory response) and il (chemoattraction of neutrophils and macrophages) is consistently found in infected nhps, with macrophages and plasmacytoid dendritic cells (pdcs) serving as the main sources of il secretion in the spleen [ , , ] . primary human monocytes and macrophages produce both il and il after infection [ ] . elevated levels of il have also been detected in marv-infected mice [ ] . increased levels of il β mrna and secreted protein were detected in primary human cells [ , ] , but contradictory data have been reported for the nhp model. one study reported elevated il β levels in final disease stages [ ] , whereas no change was observed in another study [ ] . ifnα levels were elevated in infected nhps and mice [ , , ] . however, no change in ifnα levels was detected in another study of infected nhps [ ] . it is unclear whether or not the observed differences are due to different marv variants being used for the studies. serum levels of several chemokines were also found to be elevated during marv infection of nhps and mice, including macrophage inflammatory proteins (mip) and monocyte chemotactic protein (mcp- ) [ , , ] . the involvement of multiple cell types along with the possible role of non-infected cells in the secretion of cytokines further complicates the analysis of existing data. primary human monocytes and macrophages are activated by marv infection inducing the secretion of cytokines. induction of cytokines has also been described using uv-inactivated marv, suggesting that viral replication might not be needed for the observed cytokine increase [ ] . in contrast, marv-infected mdcs show no upregulation of activation markers, do not secrete cytokines, and fail to stimulate t cells [ ] . mdcs treated with vlps containing marv vp and gp show functional mdc responses including cytokine secretion indicating that marv replication is required to inhibit mdc activation [ ] . however, infection of mdcs with marv did not prevent lps-induced tnfα production whereas dsrna-dependent ifnα secretion was inhibited [ ] , suggesting differential regulation of cytokines by marv. interestingly, pdcs in the spleen were identified as the major source of secreted ifnα in marv infected nhps, but secretion most likely occurs from non-infected cells [ ] . it has been shown for ebov that pdcs are not productively infected due to impairment of viral entry [ ] . these results suggest that secretion of cytokines by non-infected bystander cells might play an important role during marv pathogenesis. taken together, marv infection induces both an increase in the production of proinflammatory cytokines and high levels of chemokines, but the molecular mechanisms causing these changes are not well understood. to date four different animal models have been established for marv infection: nhps, mice, guinea pigs, and hamsters. the nhp model best reflects the symptoms and pathology observed in human cases (described in . clinical manifestations and reviewed in [ , ] ) with uniform lethality in cynomolgus and rhesus macaques as well as african green monkeys [ , , , , , , [ ] [ ] [ ] . the disease symptoms are generally the same for all types of nhps. the animals develop febrile illness with high fever, anorexia, weight loss and unresponsiveness. death is observed after - days and thrombocytopenia, lymphopenia, blood coagulation abnormalities and hemorrhages are observed. squirrel monkeys have also been successfully infected with marv, showing typical disease symptoms [ ] . recently, a small nhp model using marmosets has been developed recapitulating the features of human infections except for the typical maculopapular rash development that is observed in other nhps and humans infected with marv [ ] . rodents with an intact immune system do not develop disease after infection with marv. marv variants musoke and ci and ravv variant ravn were adapted to severe-combined immunodeficiency (scid) mice by serial passaging, reducing the time to death from - days to - days for all tested virus variants [ ] . further passaging of the scid mouse-adapted marburgviruses in immunocompetent mice was used to establish mouse models for ravv ravn and marv ci [ , ] . successful adaptation by serial passaging was also used to generate lethal infection models for both guinea pig [ , ] and hamster [ , ] . coagulation abnormalities, typical rash development, and hemorrhagic manifestations (especially in mice) are not as pronounced as in the nhp model [ ] ; (reviewed in [ ] ). neuropathogenicity, recapitulating the cns involvement described during the first human mvd outbreak in germany [ , , ] , has only been observed in the hamster model [ ] . it is not clear if cns pathology is developed in other animal models as no brain pathology has been observed in mice [ ] and cynomolgus macaques [ ] . nevertheless, virus has been isolated from brain from marv-infected marmosets, showing micro-hemorrhages [ ] . sequence comparison of the rodent-adapted viruses to the human marv isolates revealed several mutations. sixty-one nucleotide changes in the mouse-adapted ravv ravn variant were detected in the orfs of np, vp , vp , and vp ( amino acid changes in total) or untranslated regions (vp , vp , gp, vp ) [ ] . in a second study analyzing genome changes during mouse adaptation, nucleotide changes during adaptation of ravv ravn and changes for marv ci were described, with most amino acid changes occurring in vp [ ] . during guinea pig adaptation, only nucleotide changes were observed, resulting in four amino acid changes. one of these changes was located in vp and the other mutations were detected in the viral polymerase l [ ] . the only amino acid exchange detected in both the mouse-and the guinea pig-adapted marv is amino acid in vp (asp to asn). this was also the first mutation detected during mouse adaptation for both ravv ravn and marv ci , as analyzed by sequencing of serial passages [ ] . this is of particular interest because vp has been shown to function as an inhibitor of ifn signaling (see above, . . viral proteins, vp ) [ , ] . both ifn receptor-and stat -deficient mice develop disease using non-adapted marv, highlighting the importance of the ifn pathway for the control of mvd [ ] [ ] [ ] . virological, serological, and molecular diagnostic methods for the detection for marv are available, including virus isolation, elisa, rt-pcr, em, and immunohistochemistry (summarized in [ , ] ). during outbreak settings, mobile laboratories commonly use pcr and/or elisa analysis for rapid screening. sensitive elisa assays have been developed for detection of viral antigen or virus-specific antibodies using overexpressed marv np or gp [ ] [ ] [ ] [ ] . detection of filoviruses by pcr is the only assay currently available to distinguish between different virus variants for a variety of tissue and fluid specimens. the use of a combination of virus-specific primer sets for conventional rt-pcr makes the detection of all know filoviruses in a single assay possible [ ] . for more sensitive and quantitative detection, real-time rt-pcr-based assays have been developed for the detection of marv [ , [ ] [ ] [ ] [ ] [ ] . feasibility of real-time rt-pcr in the field was successfully proven during the marv outbreak in uíge, angola using an improved field laboratory-adapted rna isolation protocol [ ] . a network of european bsl- facilities in collaboration with a company (qiagen) developed the first commercial prototype of a real-time-rt-pcr assay for the detection of filoviruses [ ] . a recently developed assay, rt loop-mediated isothermal amplification (lamp), has the potential to significantly improve field diagnosis of marv infections, by eliminating the need of pcr machines [ ] . initial approaches using inactivated virus to develop a vaccine against marv were unsuccessful or had contradictory results [ ] . in addition, successful protection of rodents did not always translate into protection of nhps. for example, inactivated marv protects guinea pigs from lethal marv challenge but only % of challenged nhps survived [ , [ ] [ ] [ ] . a panel of different approaches has been used in order to develop successful vaccines for marv (reviewed in [ , ] ). recombinant gp expressed from insect cells or a dna vaccine based on gp only partially protected guinea pigs, but use of a combination of both vaccines resulted in % survival of guinea pigs [ ] . in another study, complete protection of guinea pigs using a different gp dna vaccine was reported, but only four of six vaccinated nhps survived the challenge with marv, showing incomplete protection [ ] . increased doses of a codon-optimized dna vaccine resulted in % survival of nhps, although some animals developed symptoms before recovering. in comparison to other vaccine candidates, a poor induction of virus-specific antibodies was observed using a dna vaccine [ ] . a codon-optimized dna vaccine elicited a strong antibody response and resulted in complete protection of mice with no clinical symptoms observed [ ] . vaccine candidates based on the venezuelan equine encephalitis virus (veev) replicon system expressing either marv gp along with np or gp alone completely protected guinea pigs and nhps [ ] . a vaccine based on vlps represents an additional candidate for protection against marv [ ] . complete protection of guinea pigs has been demonstrated with a vlp-based vaccine containing marv gp, with induction of virus-specific antibodies. protection with this vaccine relied on a functional cd + t cell response, whereas depletion of cd + t cells did not ablate the protective response [ ] . vlps containing marv musoke gp provided cross-protection in animals challenged with marv ci or ravv ravn in both guinea pigs and nhps [ , ] . another approach to marv vaccines is the use of viral vectors expressing marv gp. to date, two different systems have been established based on replication-defective adenoviral vectors or recombinant vsv expressing marv gp. the adenovirus-based vaccine successfully protects guinea pigs and nhps, and provides cross-protection. high levels of cross-reactive marv-specific igg and t cell responses are induced, indicating an induction of an immune response [ , ] . preexisting immunity against the adenovirus strain ad might pose a problem for its successful use in humans (reviewed in [ ] ). the vsv-based vaccine completely protects nhps and additionally has proven successful in post-exposure treatment (reviewed in [ ] ). a single immunization with recombinant vsv expressing marv musoke gp resulted in % protection of cynomolgus macaques challenged by intramuscular injection or aerosol exposure and protected against ravv ravn and marv angola [ , , ] . although marv-specific igg were produced, only low levels of neutralizing antibodies were detected [ , ] . surprisingly, t cell-mediated responses were not observed in nhps vaccinated with recombinant vsv expressing marv gp [ , ] . safety is a concern for this vaccine, especially for immunocompromised individuals, as it is a replication-competent vsv vector. however, in all vsv-based filovirus vaccine studies vsv viremia was observed only shortly after immunization. additionally, the vsv-based filovirus gp vaccine was well tolerated and protective in immunocompromised mice and nhps and lacked neurovirulence in nhps [ ] [ ] [ ] (reviewed in [ ] ). cross-protection has not been observed in animals vaccinated with marv-based vaccines and subsequently challenged with ebov, while combined marv and ebov vaccines have been successful in protection against both viruses [ , , ] . to date no approved treatment is available for marv infection. supportive care (fluids, anti-microbials, blood transfusions) has been the primary treatment of patients during mvd outbreaks. in the guinea pig model various treatments had some success as reflected by prolonged survival or increased survival rates. applied treatments included cytokine inhibition, ifn treatment, or antibody transfer. the tested treatments were unsuccessful in the nhp model (reviewed in [ , ] ). a third of ebov-infected nhps survived, however, following treatment with recombinant nematode coagulant protein , while only one of six marv-infected animals survived [ , ] . treatment using antisense technology to block viral protein expression using phosphorodiamidate morpholino oligomers (pmo) beginning to minutes after marv infection completely protected nhps [ ] . additionally, a small molecule inhibitor showed complete protection of marv-infected mice when administered h after infection but has not been tested in nhps [ ] . the vsv-based vaccine expressing marv gp has also been demonstrated to be effective as a post-exposure treatment. a hundred percent survival of nhps was 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of the l gene and ' trailer region of ebola virus the ebola virus genomic replication promoter is bipartite and follows the rule of six marburg virus gene encodes the virion membrane protein, a type i transmembrane glycoprotein gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer a new ebola virus nonstructural glycoprotein expressed through rna editing intracellular transport and processing of the marburg virus surface protein in vertebrate and insect cells acylation of the marburg virus glycoprotein the marburg virus surface protein gp is phosphorylated at its ectodomain carbohydrate structure of marburg virus glycoprotein characterization of filoviruses based on differences in structure and antigenicity of the virion glycoprotein proteolytic processing of marburg virus glycoprotein the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus tyro family-mediated cell entry of ebola and marburg viruses human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry different potential of c-type lectinmediated entry between marburg virus strains t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain crystal structure of the marburg virus gp core domain in its post-fusion conformation role of the transmembrane domain of marburg virus surface protein gp in assembly of the viral envelope the cytoplasmic domain of marburg virus gp modulates early steps of viral infection conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor characterization of marburg virus glycoprotein in viral entry an interferon-alpha-induced tethering mechanism inhibits hiv- and ebola virus particle release but is counteracted by the hiv- vpu protein. cell host microbe broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin the gp-protein of marburg virus contains the region similar to the 'immunosuppressive domain' of oncogenic retrovirus p e proteins implication of a retrovirus-like glycoprotein peptide in the immunopathogenesis of ebola and marburg viruses ectodomain shedding of the glycoprotein gp of ebola virus sorting of marburg virus surface protein and virus release take place at opposite surfaces of infected polarized epithelial cells interactions with the host cell tsg is recruited by a late domain of the nucleocapsid protein to support budding of marburg virus-like particles phosphorylation of marburg virus matrix protein vp triggers assembly of nucleocapsids with the viral envelope at the plasma membrane establishment and application of an infectious virus-like particle system for marburg virus vp , the matrix protein of marburg virus, is associated with membranes of the late endosomal compartment interactions of marburg virus nucleocapsid proteins the matrix protein of marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway generation of marburg virus-like particles by co-expression of glycoprotein and matrix protein multivesicular bodies as a platform for formation of the marburg virus envelope vacuolar protein sorting pathway contributes to the release of marburg virus oligomerization and polymerization of the filovirus matrix protein vp interaction of tsg with marburg virus vp depends on the pppy motif, but not the pt/sap motif as in viruses , the case of ebola virus, and tsg plays a critical role in the budding of marburg virus-like particles induced by vp , np, and gp regulation of marburg virus (marv) budding by nedd . : a different ww domain of nedd . is critical for binding to marv and ebola virus vp conserved motifs within ebola and marburg virus vp proteins are important for stability, localization, and subsequent budding of virus-like particles identification of amino acids in marburg virus vp that are important for virus-like particle budding marburg virus vp antagonizes 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dendritic cells without inducing the production of cytokines and full maturation a c-terminal basic amino acid motif of zaire ebolavirus vp is essential for type i interferon antagonism and displays high identity with the rna-binding domain of another interferon antagonist, the ns protein of influenza a virus phosphorylation of vp impairs ebola virus transcription phosphorylation of marburg virus vp at serines and is critical for its interaction with np inclusions ebola virus transcription activator vp is a zinc-binding protein oligomerization of ebola virus vp is essential for viral transcription and can be inhibited by a synthetic peptide crystal structure of the c-terminal domain of ebola virus vp reveals a role in transcription and nucleocapsid association the ebola virus vp is an rna binding protein the ebola virus ribonucleoprotein complex: a novel vp -l interaction identified ebola virus vp -mediated transcription is regulated by rna secondary structure formation role of ebola 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with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha association of the caveola vesicular system with cellular entry by filoviruses folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity ebola virus enters host cells by macropinocytosis and clathrinmediated endocytosis the tyro receptor kinase axl enhances macropinocytosis of zaire ebolavirus the ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner cellular entry of ebola 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filoviruses virus-like particles exhibit potential as a pan-filovirus vaccine for both ebola and marburg viral infections monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple marburg viruses de novo syntheses of marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses recombinant vesicular stomatitis virus-based vaccines against ebola and marburg virus infections vesicular stomatitis virusbased vaccines protect nonhuman primates against aerosol challenge with ebola and marburg viruses live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of ebola virus hemorrhagic fever vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys advanced antisense therapies for postexposure protection against lethal filovirus infections antiviral activity of a small-molecule inhibitor of filovirus infection postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors thank s. carroll and j. towner, cdc, atlanta, ga for providing the phylogenetic tree analysis shown in figure b , bobbie rae erickson, cdc, atlanta, ga for providing the photograph shown in figure , and w. slenczka, university of marburg, germany for providing the electron micrograph shown in figure . the picture in figure a was taken from [ ] the photographs shown in figure b were taken from [ ] . this work was supported by national institutes of health (nih) grants u -ai and ai (new england regional center of excellence-kasper, subaward - ). the authors declare no conflict of interest. key: cord- - l knu authors: elfiky, abdo a. title: ebola virus glycoprotein gp —host cell-surface hspa binding site prediction date: - - journal: cell stress chaperones doi: . /s - - -z sha: doc_id: cord_uid: l knu ebola virus (ebov) infection is a widespread infection that has created a bad memory in africa. in the and outbreak, more than , infections were reported by the world health organization, with about , deaths in guinea, liberia, and sierra leone. heat shock protein a (hspa ), termed also grp , is a host cell chaperone protein responsible for the unfolded protein response in the endoplasmic reticulum. under stress, hspa is upregulated and becomes cell-surface exposed. recent studies report the association of cell-surface hspa with ebov glycoproteins gp and gp . in this study, structural and sequence analysis and molecular docking are used to predict the possible binding site between the cell-surface hspa and ebov gp . the results show a promising binding site that supports the hypothesis of hspa selectivity for binding to a specific peptide sequence (pep ). this study paves the way to suggest possible inhibitors to stop viral association with cell-surface receptors and subsequently reduce viral infection. electronic supplementary material: the online version of this article ( . /s - - -z) contains supplementary material, which is available to authorized users. ebola virus (ebov) is one of the re-emerging viruses with a high mortality rate of up to % (bhattacharyya and hope ; shurtleff et al. ) . ebov belongs to the filovirus family and affects the liver (pallesen et al. ) . ebov requires different host factors during the life cycle (cantoni and rossman ; ibrahim et al. ) . the outbreak of ebov caused more than , deaths with hemorrhagic fever as the main characteristic effect (el gohary et al. ). the ease with which the virus spread (via body fluids) and its high mortality rate made ebov a global health threat of international concern (el gohary et al. ). the current development in drug design to eradicate the virus, using direct-acting antivirals (daa), has reduced the momentum of viral spreading (elfiky ; gonzalez-grande et al. ; yang et al. ) . the upregulation of specific cellular proteins that mediate the alleviation mechanisms to reduce stress is induced by the unfolded protein response (upr) mechanism in stressed cells. heat shock proteins (hsp), the chaperones, are among those proteins that are upregulated under stress, as in viral infection or some types of cancers (ibrahim et al. ) . glucose-regulated protein (grp ), a member named hspa (kampinga et al. ) of the hsp chaperone family is termed the master of the upr mechanism in the lumen of the endoplasmic reticulum (er) (gething and sambrook ; ibrahim et al. ; lee ; li and lee ; quinones et al. ; rao et al. ) . under cellular stressors, hspa releases activating transcription factor (atf ), protein kinase rna-like endoplasmic reticulum kinase (perk), and inositol-requiring enzyme (ire ) because of the accumulation of unfolded proteins. the released enzymes cause inhibition of protein synthesis and enhancement of refolding mechanisms (ibrahim et al. ; shen et al. ) . subsequently, hspa is upregulated and succeeds in escaping the er retention (detected in the cytoplasm and over the cell membrane (cell-surface hspa )) (ibrahim et al. ; wu et al. ) . cell-surface hspa is susceptible to pathogen recognition by viral envelope glycoproteins or fungal coat proteins (gebremariam et al. ; ibrahim et al. ). pep , a cyclic -residues peptide (ctvalpggyvrvc) targets specifically cell-surface hspa in vivo (kim et al. ) . it is used to deliver the chemotherapeutic, doxorubicin, to cancer cells presenting cell-surface hspa (ibrahim et al. ; martin et al. ) . additionally, hspa is reported to be associated with some viral proteins like spike protein in coronaviruses and e in human papillomavirus while the binding site was predicted (elfiky ; ibrahim et al. ) . it was reported that hspa is associated with ebov in er (shurtleff et al. ) . it was suggested that hspa could be used as a drug target to stop ebov infection (shurtleff et al. ). in addition, ebov glycoproteins gp and gp have been shown to accumulate in the er of the infected cells, causing stress response (bhattacharyya and hope ) . in this work, the binding site between cell-surface hspa and viral gp protein is predicted based on sequence, and thus, pep is an example of a class of drugs that may reduce ebov infections. protein/peptide and protein/protein docking are employed to explore such binding using the protein/ protein docking software "haddock" which utilizes solvation and molecular dynamics simulation (mds) in refining the interacting residues (binding site) for the interactions formed to be trustful (van dijk and bonvin ) . in addition, hpepdock constructs different possible conformations of the peptide and tests the binding affinity of each of the predicted structures against the target protein (zhou et al. ). nine solved structures for ebov glycoproteins are found in the protein data bank (berman et al. ) with the following codes: jq , qd , qd , mam, ea , kel, ken, ea , and kem with a resolution of . , . , . , . , . , . , . , . , and . Å, respectively (ehrhardt et al. ; pallesen et al. ; west et al. west et al. , . four of these structures are solved by x-ray crystallography ( jq , mam, ea , and ea ), while the rest are solved by cryo-electron microscopy. all the solved structures have been released during the past years. the pdb files and their corresponding fasta sequence files are downloaded from the protein data bank database (pdb). by the aid of pymol software (delano ), water, ions, and protein chains other than ebov gp are removed from the pdb files. the same is done with the only available wild-type and fulllength hspa (x-ray crystallography solved structure pdb id: e with . Å resolution) in the open conformation (yang et al. ; yang et al. ) . it is important to use the open conformation of the hsp a in which the sbdα and sbdβ are apart from each other, and the substrate affinity to sbdβ is higher (chiappori et al. ). the available sequences for ebov gp found in the protein database of the national center for biotechnology information (ncbi) are downloaded (ten sequences found in the identical protein group database), and clustal omega is utilized to perform multiple sequence alignment (msa) with the pep sequence ctvalpggyvrvc (sievers et al. ) . espript . is used to represent the msa (gouet et al. ) . the c -c region of ebov gp protein that fits well with pep ( . % identity) is analyzed by the protscale web server (expasy bioinformatics resource portal) (garg et al. ; gasteiger et al. ) . pymol is used to superimpose all the solved structures to show the structural conservation of the c -c region of ebov gp . haddock web server (van dijk and bonvin ) was utilized to test the recognition of the full-length ebov gp (pdb id: jq : a, qd : a, qd : a, mam: g, ea : a, kel: a, ken: a, ea : a, and kem: a) by hspa sdbβ (pdb id: e : a). for hspa , the active residues are selected to be i , t , v , v , t , f , s , v , and i (yang et al. ) . in addition, ebov gp active residues are chosen to be the c -c region. protein-ligand interaction profiler (plip) web server (salentin et al. ) was selected to assess the binding pattern after docking. pep cyclic peptide (ctvalpggyvrvc) model was built using i-tasser web server (zhang ) . the protein/peptide docking software hpepdock (zhou et al. ) and haddock were utilized to dock the cyclic pep model into the binding site of hspa . a blind rigid docking scheme is used in hpepdock without the binding site determination for hspa to scan the possible binding sites. the easy interface of haddock is utilized to dock the cyclic pep peptide (all residues are treated as active) into hspa sbdβ (i , t , v , v , t , f , s , v , and i are selected to be the active residues). the cyclic peptide pep was reported to selectively target cell-surface hspa (which appears in some types of cancer) and used as a drug carrier for the anti-cancer agent, doxorubicin (ibrahim et al. ; yoneda et al. ) . figure a shows part of the multiple sequence alignment (msa) between the pep sequence ctvalpggyvrvc and the ten ebov gp sequences downloaded from the ncbi protein database. a complete msa is found in the supplementary file (fig. s ). five identical residues (compared with the pep sequence) were found in the msa of ebov gp sequences, which are c , a , p , g , and c (the numbering is based on pdb id: jq ). interestingly, both c and c form disulfide bonds (see the green numbers at the bottom of fig. a ) with c and c , respectively. the presence of these disulfides suggests its contribution to the folding of gp and gp , as mentioned in earlier work (cantoni and rossman ) . moreover, the cyclic peptide (pep ) can target cs-hspa . the c -c region of ebov gp is found in the β-turn region and the loop separating between the β and β secondary structure (see the top panel of fig. a) . figure b shows the hydrophobicity as a function of amino acid residues of both the pep and c -c regions of ebov gp sequences. kyte and doolittle hydropathic parameters (kyte and doolittle ) are used by protscale web server of the expasy bioinformatics resource portal. a high degree of similarity in the hydrophobicity values for the ebov gp c -c region supports the msa data. the sequence, and thus the structural similarity of ebov gp compared with pep , indicates its ability to bind cellsurface hspa substrate-binding domain β (sbdβ). in addition, fig. c shows the molecular surface of the peptide pep and the c -c region of ebov gp . three colors are used to represent the residues: highly hydrophobic residues in red (such as c, i, l, a, and v), low hydrophilicity residues in yellow (such as g, p, and t), and hydrophilic residues in green (such as d and r). as shown, the two structures show some hydrophobic patch that was reported previously to be the target regions for hspa . these hydrophobic residues of ebov gp (c , l , a , a , i , f , and c ) are suggested to be the binding site for cell-surface hspa . figure a shows the structural conservation of the ebov gp c -c region (red-colored cartoon) in all the solved structures (colored cartoons) found in the protein data bank (pdb ids: jq , qd , qd , mam, ea , kel, ken, ea , and kem). the root mean square deviation (rmsd) values of the superposition range from . Å up to . Å. as shown in the cartoon (lower) and surface representation (upper) of fig. b ( °f ig. a multiple sequence alignment (msa) between pep and ebov gp sequences downloaded from the ncbi protein database. the full msa is presented in the supplementary fig. s . alignment is made using the clustal Ω web server and represented by espript software. red highlights indicate identical residues found, while residues written in red are conserved. b hydrophobicity plot (kyte and doolittle) for both pep (orange) and ebov gp (c -c region) (gray) peptides. c the surface representation of both the pep cyclic peptide and ebov gp c -c region. hydrophobic residues are colored red, hydrophilic residues in green, and residues with weak hydrophilicity are colored yellow rotation on the x-axis of fig. a) , the c -c region is surface exposed (encircled). it can interact with chaperone proteins with its hydrophobic residues l , i , and f (see the enlarged panel of fig. a for jq ) ebov gp -hspa docking table lists the established interactions upon docking ebov gp solved structures (pdb codes are listed) into hspa sbdβ structure and the docking scores of haddock (average values with the standard deviations calculated for the topranked cluster for each ebov gp solved structure). mainly, two types of interactions are established between ebov gp and hspa , h-bonding and hydrophobic interactions. the ea structure only has two salt bridges formed (e :k and e k for ebov gp -hspa ). on average, six hbonds are formed, and four hydrophobic interactions are established upon docking ebov gp into hspa . the primary residues of the ebov gp that form h-bonds are a , a , p , d/e , g , and r . g forms only h-bonds ( interactions), while a , a , d/e , and r form both h-bonding and hydrophobic interactions with ratios : , : , : , and : , respectively. moreover, the main interactions formed by p are hydrophobic ( out of ) interactions. on the other hand, t , v , q , f , s , v , k , and v are the primary interacting residues from the hspa . s and k only interact through h-bonding ( and interactions, respectively). f and v form hydrophobic interactions only ( interactions for each of them). meanwhile, the other residues, t , v , q , and v , form both hbonds and hydrophobic contacts with ebov gp ( : , : , : , and : , respectively). i and i both form few ( and , respectively) hydrophobic interactions with ebov gp , while t show three h-bonds, and t forms two h-bonds and two hydrophobic contacts with hspa . figure a and b show the docking pose of the ebov gp structure ( jq ) into hspa . in fig. a , hspa is represented in the green cartoon, while ebov gp is represented in a cyan cartoon with the c -c region in red. the residues involved in the interactions are in line representation and labeled in the enlarged panel. figure b shows the molecular surface of hspa and ebov gp in cartoon representation. the docking pose shows that ebov gp fits perfectly inside the substrate-binding domain β pocket of hspa with a predicted binding affinity of − . ± . kcal/mol (haddock score). table shows the docking analysis made by plip for the pep peptide after docking into hspa sbdβ. the docking interactions were found in the haddock trial with i , t , v , f , and i of the hspa , while only five hydrophobic contacts were established in the case of the hpepdock experiment (t , v , v , and i of the hspa ). compared with ebov gp , the peptide pep has the same pattern of interactions with hspa (s and k for h-bonding while t , v , and f for hydrophobic contacts). this illustrates the possibility of recognition of the ebov gp for cell-surface hspa overexpressed on cancer cells or cells under other stressors. the present in silico study predicts the binding mode of ebov gp into the hspa chaperone. molecular dynamics simulation is suggested as future work to study the dynamics at the binding site and to test some binding inhibitors. the ebola virus, ebov, is one of the most deadly viral infections in west-african countries. the viral protein gp is the crucial host-cell recognizing protein that enables viral entry. hspa overexpression is reported to increase the infectivity of ebov. inhibiting hspa / ebov gp binding would help in lowering the viral infection. the present work suggests the binding site of the viral/host-cell receptor utilizing in silico methods. this work paves the way for exploring hspa /ebov gp binding inhibitors. further experimental work is required to prove the suggested binding site and to test some inhibitors. author contributions a. e. designed the research study, performed the docking calculations, analyzed the data, and wrote the paper. a. e. has read and approved the final manuscript. data availability the docking structures are available upon request from the corresponding author compliance with ethical standards announcing the worldwide protein data bank full-length ebola glycoprotein accumulates in the endoplasmic reticulum ebolaviruses: new roles for old proteins an atomistic view of hsp allosteric crosstalk: from the nucleotide to the substrate binding domain and back the pymol molecular graphics system. delano scientific, san carlos ehrhardt sa et al ( ) polyclonal and convergent antibody response to ebola virus vaccine rvsv-zebov ebola virus l polymerase rdrp sequence and phylogenetic analysis novel guanosine derivatives as anti-hcv ns b polymerase: a qsar and molecular docking study human papillomavirus e -host cell receptor, grp , binding site prediction protein identification and analysis tools on the expasy server coth mediates fungal invasion of host cells during mucormycosis protein folding in the cell new approaches in the treatment of hepatitis espript: analysis of multiple sequence alignments in postscript grp : a cell's response to stress covid- spike-host cell receptor grp binding site prediction guidelines for the nomenclature of the human heat shock proteins targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic grp ligand a simple method for displaying the hydropathic character of a protein the er chaperone and signaling regulator grp /bip as a monitor of endoplasmic reticulum stress stress induction of grp /bip and its role in cancer targeting grp to enhance melanoma cell death structures of ebola virus gp and sgp in complex with therapeutic antibodies ebola virus glycoprotein gp -host cell-surface hspa binding site prediction grp : a chaperone with diverse roles beyond the endoplasmic reticulum coupling endoplasmic reticulum stress to the cell death program: role of the er chaperone grp plip: fully automated protein-ligand interaction profiler er stress regulation of atf localization by dissociation of bip/grp binding and unmasking of golgi localization signals hspa is an essential host factor for ebola virus infection fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega structural basis of pan-ebolavirus neutralization by a human antibody against a conserved, yet cryptic epitope structural basis of broad ebolavirus neutralization by a human survivor antibody glucose-regulated protein mediates hormoneindependent prostate cancer progression and metastasis through maspin and cox- expression anti-hcv drugs in the pipeline close and allosteric opening of the polypeptide-binding site in a human hsp chaperone bip conformation transitions of the polypeptide-binding pocket support an active substrate release from hsp s a cell-penetrating peptidic grp ligand for tumor cell-specific prodrug therapy ) i-tasser server for protein d structure prediction hpepdock: a web server for blind peptide-protein docking based on a hierarchical algorithm publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the author declares that he has no competing interest. key: cord- -oz hras authors: nelson, elizabeth a.; barnes, alyson b.; wiehle, ronald d.; fontenot, gregory k.; hoenen, thomas; white, judith m. title: clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: oz hras the outbreak of ebola virus (ebov) in western africa highlighted the need for anti-ebov therapeutics. clomiphene is a u.s. food and drug administration (fda)-approved drug that blocks ebov entry and infection in cells and significantly protects ebov-challenged mice. as provided, clomiphene is, approximately, a : mixture of two stereoisomers, enclomiphene and zuclomiphene. the pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which ebov can persist. here we compared the ability of clomiphene and its isomers to inhibit ebov using viral-like particle (vlp) entry and transcription/replication-competent vlp (trvlp) assays. clomiphene and its isomers inhibited the entry and infection of vlps and trvlps with similar potencies. this was demonstrated with vlps bearing the glycoproteins from three filoviruses (ebov mayinga, ebov makona, and marburg virus) and in two cell lines ( t/ and vero e ). visual problems have been noted in ebov survivors, and viral rna has been isolated from semen up to nine months post-infection. since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-ebov agent, for example, to potentially help ameliorate symptoms in ebov survivors. the epidemic of ebola virus (ebov) that swept through western africa beginning in december , was the worst outbreak of this deadly hemorrhagic fever virus in recorded history. over , individuals were infected, over , died, and there are currently an estimated , survivors [ , ] . in addition to the tragic loss of life, many survivors are now suffering from sequelae that arose after they recovered from their ebov infections; these sequelae include visual problems, hearing loss, body aches, severe fatigue, and memory loss [ , ] . moreover, infectious ebov has been shown to persist in semen for six months, ebov rna has been found in semen as late as nine months post-infection, and at least one case of sexual transmission has been reported [ , ] . currently there are no approved drugs with which to treat ebov patients. consequently, during the recent outbreak several agents were administered to patients on a compassionate care transcription/replication-competent viral-like particles (trvlps) were prepared (under biosafety level (bsl ) conditions) as described in [ , ] . briefly, to prepare a p stock, % confluent hek t/ cells, seeded h prior in six well plates, were transfected with pcaggs-np, pcaggs-vp , pcaggs-vp , pcaggs-l, a tetracistronic minigenome plasmid, and pcaggs-t polymerase using transit-lt (mirus, madison, wi, usa). the minigenome plasmid encodes renilla luciferase, as well as the matrix proteins vp and vp and the gp envelope protein from ebov. h post transfection, the medium in each well was replaced with ml fresh growth medium containing % fbs. h after transfection, the medium (containing trvlps harboring the renilla luciferase-containing mini-genome) was harvested, pooled, and cleared of cellular debris by centrifugation for min at ˆg. p and p stocks were prepared by serial passaging of p stocks on hek t/ target cells freshly transfected with pcaggs-np, pcaggs-vp , pcaggs-vp , pcaggs-l, and pcaggs-tim . trvlps (p , p , and p ) were stored on ice and used within two weeks. entry reporter viral-like particles (vlps) bearing gps from mayinga and makona ebov, and from marburg virus (marv; angola isolate, gift of dr. christopher broder (uniformed services university of the health sciences, bethesda, md, usa) were prepared essentially as described in previous work [ , , , ] . in brief, % confluent hek t/ cells were transfected with cdnas encoding ebov or marv gp, vp , mcherry-vp , and β-lactamase-vp (βlam-vp ) (although not utilized in this study, mcherry-vp in the vlps can be used to monitor vlp binding to and trafficking within cells as described in [ , ] ). the cell medium was collected and h post-transfection and was cleared of cellular debris. vlps in the cleared medium were pelleted through a % sucrose cushion by centrifugation, resuspended in hm buffer ( mm hepes, mm mes, mm nacl, ph . ), and repelleted. the final vlp pellet was resuspended ( : starting volume of medium) in % sucrose-hm. the total protein concentration of the vlps was determined by bicinchoninic acid (bca) assay. all entry-reporter vlp preparations were assessed by western blot analyses (for the presence of the indicated glycoprotein as well as ebov vp ) and titered on vero e cells to confirm entry competency. entry-reporter vlps were frozen in single use aliquots at´ ˝c and were used within four months. infection of hek t/ cells by trvlps was assayed as described [ , ] with minor modifications. in brief, to prepare target cells, hek t/ cells were seeded in parallel opaque white -well plates (bd falcon, thermofisher scientific, waltham, ma, usa) for trvlp and cell viability assays and in clear-bottom -well plates to assess cell density and transfection efficacy. at - h post seeding, when the cells were approximately % confluent, the cells were transfected with (per well) . ng pcaggs-np, . ng pcaggs-vp , . ng pcaggs-vp , . ng pcaggs-l, and . ng pcaggs-tim using . µl transit-lt . these plasmids are necessary for the target cells to support entry and replication of incoming trvlps. to assess transfection efficacy and to provide a negative control, wells on each plate were transfected as above, but with a green fluorescent protein (gfp) expression plasmid in place of pcaggs-l. - h post transfection, the medium was removed and these target hek t/ cells were pre-treated with the indicated concentration(s) of the indicated drug(s) (dmso for mock) diluted in opti-mem (omem, gibco life technologies via university of virginia tissue culture facility, charlottesville, va, usa) for h at ˝c in a % co incubator. to assess trvlp infection, the pretreatment solution was removed and replaced with µl or µl trvlps diluted to µl in growth medium containing % scs and the indicated concentration(s) of the indicated drug(s) (dmso for mock). the cells were then incubated for h at ˝c in a % co incubator, after which the medium was replaced with µl of fresh growth medium containing % scs. µl of renillaglo substrate (promega, madison, wi, usa) was then added to each well and the plate was immediately analyzed on a glomax plate reader. to assess cell viability, the pretreatment solution was removed and replaced with µl fresh growth medium containing % scs and the indicated concentration(s) of the indicated drug(s) (dmso for mock). the cells were then incubated for h at ˝c in a % co incubator, after which the medium was replaced with µl of fresh growth medium containing % scs. µl of celltiter-glo . (promega) was then added to each well and the plate placed on a jitterbug orbital shaker (boekel scientific, thermofisher scientific, waltham, ma, usa) set at rpm for min at room temperature (rt). the plate was then incubated at rt for min, after which the luminescent signal was detected using a synergy ht (biotek, winooski, vt, usa) plate reader. vlp entry assays were performed as described [ , , ] with minor modifications. in brief, - , vero e or t/ cells were seeded per well of a clear -well plate. at - h post seeding, when the cells were~ %- % confluent, the cells were treated with the indicated drug(s) at the indicated concentration(s) (dmso for mock) diluted in omem for h at ˝c in a % co incubator. vlps diluted in omem in the presence of the indicated drug(s) at the indicated concentration(s) (dmso for mock) were bound to the cells by spinfection ( ˆg) for h at ˝c. the cells were then incubated for h in a ˝c, % co incubator. the βlam substrate ccf -am (life technologies, thermofisher scientific, waltham, ma, usa) was then loaded into the cells as previously described, but with mm instead of mm probenecid (mp biomedicals, thermofisher scientific, waltham, ma, usa) in the loading buffer. the cells were incubated overnight at rt and then fixed and analyzed by flow cytometry as described [ , , ] . to measure cell viability, - , vero e cells, seeded and grown as above but in -well opaque white plates were treated (as above) for vlp entry, but vlps were not added and ccf -am was not loaded. after the overnight incubation at rt (see above), the medium was removed from the cells and replaced with µl of fresh medium per well. microliters (per well) of celltiter-glo . was then added. the plate was placed on a jitterbug orbital shaker ( rpm) for min at rt. the plate was then incubated at rt for min, after which the luminescent signal was detected using a biotek synergy ht plate reader. for both trvlp infection and vlp entry, raw data were normalized to mock infection/entry signals, and percent inhibition of infection/entry was determined. graphpad prism . was used to perform non-linear regression analyses (log (agonist) vs. response; variable slope) of the percent inhibition values. inhibitory concentration, % (ic ) and % (ic ) values were interpolated based on the regression curve, and ec values were automatically generated. the graphpad program "ecanything" [ ] was used to determine ec values, which are based on the ec and hill slope values from the regression analysis. clomiphene is a~ : mixture of two stereoisomers: enclomiphene and zuclomiphene. their primary metabolites are -hydroxy-enclomiphene and, to a lesser extent, -hydroxy-zuclomiphene. we first compared the ability of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxy-zuclomiphene to block ebov using the trvlp life cycle modeling system described by watt and hoenen [ , ] . as seen in figure a , both isomers of clomiphene (enclomiphene and zuclomiphene) as well as the -hydroxy metabolites of each ( -hydroxy-enclomiphene, and -hydroxy-zuclomiphene) displayed dose-dependent inhibition of trvlp infection similar to that seen with the parent mixture (clomiphene) when cells were pretreated for h with increasing concentrations of compound. at a dose of µm, all five forms of clomiphene showed approximately equal potency, reducing the trvlp (renilla luciferase) signal by approximately -fold. parallel sets of cells showed no evidence of cytotoxicity by any form of clomiphene ( figure b ). our previous findings indicated that clomiphene blocks ebov infection by blocking entry of viral particles into the cell cytoplasm [ , ] ; this effect appears to be at the level of fusion between the membrane of the viral particle and the limiting membrane of an niemann-pick disease, type c positive (npc + ) endolysosome, the site of ebov fusion [ ] [ ] [ ] . we, therefore, compared the effects of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxyzuclomiphene on ebov entry using entry reporter vlps, as described previously [ , , ] . at a concentration of μm, clomiphene, both of its isomers (enclomiphene and zuclomiphene), as well as the -hydroxy metabolite of each isomer blocked vlp entry (figure a) , with no evidence of cytotoxicity ( figure. b ). in this system, -hydroxy-zuclomiphene appeared more potent than the our previous findings indicated that clomiphene blocks ebov infection by blocking entry of viral particles into the cell cytoplasm [ , ] ; this effect appears to be at the level of fusion between the membrane of the viral particle and the limiting membrane of an niemann-pick disease, type c positive (npc + ) endolysosome, the site of ebov fusion [ ] [ ] [ ] . we, therefore, compared the effects of clomiphene, enclomiphene, zuclomiphene, -hydroxy-enclomiphene, and -hydroxy-zuclomiphene on ebov entry using entry reporter vlps, as described previously [ , , ] . at a concentration of µm, clomiphene, both of its isomers (enclomiphene and zuclomiphene), as well as the -hydroxy metabolite of each isomer blocked vlp entry (figure a) , with no evidence of cytotoxicity ( figure b ). in this system, -hydroxy-zuclomiphene appeared more potent than the other forms of clomiphene ( figure a ), but this is likely an assay-dependent result, as -hydroxy-zuclomiphene was not more potent in the trvlp assay ( figure a ). other forms of clomiphene ( figure a ), but this is likely an assay-dependent result, as -hydroxyzuclomiphene was not more potent in the trvlp assay ( figure a ). as elaborated in sections and , enclomiphene and zuclomiphene exhibit different pharmacological properties. notably, zuclomiphene has a longer half-life [ , ] , and evidence suggests that the zuclomiphene component is responsible for more of the adverse side effects seen in mice treated with clomiphene [ ] . we, therefore, compared clomiphene, enclomiphene, and zuclomiphene in more detail for their effects on trvlp infection and vlp entry. we first compared clomiphene and enclomiphene in the trvlp infection and vlp entry systems using eight-point dose response curves. as seen in figure , clomiphene and enclomiphene showed similar dose-dependent inhibition of trvlp infection. in this experiment the calculated ic for clomiphene was . μm and that for enclomiphene was . μm. clomiphene and enclomiphene also showed similar dose-dependent inhibition in the vlp entry system; the ic for clomiphene was . μm and for enclomiphene was . μm (figure ). similar results were seen when comparing eight doses of clomiphene and zuclomiphene in trvlp infection ( figure a ) and vlp entry ( figure b ) as elaborated in sections and , enclomiphene and zuclomiphene exhibit different pharmacological properties. notably, zuclomiphene has a longer half-life [ , ] , and evidence suggests that the zuclomiphene component is responsible for more of the adverse side effects seen in mice treated with clomiphene [ ] . we, therefore, compared clomiphene, enclomiphene, and zuclomiphene in more detail for their effects on trvlp infection and vlp entry. we first compared clomiphene and enclomiphene in the trvlp infection and vlp entry systems using eight-point dose response curves. as seen in figure , clomiphene and enclomiphene showed similar dose-dependent inhibition of trvlp infection. in this experiment the calculated ic for clomiphene was . µm and that for enclomiphene was . µm. clomiphene and enclomiphene also showed similar dose-dependent inhibition in the vlp entry system; the ic for clomiphene was . µm and for enclomiphene was . µm (figure ). similar results were seen when comparing eight doses of clomiphene and zuclomiphene in trvlp infection ( figure a ) and vlp entry ( figure b ) assays. the ic and ic values from all of the eight-point dose response curves (figures - ) are given in table . table . previous work using authentic ebov in a bsl lab showed that clomiphene blocks infections in multiple cell types including vero e (kidney), hepg (liver), and huvec (endothelial) cells, and by all strains of filoviruses tested: three strains of ebola virus and two of marburg virus [ , ] . here we used assays that can be conducted in a bsl laboratory to begin to test whether enclomiphene shows similar breadth as a filovirus inhibitor. we first used the trvlp system to compare the efficacy of clomiphene and enclomiphene in vero e cells. the trvlp experiments presented thus far were conducted in hek t/ cells, which are optimal for this assay because they are highly transfected with the multiple plasmids needed to produce trvlps and to assay their lifecycle capacity [ , ] . vero e cells are also good targets for this assay. as seen in figure , clomiphene and enclomiphene blocked trvlp infection in vero e cells (with similar dose profiles). treated samples; and (b) hek t/ were seeded, treated with clomiphene or zuclomiphene, and vlps bearing ebov gp (mayinga strain) were bound to the cells as described in the legend to figure . cytoplasmic entry was assayed as described in the legend to figure . data ± sd are from triplicate samples normalized to the average % entry in mock treated cells ( . % and . % for the two sets of drugs). previous work using authentic ebov in a bsl lab showed that clomiphene blocks infections in multiple cell types including vero e (kidney), hepg (liver), and huvec (endothelial) cells, and by all strains of filoviruses tested: three strains of ebola virus and two of marburg virus [ , ] . here we used assays that can be conducted in a bsl laboratory to begin to test whether enclomiphene shows similar breadth as a filovirus inhibitor. we first used the trvlp system to compare the efficacy of clomiphene and enclomiphene in vero e cells. the trvlp experiments presented thus far were conducted in hek t/ cells, which are optimal for this assay because they are highly transfected with the multiple plasmids needed to produce trvlps and to assay their lifecycle capacity [ , ] . vero e cells are also good targets for this assay. as seen in figure , clomiphene and enclomiphene blocked trvlp infection in vero e cells (with similar dose profiles). the current trvlp system utilizes plasmids encoding proteins from the mayinga ( ) isolate of ebov [ , ] , an oft-used reference strain. we, therefore, utilized entry reporter vlps, which can accommodate glycoproteins from other viruses [ , ] , to ask whether the ability of enclomiphene to block ebov entry extends to other filoviruses. as seen in figure a , enclomiphene exerted a similar effect as clomiphene in blocking entry mediated by the gp from makona ebov, the isolate that caused the - outbreak in western africa, and, as seen in figure b , enclomiphene inhibited entry mediated by a marburg virus gp, representing a different genus within the filovirus family, similar to the effects of clomiphene. the current trvlp system utilizes plasmids encoding proteins from the mayinga ( ) isolate of ebov [ , ] , an oft-used reference strain. we, therefore, utilized entry reporter vlps, which can accommodate glycoproteins from other viruses [ , ] , to ask whether the ability of enclomiphene to block ebov entry extends to other filoviruses. as seen in figure a , enclomiphene exerted a similar effect as clomiphene in blocking entry mediated by the gp from makona ebov, the isolate that caused the - outbreak in western africa, and, as seen in figure b , enclomiphene inhibited entry mediated by a marburg virus gp, representing a different genus within the filovirus family, similar to the effects of clomiphene. clomiphene emerged from two independent screens of fda-approved drugs for anti-ebov activity in tissue culture cells [ ] [ ] [ ] , and was also found to provide %- % protection in female mice challenged with a lethal dose of ebov [ , ] . further analyses demonstrated that clomiphene blocks ebov entry into the host cell cytoplasm after virus particles are transported to endolysosomes [ , ] , the site of ebov fusion and cytoplasmic entry [ ] [ ] [ ] . hence, clomiphene interferes with the process of ebov fusion with the endolysosomal membrane. clomiphene, as used to treat female infertility, is a ~ : mixture of two stereoisomers, enclomiphene and zuclomiphene. since zuclomiphene is thought to impart more side effects [ ] , enclomiphene is being developed for the treatment of secondary hypogonadism (low testosterone) [ ] . the main objective of this study was to assess the abilities of enclomiphene and zuclomiphene to block ebov infection, as the individual isomers might offer (different) advantages in different clinical settings. we found that enclomiphene and zuclomiphene were highly similar to clomiphene in their ability to block trvlp infection and vlp entry, which are validated surrogate assays for ebov infection and ebov entry, respectively. clomiphene, enclomiphene, and zuclomiphene inhibited vlp entry and trvlp infection with similar ic values. where tested, they were equally effective to each other in two cell types and for entry mediated by three filoviral gps. -hydroxy-enclomiphene, and -hydroxy-zuclomiphene also blocked ebov trvlp infection and vlp entry under the clomiphene emerged from two independent screens of fda-approved drugs for anti-ebov activity in tissue culture cells [ ] [ ] [ ] , and was also found to provide %- % protection in female mice challenged with a lethal dose of ebov [ , ] . further analyses demonstrated that clomiphene blocks ebov entry into the host cell cytoplasm after virus particles are transported to endolysosomes [ , ] , the site of ebov fusion and cytoplasmic entry [ ] [ ] [ ] . hence, clomiphene interferes with the process of ebov fusion with the endolysosomal membrane. clomiphene, as used to treat female infertility, is a~ : mixture of two stereoisomers, enclomiphene and zuclomiphene. since zuclomiphene is thought to impart more side effects [ ] , enclomiphene is being developed for the treatment of secondary hypogonadism (low testosterone) [ ] . the main objective of this study was to assess the abilities of enclomiphene and zuclomiphene to block ebov infection, as the individual isomers might offer (different) advantages in different clinical settings. we found that enclomiphene and zuclomiphene were highly similar to clomiphene in their ability to block trvlp infection and vlp entry, which are validated surrogate assays for ebov infection and ebov entry, respectively. clomiphene, enclomiphene, and zuclomiphene inhibited vlp entry and trvlp infection with similar ic values. where tested, they were equally effective to each other in two cell types and for entry mediated by three filoviral gps. -hydroxy-enclomiphene, and -hydroxy-zuclomiphene also blocked ebov trvlp infection and vlp entry under the conditions tested. therefore, we propose that, like clomiphene, enclomiphene and zuclomiphene have potential as pan-filoviral inhibitors in multiple cell types. approximately , individuals died during the recent outbreak of makona ebov, and another~ , have survived their infection [ ] . tragically, many of these survivors are displaying post ebola virus disease syndromes, including visual problems, hearing loss, and excessive fatigue [ , ] . in addition, infectious ebov has been found in semen for up to six months [ ] , raising concerns of sexual transmission [ ] . hence, drugs that accumulate in tissues of the eye and male reproductive tract could be particularly helpful for certain ebola virus survivors. in this respect it is interesting that, in mice, both enclomiphene and zuclomiphene persist in the eye and male reproductive tract longer than in other tissues, with zuclomiphene maintained at higher levels than enclomiphene. zuclomiphene was also found to persist in the brain, whereas enclomiphene was not [ ] . clomiphene provided up to % protection in the lethal mouse model of ebov infection when administered to female mice at a dose of mg/kg on days , , , , , and post-infection, with no apparent ill effects on surviving mice. the maximum serum concentration (c max ) for a mg/kg dose (intra-peritoneal administration, ip) of clomiphene to female mice was . µm (lisa johansen, personal communication) and, hence, above the ic for blockade of ebov infection in tissue culture cells [ , ] and above the ic values reported here for clomiphene and its isomers in blocking ebov vlp entry and trvlp infection ( table ). the standard dose of clomiphene to treat female infertility is mg/day (per os). with this (oral) dose, reported c max values (summed for enclomiphene and zuclomiphene) range from . to . µm (table ) ,~ - -fold below the ic ( . µm in vero e cells) for anti-ebov activity [ , ] . therefore, for potential treatment of acute ebov infections, higher doses of clomiphene would certainly be needed, likely in combination with other anti-ebov drugs. the human equivalent dose (to the mouse dosing employed in [ ] ) for an adult weighing kg would be mg clomiphene every other day [ ] . in this respect it is noteworthy that higher doses of clomiphene (up to mg/day) have been administered to non-responding (anovulating) females [ , ] , and that considerably higher c max values were obtained when patients were administered the standard dose of clomiphene ( mg), but by intravenous administration (iv) instead of the oral route (table ) [ ] . hence, it is conceivable that higher doses of clomiphene could be administered (perhaps as part of a cocktail) to treat acute ebov infections. irrespective of the utility of clomiphene for acute ebov infections, given the persistence of the clomiphene isomers in the eye and male reproductive tract [ ] , clomiphene (alone or in a combination) may have utility in the management of post ebola virus disease symptoms in ebov survivors. for example, enclomiphene and zuclomiphene accumulate~ -to~ -fold, respectively, in the harderian gland (located in the orbit) and the uveal tract of the mouse eye. similar accumulation of zuclomiphene was seen in tissues of the male mouse reproductive tract [ ] . if such accrual occurs in humans, the standard dose ( mgs per day, per os) of clomiphene, which contains both isomers, could be sufficient to reach anti-viral levels in the eye and male reproductive tract; the levels would more likely be sufficient if clomiphene were given at a higher dose and/or in combination with another anti-ebov agent. interestingly, no sperm were seen in the testes and epididymes of male mice treated for days with mg/kg/day of zuclomiphene [ ] . since the two isomers of clomiphene and their hydroxylated metabolites all blocked ebov vlp entry and replication in cell cultures, the protective effect of clomiphene in the mouse model [ , ] could have been due to any or all of these forms. the metabolism of en-and zuclomiphene in humans is well-characterized [ , ] , and differences between the isomers have been shown in mice [ , ] . it will, therefore, be interesting to compare the efficacies of the individual clomiphene isomers in the mouse model of ebov infection. studies on their relative efficacies in the eyes and reproductive tract will be especially revealing. table in reference [ ] ; c values deduced from figure of reference [ ] ; anov., anovulating; h, hour; n/a, not applicable; p , anovulatory patient ; p , anovulatory patient ; itt, intention-to-treat (i.e., healthy males with low testosterone levels). we have shown that, like the parent mixture, the two isomers of clomiphene (and their hydroxylated metabolites) block entry and replication of ebov vlps in cell cultures. the activity of the isomers was shown in two cell types and against three filoviral gps. both isomers of clomiphene accumulate in tissues of the eye and male reproductive tract. hence we propose that clomiphene, an fda-approved drug that provided up to % protection in the mouse model of ebola virus disease, remain in consideration as an anti-ebov agent, 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pharmacokinetic study of clomiphene citrate isomers in anovular patients with polycystic ovary disease dose translation from animal to human studies revisited monitoring plasma concentrations to individualize treatment with clomiphene citrate serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women pharmacokinetics of intravenous clomiphene isomers the work was supported by a grant from the nih to jmw (ro ai ) and in key: cord- -bmfj fb authors: malin, jakob j.; suárez, isabelle; priesner, vanessa; fätkenheuer, gerd; rybniker, jan title: remdesivir against covid- and other viral diseases date: - - journal: clin microbiol rev doi: . /cmr. - sha: doc_id: cord_uid: bmfj fb patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus (sars-cov- ) pandemic. remdesivir (gs- ) is the first approved treatment for severe coronavirus disease (covid- ). it is a novel nucleoside analog with a broad antiviral activity spectrum among rna viruses, including ebolavirus (ebov) and the respiratory pathogens middle east respiratory syndrome coronavirus (mers-cov), sars-cov, and sars-cov- . first described in , the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic rna viruses. in vivo, remdesivir showed therapeutic and prophylactic effects in animal models of ebov, mers-cov, sars-cov, and sars-cov- infection. however, the substance failed in a clinical trial on ebolavirus disease (evd), where it was inferior to investigational monoclonal antibodies in an interim analysis. as there was no placebo control in this study, no conclusions on its efficacy in evd can be made. in contrast, data from a placebo-controlled trial show beneficial effects for patients with covid- . remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. although this is an important milestone in the fight against covid- , approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable. tients with covid- . remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. although this is an important milestone in the fight against covid- , approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable. i n december , a novel coronavirus (ncov), severe acute respiratory distress syndrome coronavirus (sars-cov- ), emerged in wuhan, central china. like in sars-cov, infections with the closely related sars-cov- cause a respiratory disease that can progress to viral pneumonia and acute respiratory distress syndrome (ards) ( ) . because of its onset in december , the associated disease was called coronavirus disease . through june , the ongoing pandemic caused more than million confirmed covid- cases and nearly , deaths globally ( ). in the light of an uncontrolled expansion and the steadily increasing covid- fatalities in january and february , huge efforts were put into the identification of effective antiviral agents against covid- . nucleoside/nucleotide analogs are one of the most promising antiviral drug classes in general, and significant drug discoveries emerged from this class that today form the basis for treatments against infections with several herpesviruses, human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv). remdesivir or gs- is a prodrug of a nucleoside analog with direct antiviral activity against several single-stranded rna viruses, including sars-cov and middle east respiratory syndrome coronavirus (mers-cov). the first cell-based studies of remdesivir also showed antiviral activity against the novel sars-cov- ( , ) . in the absence of any effective treatment options against covid- , remdesivir has been applied under compassionate use. recently, preliminary data from a randomized placebo-controlled clinical trial showed that remdesivir reduces the time to recovery in patients with covid- ( ) , leading to an emergency-use authorization (eua) by the u.s. food and drug administration (fda) only days after the first press release from the national institute of allergy and infectious diseases (niaid) ( ) . on july, the european medicines agency (ema) granted a conditional marketing authorization for remdesivir, now being the first approved antiviral treatment against covid- ( ) (fig. ). here, we provide a comprehensive review of results from preclinical and clinical studies on this important novel antiviral drug to understand its clinical significance. in addition, we briefly describe the discovery and molecular mechanism of viral inhibition. nucleoside and nucleotide analogs as small-molecule-based antivirals have been explored for many years and form the backbone of treatment against viral infections, including hiv, hepatitis b virus, and herpesvirus infections ( ) ( ) ( ) . in , the nucleotide analog sofosbuvir was approved by the fda for the treatment of chronic hepatitis c virus infections. the novel compound that targets the rna-dependent viral polymerase (ns b) revolutionized hcv treatment, as it is able to cure the formerly lifelong chronic progressive disease when combined with other antivirals ( ) . in the past years, nucleoside/nucleotide analogs were increasingly recognized as potential antivirals targeting other positive-stranded rna viruses such as members of the flaviviridae, picornaviridae, caliciviridae, and coronaviridae families, as they share relevant amino acid sequences with hcv ( ) , and the rna-dependent polymerases are closely related phylogenetically ( , ) . this supported the assembly of antiviral compound libraries that could be screened against emerging rna viruses. in the past years, several pharmacological advances in the development of nucleoside analogs were made based on structure-to-activity relationship (sar) studies that improved pharmacokinetics, antiviral activity, and selectivity ( ) ( ) ( ) . a comprehensive overview of the medicinal chemistry and pharmacological evolution of antiviral nucleoside analogs can be found elsewhere ( , ) . nucleoside analogs require intracellular activation by phosphorylation in order to become their active metabolites. one of the most important milestones was the addition of a monophosphate prodrug to the nucleoside, which significantly improved intracellular delivery and activation ( ) ( ) ( ) . this so-called pro-tide approach, developed by mcguigan et al. ( , ) , was also used to optimize the precursor of remdesivir named gs- . the parent molecule of remdesivir, gs- , was derived from a small-molecule library of around , diverse nucleoside and nucleoside phosphonate analogs that were assembled over many years of antiviral research based on their potential ability to target emerging rna viruses such as sars-cov and mers-cov of the coronaviridae or zika and dengue viruses of the flaviviridae family ( ) . following the ebolavirus (ebov) epidemic in west africa from to , a selection of promising leads from this library underwent intensive testing against different types of ebov in collaboration with the centers for disease control and prevention (cdc) and the u.s. army medical research institute of infectious diseases (usamriid), which included studies in nonhuman primates (nhps) ( ) . these efforts finally led to the identification of gs- , a monophosphate prodrug version of gs- , as the most promising lead against ebov. gs- , later renamed remdesivir, had a broad antiviral spectrum, including ebov, marburg virus, respiratory syncytial virus (rsv), hcv, and several paramyxoviruses ( , , ) , in vitro. in addition, it demonstrated activity against mers-cov ( - ) and sars-cov ( , ) . favorable in vitro results stimulated further evaluation in ebov-infected macaques, where remdesivir suppressed viral replication and improved survival, clinical signs of the disease, and pathophysiological blood markers ( ) . after its discovery, remdesivir was administered under compassionate use to patients with ebolavirus disease (evd) but stopped after an interim analysis of the first randomized controlled clinical trial (rct) showed an inferiority of remdesivir to treatments with monoclonal antibodies (mab and regn-eb ). the trial evaluated the efficacies of , , , , and - .) different investigational therapeutics against evd. following the interim analysis, the remdesivir arm was halted for the remainder of the trial ( ) . in december , a novel coronavirus, sars-cov- , emerged and caused a pandemic that is still ongoing. there were strong arguments for the antiviral effect of remdesivir against coronaviruses emerging from multiple cell-based in vitro models, including primary human airway epithelial (hae) cell cultures ( ) , and, for mers-cov, from a mouse model of pulmonary infection ( ) . in addition, in a rhesus macaque model of mers-cov infection, remdesivir demonstrated strong prophylactic properties, and administration was associated with clinical benefits for treated subjects ( ) . the global hazards caused by the pandemic with the novel sars-cov- prompted the identification of potential treatment options. given the solid preclinical data, remdesivir was considered one of the most promising candidates that went into clinical testing against covid- . remdesivir is a monophosphoramidate nucleoside prodrug that undergoes intracellular metabolic conversion to its active metabolite nucleoside triphosphate (ntp). as described for several other direct-acting antivirals, the active metabolite of remdesivir (remdesivir triphosphate [remdesivir-tp] or gs- ) subsequently targets the machinery responsible for the replication of the viral rna genome, a highly conserved element of the viral life cycle. nucleoside analogs are synthetic compounds that work by competition with endogenous natural nucleoside pools for incorporation into replicating viral rna. while these compounds mimic their physiological counterparts, the incorporation of the analog molecule disrupts subsequent molecular processes. the drug target and the exact processes that lead to the inhibition of viral replication have been studied extensively in ebolavirus ( , ) . the suggested drug target, the ebov rna-dependent rna polymerase (rdrp) complex, was only recently biochemically purified, which allowed for in-depth molecular analyses. viral rdrp is the target protein for the active metabolite remdesivir-tp. remdesivir-tp acts as the substrate for rdrp where it competes with atp for incorporation into new strands. inhibition of ebov rdrp most probably results from delayed chain termination, a mechanism that is known from approved antivirals against human immunodeficiency virus type (hiv- ) and hbv ( ) ( ) ( ) ( ) . in the case of ebov, the incorporation of remdesivir-tp into replicating rna was observed to cause chain termination predominantly at five positions downstream (i ϩ ) ( ) . importantly, the activity of human rna polymerase is not inhibited in the presence of remdesivir-tp ( ) . in sars-cov and mers-cov, remdesivir-tp interferes with the nsp polymerase, which is a multisubunit rna synthesis complex of viral nonstructural proteins (nsp's) produced as cleavage products of viral polyproteins. as nsp is highly conserved across the coronavirus family, it is most likely that the mechanism of action (moa) of remdesivir does not differ significantly among covs ( , ) . like in ebov, remdesivir-tp efficiently inhibits the replication of sars-cov and mers-cov by causing delayed chain termination when being incorporated into the replicating rna ( ) . a recent biochemical analysis revealed that in sars-cov- , remdesivir-tp causes the termination of rna synthesis at three positions after the position where it is incorporated (i ϩ ). this mechanism was nearly identical in rdrps of sars-cov and mers-cov ( ) . the premature termination of rna synthesis ultimately abrogates further transcriptional and translational processes needed for the generation of new virions (fig. ) . the resulting antiviral effects of remdesivir have been studied in different cell-based models. remdesivir has been tested against various viruses in different cell-based systems and target cells (table ) . when comparing the results of antiviral assays, one has to take into account that different assay methodologies and parameters such as the target cell type and virus input used may have significant impacts on the efficacy outcome ( ) . a variety of methods are available to assess the antiviral activity of candidate compounds in cell-based models. simplified, susceptible target cells allowing viral replication are infected and subsequently exposed to serial concentrations of the test compounds. historically, compounds were analyzed for their ability to reduce the amount of virus pfu on cellular layers ( ) . although this method has the advantage of addressing the complete viral life cycle, it has been widely replaced by molecular methods that allow automated quantification. the antiviral effects of test compounds can be assessed by monitoring viral replication by either quantification of viral rna using real-time pcr (rtpcr) or measurement of fluorescent reporter gene expression (rge) from genetically modified virus strains. a special type of reporter gene assay is viral replicon (repl) assays using genetically modified virus genomes that undergo transcriptional and translational processes inside the target cell but do not yield infectious progeny. often, genes encoding structural proteins are exchanged by reporter genes that enable the monitoring of viral replication. this approach has been used to study the inhibition of hcv replication. alternatively, viral antigens (ags) can be quantified by fluorescence-or chemiluminescence-based immunostaining. antiviral effects can also be measured indirectly by assessing virus-induced cytopathic effects (cpe) in the presence of test compounds. assays measuring cpe can display not only direct antiviral effects but also beneficial effects of compounds with antivirulence properties. this is an advantage for high-throughput screening due to a gain of signal ( ) . in addition, cpe can be used to measure antiviral activity when no robust rge-or rtpcr-based assay is available. it is unclear which type of assay provides the best information that can predict in vivo efficacy. however, the comparability of results from direct methods measuring viral replication and from cpe-based assays is limited by the fact that viral loads and related cpe do not automatically have a linear association. the choice of target cell displays another important factor. activity against filoviruses is classically assessed in vero e cells that were derived through the immortalization of african green monkey kidney cells. this cell line is known to highly express the angiotensin-converting enzyme (ace- ) receptor, which is required for viral entry of both sars-cov and sars-cov- into the target cell ( ) . in addition, vero e cells support the replication of sars-covs to high titers, which made them a standard cell model to study related pathogens ( ) ( ) ( ) ( ) . besides common target cells, the antiviral activity of remdesivir was evaluated in human cell lines and primary cells that represent more clinically oriented in vitro systems. activity against filoviruses was tested in a human liver cancer cell line (huh- ) and human primary macrophages (hpms). anti-cov activity was evaluated in a human lung epithelial cell line (calu- ), primary hae cells, and immortalized human foreskin microvascular endothelial cells (hmvecs). in contrast to sars-cov experiments that were conducted in hae cells, assays against sars-cov- were conducted in vero e cells, which might explain the -log-lower antiviral efficacy measured for sars-cov- ( , , ) . a recent comparison of replication kinetics and cpe of sars-cov and sars-cov- in vero e cells concluded that there were no significant differences in drug sensitivities to remdesivir, thereby supporting this hypothesis ( ). in , warren et al. ( ) tested the small-molecule nucleoside analog remdesivir (gs- ) against ebov. the half-maximum effective concentrations (ec s) for ebov inhibition were between . and . m in different cell types, including human macrophages and endothelial cells. it was also shown that remdesivir inhibits the replication of other pathogenic rna viruses such as rsv (ec , . m) and mers-cov (ec , . m) while having low cytotoxicity in a wide range of human primary cells and cell lines ( ) . the characterization of the antiviral spectrum of remdesivir in vitro was subsequently reevaluated and expanded across multiple virus families, including representatives of the filo-, paramyxo-, pneumo-, arena-, rhabdo-, flavi-, and coronavirus families, including zoonotic and epidemic covs ( , , , , ) . remdesivir effectively inhibited ebov (ec , . to . m) and rsv (ec , . ), with results being comparable to those reported by warren et al. ( ) . in addition, efficacy against marburg virus (ec , . to . m) and several paramyxoviruses (ec , . to . m) was demonstrated in cell-based assays ( ) . the ec s for mers-cov and ( ) demonstrated that the antiviral spectrum of remdesivir also includes porcine covs and endemic human covs (hcovs) that are associated with the common cold (hcov-oc and - e). after the outbreak of sars-cov- in january , remdesivir was rapidly tested in a vero e cell-based model that made use of direct viral quantification by rtpcr along with the antimalaria and immune-modulating drug chloroquine and known antivirals such as ribavirin and penciclovir. remdesivir was active against sars-cov- , with an ec of . m, which was slightly lower than that of chloroquine (ec , . m) and remarkably lower than those of other tested antivirals (ec , . to . m). more recently, a comprehensive comparison of the antiviral effects of remdesivir on the replication kinetics and cytopathology of clinical isolates of sars-cov and sars-cov- was made in vero e cells. here, both viruses showed similar sensitivities to remdesivir, with ec s of . and . m, respectively, when measuring the % cytopathologic effect ( ) . these values are substantially higher than those reported in previous studies that made use of reporter gene or rtpcr assays, which seems to have a methodological background. however, the higher values in this study may also reflect significantly lower susceptibilities of sars-cov and sars-cov- clinical isolates to remdesivir. choy et al. ( ) recently determined the ec ( . to m) for another sars-cov- clinical isolate (hong kong/vm / ) using three different vero e cell-based assays. however, the comparability of these results to those of previous studies is limited by viral load calculations fitted to logarithmic scales (log rna copies per milliliter) in order to estimate the effect of increasing drug concentrations. finally, efficacy data were also reported by the manufacturer. gilead's fact sheet on remdesivir reports an ec of . m (preliminary data), but no information is given on the specific strain that was tested by the china cdc in collaboration with gilead sciences ( ) . a complete overview of (peer-reviewed) published in vitro efficacy data is given in table . nucleoside analogs such as remdesivir are generally expected to have a higher barrier to antiviral resistance than other antivirals such as neuraminidase inhibitors due to their well-conserved and vulnerable drug target ( ) . however, a general obstacle in the development of antiviral nucleoside analogs against covs is the presence of a potent exoribonuclease (exon)-mediated proofreading function of the rdrp subdomain nsp , which causes resistance against ribavirin and -fluorouracil ( ) . exon is able to identify and remove incorporated nucleoside analogs. an important observation was that virus mutants lacking exon are -fold more sensitive to remdesivir (ec , . m) than the wild type with intact proofreading, implying that remdesivir is prone to exon-mediated proofreading. nevertheless, this effect was shown to be concentration dependent, and even with intact exon proofreading, remdesivir inhibits viral replication still at submicromolar concentrations in direct antiviral assays ( ) . the relatively modest effect of exon on remdesivir susceptibility was attributed to the fact that the incorporation of remdesivir into replicating rna occurs efficiently in comparison to natural nucleotides and to the mechanism of delayed chain termination that causes nsp inhibition by remdesivir ( , , ) . the additional natural nucleotides that are subsequently incorporated may have protective effects against exon activity ( ) . although nsp rdrp is highly conserved among covs, the amino acid identity still varies between and % when including human and zoonotic covs ( ) , and variations in the amino acid sequence could have effects on viral susceptibility to remdesivir. therefore, brown et al. ( ) tested the antiviral activity of remdesivir in strains with the most divergent rdrp compared to sars-and mers-cov. they included endemic human covs ( e and oc ) and a porcine cov known to harbor a native residue in the nsp subunit that confers antiviral resistance in betacoronaviruses. they found that the activity of remdesivir includes both contemporary human and highly divergent zoonotic covs and that natural variations in wild-type rdrp do not confer remdesivir resistance. another important factor regarding the remdesivir drug target is that mutations leading to changes in neighboring amino acids of the nsp subunit will most probably result in a substantial loss of viral fitness. agostini et al. ( ) demonstrated that mutations in nsp can be induced in wild-type murine hepatitis virus (mhv) by serial passage in the presence of increasing concentrations of gs- . these mutations (f l and v l) led to decreased remdesivir susceptibility, with a . -fold or -fold shift in the ec ( . to . m). the introduction of these substitutions into the sars-cov genome had a similar effect on remdesivir susceptibility, but mutants were unable to compete against the wild-type strain in coinfection passages without selective pressure. furthermore, variants carrying f l or v l were attenuated in vivo, as shown by decreased lung titers in a mouse model of sars-cov infection. in summary, remdesivir has a high genetic barrier to resistance development, and known resistant variants suffer from a loss of competitive fitness. this may suggest that these mutations will most likely not be maintained in nature and do not favor an uncontrolled spread of remdesivir-resistant variants. compounds that are able to block exon-mediated proofreading would be of interest for combination therapy as they significantly increase virus susceptibility to remdesivir in vitro. ebolavirus disease. the pro moiety of remdesivir can be degraded by serum esterase, which negatively affects efficacy and the pharmacokinetic profile. because of high serum carboxylesterase activity in most rodents primarily used for animal models, initially, there was no suitable rodent model to study the efficacy of remdesivir in vivo ( ) . the first in vivo studies were therefore performed in nhps that have lower or no serum esterase activities and are therefore comparable to humans ( ) . in an nhp model of fatal evd, rhesus monkeys were inoculated with ebov by intramuscular injection and treated for days with an intravenous (i.v.) vehicle (n ϭ ), mg/kg of body weight on day or day (n ϭ per group), mg/kg after a -mg/kg loading dose on day or day (n ϭ per group), or mg/kg of gs- once daily starting days after inoculation. of animals treated days after virus exposure ( mg/kg daily or mg/kg with a -mg/kg loading dose), % of nhps survived the -day in-life phase. in contrast, only out of subjects treated on day or survived the infection (all at mg/kg daily; received a -mg/kg loading dose). antiviral effects (reduction in plasma viral rna) and reductions in clinical signs were more pronounced in the -mg/kg treatment group. warren et al. concluded that there was substantive postexposure protection with daily dosing of mg/kg remdesivir ( ) . for mers-cov, a head-to-head assessment of remdesivir versus combinations of lopinavir, ritonavir, and interferon beta was performed in esterase-deficient mice with a humanized dipeptidylpeptidase (ddp ) receptor. humanization of the ddp receptor is required for mers-cov infection in mouse models because the wild-type ddp receptor does not enable mers-cov spike protein binding. wildtype mice are therefore not susceptible to mers-cov infections ( ) . in this study, the therapeutic and prophylactic properties of remdesivir could be reproduced, and in the overall view of the authors, remdesivir was superior to other tested antivirals and combination treatments ( ) . remdesivir was also evaluated in a rhesus macaque model of mers-cov infection. the prophylactic administration of mg/kg h before inoculation with mers-cov prevented all six tested animals from developing active disease. therapeutic administration by h postinoculation reduced clinical signs of the disease, viral replication in the lungs, and pathological lung tissue processes in all six animals of the treatment group. those authors therefore considered remdesivir to be a potential candidate against the novel sars-cov- ( ). given the limited plasma stability of remdesivir in rodents that is caused by high serum esterase activity, its antiviral activity against covs was evaluated in a mouse model of sars-cov infection using carboxylesterase c knockout (ces c Ϫ/Ϫ ) mice. here, prophylactic and early therapeutic administration of remdesivir ( h before or h after inoculation) reduced viral loads in the lungs and improved respiratory functions and clinical signs of the disease when given subcutaneously at doses of mg/kg twice daily ( ) . in comparison to studies in nhps or humans, high doses of remdesivir are necessary in mouse models, as tissue levels of the active remdesivir metabolite are approximately times lower than those in nhps, even in esterase-deficient mice. in addition, remdesivir-tp is more rapidly degraded in rodent lung tissues than in nhp lungs and primary human airway cells ( , ) . recently, the efficacy of remdesivir treatment was finally tested in a rhesus macaque model of sars-cov- infection ( ) . animals were infected with sars-cov- (n ϭ ) by combined intranasal, oral, ocular, and intratracheal inoculations and subsequently treated with intravenous placebo or remdesivir ( -mg/kg loading dose followed by mg/kg daily) for days starting at h postinfection. remdesivir-treated animals did not show signs of respiratory disease and had lower lung virus titers and less lung tissue damage than the placebo group. according to the manufacturer's information on the pharmacokinetic bridge from rhesus monkeys to humans, these doses approximate serum drug exposures that are equivalent to mg and mg, respectively ( ) . it is also important to note that the dynamics of acute sars-cov- infection progress more rapidly in animal models than in humans and that optimal treatment time points that are calculated based on expected viral load peaks cannot be directly translated to humans ( ) . although the results of this study have to be interpreted with caution until final publication, they support early treatment initiation with remdesivir for sars-cov- infections. more than , adult patients, pediatric patients, and pregnant women with covid- were treated with remdesivir through the compassionate-use program according to the manufacturer gilead sciences. liver function test abnormalities were reported in of evaluated cases ( ) . once the covid- epidemic started in china, at a time when there was no clinical trial in preparation, the first observational data for remdesivir arose from patients treated under compassionate use. in a prospective cohort study funded by gilead sciences, patients with covid- were treated with remdesivir for a -day course ( mg on day , followed by mg daily). clinical improvement was observed in ( %) of evaluable patients ( ) . another study from italy reported on patients treated with remdesivir in a general infectious disease ward. interpretations of data from this study are very limited due to the low sample size, as only patients completed a -day treatment course. the most frequent adverse events (aes) were elevations of liver transaminase levels ( / patients) and acute kidney injury ( / patients) ( ) . as there were no control groups, no efficacy statements can be made based on these studies. one approach with a simulated control group is currently under peer review and suggests reductions in mortality with remdesivir ( ) . several clinical trials were conducted to evaluate its efficacy against evd and covid- . ebolavirus disease. two studies on remdesivir were conducted in the context of ebolavirus disease, prevail iv (clinicaltrials.gov identifier nct ) and the pamoja tulinde maisha (palm) trial (clinicaltrials.gov identifier nct ). prevail iv was a small phase study in men with evidence of ebolavirus persistence in their semen, who were formerly included in the observational evd survivor study prevail iii. however, no reliable safety or efficacy data on remdesivir were derived from this study. the first randomized controlled phase / clinical trial evaluating the efficacy of remdesivir (palm) started in in the democratic republic of congo during an outbreak of evd. within months, a total of patients were enrolled in the study assessing the efficacies of four different therapeutic strategies in an open-label parallel : : : design. patients received either remdesivir, the single monoclonal antibody mab , a coformulated composition of human igg monoclonal antibodies called regn-eb , or the triple monoclonal neutralizing antibody complex zmapp (control group). the primary endpoint was death at day of enrollment. an interim analysis on august that included data from patients showed that the mortality rates with both zmapp ( / ; . %) and remdesivir ( / ; . %) were higher than those with mab ( / ; . %) and regn-eb ( / ; . %). therefore, randomization into these groups was subsequently stopped. as this study did not include a placebo control arm, no definite conclusions on the clinical efficacy of remdesivir against evd can be made. however, the observed mortality rate of approximately % is comparable to that of the natural course of the disease and thus does not suggest a substantial clinical benefit of remdesivir ( ) . covid- . the first phase randomized, double-blind, placebo-controlled trial evaluating the efficacy of remdesivir against covid- (clinicaltrials.gov identifier nct ) started in february in wuhan. hospitalized patients with severe covid- were enrolled (defined as having hypoxia and radiological signs of lung involvement) and treated for days with standard doses of remdesivir ( mg on day and mg on days to ; n ϭ ) or a placebo (n ϭ ). due to the rapidly changing dynamics of the outbreak in china, with a local decrease in new includable cases during march , the trial was stopped preterm with only patients enrolled and could not reach the calculated target enrollment size. in the final analysis of the present data set, treatment with remdesivir was not associated with significant clinical improvement in the treatment arm compared to the placebo (hazard ratio [hr], . [ % confidence interval {ci}, . to . ]). furthermore, there was no significant difference between groups regarding mortality and time to viral clearance. in a subgroup of patients who were treated early within days of symptom onset, remdesivir was associated with a numerical median reduction of days in the time to clinical improvement, but this finding was not statistically significant (hr, . [ % ci, . to . ]). those authors therefore proposed to evaluate remdesivir earlier in the course of covid- ( ) . the adaptive covid- treatment trial (actt) started at the end of february and included a total of study sites globally. a total of , hospitalized patients with all stages of covid- that included signs of lower respiratory tract involvement (hypoxia or radiological evidence) were enrolled until april . patients received either standard doses of remdesivir (n ϭ ) or a placebo (n ϭ ) for days in a double-blind design. the primary outcome was time to recovery, defined as discharge from the hospital or continued hospitalization for infection control purposes only (no further medical treatment needed) ( ). on april , the data and safety monitoring board (dsmb) concluded that there was a significant effect of remdesivir after reviewing an interim data analysis. based on the available data, the dsmb recommended enabling patients of the placebo arm to benefit from a switch to remdesivir, which required early unblinding for a limited number of patients ( ) . the preliminary data have recently been published and show that treatment with remdesivir was associated with a reduction in the time to recovery from a median of to days (recovery rate ratio [rrr], . [ % ci, . to . ] [p Ͻ . ]). this effect was independent of symptom duration prior to randomization. but subgroup analyses showed that patients with the need for oxygen therapy (ordinal score of ) benefit most from the treatment (rrr, . [ % ci, . to . ] [n ϭ ]), while no effect could be demonstrated for patients on invasive ventilation and/or extracorporeal membrane oxygenation (ecmo). the mortality rate by days was . % (remdesivir) versus . % (placebo), which was not statistically significant (hr, . [ % ci, . to . ] [p ϭ . ]) ( ) . based on the preliminary results of this trial, the fda issued an emergency-use authorization for remdesivir only days after the initial press release from the niaid. while complete results have not yet been published, another clinical trial was conducted, which evaluated the optimal treatment duration with remdesivir. the randomized open-label trial gs-us- - (previously simple) started in march and evaluated the efficacy of a -day treatment regimen (n ϭ ) versus a -day regimen (n ϭ ) of remdesivir in severe covid- . the results were recently published and suggest similar effects of -day and -day treatments when adjusting for baseline clinical status. clinical improvement, clinical recovery, and mortality by day were assessed. due to the absence of a control group, these results do not permit an overall assessment of the efficacy of remdesivir ( ) . in addition to the study of severe covid- cases, another open-label trial in patients with moderate covid- is ongoing (table ) . gilead sciences, the manufacturer of remdesivir, conducted four phase clinical trials to evaluate the safety, tolerability, and pharmacokinetics of remdesivir (gs-us- - , - , - , and - ) in a total of patients, of whom received remdesivir and received a placebo. overall, the drug is generally well tolerated. adverse events (pooled data) occurred in only a few cases and included phlebitis ( subjects), constipation ( ), headache ( ), ecchymosis ( ), nausea ( ) , and pain in extremities ( ) . only a few grade and laboratory abnormalities were detected: transient elevations of alanine aminotransferase (alt)/aspartate aminotransferase (ast) levels from day until day ( subjects), mild reversible prolongation of the prothrombin time without changes in international normalized ratio (inr) ( subjects), and mild hyperglycemia ( subjects). there were no signs of nephrotoxicity in healthy subjects and no patterns of clinically relevant changes in vital signs or electrocardiograms ( ) . the available safety data from phase / studies are provided in a fact sheet for health care providers that was published in the context of the emergency-use authorization issued by the fda and can be downloaded ( ) . in the first phase trial of remdesivir that was conducted in the context of evd, one event of hypotension occurred in the remdesivir arm that was judged as not being related to underlying evd by the site investigators. the event occurred during the administration of the loading dose and led to a fatal cardiac arrest. an independent pharmacovigilance committee concluded that the death could not be readily distinguished from underlying fulminant evd ( ) . safety data from the compassionate-use study are not conclusive as there was no control group. however, the most frequently reported adverse events in patients treated with remdesivir were increases in hepatic enzyme levels ( patients; %) and diarrhea ( patients; %) ( ) . in the chinese phase trial where patients with covd- received remdesivir, no deaths occurred that were judged as being possibly related or related to the study drug. the frequencies of adverse events (most frequently constipation, hypoalbuminemia, hypokalemia, and anemia) were virtually identical in the treatment and placebo groups ( ) . preliminary data from the actt study do not change this picture. the incidences of most adverse events were not found to be significantly different among the treatment and placebo groups. grade to adverse events in general and some adverse events such as anemia or increased transaminase levels occurred slightly more often in the placebo group (grade to aes in versus with remdesivir). other adverse events occurred slightly more often in the remdesivir group (increased creatinine levels, pyrexia, and hyperglycemia) ( ) . in the openlabel trial on patients with severe covid- , the most common adverse events were nausea ( %), worsening of respiratory failure ( %), elevated ast levels ( %), and constipation ( %) ( ) . taken together, at this time, there is no evidence for grade to or even severe adverse events resulting from once-daily doses of remdesivir ( mg up to mg i.v.) for treatment durations of up to days. the drug seems to be well tolerated. grade and adverse events have been described in healthy volunteers and patients with covid- treated with remdesivir and mainly refer to transient elevations of alt or ast levels. there are not sufficient data on the safety of remdesivir in patients younger than nucleoside analogs require active cellular uptake by nucleoside transporters and intracellular activation by cellular and viral kinases to become their active ntp metabolite. this activation process requires three phosphorylation steps, of which the first step is most often inefficient and rate limiting ( ) . a common problem of nucleoside analogs is that they yield suboptimal levels of ntp at the site of infection ( ) . remdesivir is a monophosphoramidate prodrug of a =-cyano-substituted adenosine nucleoside analog that is able to bypass the rate-limiting first phosphorylation step to effectively deliver intracellular ntp ( ) . the prodrug component is necessary to mask the negatively charged phosphonate group, which allows faster entry into target cells independently of membrane transporters. phosphonate-containing pronucleotides have the disadvantage that their diacids are deprotonated at a physiological ph ( ) . remdesivir has suboptimal oral bioavailability and therefore can be administered only by intravenous infusion in the actual formulation. however, there might be pharmacological approaches to solve this problem. one example of a clinically approved nucleoside phosphonate with an oral formulation is the nucleoside reverse transcriptase inhibitor (nrti) tenofovir ( ) , which is one of the drugs most frequently used for therapy of hiv infections. the prodrug remdesivir (gs- ) has a relatively short systemic half-life (ϳ . h) and is rapidly converted intracellularly into several intermediate metabolites (gs- and gs- ) before being converted into the more stable and active tp metabolite (gs- ) ( , ) . plasma concentrations of remdesivir that are reached by the administration of therapeutic doses are several times higher than the concentrations required to inhibit sars-cov- replication in vitro ( , , ) . a dose of mg remdesivir yields a maximum concentration of drug in serum (c max ) of . m (area under the concentration-time curve [auc] of . m), and subsequent dosing of mg daily reaches a c max of . m (auc of . m) on day ( ) . data on tissue distributions are available from studies with cynomolgus monkeys, where remdesivir and its metabolites were detectable in testes, eyes, and brain h after a -mg/kg dose, which is comparable to mg in humans. unfortunately, no data on pulmonary drug delivery were reported in this publication ( ) . distribution into lung tissues was recently studied in six rhesus macaques, where the intermediate metabolite gs- , which was used as a surrogate for tissue loading, could be detected in all samples h after injection of remdesivir. the intermediate metabolite gs- was not detectable in lung tissue samples ( ) . it remains unclear how plasma concentrations of remdesivir and its metabolites correlate with pulmonary drug delivery in humans, and there are speculations of suboptimal exposure in respiratory target cells of sars-cov- ( ). thus, therapeutic strategies that improve pulmonary drug exposure might be helpful to further improve the clinical efficacy of remdesivir. remdesivir is administered by intravenous infusion over to min. the standard dose for adults and pediatric patients weighing kg and higher is a loading dose of mg followed by once-daily doses of mg. dose adjustments are necessary for pediatric patients weighing less than kg. it is not known if dose adjustments based on kidney or liver function are necessary. administration in patients with a glomerular filtration rate (gfr) below ml/min is not recommended based on the potential accumulation of sulfobutylether-␤-cyclodextrin sodium salt present in both formula-tions of remdesivir ( ) . the optimal treatment duration for covid- is still unknown. in phase trials, a treatment course of or days was investigated. based on these data, the actual recommendations in the context of emergency authorizations are days for patients who do not require mechanical ventilation, which can be extended up to days if patients do not demonstrate clinical improvement. for patients on mechanical ventilation, the actual recommended treatment duration is days ( ) . the in vitro activity of remdesivir against ebov has been demonstrated in various cell-based models and for many different ebov strains ( , , ) . in addition, it showed therapeutic and prophylactic effects in a rhesus monkey model of lethal evd ( ) . however, in the palm study, remdesivir was less efficacious than other investigational drugs. as this study did not include a placebo control group, no definite conclusions on the clinical efficacy of remdesivir in evd can be made. the mortality rate among patients treated with remdesivir was approximately %, which was similar to that of the control group treated with the triple monoclonal antibody zmapp but significantly higher than those with mab ( . %) and regn-eb ( . %) ( ) . in a previous trial on evd, the mortality rates were % among zmapp-treated patients and about % among patients who received the standard of care only ( ) . the reason for these differences in mortality rates is unclear and may result from differences in the virulence of ebov, sample size, patient population, or standard-of-care practices. however, a final interpretation of the clinical effects of remdesivir remains elusive, and based on the results of the palm trial, it is unlikely that remdesivir will be clinically reevaluated for the treatment of evd. remdesivir is active in vitro against various covs, including sars-cov- ( , , , ) , and its mechanism of action has been studied extensively. animal studies that included nonhuman primate models of mers-cov and, recently, sars-cov- support its efficacy, especially when administered early in the course of the disease ( , ) . finally, a phase trial showed beneficial clinical effects of remdesivir in patients who require supplemental oxygen, while clinical efficacy for critically ill patients who require mechanical ventilation could not be demonstrated ( ) . remdesivir reduces the time to recovery by %, which is a relatively modest but clearly therapeutic effect ( ) . besides these beneficial effects on patients, this may help to reduce the number of inpatient days, with positive effects on hospital costs and capacity issues that have emerged during the covid- pandemic in several countries. with regard to mortality, a lower -day mortality rate of patients treated with remdesivir was reported from the actt study, which may indicate a beneficial but not statistically significant effect. taking into account that the study was not powered to evaluate mortality, this is still a positive signal that must be further evaluated in large-scale studies. a meta-analysis with pooled data from the two available rcts that is still under peer review concludes a statistically significant reduction in mortality (risk ratio [rr], . [ % ci, . to . ]) ( ) . although the clinical data on remdesivir are not fully published yet, the emergency authorizations and recent approval by the ema are encouraging, as most other investigational drugs have failed until now ( , ) , leaving remdesivir the only antiviral with clinically proven efficacy against covid- to date. complete results of the actt study, including impacts on viral loads and mortality by day , as well as results from ongoing trials and meta-analyses will provide more information on the clinical efficacy of remdesivir. after decades of research on direct-acting antiviral drugs, remdesivir is the first nucleoside analog that can be used to treat infections caused by a respiratory virus. in the light of its beneficial clinical effects, its favorable safety profile, and the absence of alternatives to treat covid- , remdesivir will increasingly be used outside the context of clinical trials or compassionate-use programs. the drug is already available in the united states and japan based on emergency-use authorizations and was recently approved in europe. however, treatment with the antiviral drug remdesivir alone will not be sufficient to reliably save the lives of patients suffering from covid- or to solve the hazardous public health issues caused by the ongoing covid- pandemic. antiviral therapy in hospitalized patients cannot prevent the virus from being transmitted among communities and cannot reverse pathophysiological processes that have occurred already at the time of diagnosis. in general, prophylactic measures would be much more efficient in reducing covid- -associated morbidity and mortality as well as economic implications ( ) . the prophylactic use of remdesivir might be effective, as it completely protected exposed macaques from mers-cov-induced clinical disease ( ) . prophylactic effects are also known from other virostatic-acting drugs like neuraminidase inhibitors that may prevent influenza virus infections and can also be used as postexposition prophylaxis ( ) . however, the prophylactic use of remdesivir is generally hampered by its poor oral bioavailability and the absence of an oral formulation. further pharmacological efforts are needed to make the drug accessible to an outpatient population. recently, the manufacturer announced in an open letter that a phase trial with remdesivir inhalation is being planned and already accepted by the fda ( ) . interestingly, another sars-cov- active nucleoside analog called eidd- , which is orally bioavailable, is currently in preclinical evaluation ( ) . finally, it should be mentioned that drug pricing will also have significant implications for the possibility of applying remdesivir with a broader scope. the therapeutic efficacy of remdesivir might be improved by the addition of other antivirals or immunomodulatory agents. it has recently been shown that glucocorticoids are able to improve clinical outcomes in cases of severe and critical covid- ( ) . based on these data, it can be expected that physicians will use both remdesivir and glucocorticoids to treat patients with severe or critical covid- . however, combination therapy should be used with caution, as drug interactions may occur. in vitro, remdesivir acts as the substrate or inhibitor of several drug-metabolizing enzymes (e.g., cyp a ), which could influence the exposure levels of other therapeutic agents. in addition, these agents may interfere with the pharmacokinetics of remdesivir. the immunomodulatory drug hydroxychloroquine, for example, seems to reduce the antiviral activity of remdesivir by impairing its intracellular metabolic activation ( ) . another approach that may improve clinical outcomes could be combination therapy with direct antiviral drugs that target several processes within the viral life cycle. although this strategy is highly effective in the therapy of chronic infections with hiv and hcv, it is unclear if this is true for acute infections with sars-cov- . clinical 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paramyxoviruses aryl phosphoramidate derivatives of d t have improved anti-hiv efficacy in tissue culture and may act by the generation of a novel intracellular metabolite application of phosphoramidate protide technology significantly improves antiviral potency of carbocyclic adenosine derivatives therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease palm consortium study team. . a randomized, controlled trial of ebola virus disease therapeutics comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir delayed chain termination protects the anti-hepatitis b virus drug entecavir from excision by hiv- reverse transcriptase presteady-state kinetic studies establish entecavir =-triphosphate as a substrate for hiv- reverse transcriptase in vitro inhibition of hepadnavirus polymerases by the triphosphates of bms- and lobucavir identification of bms- as a potent and selective inhibitor of hepatitis b virus structure of the rna-dependent rna polymerase from covid- virus structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors remdesivir is a direct-acting antiviral that inhibits rnadependent rna polymerase from severe acute respiratory syndrome coronavirus with high potency testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs mouse hepatitis virus (mhv- ). plaque assay and propagation in mouse cell line dbt cells cell-based assays to identify inhibitors of viral disease sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor isolation, sequence, infectivity, and replication kinetics of severe acute respiratory syndrome coronavirus enhanced isolation of sars-cov- by tmprss -expressing cells exogenous ace expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication sars-associated coronavirus replication in cell lines broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro fact sheet for health care providers: emergency use authorization (eua) of veklury (remdesivir)-revision . us food and drug administration the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites species difference of esterase expression and hydrolase activity in plasma a mouse model for mers coronavirus-induced acute respiratory distress syndrome clinical benefit of remdesivir in rhesus macaques infected with sars-cov- remdesivir: review of pharmacology, pre-clinical data and emerging clinical experience for covid- fact sheet for health care providers: emergency use authorization (eua) of veklury (remdesivir) compassionate use of remdesivir for patients with severe covid- icu) and non-icu patients: clinical outcome and differences in post-treatment hospitalisation status efficacy of remdesivir in covid- patients with a simulated two-arm controlled study a randomized, controlled trial of zmapp for ebola virus infection remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial the national institute of allergy and infectious diseases decision to stop the adaptive covid- trial: on solid ethical and scientific grounds remdesivir for or days in patients with severe covid- remdesivir (gs- ) investigator's brochure version . advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases inhibition of viral rna polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand rna viruses beyond hepatitis c virus the mechanism of action of ␤-d- =-deoxy- =-fluoro- =-c-methylcytidine involves a second metabolic pathway leading to ␤-d- =-deoxy- =-fluoro- =-c-methyluridine =-triphosphate, a potent inhibitor of the hepatitis c virus rna-dependent rna polymerase medicinal chemistry of nucleoside phosphonate prodrugs for antiviral therapy safety, tolerability, and pharmacokinetics of remdesivir, an antiviral for treatment of covid- , in healthy subjects remdesivir for treatment of covid- : combination of pulmonary and iv administration may offer aditional [sic] benefit clinical benefit of remdesivir in rhesus macaques infected with sars-cov- remdesivir use in patients with coronavirus covid- disease: a systematic review and meta-analysis a trial of lopinavir-ritonavir in adults hospitalized with severe covid- effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial neuraminidase inhibitors for preventing and treating influenza in healthy adults and children an open letter from daniel o'day, chairman & ceo an orally bioavailable broad-spectrum antiviral inhibits sars-cov- in human airway epithelial cell cultures and multiple coronaviruses in mice dexamethasone in hospitalized patients with covid- -preliminary report this research received no specific financial support. j.j.m. and i.s. receive funding from german center for infection research (dzif) stipends ti . _malin_ and ti . _suarez_ . j.r. receives funding from the dzif thematic translational unit tuberculosis (ttu tb) (grant numbers ttu . and . ) and the german research foundation (dfg ry ). the funders had no role in data collection, interpretation, or the decision to submit the work for publication.we acknowledge patrick lane, sceyence studios, for graphical enhancement of the presented figures.g.f. has served as an advisor to gilead sciences and has conducted clinical research supported by gilead sciences. j.j.m., v.p., i.s., and j.r. declare no potential conflicts of interest.all authors critically discussed the present study results and their clinical significance. j.j.m. wrote the original draft and was responsible for conceptualization, investigation, and visualization. i.s. and v.p. were involved in reviewing and editing the jakob j. malin (m.d.) obtained an m.sc. in medicine at maastricht university and a doctoral degree in the field of viral infectious diseases at the university of bonn. he works as a medical doctor and clinician-scientist at the university hospital of cologne, completing a clinical residency in internal medicine and infectious diseases. he is involved in coordinating and conducting clinical trials on anti-infectives, including investigational drugs against covid- . moreover, he contributes to the development of clinical guidelines on covid- pharmacotherapy released by the german society of infectious diseases (dgi). dr. malin has a strong translational research focus on antimicrobial drug discovery. since , he has been working on antimicrobial compounds against several pathogens, including opportunistic mycobacteria. he is affiliated with the translational research unit for infectious diseases (tru-id) at the center for molecular medicine cologne (cmmc), where he works as a postdoctoral researcher and is currently expanding his translational working field for sars-cov- . key: cord- - p n tjx authors: lambe, teresa; bowyer, georgina; ewer, katie j title: a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines? date: - - journal: philos trans r soc lond b biol sci doi: . /rstb. . sha: doc_id: cord_uid: p n tjx sporadic outbreaks of ebola virus infection have been documented since the mid-seventies and viral exposure can lead to lethal haemorrhagic fever with case fatalities as high as %. there is now a comprehensive body of data from both ongoing and completed clinical trials assessing various vaccine strategies, which were rapidly advanced through clinical trials in response to the – ebola virus disease (evd) public health emergency. careful consideration of immunogenicity post vaccination is essential but has been somewhat stifled because of the wide array of immunological assays and outputs that have been used in the numerous clinical trials. we discuss here the different aspects of the immune assays currently used in the phase i clinical trials for ebola virus vaccines, and draw comparisons across the immune outputs where possible; various trials have examined both cellular and humoral immunity in european and african cohorts. assessment of the safety data, the immunological outputs and the ease of field deployment for the various vaccine modalities will help both the scientific community and policy-makers prioritize and potentially license vaccine candidates. if this can be achieved, the next outbreak of ebola virus, or other emerging pathogen, can be more readily contained and will not have such widespread and devastating consequences. this article is part of the themed issue ‘the – west african ebola epidemic: data, decision-making and disease control’. the - epidemic of ebola virus disease (evd), in west africa, was unprecedented in both scale and spread. the case count and death toll has surpassed the total number of cases for all previous known outbreaks. this epidemic has caused more than deaths with more than cases: figures widely thought to be underestimated [ ] . development and deployment of efficacious evd therapeutics and vaccines could have played a more significant role in limiting the - outbreak and remain a priority for the prevention of future evd epidemics [ ] . the family filoviridae includes marburg and five ebolaviruses, named for their location of recognition: zaire (now known as ebola virus, ebov), sudan (sudv), reston (restv), taï forest (tafv) and bundibugyo virus (bdbv) [ ] . the single protein expressed on the surface of filoviruses, the glycoprotein (gp), is antigenically and immunologically important and is frequently therapeutically targeted. however, bioinformatics analysis, at the structure-based and amino acid level, demarcates limited regions of homology in gp [ ] and also large areas of divergence particularly in the head domain of gp. it is therefore unclear if exposure to one member of the family filoviridae confers cross-protective immunity. it is unclear if the immune response involved in ebola virus clearance is dependent on a single aspect of the immune system (cellular or humoral immunity) or if a multifaceted response is a prerequisite and at present there is no clear correlate of protection for evd. indications from experimental infection of vaccinated non-human primates suggest a role for gp-specific igg and cd þ t cells [ , ] , and the limited analyses of convalescent samples suggest that the presence of ebola virus-specific igg and intact cell-mediated immunity (cmi) are associated with survival from natural infection [ ] [ ] [ ] . these observations are supported by immunological analysis of survivors from the current outbreak, which indicates a multi-faceted response is induced post-infection with strong humoral immunity and significantly pronounced transcriptional changes in cd þ t cells in response to several ebola virus proteins [ ] . evd vaccine development has been well established since the discovery of ebola virus and a number of different vaccine platforms have been used, resulting in at least one efficacious vaccine. these vaccine platforms include dna, recombinant or subunit proteins, virus-like particles (vlps) and recombinant viral vectors. a number of these vaccines have advanced past pre-clinical testing and are undergoing clinical testing; generally, those modalities that have demonstrated efficacy or high levels of immunogenicity in pre-clinical models. the most clinically advanced vaccines against evd are based on generating immune responses toward gp with trials ongoing in europe, the usa and africa. there are eight vaccines in clinical trials, all targeting the ebola virus gp, with some regimens employing a single dose and others using two vaccines in a heterologous prime-boost approach. prime-boost regimens have been shown to be more immunogenic than single-dose vaccinations for diseases such as malaria; however, there are financial and logistical implications associated with administering two vaccines [ ] . the ebola virus vaccines that are currently being assessed differ in the qualitative immune response postvaccination, which may be due to the use of alternate vaccine platforms. indeed, while humoral immunity may be critical for protection post vesicular stomatitis virus (vsv)-vectored vaccines in non-human primates (nhps), cmi may be key post single-shot adenovirus vectored vaccines [ ] . as both humoral and cellular immunity have been demonstrated to be protective in nhp, and are induced in man post-ebov infection, a vaccine regimen which induces long-lived and sustainable levels of cmi and antibodies is desirable. primarily, and finally, only those vaccines that are safe, efficacious and deployable will be field-effective, therefore only vaccine candidates that have progressed to published phase i studies will be reviewed here. clinical trials where results were published in peer-reviewed journals by july are summarized in table . ebolavirus disease the earliest clinical studies of vaccines against evd used plasmid dna encoding the nucleoprotein from the zaire ebolavirus and the glycoproteins from ebov and sudv [ , ] . although well tolerated with mostly mild and short-lived adverse events (aes), dna vaccines tend to be poorly immunogenic and have been largely superceded by subunit proteins, recombinant viral vectors and vlps in several vaccine development fields, including ebola virus [ ] . the vaccines most progressed for the prevention of evd use viral vector platform technology and use either the adenovirus, modified vaccinia virus ankara or vesicular stomatitis virus backbones. recombinant human serotype adenovirus (adhu ) vectors encoding gp have been evaluated in single-dose studies and have shown very acceptable safety profiles. unfortunately, adhu -seropositive volunteers demonstrated statistically significantly lower gp-specific igg titres [ ] . increasing the administered dose of vaccine appears to overcome pre-existing immunity to this serotype; however, this is associated with increased reactogenicity [ ] . unfounded concerns also still persist about a potential increase in hiv- infection rates among adhu -seropositive vaccinees, based on data from two phase ii studies of the merck rad hiv- gag/pol/nef vaccine (the step and phambili trials) [ ] [ ] [ ] . use of rarer serotypes of human adenoviruses, such as ad and adhu , can bypass pre-existing immunity [ ] and have demonstrated acceptable safety and tolerability profiles. the most widely evaluated adenoviral vector evd vaccines are based on a simian adenovirus serotype backbone (chad ) encoding either ebolavirus glycoprotein alone [ , ] and administered as a single shot or as a mixture of chad ebov and chad sudv vaccines [ ] . between september and january , four phase i studies of these vaccines were undertaken, one each in the us, uk, switzerland and mali. replication-deficient chimpanzee adenovirus vectors have been extensively evaluated as candidate vaccines for malaria, hiv, hcv, rsv, influenza and tuberculosis, in adults, children and infants, with no safety concerns [ ] . safety profiles were similar for candidate ebola virus vaccines, with higher doses eliciting more aes; however, aes were mostly graded as mild, resolving within h of vaccination. an additional vaccine modality being tested is modified vaccinia ankara (mva); both unmodified and recombinant mva vectored vaccines have been evaluated extensively as vectors for smallpox, malaria, flu and tb vaccines with excellent safety demonstrated in immunocompetent and hiv-infected participants [ , ] . in pre-clinical trials, chad was observed to provide % protection when nhps were challenged with ebola virus shortly after vaccination; however, an mva boost was required to enable uniform protection when challenge occurred months after priming vaccination. this increased durability of protection was attributed to the superior effector and memory cd þ t cell responses elicited by the mva boost [ ] . owing to the superior durability of protection demonstrated by the prime-boost regimen in preclinical trials, several phase i clinical trials were initiated to test this regimen in humans. mva-bn filo is a recombinant, replication-deficient mva vector, encoding ebov, sudv and marburg glycoproteins as well as tafv nucleoprotein. two clinical trials have assessed a chad prime with mva-bn filo boost, demonstrating acceptable safety profiles and significantly enhanced cellular and humoral immunogenicity compared with a single-shot chad vaccination [ , ] . the other vaccine modality being assessed as an ebola vaccine candidate is the recombinant vesicular stomatitis virus encoding ebov glycoprotein (rvsv-zebov), which is an attenuated replication-competent viral vector. unmodified vsv causes either asymptomatic infection or a mild, flu-like illness in humans [ ] ; however, truncation of the vsv glycoprotein results in an attenuated, avirulent form that has been shown to be safe in pre-clinical studies in macaques and mice with no evidence of viral shedding [ ] . a rvsv hiv- gag vaccine has also generated acceptable safety data in two phase i studies [ , ] . candidate vaccines using these vectors also elicited protection against both ebola and marburg viruses in a nhp model where several rvsv each encoding a single filovirus glycoprotein were mixed and injected in a single dose that elicited protection against a lethal challenge [ ] . six phase i studies of rvsv-zebov were initially performed between october and january , two in the us and one each in kenya, gabon, switzerland and germany [ , ] . almost all participants displayed transient rvsv viraemia, starting at day , and lasting beyond day in around % of participants, with mostly mild and moderate aes lasting up to h on average. in total, % ( / ) of participants across the six trials had documented fever after vaccination, significantly higher than the % observed after vaccination with chad ( / , p ¼ . , two-tailed fisher's exact (prism version , graphpad software)). although both vaccines have been evaluated across a range of doses, post-immunization fever levels are an important consideration for a vaccine that may be deployed in an outbreak of a highly febrile illness, such as evd. in the geneva trial, of participants developed arthritis lasting a median of days, with virus subsequently identified in synovial joint fluid, indicating peripheral viral replication; two cases of shorter duration were identified among participants in the other studies. a further study in geneva where a much lower dose of rvsv-zebov was evaluated in a further volunteers resulted in much lower total igg and neutralizing antibody titres and was again associated with arthritis in % of participants [ ] . transient arthritis is often observed after immunization with commonly used live vaccines, such as rubella [ ] ; however, when rvsv-zebov progressed to a large-scale phase iii cluster-randomized efficacy trial, this particular adverse event was not reported and, in addition, in an interim analysis the vaccine elicited high levels of efficacy in evd-exposed participants [ ] . incidence of post-vaccination fever in that study has not yet been reported. in summary, all of the candidate vaccines for evd that progressed to phase i studies in response to the - outbreak demonstrated acceptable safety profiles; therefore, describing the magnitude and characteristics of vaccine-induced immunity was the next scientific aim. in the absence of any gold standard for measuring humoral immunity against ebola virus, a wide variety of assays were used to evaluate antibody responses to vaccines during the recent evd outbreak. binding assays were used to quantify antibody binding to recombinant protein or inactivated whole virion while neutralization assays were used to assess the functional capacity of antibodies. binding assays, particularly enzyme-linked immunosorbent assays (elisas), are commonly used to assess the quantity of antigen-specific antibody induced post-vaccination and can additionally be used to measure qualitative aspects of the humoral immune response such as isotype profiles and in vitro avidity. ebolavirus outbreaks occur sporadically and have previously been considered too limited in size to enable assessment of vaccine efficacy prior to regulatory approval. in , the usa food and drug administration (fda) introduced the 'animal rule' enabling data from pre-clinical trials to be used to demonstrate efficacy when human trials were not possible as an alternative licensing route for drugs and vaccines against highly lethal diseases [ ] . despite the wide use of elisa assays to measure antiglycoprotein igg responses, the majority of phase i studies used an array of different antibody binding assays to assess the quantity and quality of the antigen-specific antibody response induced by vaccination (table ). the use of different assays conducted in different laboratories following different protocols hinders the comparison of humoral immunogenicity induced by different vaccine candidates. key differences in these assays that affect comparability of results include the use of glycoprotein from different strains of zaire ebola virus (kikwit, makona or mayinga), use of whole virion or recombinant protein and the use of different read-outs or units. centralized standard assays or readily available biological standards for a range of emerging pathogens would be useful tools to aid decision makers in choosing the most promising candidate to take forward for further testing and rapid deployment during outbreaks. a who reference reagent for anti-ebov igg has been established by the who expert committee on biological standardisation (ecbs) for use as a reference standard in humoral immunoassays including neutralization and elisas against ebola virus [ ] . it is now freely available from the national institute for biological standards and control (nibsc, uk) and should facilitate retrospective comparison of responses between different trials. to aid in the comparison of immunogenicity across trials, samples from a recent phase i trial of chad mva-bn filo [ ] were run on a large number of these assays and correlation analyses were conducted (table ) . a lack of correlation between some of these assays may indicate that they measure different aspects of the humoral immune response and may therefore correlate differently with protection. for example, the competition elisa used by ewer and co-workers did not correlate with neutralizing titre to live ebola virus mayinga strain or several of the glycoprotein elisas. the competition elisa measures the ability of anti-ebov antibodies in a sample to compete with the monoclonal ebov antibody (mab) g and therefore only detects activity against a single gp epitope. the lack of a correlation with neutralizing titre may indicate that antibodies binding to this epitope are non-neutralizing and that the protection that has been observed when this mab has been used in a mab cocktail in nhp models is conferred by an alternative mechanism [ ] . many of the other binding assays used in these phase i trials correlate strongly and, in particular, it is useful that the standardized glycoprotein elisa and pseudotyped lentivirus assays each correlate strongly with neutralizing titres against live ebola virus as these assays avoid the need to work at high containment levels, but may be used to indicate the presence of antibodies with neutralizing activity. in a pre-clinical nhp model, igg responses after immunization with adhu -based ebola virus vaccines were measured using an elisa against gp, where % protection against a lethal challenge was predicted by titres of or greater, while a titre of around predicted % survival. at an interim analysis of the phase iii ring vaccination study of rvsv-zebov in guinea estimated vaccine efficacy in this trial to be % [ ] . no immunogenicity data were published for this trial, due to logistical difficulties associated with collecting biological samples, therefore, it is unclear which components of the immune response to vaccination mediated this protective effect. however, phase i trials of the same vaccine at the same dose, in the usa, induced igg against zaire-mayinga gp with a geometric mean titre (gmt) of ( % ci: - ) [ ] at day post-vaccination, which is probably higher than the level achieved - days post-vaccination by which time no new cases were observed in the rvsv-zebov ring vaccination trial. a gmt of is well below the titre required to protect % of non-human primates in the experimental model. chad zebov given at a dose of  viral particles (vp) induced titres comparable to those induced by rvsv-zebov, with day gmts of ( % ci: - ) in us adults and ( - ) in malian adults [ ] . peak titres after chad is boosted with mva zebov were significantly increased to ( - ) [ ] . although the use of different assays (summarized in table ) limits comparison of immunogenicity across clinical trials, there is still much to be learnt from comparisons of groups within trials. desantis and co-workers showed a limited dose effect on anti-gp igg titres, as assessed through elisa, when chad ebov was tested at .  vp and  vp. peak titres and response rates were similar in these two groups both at peak and at day , indicating that durability was also comparable [ ] . however, in a trial by ledgerwood et. al., which compared a mixture of chad zebov and chad sudv in a : ratio, each at  or  vp, indicated that a -fold higher dose induced significantly higher geometric mean elisa titres of anti-gp igg despite similar response rates [ ] . in the same trial, % of volunteers in the high dose group developed antibody responses against the vaccine strain of zaire glycoprotein (mayinga), while only % developed responses against glycoprotein from the outbreak strain (guinea). additionally, the response rates induced against the vaccine strain sudan glycoprotein ( % in the high dose group and % in the low dose group) were lower than for zaire ( and %) despite receiving the same dose of each. this may reflect the different sensitivities of the two assays. the rapid development of multiple vaccine candidates in parallel during the recent outbreak has highlighted the need for standardization of assays that measure humoral immunogenicity. correlating responses measured by different assays and the inclusion of reference standards can aid comparisons of the quality and quantity of the humoral response induced by different vaccine candidates and support decisions on which to take forward for further development. there is a lack of discrimination between neutralizing and non-neutralizing antibody levels through elisa output and while non-neutralizing antibodies play an important role in curbing infection, there is also a concerted effort to identify neutralizing antibodies (nab) as a key determinant of infection control in many disease settings [ , ] . at present, there are data suggesting that both neutralizing and non-neutralizing antibodies are important in the evd setting and as such measuring both aspects of humoral immunity through elisa and neutralization assays will be informative. virus neutralization assays (e.g. fluorescent antibody virus neutralization (favn) assay or plaque reduction neutralization test (prnt)) can measure nab responses. however, there is a prerequisite for highly trained staff and access to containment level facilities to work with ebolavirus, which are not widely available: for example, a single laboratory based in marburg analysed samples from most of the phase i studies. additionally, these assays necessitate careful and considered planning resulting in low-throughput, expensive and timeconsuming assays to prevent accidental exposure [ , ] . to circumvent these restrictions, assays using non-pathogenic, replication-defective pseudotyped viruses (pvs) have been developed that facilitate the study of highly pathogenic viruses in standard containment level laboratories. pvs are chimeric virions, which comprise the structural and enzymatic core of one virus with a heterologous envelope protein, e.g. ebov table . relationships between different assays used to assess humoral responses to ebola vaccine candidates. spearman's rank and p-values for correlations between each of the assays tested. the same samples were run on each assay. samples were serum from healthy uk volunteers in a phase i trial of chad _mva ebo z conducted at the university of oxford [ ] . all samples were from the same time point-two weeks after mva boost. adi elisa, pseudotyped lentivirus neutralization assay and standardized elisa were carried out at the university of oxford, competition elisa at phe, neutralization assay and whole virion elisa at institute for virology, philipps university, marburg, and the nih elisa at the nih. glycoproteins [ ] . typically, retroviruses (lentiviruses and gammaretroviruses) or rhabdoviruses (vesicular stomatitis virus) are used as pseudotype cores. transduction of permissive target cells, facilitating genome transfer and subsequently reporter protein expression, allows a quantifiable read-out of transduction and produces a quantitative read-out. however, pre-incubation of pv with nab which bind an envelope protein, and can inhibit cell entry, will result in a lower level of quantifiable reporter protein expression. there are many pv assays which have been developed to assess neutralizing antibodies toward the filovirus family members and indeed a number of these assays have been used in a who collaborative study to establish a reference standard for antibodies to ebola virus [ ] . the data from this study suggest that pv assays, while offering an obvious improvement to live nab assays, may generate false positives and as such any new filovirus pv assay requires stringent standardization using validated negative and positive controls. there have been a limited number of pv assays used in recent clinical trials, which may reflect the urgent need to rank vaccine modalities during the - ebola outbreak and the concerted effort to screen using live neutralization assays. however, there are a number of informative comparisons between live ebov neutralization and pv neutralization during some of the more recent clinical trials which will help establish the usefulness and early application of these assays in screening and ranking vaccine candidates (table ) . in , neutralizing antibodies, as measured with a singleround infection assay with ebov gp-pseudotyped lentiviruses, were measured following a single im vaccination in a phase i clinical trial of adhu vaccine expressing ebov gp antigen. with the exception of one subject, sera did not inhibit virus entry [ ] . the same team also used ebov or marv gp-pseudotyped lentiviruses to measure neutralizing antibody activity after or dna vaccinations. no significant marburg virus neutralizing activity was observed following the dna vaccines, and only low-level ebov neutralizing activity was measured following the fourth dna vaccination [ ] . these data have helped evaluate vaccine regimens and have identified those vaccines and regimens which yield improved neutralizing responses. in a recent phase i clinical study in oxford, seven different assays of humoral immunity were undertaken on samples from vaccinees, providing a unique opportunity to assess the relationship between different measures of immunogenicity (table ) . volunteers were administered a single dose of chad ebov gp and a subset were then administered a booster dose of a mva strain, encoding the same ebola virus glycoprotein. two assays were used to measure neutralizing antibodies; direct neutralization of live ebov (mayinga strain) and a pseudotyped lentivirus expressing the glycoprotein from the mayinga strain, with a read-out of % inhibitory concentration (ic ). neutralizing antibody titres to live ebov (mayinga strain) were low at days after the chad dose while the levels were significantly higher days after the mva boost vaccine. again, low-level inhibition of infection was observed post-prime, using a pseudotyped lentivirus expressing the gp from the mayinga strain of ebov, and the levels of nab increased significantly post-boost. importantly, neutralizing antibody titres in the live ebov and pv assay correlated positively with each other (r ¼ . , p ¼ . ) as did titres measured by elisa and live ebov (mayinga strain) neutralization assay (r ¼ . , p ¼ . ) [ ] . nab were also assayed in vaccinees who received rvsv-zebov, through the use of vsv pseudovirions expressing the glycoprotein from the ebov kikwit strain, and a live neutralization assay with infectious ebov isolate mayinga. nab have been detected in vaccinees receiving as little as  pfu rvsv-zebov, and the two nab assays showed significant increases in neutralizing antibodies after all doses of rvsv-zebov (ranging from  pfu to  pfu) [ ] . a strong correlation between antibody titres as assessed with glycoprotein elisa and vsv pseudovirions or live virus neutralization titres were demonstrated. a significant correlation between vaccine dose and neutralizing antibody titres was also identified using the vsv pseudovirions but such correlations were not observed using whole virions or infectious ebola virus. this may reflect the lower sensitivity and thus weaker discriminatory capacity of whole virions or infectious ebola virus assays. the increased interest in measuring nab post vaccination toward highly pathogenic viruses and the suggested increased sensitivity in established and standardized pv assays will underpin the continued development and use of pv assays for broad-spectrum detection of post-vaccination humoral responses. baize and co-workers analysed samples from patients in two large outbreaks in in gabon and compared cellular and humoral responses between survivors and patients who succumbed to infection to determine the relative contribution of different immune parameters to evd outcome [ ] . survival was associated with early and rapid rises in igg directed mainly toward the viral nucleoprotein, in addition to activation of cd þ t cells with upregulation of cd , fasl, perforin and ifng. cd þ t cells specific to the ebola virus nucleoprotein are also associated with protection in a mouse model of evd [ ] and t cells induced by rad zebov gp vaccination in nhp afford protection in a lethal challenge model [ ] , while in vivo depletion of cd þ t cells using a monoclonal antibody abrogated protection in four out of five nhp, whereas passive transfer of igg did not induce protection. therefore, enumeration of antigen-specific t cell responses to candidate ebola vaccines is an important aspect of the immunological outcomes for studies in humans. many clinical trials of replicationdeficient viral vectored vaccines have reported t cell responses as measured by flow cytometry with intracellular cytokine staining (ics) using pools of peptides spanning the glycoprotein to stimulate peripheral blood mononuclear cells (pbmc). comparisons across studies are impeded by differences in sample types used (freshly isolated versus cryopreserved pbmc), variation in denominator used to report responses (memory versus total cd þ ) and differences in data analysis methodology [ , , , ] . some groups have also reported glycoproteinspecific t cell responses by ifng elispot in addition to ics; however, again differences in responses between fresh and frozen pbmc make broad comparisons difficult. although comparisons between all the trials are unreliable, limited comparisons are possible; e.g. when using fresh pbmc with ifng elispot, radhu and chad vectors encoding ebov gp induced antigen-specific t cell responses of a similar magnitude [ , ] . for the vaccine regimen where chad vectors encoding ebov and sudv gp were mixed, only the rstb.royalsocietypublishing.org phil. trans. r. soc. b : responder frequency and not the magnitude of response was reported; however, responder frequency to the mixture of vectors was similar to that observed to chad ebov gp alone. however, and possibly more importantly, comparisons between groups within the same study have yielded interesting insights into the use of viral vectors that have broader relevance beyond ebola virus vaccines. for example, a large trial in oxford comparing mva-bn filo priming and adhu zebov boosting demonstrated for the first time in humans that priming with the mva vector and boosting with an adenovirus is at least as effective at inducing antigen-specific cd þ t cells as the conventional delivery of ad-prime followed by mva boost [ ] . surprisingly, the rvsv-zebov vaccine, which to-date has been the only vaccine for which field efficacy has been assessed, has not yet been described for t cell-inducing capability [ , ] . in cynomologous macaques immunized with rvsv-zebov, cytokine-secretion from cd þ t cells stimulated with gp peptides was not detectable after immunization; however, responses were detected in vaccinated animals after ebov challenge, suggesting that immunization may have primed a response [ ] . if t cell responses to the rvsv-zebov have been assessed in phase i studies, then the absence of cmi would be of interest to the vaccine development field as maintenance of humoral immunity is obviously dependent on cmi, yet the failure to detect such a response peripherally is both surprising and interesting from the immunologist's perspective. as the immediate response to the - evd outbreak in west africa begins to wind down, scientists and policy makers involved in vaccine development can reflect on both the progress made by the field in this difficult time as well as implications for future outbreaks. notable gains and successes have been achieved, including rapid approval and completion of clinical studies for both existing and newly produced candidate evd vaccines which underpins the capacity of both ethical and regulatory authorities to expedite approvals in the context of public health emergencies. publication of data from studies undertaken in response to the outbreak has also been rapid with many journals setting aside policies relating to prior disclosure of data to allow data sharing with who and other bodies, without prejudice of future publication. that vaccines against evd existed at all is partly due to north american biodefense priorities, rather than a planned strategy of preparedness for rapid response to emerging pathogens. the efficacy of the rvsv-zebov vaccine in the phase iii trial in guinea in the context of a primarily iggmediated response suggests that an efficacious evd vaccine may be comparatively easy to produce, compared with efforts to generate vaccines for diseases such as malaria, hiv and tuberculosis. indications from the nhp vaccine model with rad ebov gp and analysis of immune responses in evd survivors indicated that a cd þ t cell component would be a prerequisite of an effective evd vaccine. yet the rvsv-zebov vaccine may not induce a significant cmi component based on pre-clinical data. an additional consideration is that passive transfer of ebola-specific antibody can fail to protect nhps from lethal ebola virus challenge, indicating that antibody titres may be a nonmechanistic correlate of protection and that qualitative aspects of the antibody response and/or cellular immunity that are not readily measured also play key roles in protection [ ] . it is likely that the mechanism of protection and protective level of a particular immune component may well differ in an experimental model in nhps, challenged in a manner that may not accurately reflect natural exposure and in humans exposed to natural infection. this highlights the importance of not being over-reliant on immunological correlates derived from pre-clinical testing. clearly, different vaccine delivery methods induce qualitatively different immune responses; induction of long-lasting humoral immunity in the absence of t cells seems unlikely, so the ability of prime-boost regimens to induce durable cmi and humoral immunity may be desirable for longevity of protection. in december , who held a workshop on the prioritization of pathogens, generating a list of seven diseases requiring urgent r&d, with a further three diseases requiring action as soon as possible [ ] . this list encompasses emerging diseases with potential to generate a public health emergency and for which no, or insufficient, preventative or curative solutions exist. crimean congo haemorrhagic fever (cchf), evd, marburg haemorrhagic fever, lassa fever, mers, sars, nipah and rift valley fever virus were identified as priorities for emerging pathogen research, with zika, chikungunya and severe fever with thrombocytopaenia syndrome (sfts) determined to be serious and necessitating further action as soon as possible. this evd outbreak has focused attention on emerging pathogens, shifting their place in the public consciousness from a theoretical emergency to a very tangible threat to global health. vaccine developers must now think in terms of addressing emerging pathogens as an entirety rather than a collection of individual highly virulent pathogens. to this end, the recent outbreak has yielded some valuable insights. firstly, in the context of an ongoing outbreak, a single dose of a viral vector vaccine could be sufficient to induce at least short-term protective immunity against filoviruses. this can usefully protect front-line healthcare workers and communities, thereby reducing the spread of an epidemic in the earliest stages [ ] . where the outbreak pathogen is readily identified a single dose of a recombinant adenoviral or vsv vector may suffice for outbreak control. however, to counter the threat of infectious outbreaks which may emerge unpredictably, large-scale prophylactic immunization of healthcare workers would be a useful first line of defence. a single-dose multivalent vaccine encoding antigens from several putative emerging pathogens could be particularly valuable in such a scenario. for example, recombinant mva would be suited to this purpose, given the potential to insert various foreign genes at numerous sites in the genome under a number of promoters [ ] , in contrast with adenovirus vectors that have a limited cloning capacity to carry foreign genes. immunity could subsequently be boosted with an adenoviral vector expressing a specific transgene, once the outbreak pathogen is identified. a study of an evd vaccine regimen employing mva as a priming vector and adhu as a boost has demonstrated that this is a feasible approach [ ] . speculatively grouping rstb.royalsocietypublishing.org phil. trans. r. soc. b : emerging pathogens according to geographical incidence may be one strategy for determining components of multivalent vaccines and an example of a potential strategy for designing vaccines using this approach is suggested in figure . almost all of the current evd vaccine candidates use gp as the antigen due to abundant expression on the surface of the virus, and there is extensive pre-clinical data reflecting promising immunogenicity and efficacy against challenge for this target antigen. studies of patients with evd in two outbreaks in gabon revealed that in survivors, igg responses were largely directed against nucleoprotein [ ] . the same observation was made in a small study of evd cases in the usa [ ] . characterization of immune responses in natural ebolavirus infection have also revealed that exposure can induce cross-reactive antibodies capable of neutralizing multiple ebolavirus species [ ] . this study of naturally acquired immunity in survivors of a bdbv outbreak in uganda in demonstrated for the first time that potent neutralizing antibodies to the glycan cap of gp could inhibit several ebolaviruses, including sudv, and protect guinea pigs from a heterologous challenge with ebov which may offer insight into immunogen design to confer heterosubtypic immunity across multiple ebolaviruses. given the very large number of survivors of the - outbreak, significant efforts should be made to characterize the immunity that afforded protection in the face of infection with such a highly lethal pathogen, to help inform antigen selection for future vaccines and advance our understanding of post-evd immunology. whether vaccines need to emulate these naturally occurring mechanisms remains to be seen, but these observations are of critical importance to help advance the field. closer examination of the priority pathogens highlighted by the who may facilitate development of broad-spectrum vaccines; viruses with small genomes that have obvious vaccine candidate antigens or where pre-clinical data are highly indicative [ ] . in this setting, data from the one health approach, which combines human, animal and environmental considerations to address global health challenges, are invaluable; for example, immunogenicity data derived from studies in zoonotic reservoirs can be applied to vaccine development [ , ] . furthermore, studies of families of viruses using technological advances such as cryo-electron microscopy, b cell cloning and antibody repertoire sequencing may yield novel targets that may be effective for tackling emerging pathogens. phylogenetically related viruses that share receptor-binding domains, and epitopes within envelope proteins may well share therapeutic and vaccine targets [ ] . as discussed above, great efforts have been made to characterize the magnitude of neutralizing antibody titres induced by vaccination, yet the detail of these entry-blocking mechanisms has not yet been fully utilized by vaccinologists. detailed understanding of host-pathogen interactions has yielded numerous opportunities for vaccines against other diseases, including malaria and hiv [ ] . in-depth understanding of the processes associated with inhibition of receptor binding, such as conformational rearrangement of glycoproteins required for viral fusion, could possibly identify ubiquitous targets for mabs that can prevent infection of multiple virus species [ ] : for example, the entry of all filoviruses into mammalian cells utilizes the same endosomal receptor [ ] , the niemann-pick c protein, presenting an obvious target for inhibition of virus entry. outbreaks of highly pathogenic diseases such as evd are devastating for the populations affected and will always focus attention on control strategies to prevent future occurrences. while it is widely acknowledged that the global public health response to the - was tragically slow to begin, immunologists and vaccinologists now have a valuable opportunity to learn as much as possible from this disaster. as well as probing the vaccine-induced responses from the many clinical trials that have taken place, detailed characterization of naturally acquired immunity in both survivors of evd and asymptomatic, ebov igg-seropositive members of communities should be systemically compiled. previous data from gabon have illustrated that substantial proportions of rural populations have evidence of ebov-specific immunity [ ] . we now have a collective responsibility to assimilate all the information that this outbreak has generated in order to be best placed when the next epidemic comes, so that we can respond effectively and robustly to curb an outbreak in its infancy. who. situation report: ebola virus disease emergency ebola response: a new approach to the rapid design and development of vaccines against emerging diseases virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cdna structural basis for marburg virus neutralization by a cross-reactive human antibody chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge cd þ cellular immunity mediates rad vaccine protection against 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targeting emerging viruses: advancements and mechanisms a pfrh -based vaccine is efficacious against heterologous strain blood-stage plasmodium falciparum infection in aotus monkeys broadly neutralizing alphavirus antibodies bind an epitope on e and inhibit entry and egress ebola viral glycoprotein bound to its endosomal receptor niemann-pick c high prevalence of both humoral and cellular immunity to zaire ebolavirus among rural populations in gabon authors' contribution. all authors contributed to the design, drafting and revision of the article and approved the final version for publication.competing interests. we have no competing interests. funding. k.j.e. is funded by a horizon award from the european union. g.b. is funded by the wellcome trust. t.l. is funded by the mrc. key: cord- -mwnnmwnw authors: herst, c.v.; burkholz, s.; sidney, j.; sette, a.; harris, p.e.; massey, s.; brasel, t.; cunha-neto, e.; rosa, d.s.; chao, w.c.h.; carback, r.; hodge, t.; wang, l.; ciotlos, s.; lloyd, p.; rubsamen, r. title: an effective ctl peptide vaccine for ebola zaire based on survivors’ cd + targeting of a particular nucleocapsid protein epitope with potential implications for covid- vaccine design date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: mwnnmwnw the - west africa ebov epidemic was the biggest ebov outbreak to date. an analysis of virus-specific cd + t-cell immunity in survivors showed that of those individuals had a cd + response to at least one ebov protein. the dominant response ( / subjects) was specific to the ebov nucleocapsid protein (np). it has been suggested that epitopes on the ebov np could form an important part of an effective t-cell vaccine for ebola zaire. we show that a -amino-acid peptide np - (yqvnnleei) located in a conserved region of ebov np provides protection against morbidity and mortality after mouse adapted ebov challenge. a single vaccination in a c bl/ mouse using an adjuvanted microsphere peptide vaccine formulation containing np - is enough to confer immunity in mice. our work suggests that a peptide vaccine based on cd + t-cell immunity in ebov survivors is conceptually sound and feasible. nucleocapsid proteins within sars-cov- contain multiple class i epitopes with predicted hla restrictions consistent with broad population coverage. a similar approach to a ctl vaccine design may be possible for that virus. mouse using an adjuvanted microsphere peptide vaccine formulation containing np - is enough to confer immunity in mice. our work suggests that a peptide vaccine based on cd + t-cell immunity in ebov survivors is conceptually sound and feasible. nucleocapsid proteins within sars-cov- contain multiple class i epitopes with predicted hla restrictions consistent with broad population coverage. a similar approach to a ctl vaccine design may be possible for that virus. keywords: ebola zaire vaccine, ctl vaccine, controller, yqvnnleei, covid- , sars-cov- , flow focusing development of safe and effective vaccines for some viruses such as hiv and ebov has been challenging [ ] . although vaccine development has been almost exclusively focused on eliciting a humoral immune response in the host through inoculation with whole protein antigen [ ] [ ] [ ] [ ], ctl peptide vaccines producing a t-cell response may offer an important alternative approach [ ] . for hiv and ebov and influenza in particular, the potential of ctl vaccines has been discussed [ ] [ ] [ ] . although computational prediction alone has been used for t-cell vaccine design [ ] [ ], we saw a unique opportunity to see if a preventative ebov t-cell vaccine could be successfully designed based on the specific epitopes targeted by survivors of documented ebov infection. the notion of hla restricted hiv control has been described [ ] . pereyra- heckerman conducted an analysis of virus-specific cd + t-cell immunity in individuals living with hiv [ ] . they reported that hiv controllers, individuals living with hiv not undergoing treatment who do not progress to aids, have cd + cells targeting different hla restricted class i epitopes on hiv compared with progressors, individuals with hiv who progress to aids in the absence of therapy. pereyra-heckerman suggested that this observation could guide the in-silico development of a ctl vaccine for hiv and other diseases. acquired immunity has been documented after ebov infection [ ] . antibody as well as t-cell responses have been described [ ] . sakebe et al. have shown that of subjects surviving the - ebov outbreak in west africa, cd + t-cells from of those survivors responded to at least one ebov antigen, with of the responders targeting epitopes on ebov np [ ] . one of the most commonly targeted ebov eptitopes on ebov np in the survivor group (targeted by cd + cells from four survivors) was np - (ipvyqvnnleeicqliiqaf). they also suggested that a ctl vaccine could be designed using epitopes targeted by cd + t-cells identified in these ebov controllers. human pathogen-derived peptide antigens that are also recognized by c bl/ t-cells have been previously described. these include peptides from vesicular stomatitis virus (vsv) rgyvyqgl [ ] , and human immunodeficiency virus (hiv) rgpgrafvti [ ] . the existence of such epitopes makes a range of pre- clinical vaccine experiments possible without having to rely on non-human primates and expensive and complex-to-manage humanized mouse models. wilson et al. showed that the ebov nucleoprotein (np) is an immunogen that provides protective, ctl-mediated immunity against ebov in a c bl/ mouse model and that this protection was conferred by a peptide sequence within ebola zaire: np - (vyqvnnleeic) [ ] . wilson we set out to see if we could drive ctl expansion directed against np - to occur after vaccinating c bl/ mice with ebola zaire np - (vyqvnnleeic), and to subsequently conduct an in-vivo ebov challenge study to see if this peptide was protective. we fabricated adjuvanted microspheres for this study as a room temperature stable dry powder using the flow focusing process to be µm in diameter so as to prevent more than one microsphere from being phagocytosed by any given antigen presenting cell (apc) at the same time [ ] . by loading only one peptide sequence per microsphere, we maximized the peptide payload and mitigated the possibility of multiple, different peptide sequences being delivered to the apc simultaneously, which could possibly result in competitive inhibition at the motif which could interfere with antigen presentation and subsequent t-cell expansion (supplementary material section ). we also set out to see if a similar approach to a ctl vaccine design for sars-cov- would be feasible based on an analysis of the hla binding characteristics of peptide sequences on sars-cov- nucleocapsid. we used a previously described biodegradable dry powder, plga microsphere, synthetic vaccine platform adjuvanted with tlr- and tlr- agonists for this study [ ] . in that article, we showed that the tlr- and tlr- agonists given together with a peptide in a mouse model did not produce t-cell expansion by elispot and that microencapsulation of the peptide and the tlr- ligand, with the tlr- ligand in the injectate solution, was required to elicit an immune response to the delivered peptide antigen as determined by elispot. that study also demonstrated that the microencapsulated peptides alone were insufficient to induce an adequate immune response without the presence of the tlr- and tlr- agonists administered as described. the tlr agonists used for this vaccine formulation are used in fda approved vaccines and can be sourced as non-gmp or gmp material for pre-clinical and clinical studies. we show here that the h -d b restricted epitopes vsv (rgyvyqgl) and ova (siinfekl), when administered to c bl/ mice, each produce a cd + we used this adjuvanted microsphere peptide vaccine platform to immunize c bl/ mice with np - , the ctl+ class i peptide antigen from the ebola ziare np protein identified as protective by wilson et al. [ ] . microspheres containing np - and cpg were prepared as a dry powder formulation and suspended before use in a pbs injectate solution containing mpla, and administered intradermally via injection at the base of the tail into mice as described in a previous publication [ ] . as illustrated in figure c , there was no statistically significant difference between the elispot data for the vaccinated mice versus the response seen in the negative elispot controls. wilson reported that protection seen in her experiment was due to a peptide sequence within np- - . we hypothesized that the np - epitope was inefficiently processed into mhc binding sub-sequences during antigen presentation. in order to explore possible h -d b matches for peptide sequences contained within ebola zaire np - (vyqvnnleeic), we prepared three peptide vaccine formulations, each containing one of the three possible mer sub-sequences within np - . these sequences are shown in table . we then vaccinated, via intradermal (tail) injection, three groups of mice with microspheres containing one of the three mer sub-sequences of np - ( per group). elispot analysis was performed, stimulating harvested splenocytes with the three possible mer sub-sequences. splenocytes from mice receiving the np - sub-sequence had a statistically higher elispot response than mice vaccinated with the other two possible sub-sequence mers (p < . ) as shown in figure a . this is consistent with the predicted h -d b binding affinity of yqvnnleei as shown in supplementary material table . we then loaded one population of adjuvanted microspheres with np - . this peptide has a predicted favorable h -i b binding affinity as shown in supplementary material table . we showed that vaccination of mice with the adjuvanted microsphere vaccine loaded with vg and np - showed an elispot response to np - whereas mice vaccinated with adjuvanted microspheres not loaded with peptide did not ( figure d ). we also showed that mice vaccinated with vg alone did not show an elispot response to np - ( figure a ) and, conversely, mice vaccinated with np - did not show a response to vg (figure b ). we conducted a pilot study demonstrating that intraperitoneal injection of the adjuvanted microsphere vaccine produced a statistically superior immune response by elispot compared with the same dose delivered by intradermal tail or intramuscular injection in c bl/ mice (supplementary material section ). based on the data from that study, and the fact that the volume of the intraperitoneal space would allow larger amounts of microsphere suspension to be delivered, we chose to proceed with intraperitoneal administration for the challenge portion of this study delivering mg of microspheres per dose. we dosed three groups of mice, ten mice per group, with the adjuvanted mi- table . peak mortality across all groups tested was seen in mice challenged with , pfu maebov versus pbs buffer control as shown in the survival curve in we saw what appears to be an innate immune response at the , pfu ebov exposure level. it has been suggested that ebov can mediate an innate immunity response through stimulation of tlr- [ ] . because the adjuvanted this provides some evidence that the protective effect of vaccination using this adjuvanted microsphere vaccine is reproducible. serum samples from sacrificed animals exposed to ebov who did not receive vaccine were quantitatively assayed for various cytokines using bioplex plates. animals having unwitnessed demise did not have serum samples collected. a pearson correlation analysis was performed to assess relationships between specific cytokine levels and survival. the results are shown in table . we observed low levels of il- in surviving mice. nhps infected with ebov have been determined by other researchers to have elevated levels of il- in plasma and serum [ ] [ ] . ebov infected humans have also shown elevated il- levels and these elevated levels have been associated with increased mortality [ ] . similarly, we observed low levels of mcp- , il- and gm-csf in survivors. [ ] [ ] and elevated levels of mcp- were associated with fatalities in ebov infected human subjects [ ] . human survivors of ebov have been found to have very low levels of circulating cytokines il- and elevated levels of gm-csf have been associated with fatality in humans exposed to ebov [ ] . we lating t-cells targeting sars-cov- nucleocapsid two years after initial infection. [ ] we decided to investigate the feasibility of designing a sars-cov- peptide vaccine targeting sars-cov- nucleocapsid. all available sars-cov- protein sequences were obtained from the ncbi viral genomes resource within genbank, an nih genetic sequence database [ ] . retrieved sequences were processed using multiple sequence alignment (msa) via clustal for the nucleocapsid phosphoprotein [ ] . the nucleocapsid phosphoprotein sequences were trimmed down to every possible peptide sequence dosing [ ] . predicted values of these peptides were cross referenced with actual in-vitro binding measurements from identical mer peptides when that data was available. most preventative vaccines are designed to elicit a humoral immune response, typically via the administration of whole protein from a pathogen. antibody antigens and the hla restricted nature of ctl vaccines have limited their utility to protect individuals from infectious disease [ ] . however, observations derived from individuals able to control hiv infection [ ] and ebov infection [ ] demonstrating that control may be associated with specific ctl targeting behavior, suggest that there may be an important role for hla-restricted pep- ebov can cause severe pulmonary problems in exposed subjects [ ] . these problems can be especially severe when the virus is delivered by aerosol [ ] [ ]. interaction of ebov specific antibody, nhp lung tissue and ebov delivered to nhps via aerosol can produce a more lethal effect than in nhps without circulating anti-ebov antibody exposed to aerosolized ebov (unpublished conference presentation). this suggests that a ctl vaccine may be more effective for prophylaxis against filovirus protection than an antibody vaccine if the anticipated route of ebov exposure is via aerosol. coproteins present on the surface, show a high degree of conservation. epitopes within these internal proteins often stimulate t-cell-mediated immune responses [ ] . as a result, vaccines stimulating influenza specific t-cell immunity have been considered as candidates for a universal influenza vaccine [ ] . response to sars-cov- nucleocapsid two years after infection. [ ] this suggests that the same approach could be applied to sars-cov- which has con- table and supplementary material table . the remaining sars-cov- peptides listed in could also be further qualified as potential vaccine candidates by confirming mhc binding predictions by in-vitro binding affinity and/or binding stability studies [ ] [ ] [ ] . another approach to evaluating the sars-cov- candidate vaccine peptides though in-vitro testing is also possible. as we have shown in this paper, a peptide targeted by ebov controllers could form the basis of a preventative vaccine for ebov. elispot analysis of pbmcs taken from the peripheral blood of covid- controllers and progressors to assess the presence of a differential response to the peptides could lead to a broadly applicable protective ctl vaccine against sars-cov- by incorporating peptides into the vaccine that are more commonly targeted for cd + attack by the controllers versus the progressors. a peptide vaccine for sars-cov- , unlike a typical antibody vaccine, is not limited to virus surface antigen targets. this provides opportunities to attack other targets on sars-cov- besides spike which may be prone to mutation [ ] . in addition, a peptide vaccine mitigates the risk of antibody disease enhancement (ade) seen in the context of a non-neutralizing antibody response to a whole protein vaccine [ ] [ ] . also, neutralizing antibodies directed against spike protein in sars-cov- patients have been associated with an increased risk of acute lung injury (ali) [ ] . specifically, patients succumbing to sars-cov- were found to develop a neutralizing antibody (nab) response to spike protein faster than survivors after the onset of symptoms and the nab titers were higher in the patients who died compared with those who recovered [ ] . to the extent to which antibody vaccines producing an antibody response against the spike protein in sars-cov- could increase the risk of ali, this risk could also be mitigated by a using peptide vaccine as an alternative approach. the extent of the covid- outbreak should allow many more controllers to be identified than the thirty individuals studied by sakabe and the seven individuals identified in the peng study [ ] [ ] . furthermore, sakebe and peng did not report progressor data perhaps because of the difficulty in obtaining blood samples from those patients. if researchers act now during the covid- outbreak, perhaps controller and progressor blood samples could be collected and prospectively analyzed, quickly creating a database of optimal candidate class i peptides for inclusion into a ctl vaccine with potentially broad hla coverage for subsequent rapid manufacture and deployment. it would be interesting to see the extent to which the peptides favored by controllers appear on sars-cov- nucleocapsid, making sars-cov- a second example, across two different viruses, of controllers exhibiting ctl attack preferentially on the nucleocapsid protein. all animal 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being more vulnerable to mutations among coronavirus proteins from different species recent advances in the vaccine development against middle east respiratory syndrome-coronavirus antibody responses against sars coronavirus are correlated with disease outcome of infected individuals advances in the study of hla-restricted epitope vaccines screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes sars-cov- nucleocapsid top candidate peptides with associated predicted hla restricted binding affinities peptide start key: cord- -yh x w q authors: ndungo, esther; herbert, andrew s.; raaben, matthijs; obernosterer, gregor; biswas, rohan; miller, emily happy; wirchnianski, ariel s.; carette, jan e.; brummelkamp, thijn r.; whelan, sean p.; dye, john m.; chandran, kartik title: a single residue in ebola virus receptor npc influences cellular host range in reptiles date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: yh x w q filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented ebola virus disease (evd) outbreak in western africa. one obstacle to controlling these epidemics is our poor understanding of the host range of filoviruses and their natural reservoirs. here, we investigated the role of the intracellular filovirus receptor, niemann-pick c (npc ) as a molecular determinant of ebola virus (ebov) host range at the cellular level. whereas human cells can be infected by ebov, a cell line derived from a russell’s viper (daboia russellii) (vh- ) is resistant to infection in an npc -dependent manner. we found that vh- cells are resistant to ebov infection because the russell’s viper npc ortholog bound poorly to the ebov spike glycoprotein (gp). analysis of panels of viper-human npc chimeras and point mutants allowed us to identify a single amino acid residue in npc , at position , that bidirectionally influenced both its binding to ebov gp and its viral receptor activity in cells. significantly, this single residue change perturbed neither npc ’s endosomal localization nor its housekeeping role in cellular cholesterol trafficking. together with other recent work, these findings identify sequences in npc that are important for viral receptor activity by virtue of their direct interaction with ebov gp and suggest that they may influence filovirus host range in nature. broader surveys of npc orthologs from vertebrates may delineate additional sequence polymorphisms in this gene that control susceptibility to filovirus infection. importance identifying cellular factors that determine susceptibility to infection can help us understand how ebola virus is transmitted. we asked if the ebov receptor niemann-pick c (npc ) could explain why reptiles are resistant to ebov infection. we demonstrate that cells derived from the russell’s viper are not susceptible to infection because ebov cannot bind to viper npc . this resistance to infection can be mapped to a single amino acid residue in viper npc that renders it unable to bind to ebov gp. the newly solved structure of ebov gp bound to npc confirms our findings, revealing that this residue dips into the gp receptor-binding pocket and is therefore critical to the binding interface. consequently, this otherwise well-conserved residue in vertebrate species influences the ability of reptilian npc proteins to bind to ebov gp, thereby affecting viral host range in reptilian cells. e bola virus (ebov) is the causative agent of highly lethal zoonotic infections in humans and nonhuman primates in sub-saharan africa ( ) ( ) ( ) . despite the emerging roles of ebov and related members of the family filoviridae (filoviruses) in human disease, our knowledge of the ecological host range of these agents remains limited. bats are thought to be important reservoirs for filoviruses; however, conclusive evidence in favor of this hypothesis has been obtained only for marburg virus (marv) and ravn virus (ravv), which were recently found to circulate in egyptian rousettes (rousettus aegyptiacus) ( ) ( ) ( ) ( ) . previous studies demonstrated that, whereas a broad range of mammalian and avian cell lines are susceptible to ebov and/or marv, all tested reptilian and amphibian lines are resistant to infection ( ) ( ) ( ) . these findings suggested the existence of one or more unknown determinants of filovirus host range. although the determinants of filovirus infection and disease at the organismal level are likely to be complex, it is well established that interactions between viruses and cell-intrinsic host factors, such as entry receptors, can dictate host range. for example, ortholog-specific sequence variations in angiotensin-converting enzyme (ace ) and transferrin receptor (tfr ) influence the host range of viruses for which they serve as receptors (severe acute respiratory syndrome-related coronaviruses [ , ] and new world mammarenaviruses, canine parvoviruses, and murine mammary tumor virus [ ] [ ] [ ] [ ] [ ] [ ] , respectively). jae and coworkers demonstrated that chicken cells are resistant to infection by an old world arenavirus, lassa virus, because of a single amino acid difference in the chicken ortholog of its intracellular receptor, lamp ( ) . we and others recently demonstrated that niemann-pick c (npc ), a large endo/ lysosomal membrane protein involved in cellular cholesterol trafficking, is an essential intracellular receptor for filovirus entry and infection ( ) ( ) ( ) ( ) . we also found that npc could influence the cellular host range of filoviruses-human npc conferred susceptibility to filovirus entry and infection when expressed in the nonpermissive reptilian cell line vh- , derived from a russell's viper (daboia russellii) ( ) . in that study, however, we did not establish the molecular basis of the npc -dependent block to viral entry in vh- cells. recently, we found that a single amino acid residue (position ) in the second luminal domain of npc , domain c, is under positive selection in bats and controls the susceptibility of bat cells to ebov infection in a host species-dependent manner ( , ) . here, we demonstrate that an adjacent residue, , highly conserved in domain c of npc , influences ebov host range in reptilian cells by controlling its activity as a filovirus receptor. the recently solved structure of the ebov entry glycoprotein (gp , ; hereafter referred to as gp) bound to domain c shows that these two residues are in a loop that dips into the exposed receptor-binding site ( ) . therefore, our findings identify a hot spot in npc at the ebov gp-binding interface that influences virusreceptor recognition and host cell susceptibility, suggesting evolutionary scenarios in which antagonism with filoviruses could sculpt host npc genes selectively, without compromising their ancient, and essential, function in cellular cholesterol homeostasis. the second luminal domain of the russell's viper npc ortholog binds poorly to the ebola virus glycoprotein. we postulated that ebov fails to enter and infect russell's viper vh- cells because the ebov entry glycoprotein, gp, cannot recognize the viper ortholog of the filovirus intracellular receptor, niemann-pick c (daboia russellii npc [drnpc ]). we previously showed that the second luminal domain (c) of human npc (homo sapiens npc [hsnpc ]) directly contacts a cleaved form of ebov gp (gp cl ) and that gp cl -hsnpc domain c binding is essential for filovirus entry ( , ) . accordingly, we investigated the capacity of drnpc domain c to bind to gp cl and support ebov entry and infection. we first used reverse transcription-pcr (rt-pcr) to isolate and sequence drnpc domain c. alignment of domain c amino acid sequences from hsnpc and drnpc revealed a substantial degree of conservation ( % amino acid identity), with identical arrangements of cysteine residues and similar predicted secondary structures, suggesting a similar overall fold for the two proteins ( fig. ) . to facilitate in vitro gp cl -npc -binding studies, we engineered a soluble form of drnpc domain c, as previously described for hsnpc ( ) . transfection of hek t cells with this construct afforded the secretion of an extensively n-glycosylated form of drnpc domain c ( fig. a) . as shown previously, purified hsnpc domain c could bind to recombinant vesicular stomatitis virus indiana particles bearing cleaved ebov gp (rvsv-gp cl ), as measured by enzyme-linked immunosorbent assay (elisa) ( ) ; in contrast, drnpc domain c exhibited no binding by elisa, even at the highest concentration tested (fig. b) . therefore, drnpc domain c, in contrast to its human counterpart, recognizes the ebov glycoprotein poorly or not at all. domain c can substitute for hsnpc domain c in mediating endo/lysosomal cholesterol clearance but not ebov entry and infection. while the efficient secretion of the soluble, glycosylated drnpc domain c construct suggested that it was not misfolded, it was nevertheless conceivable that subtle structural aberrations rendered this protein biologically inactive. accordingly, we assessed the capacity of drnpc domain c to support npc 's best-established cellular functionclearance of unesterified cholesterol from endo/lysosomal compartments ( fig. a domains, a, c, and i. we therefore generated and tested an hsnpc chimera in which domain c (residues to ) was seamlessly replaced with its viper counterpart (hsnpc -drc)-we previously found that replacing domain c in npc using restriction site cloning, which introduced two additional amino acid residues at each junction, resulted in proteins that were defective in localization and cholesterol clearance. the wild-type (wt) hsnpc and hsnpc -drc chimera constructs were then stably expressed in the npc -null chinese hamster ovary (cho) m cell line ( ) . as expected, immunostaining of wt hsnpc transiently expressed in a u os npc Ϫ/Ϫ cell line ( ) showed colocalization with the endo/lysosomal marker lamp (fig. a) the behavior of hsnpc -drc resembled that of wt hsnpc , indicating that it too localizes to endo/lysosomal compartments (fig. a) . these results suggest that drnpc domain c is correctly folded and does not interfere with the correct folding and trafficking of full-length hsnpc . we next monitored the cholesterol clearance activity of each protein upon stable expression in npc -null m cells (fig. b) . filipin, a fluorescent probe for free choles- terol, extensively stained the cholesterol-laden endo/lysosomal compartments of the parental m cells, as shown previously ( ) . ectopic hsnpc expression could clear this accumulated cholesterol, as previously described ( ), substantially reducing filipin staining. remarkably, hsnpc -drc could rescue cholesterol clearance as efficiently as wt hsnpc (fig. b ). these findings affirm that drnpc domain c is biologically active and competent to perform a major housekeeping function of its human counterpart, despite its divergence from the latter at out of amino acid positions ( fig. ). finally, we challenged m cell lines expressing wt hsnpc or hsnpc -drc with authentic ebov (fig. c ). replacement of human domain c with its russell's viper ortholog reduced ebov infection by almost orders of magnitude. similar results were obtained in infections with rvsv-ebov gp (fig. d) , confirming that the drnpc domain c-imposed infection block occurs at the viral entry step. taken together, these observations afford two conclusions. first, the failure of drnpc to support ebov entry and infection arises at least in part because its domain c cannot bind to ebov gp cl . second, one or more differences between the domain c sequences of hsnpc and drnpc render drnpc bereft of viral receptor activity without perturbing its normal function in cellular cholesterol homeostasis. to uncover the molecular basis of drnpc 's defective viral receptor function, we engineered and tested a panel of mutant, soluble npc domain c constructs in both hsnpc and drnpc backgrounds. we first considered the possibility that one or more differences in n-linked glycosylation sites determine the hsnpc -drnpc difference, because it is either required for gp cl -hsnpc binding or deleterious for gp cl -drnpc binding (fig. ) . six sequons are conserved between the two proteins, but drnpc and hsnpc domains c contain two and one unique sequons, respectively. accordingly, we generated soluble domain c proteins containing or lacking each unique sequon and tested these putative gain-of-function and loss-offunction mutants for binding to ebov gp cl . "humanized" drnpc domain c proteins engineered to lack their unique sequons at position or (hsnpc numbering) or to gain the sequon at position remained defective at ebov gp cl binding in the elisa. conversely, hsnpc domain c proteins engineered to resemble drnpc at each of these three positions remained fully competent to bind to ebov gp cl . therefore, differences in n-linked glycosylation between the domains c of hsnpc and drnpc do not account for the defective ebov receptor activity of drnpc . a single point mutation renders drnpc domain c competent to bind to ebov gp cl . having ruled out a role for variations in n-glycosylation, we next adopted a systematic approach to identify determinative sequences in npc domain c. we expressed a series of soluble hsnpc -drnpc domain c chimeras and measured their activity in the gp cl -binding elisa (fig. ) . however, only chimera , drnpc domain c containing hsnpc residues to , afforded gp cl -npc binding (fig. a) . chimera introduced russell's viper¡human amino acid changes into drnpc . to further dissect their roles, we generated and tested three additional chimeras containing subsets of these amino acid changes (chimeras to , fig. b ) in the gp cl -binding elisa. the subregion chimera fully reconstituted gp cl -drnpc domain c binding, providing evidence that one or more of the hsnpc residues in this construct confer gain of function on drnpc . to assess the individual contributions of the six russell's viper¡human amino acid changes in chimera , we separately introduced these changes into soluble drnpc domain c and tested the capacity of each point mutant to bind to ebov gp cl (fig. ) . a single conservative mutation, y ¡f, fully restored gp cl -drnpc domain c binding, whereas the other mutations had no discernible effect. thus, the presence of y instead of f at position appears to completely explain the failure of drnpc to bind to ebov gp cl . f↔y sequence change at residue controls npc 's function as an ebov entry receptor without affecting its housekeeping function. we postulated that the f↔y sequence change at residue might influence ebov gp cl -npc binding in a bidirectional manner. accordingly, we expressed and purified the reciprocal drnpc (y f) and hsnpc (f y) domain c mutants and tested them in the gp clbinding elisa (fig. ) . purified drnpc (y f) domain c bound almost as well as its human counterpart to ebov gp cl ( % effective concentration [ec ] for binding, cho-m cells stably expressing hsnpc (f y) or hsnpc -drc(y f) (fig. b ). therefore, the f y and y f mutations do not substantially affect the folding, endosomal delivery, and cholesterol clearance function of npc . finally, we challenged cell lines expressing the (f↔y) npc mutants with authentic ebov and rvsv-ebov gp (fig. c) . the capacities of both authentic and surrogate viruses to enter and infect these cells were fully congruent with the results of the gp-binding experiments. the viper¡human y f mutation afforded the complete restoration of viral infection in cells expressing the hsnpc -drc chimera (Ϸ log unit increase). reciprocally, the human¡viper f y mutation reduced viral infection in cells expressing hsnpc by Ϸ log units. thus, the infection data correlate with the gp cl -domain c-binding data, demonstrating that switching the residue at position changes the ability of human and russell's viper npc domain c to bind ebov gp cl , thereby determining the ability of these npc proteins to be used as ebov receptors. a bulky, hydrophobic amino acid residue at position favors ebov gp cl -npc domain c binding. to determine the mechanism by which the change in npc residue controls binding of hsnpc to ebov gp cl , we engineered a series of npc domain c proteins bearing amino acid residues with divergent physicochemical properties at position . examination of these mutants by gp cl -binding elisa revealed that binding avidity was generally correlated with amino acid size and polarity (fig. a) . specifically, residues with bulky, hydrophobic side chains (l and w) afforded gp cl -npc binding at wt levels, whereas residues with polar side chains (d, h, and s) abrogated binding. binding was greatly reduced, but detectable, with a and t at residue . the recently solved structure of ebov gp cl bound to npc domain c shows that residue inserts into the hydrophobic trough of ebov gp cl ( , ) , similarly to residue f of the ebov glycan cap (fig. b and c) ( ) . finally, we asked if our findings had implications for host cell range in other vertebrates, especially reptiles, which appear to be refractory to infection by ebov ( , ). an alignment of available npc domain c sequences from a panel of vertebrate species revealed that, although there exist a number of differences in amino acid sequence around residue , the f at this position is itself very well conserved among vertebrates, with only two npc orthologs-those of the russell's viper and king cobra (ophiophagus hannah)-encoding a y at this position (fig. a) . interestingly, the predicted npc polypeptide sequences of two additional snakes, the burmese python (python bivittatus) and the common garter snake (thamnophis sirtalis), show an f at position (fig. a) . to investigate the gp cl -binding capacities of the snake npc orthologs, we expressed and purified soluble npc domain c proteins for the king cobra and burmese python and tested them for binding to ebov gp cl . the capacity of these proteins to bind to ebov gp cl was concordant with the identity of the residue at npc codon . thus, king cobra npc domain c(y ) resembled viper npc domain c in its inability to bind to ebov gp cl , whereas burmese python npc domain c(f ) readily bound to ebov gp cl (fig. b) . we tested two more reptilian npc orthologs-from chinese softshell turtle and carolina anole (both carrying f )-and found that they could all bind to ebov gp cl (fig. c) . these findings provide additional evidence that npc -encoded residue influences the cellular host range of ebov at the level of virus-receptor recognition and raise the possibility that sequence differences at this position influence the susceptibility of reptiles to filovirus infection in nature. the essential entry receptor npc is the first known molecular determinant of the cellular host range of ebov and other filoviruses ( , ) . in this study, we uncover one mechanism by which npc imposes a species-specific barrier to ebov infection. we show that reptilian cells derived from the russell's viper, daboia russellii, are largely resistant to ebov entry and infection because of the presence of a y residue at position in npc , whereas the npc orthologs of most other types of animals, include humans, carry a highly conserved f residue. unexpectedly, toggling this residue between f and y in either human or russell's viper npc backgrounds switched each protein's ability to act as an ebov receptor. npc 's crucial housekeeping functiondistribution of cholesterol from the endo/lysosomal compartment to other cellular membranes-remained unaffected by these changes. thus, our work identifies a genetic determinant in npc that controls its viral receptor function, and consequently host susceptibility to ebov infection, in a manner that is selective and yet transferable between highly divergent npc orthologs. the determinative f ¡y change is located in npc 's second luminal domain (c), which directly binds to a cleaved form of the ebov entry glycoprotein (gp cl ) during viral entry ( , , ) . here, we found that f ¡y renders cells nonpermissive to ebov infection because it reduces the apparent binding affinity of gp cl for npc domain c by more than , -fold. what mechanism might account for this extraordinary effect of a single hydroxyl group on virus-receptor interaction? the recently solved structure of the ebov gp cl bound to npc domain c reveals that f in human npc domain c inserts deeply into the hydrophobic gp cl trough during gp-npc interaction, in a manner that resembles the interaction of f in the gp glycan cap with the gp cl trough in uncleaved gp ( fig. b and c) ( ) . the introduction, at position , of a polar hydroxyl group (y) or other polar side chains (d, h, s, and g) is likely to be energetically unfavorable, thereby reducing the affinity of gp cl -npc binding. we recently demonstrated that residue in npc was under positive selection in bats and was responsible for the reduced susceptibility of african straw-colored fruit bat cells to ebov infection ( , ) . since none of the bat species genes encodes a y at position in npc , there was no observed signature of positive selection at this residue. the structure rationalizes the effect of these residues on gp-npc binding, as both are located in the ␣ -␣ loop of npc domain c that directly interacts with ebov gp cl ("loop " [ ] ) (fig. c) . it is unclear what relationships, if any, exist (or have existed) in nature between filoviruses and snakes or other reptiles. experimental infections of wild-caught reptiles and amphibians by swanepoel and colleagues ( ) showed a general refractoriness to ebov infection or replication, but minimal titers were recovered on a few occasions from the brown house snake (lamprophis fuliginosus). following outbreaks of the ebola virus relative marburg virus (marv) at the mine in kitaka cave, the nearby "python cave" in queen elizabeth national park in uganda ( , ) , and the goroumbwa mine in the democratic republic of the congo ( ), a number of egyptian fruit bats were found to be infected with marv ( , , , ) . unfortunately, though the african rock python (python sebae) and forest cobra (naja melanoleuca) are part of the fauna in these locations, there were no reports on investigations of snakes from these caves for filovirus infection ( , , ) . nevertheless, our finding that two snake npc orthologs are nonpermissive to filovirus entry and infection due to a single amino acid change leads us to speculate that this change was an adaptation to reduce infection by a filovirus, thereby increasing host survivability. more-extensive wildlife sampling coupled with genetic and functional analysis of host-virus interactions associated with filovirus infection may uncover additional evidence for evolutionary arms races between filoviruses and multiple types of animals (bats, reptiles, and rodents). cells. vero grivet hek t and u os cells were maintained in high-glucose dulbecco's modified eagle's medium (dmem; thermo fisher scientific, waltham ma) supplemented with % fetal bovine serum (fbs; atlanta biologicals, flowery branch, ga) and % penicillin-streptomycin (thermo fisher scientific) at °c and % co . u os npc Ϫ/Ϫ cell lines were generated by crispr/cas genome editing as previously described ( ) and transiently transfected with npc constructs for colocalization experiments. chinese hamster ovary (cho) cells were maintained in dmem-ham's f- medium ( / mix) (thermo fisher scientific) supplemented with % fbs at °c and % co . cell lines were generated by a retroviral transduction system, as previously described ( ) , to stably overexpress the npc constructs in cho-m cells, which contain a deletion in the npc locus ( ) . freestyle -f cells were maintained in gibco freestyle expression medium (thermo fisher scientific) at °c and % co . npc constructs. npc domain c sequences (residues to ) flanked by sequences that form antiparallel coiled coils as previously described ( ) were cloned into the pcdna . (ϩ) vector. constructs made included glycosylation mutants in hsnpc domain c (l nϩd t, k nϩg s, and n a), while those in drnpc domain c were n a, n a, and r t. drnpc domain c chimeras were made by substituting these residues for human residues to (chimera ), to (chimera ), to (chimera ), to (chimera ), to (chimera ), and to (chimera ), and the point mutations made were e d, y f, i v, h y, f y, and s t. the constructs were then transiently transfected into hek t cells, and the supernatant with secreted protein was harvested after h and used in elisas. purified proteins were made by transfecting freestyle -f cells in suspension, harvesting cells h posttransfection, and purifying them by incubation with his- nickel resin. the proteins were eluted at mm imidazole and ph . and dialyzed into mm -(nmorpholino)ethanesulfonic acid (mes), mm nacl, ph . . domain c chimeras in the full-length npc were generated by seamlessly replacing the domain c sequences in hsnpc . the constructs were subcloned into the pbabe-puro retroviral vector and stably transfected into cho-m cells by retroviral transduction, as previously described ( ) . all constructs possessed n-terminal flag tags. vsv pseudotype infections. replication-incompetent vesicular stomatitis virus indiana (vsv) pseudotypes encoding enhanced green fluorescent protein (egfp) in the first position and ebov gp in place of vsv g were made as previously described ( , ) . ebov gpΔmuc matches the ebov/h.sapiens-tc/ cod/ /yambuku-mayinga isolate amino acid sequence (genbank accession number af ) but lacks the mucin-like domain (Δ - ; Δmuc) ( ) . unless otherwise indicated, virus titers were determined on vero grivet monkey cells by manual counting of egfp-positive cells. cleaved ebov gp (gp cl ) was generated in vitro using the bacterial protease thermolysin ( g/ml) (sigma-aldrich, st. louis, mo) for h at °c as described previously ( , ) , and the reaction was stopped by adding the metalloprotease inhibitor phosphoramidon ( mm) (sigma-aldrich). authentic ebola virus infections. cho cells, seeded in black cellcoat -well plates (greiner bio-one, north america, monroe, nc) were incubated with ebola virus/h.sapiens-tc/cod/ /kikwit- at the indicated multiplicity of infection in a biosafety level (bsl- ) laboratory located at usamriid. following a -h absorption, virus inoculum was removed and cells were washed once with phosphate-buffered saline (pbs). cells were then incubated at °c, % co , and % humidity for h, at which time the cells were washed once with pbs and submerged in % formalin prior to removal from the bsl- laboratory. formalin was removed, and cells were washed times with pbs. cells were blocked by adding % bovine serum albumin (bsa)-pbs to each well and incubating the cells at °c for h. cells were incubated with ebov gp-specific monoclonal antibody (mab) kz , diluted to g/ml in % bsa-pbs, at room temperature for h. cells were washed times with pbs prior to addition of goat anti-human igg-alexa fluor (thermo fisher scientific) secondary antibody. following a -h incubation with secondary antibody, cells were washed times prior to addition of hoechst (thermo fisher scientific) diluted in pbs. cells were imaged and percentages of virus-infected cells were calculated using the operetta high-content imaging system (perkinelmer, waltham, ma) and harmony high-content imaging and analysis software (perkinelmer). gp cl -npc domain c capture elisa. normalization of npc domain c supernatants and proteins was carried out as previously described ( ): resolution on sds-page gels followed by immunoblotting with anti-flag primary antibody (sigma-aldrich) and anti-mouse alexa- secondary antibody (thermo fisher scientific) and quantification on the li-cor odyssey imager (li-cor biosciences, lincoln, ne). capture elisas were also performed as previously described ( , ) . briefly, high-binding -well elisa plates (corning, corning, ny) were coated with kz ( ) ( g/ml in pbs) and then blocked using pbs containing % bovine serum albumin (pbsa). pseudotyped ebov was cleaved with thermolysin ( g/ ml) at °c for h and captured on the plate. unbound virus was washed off, and serial dilutions of either flag-tagged purified soluble npc domain c (domain c; to g/ml) or supernatants from transient transfections of the npc constructs on hek t cells were added. bound domain c was detected by a horseradish peroxidase-conjugated anti-flag antibody and ultra-tmb substrate (thermo fisher). ec values were calculated from binding curves generated by nonlinear regression analysis using prism (graphpad software, la jolla, ca). binding elisas were done in duplicate and in at least two independent experiments. all incubation steps were done at °c for h or at °c overnight. immunofluorescence. imaging was performed in u os or cho cells grown on -mm coverslips and fixed with % paraformaldehyde. for antibody staining, the coverslips were incubated with an anti-flag antibody (sigma-aldrich) in pbs containing . % triton x- and % bsa. detection was by incubation with alexa -conjugated secondary antibodies (thermo fisher scientific). for filipin staining, the coverslips were stained with g/ml of streptomyces filipinensis filipin iii complex (sigma-aldrich) in pbs for h. coverslips were mounted on glass slides using prolong antifade reagent (thermo fisher scientific), and images were acquired with an inverted fluorescence microscope equipped with a ϫ high-numerical-aperture oil objective. emergence of zaire ebola virus disease in guinea ebolavirus evolution: past and present discovery of an ebolavirus-like filovirus in europe fruit bats as reservoirs of ebola virus marburg virus infection detected in a common african bat isolation of genetically diverse marburg viruses from egyptian fruit bats ebola haemorrhagic fever growth of lassa and ebola viruses in different cell lines ebola virus haemorrhagic fever a system for functional analysis of ebola virus glycoprotein characterization of ebola 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cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry requires the host-programmed recognition of an intracellular receptor niemann-pick c (npc )/npc -like chimeras define sequences critical for npc 's function as a filovirus entry receptor niemann-pick type c (npc ) overexpression alters cellular cholesterol homeostasis filovirus receptor npc contributes to species-specific patterns of ebolavirus susceptibility in bats ebola viral glycoprotein bound to its endosomal receptor niemann-pick c direct visualization of ebola virus fusion triggering in the endocytic pathway structure of the ebola virus glycoprotein bound to an antibody from a human survivor host-primed ebola virus gp exposes a hydrophobic npc receptor-binding pocket, revealing a target for broadly neutralizing antibodies experimental inoculation of plants and animals with ebola virus international scientific and technical committee for marburg hemorrhagic fever control in the democratic republic of congo outbreak of marburg hemorrhagic fever among miners in kamwenge and ibanda districts, uganda response to imported case of marburg hemorrhagic fever, the netherland seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection niemann-pick type c function requires lumenal domain residues that mediate cholesterol-dependent npc binding endosomal proteolysis of the ebola virus glycoprotein is necessary for infection covalent modifications of the ebola virus glycoprotein role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry ebola virus can be effectively neutralized by antibody produced in natural human infection we thank tyler krause, cecelia harold, and tanwee alkutkar for technical support. we are grateful to zachary a. bornholdt for useful discussions on the gp structure and jens h. kuhn for comments on a preliminary version of the manuscript. we thank daniel s. ory for his generous gift of cho-m cells. key: cord- - gkdotvt authors: liu, william j.; shi, weifeng; zhu, wuyang; jin, cong; zou, shumei; wang, ji; ke, yuehua; li, xiaofeng; liu, mi; hu, tao; fan, hang; tong, yigang; zhao, xiang; chen, wenbin; zhao, yuhui; liu, di; wong, gary; chen, chengchao; geng, chunyu; xie, weiwei; jiang, hui; kamara, idrissa laybor; kamara, abdul; lebby, matt; kargbo, brima; qiu, xiangguo; wang, yu; liang, xiaofeng; liang, mifang; dong, xiaoping; wu, guizhen; gao, george f.; shu, yuelong title: intra-host ebola viral adaption during human infection date: - - journal: biosaf health doi: . /j.bsheal. . . sha: doc_id: cord_uid: gkdotvt the onsite next generation sequencing (ngs) of ebola virus (ebov) genomes during the – ebola epidemic in western africa provides an opportunity to trace the origin, transmission, and evolution of this virus. herein, we have diagnosed a cohort of ebov patients in sierra leone in , during the late phase of the outbreak. the surviving ebov patients had a recovery process characterized by decreasing viremia, fever, and biochemical parameters. ebov genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection, including the previously described short stretches of serial t>c mutations. remarkably, within individual patients, samples collected during the early phase of infection possessed ts at these nucleotide sites, whereas they were replaced by cs in samples collected in the later phase, suggesting that these short stretches of t>c mutations could emerge independently. in addition, up to a total of nucleotide sites spanning the ebov genome were mutated coincidently. our study showed the dynamic intra-host adaptation of ebov during patient recovery and gave more insight into the complex ebov-host interactions. the - epidemic of ebola virus disease (evd) in western africa is the largest evd outbreak to date, with , confirmed, probable, and suspected cases and , deaths as of june , (http:// who.int/csr/disease/ebola/en/). during this epidemic, the ability to perform next-generation sequencing of ebola virus (ebov) from patient specimens in the field [ , ] has generated ebov genomes, which helped to trace the origin, evolution, and transmission of ebov in west africa [ ] [ ] [ ] [ ] [ ] [ ] [ ] . based on the phylogenetic analysis, all of the ebov in this outbreak can be traced back to a few cases in guéckédou, guinea. subsequently, the virus spread to neighboring countries, such as sierra leone, liberia, nigeria, senegal, and mali [ , , ] . along with their geographical spread in west africa, ebov formed different lineages [ ] . these lineages are characterized with different single nucleotide polymorphisms (snps) emerging from different time points during the outbreak [ , [ ] [ ] [ ] , ] . these snps occur in both the non-coding and encoding regions, containing indispensable phylogenetic and evolutionary information [ , , ] . previous research demonstrated that the hotspots for non-synonymous substitutions are likely located in regions with a lower level of functional constraint of the encoded viral proteins [ ] . moreover, the intra-host selection for ebov to escape from a developing humoral immune response may drive the diversifying selection of glycoprotein (gp) mucin-like domain, as shown by the enrichment of mutations within the b-cell epitopes of gp [ ] , although this was not observed in ni's study [ ] . on the other hand, short stretches of intra-hostt to c (tnc) mutations were also observed [ , ] . this was speculated as a result of adenosine deaminases acting on rna (adars), which is yet unclear [ ] . it has been reported that viruses with the tnc mutations (genome positions - ) continued to circulate in the magazine wharf area, freetown, sierra leone, causing several infections [ ] . in particular, intra-host single nucleotide variations (isnvs) appeared during the course of the epidemic, within the b cell epitopes of gp and non-coding regions across the ebov genome [ , ] . interestingly, several isnvs were shared by two or more patients, which represented a combination of human-to-human transmission and recurrent mutations [ ] . these isnvs were used to estimate the effective viral population size within a single patient, during a transmission bottleneck [ ] , and to identify human-to-human transmission chains [ , , ] . furthermore, two isnvs were able to influence the transcription level of an adjacent gene of nucleocapsid protein (np) by two-fold [ ] . recent studies also showed that, during the epidemic, the ebov isolates from early in the outbreak with amino acid substitutions in the gp protein possessed increased tropism for human cells, indicating human adaptation of ebola virus during human-to-human transmission [ ] [ ] [ ] . however, only a few of the previous studies described longitudinal sequence data from a single patient [ , ] , and the intra-host dynamic evolution of ebov during disease progression is still largely unknown. at the sierra leone-china friendship biological safety laboratory (sle-chn bio-safety lab) [ ] , we cared for a cohort of ebov-infected patients (n = ) in the ebola treatment units (etus) in freetown, sierra leone, from mid-march to late june . the dynamic viremia [ ] and biochemical features during disease progression were characterized. by utilizing a deep sequencing platform in the field [ ] , we successfully generated virus genomes from longitudinally collected blood samples from some of the patients. surprisingly, we observed coincident emergence of serial nucleotide variations, including the previously defined short stretch of tnc mutations during the recovery process. in a single patient, genome sequences obtained from samples during earlier stages of the acute infection phase possessed ts at the tnc positions, whereas cs were found from samples collected during the recovery process. phylogenetic analyses showed that after the tnc mutations occurred, all strains possessing such mutations were grouped together, but were not clustered together with their earlier sequences without such tnc mutations from the same patient. our results suggested that such tnc mutations could arise independently within single patients, reflecting the host-adaptation of ebov during infection. we undertook a cohort study of patients admitted to jui ebola treatment centre (sierra leone-china friendship hospital) between march , and june [ ] . we used a standard case definition consistent with who guidelines. all patients were included in the study except for those who died upon arrival, or those who had no blood results within h of admission. primary survival outcome measure was collected from the ebov treatment centre. samples were tested at the on-site laboratory sle-chnbio-safety lab for the presence of ebov rna by real-timert-pcr against the glycoprotein (gp) and nucleoprotein (np) gene targets [ ] , after inactivation and manual rna extraction. positive results were reported as cycle threshold values. all patients received a rt-pcr test upon admission. the piccolo express system (abaxis, ca, usa) was used to generate metabolic and liver function profiles. amylyte was used to assay for blood biochemistry and liver function profile in the same day as the sample collection. all data were collected as part of routine patient care, and recorded on standardized forms, which were kept securely. the clinical data extracted for research purposes were anonymized and stored on a password-protected database. the sierra leone ethics and scientific review committee provided approval for the study. this work was conducted as part of the surveillance and public health response to contain the evd outbreak in sierra leone. blood samples from suspected individuals and oropharyngeal swab samples from corpses were collected for evd testing and outbreak surveillance, with a waiver to provide written informed consent during the evd outbreak under the agreement between the sierra leone and chinese governments. the activities were coordinated by the emergency operations centre in the charge of sierra leone ministry of health and sanitation during - epidemic of ebola virus disease (evd) in western africa, the ebola virus (ebov) genomes generated from the patient specimens based on next-generation sequencing platforms in the field helped to quickly trace the origin, evolution, and transmission of ebov. in particular, intra-host single nucleotide variations (isnvs) appeared during the course of the epidemic across the ebov genome. whether and how these isnvs could emerge from a single patient during the disease progression, are still largely unknown. evidence before this study some of the isnvs, such as short stretches of intra-host t to c (tnc) mutations were shared by two or more patients, which represented a combination of human-to-human transmission and recurrent mutations. furthermore, isnvs could appear in the key sites, such as the b cell epitopes of gp and non-coding regions across the ebov genome, which may influence the transcription level of an adjacent gene. in a cohort of ebov-infected patients in the ebola treatment units (etus) in freetown, sierra leone, the recovery processes of the patients were represented by the dynamic viremia and biochemical features. by utilizing longitudinally collected samples during the recovery process, we successfully generated series of virus genomes from the patients. we observed coincident emergence of serial nucleotide variations, including the previously defined short stretch of tnc mutations during the recovery process. phylogenetic analyses showed that after the tnc mutations occurred, all strains possessing such mutations were grouped together, but were not clustered together with their earlier sequences without such mutations from the same patient. our results suggested such tnc mutations could arise independently within single patients, reflecting the host-adaptation of ebov during infection. our data indicate that short stretches of tnc substitutions are part of the convergent evolution during the infection process of evd patients, shedding light on the dynamic intra-host genomic variation of ebov during the - epidemic. and who. all the information regarding individual persons has been anonymized in the report. the genome sequencing was performed as described previously [ ] . briefly, rna samples extracted from whole blood (two positive oropharyngeal swab samples with high ct values were not involved) from evd patients were reverse transcribed to cdna. pcr amplifications were performed with ebov-specific primer pairs with overlaps. amplicons from one patient were pooled for library preparation. ngs was performed using the bgiseq- (ion proton) platform. all sequenced reads were filtered to remove the low quality and short reads. the genome sequences of the viruses were assembled by mapping the filtered reads to the ebov consensus sequence (genbank: kt ) using roche newbler version . (roche), and the mutation site was manually checked with original sequencing data. clean reads were mapped to the ebov genome (kj ) by tmap . . . we then scanned the ebov genome site-by-site and determined the nucleotides for each genomic site according to mapping results. finally, the ratios of four nucleotides at each site were obtained. among the samples sequenced, full-length or nearly full-length ebov genomes were successfully generated in this study (table s ). the other samples with only short partial of the ebov genomes were not analyzed in this study. all of the mutation sites were counted for each released ebov fulllength genome, using the zaire ebolavirus isolate h.sapiens-wt/gin/ /makona-kissidougou-c (genbank accession no. kj ) as the reference sequence. the existence of substitutions within a genome was screened within the ebov genomes publicly available from genbank. a "c strain" was defined as the existence of or more t-c substitutions within a genome window width of nt. our dataset included full-length or nearly full-length ebov genomes sequenced in this study and ebov genomespublicly available from genbank. amaximum-likelihood phylogenetic tree was inferred using the software raxml, with the gtrgamma model and bootstrap replicates. the genomes and ngs data of the ebov viruses were deposited in genbank with the access numbers mf -mf (table s ). from march th to june th, , patients who had symptoms meeting the definition of suspected evd were admitted to the sierra leone-china friendship hospital in freetown. the blood specimens were collected and delivered by the sample center of ministry of health and sanitation (mohs), sierra leone. the sle-chn bio-safety lab tested the blood samples for ebov through a double-channelreal-time rt-pcr detection kit targeting both gp and np genes of ebov. a total of ( . %) patients were confirmed to have evd ( table ). the first blood samples collected from these patients after hospitalization were tested for laboratory confirmation, and the ct values from the gp and np channels matched well with a high linear correlation ( figure a ). of these patients, the most common clinical features at presentation included fever in patients ( . %; mean temperature, . °c) and gastrointestinal symptoms, e.g. abdominal pain, in patients ( . %) and anorexia/loss of appetite in patients ( . %) ( table ) . ocular signs were also common in the patients ( with conjunctivitis [ . %] and with pain behind the eyes [ . %]). the mean age of the patients was years (range, to ) and patients ( . %) were male ( table ). the case fatality rate of the evd patients was . % ( / ), similar to the overall ratio of . % ( , / , ) during this ebov epidemic. there was no significant difference (p = . ) in the average interval from symptom onset to presentation between survivors and non-survivors( figure b) . the mean body temperature on the day of hospitalization was . °c among survivors, significantly lower (p = . ) than that of nonsurvivors, . °c ( figure c ). the initial viremia of survivors and nonsurvivors was also significantly different, with non-survivors possessing lower mean ct values for both gp ( . for non-survivors vs . for survivors, p = . ) and np ( . for non-survivors vs . for survivors, p = . ) ( figure d ). .when there are two longitudinally-collected blood samples with ebola rna negative, the patient will be discharged. the corresponding temperatures (purple) during the blood sampling. blood samples from patients ( survivors and fatality ) were available for longitudinal collection during the hospitalization. as in the previous study [ ] , the viremia of the survivors ameliorated after the presentation, as revealed by increased ct values (figure ). interestingly, in our study, two different trends in the variation of body temperatures of the patients after hospitalization were observed. consistent with viremia, body temperatures of patients , , , and decreased after hospitalization. on the other hand, in patients , , , , , , and , a transient increase in body temperature after hospitalization was detected. especially in patient , the ct value had a dramatic decrease from . on day (hospitalization) to . on day ( days after hospitalization), and the body temperature of the patient elevated from . °c on day to . °c on day . on day , the viremia and fever of this patient decreased. in contrast to the survivors, the moribund patient had a continuous viremia and the body temperature increased from . °c on day to . °c on day . the patient died on day . blood biochemical parameters were also tested in the sle-chn biosafety lab in the field (figure ). hematological and biochemical abnormalities were observed in the patients upon hospitalization, which was also reported in the previous studies (biochemical testing in a laboratory tent and semi-intensive care of ebola patients on-site in a remote part of guinea: a paradigm shift based on a bleach-sensitive point-ofcare device. clinical, virological, and biological parameters associated with outcomes of ebola virus infection in macenta, guinea.). hyperglycemia occurred in patients , , upon hospitalization and ameliorated afterwards. abnormal concentrations of blood urea nitrogen, potassium, sodium, and chlorine were observed in the patients, especially in patients and . hematological abnormalities detected during the disease progression included reduced concentrations of hemoglobin and hematocrit. these biochemical abnormalities alleviated gradually during the recovery process. finally, full-length and four nearly full-length ebov genomes from patients were obtained based on the ngs platform in the field. through analysis of the ngs data of longitudinally collected blood samples from the same patient, we found that the previously reported short stretch of tnc mutations within positions - appeared in the late blood samples of the patients (day ) and (days and ), while these patients still possessed ts in early blood samples (day for patient and day for ) ( figure a ). in particular, in the day sample of patient , almost no cs were observed at the positions, whereas cs accounted for approximately % on day and~ % on day at all of the tnc nucleotide sites ( figure a ), indicating that day may be an intermediate status between days and . the similar ratios of t:c at the different tnc nucleotide sites on the same day may indicate that these tnc mutations occurred concurrently, though not all these mutations can be found on the same nucleic acid strands (reads). however, in patient , dominant percentages of cs (n %) were already observed on day , and continued increasing in percentage on day (~ %) ( figure a) . meanwhile, we found additional coincident substitutions distributed across the entire ebov genome (table and figure ) , which has the similar ratios for the substitutions as in the short stretch of the tnc mutations between positions and . this may also indicate that all these substitutions were correlated to each other. these nucleotide variations not only included tnc substitutions, but also other types of substitutions, e.g. cnt, gna, ang, ant, tna, and even one tndeletion mutation at site (table s ). these nucleotide variations occurred coincidently among different patients during the acute infection phase. the intra-host adaptation of ebov was also illustrated as the continuous change of isnv substitution ratios (the ratios of latter dominant isnv/former dominant isnv, for instance, ratios of c/t for the tnc nucleotide sites) across longitudinal sampling points in patients , , and ( figures b-d) . figure c ). for patient , substitution ratios remained n , indicating a dominant role of the latter isnv on both day and day ( figure d) , however, the increasing trend of the isnv substitution ratios could still be observed from day to day . however, there were still some exceptional sites which had consistent isnvs during the disease process (supplementary figure s ). all exceptional sites possessed the latter form at the target sites (e.g. site of viral genome had already mutated to c in patient on day ), indicating early substitution events at these sites. phylogenetic analysis of the novel and publicly released fulllength ebov genome sequences from genbank showed that the novel ebov sequences did not cluster together ( figure a ). alternatively, they formed four small independent clusters scattered across lineage sl , which was circulating during the late stage of the outbreak in . we further analyzed all ebov genomes available online and identified the strains with multiple tnc substitutions within a short genomic region in different positions of the genomes (termed as "c strains") ( figure b and table s ). a total of strains, including six sequenced in this study, possessed multiple tnc nucleotide substitutions (table s ). these "c strains" could be classified into at least different types according to the number of tnc substitutions and their positions ( figure b ). interestingly, apart from the well-known stretch with serial tnc mutations at genome position - , stretches with and serial tnc mutations were also found in the intergenic region between vp and gp in strains g . (genbank no. kr ) and libr (genbank no. kt ) (table s ) , respectively. to study the phylogenetic association of these "c strains", we mapped them onto the tree ( figure b and supplementary figure s ). however, these "c strains" scattered across the entire tree, with little phylogenetic association ( figure b ), indicating multiple independent origins of these tnc substitutions. however, some strains possessing the same tnc substitution type were clustered together (supplementary figure s ). based on current evidence, human-to-human transmission is the most plausible reason for these cases. in particular, ten strains possessed the tnc mutations within positions - ( figure c ). the prototype strain with the tnc mutations was j (kp ), sequenced from the magazine wharf area, freetown, sierra leone in november [ ] . it has been reported that j -like ebov continued to circulate in this region and had infected at least three additional patients by july . however, the strain sequenced from patient on days and the ratio of the reads of latter/former. c " " means that the corresponding data of the survivor was shown in figure b -d. d " " means that the corresponding data of the survivor was shown in supplementary figure s . e the ratio is termed as " " when the read of the former nucleotide is . f "n/a" means that the reading depths of the ngs was lower than . g "vp -vp " means that the substitutions locate in the non-coding region between vp and vp . possessed the tnc mutations and clustered together with other such "c strains". surprisingly, on day of patient , the virus possessed ts at all of the positions, and did not fall within this cluster. this was also observed with patient ( figure c ). virus samples collected on day from patient was located in a different cluster as the virus sequenced from the day sample. we further identified eight ebov strains sharing the previously described tnc mutations within positions - (the last column in table ). we called all the other viruses ( strains) as "t strains ( - )". interestingly, we found that almost all coincident substitutions that happened in our longitudinally sequenced ebov genomes (table ) had already mutated in these eight "c strains ( - )". in contrast, none of the other ebov "t strains ( - )" without the tnc mutations at position - have these substitutions. these data suggest that all substitutions distributed within the whole genome of ebov, including the short stretch of tnc mutations within position - , had a coincident evolution trend during the recovery process of the patients. in this study, we sequenced longitudinally-collected blood samples from ebov patients based on an in-fieldngs platform at the sle-chnbio-safety lab during the - ebov epidemic. although more a thousand of ebov genomes have been sequenced during the - ebov epidemic in west africa, most of the viruses were from different patients during the early days of the acute phase during the infection [ ] [ ] [ ] [ ] [ ] , , , ] . phylogenetic analysis showed that the genomes sequenced in this study were not grouped together. this suggested that in the late stages of the - ebov outbreak, the virus became diversified and various minor ebov lineages had been cocirculating in sierra leone. previous phylogenetic studies indicated that snps carried by different ebov lineages were important molecular markers to study the virus transmission among humans [ , , ] . short stretches of the tnc substitutions appeared sporadically without phylogenetic association as shown in our analysis ( figure b ). although the exact reason is yet unclear, this was speculated to be the result of adenosine deaminases acting on rna (adars) [ ] . thus, it is understandable that the tnc substitutions can occur in both the coding regions and the non-coding regions. in the present study, we found several related isnvs across the ebov genome occurring in the late acute phase of ebov infection, including a short stretch of tnc mutations within positions - . however, these mutations were not found in samples collected during the early infection phase. in one patient, we even observed the intermediate stage for these tnc mutations. these data suggest that the serial tnc mutations were emerged coincidently, and that the mutations could arise independently in different ebov patients. since only a few tnc mutations are able to change the transcription level of adjacent genes [ ] , such serial tnc mutations in our study, though some in non-coding regions, might be functional during ebov infection in humans, which should be a subject of further investigation. meanwhile,further analysis should be performed to investigate whether all these mutations can be found on the same nucleic acid strands (reads). phylogenetic analysis showed that earlier samples without the tnc mutations were not grouped together, whereas later samples with the tnc mutations were clustered together and formed a separate cluster. we . the schematic diagram for the dynamic intra-host substitution and inter-host transmission of ebov. base on the dynamic adaptation of ebola virus in the patients we described herein, we further proposed the schematic model for the relationship of the human to human transmission and the dynamic adaptation of the ebola viruses. panel a shows the disease process of one survived patient a from the preclinical period to symptom presentation, and then to recovery. during this process, the dominant viruses in the patient are the t strain which possess the former nucleic acids in the sites (table ), e.g. t in the tnc stretch ( - ). there also will be emergence of the c strains during the recovery process, which possess the latter nucleic acids in the sites (table ), e.g. c in the tnc stretch ( - ) due to unknown reasons as we indicated in our patients. t strain virus dominates in the acute detoxification period of patients and certain patients died during this period. thus in the general human to human transmission of ebola virus (patient a to patient c), the transmitted viruses are t strain virus, which takes the dominates in the ebov genomes of the west africa outbreak publicly available from genbank. however, we cannot exclude the possibility of the sporadic transmission of c strains in humans with close contact (a to b). patient b, who was infected by the c strain of ebola virus, may have a mixed quasi-species of the virus during the diseases process. this possibility requires further exploration. also identified a total of strains with serial tnc substitutions that belonged to different tnc mutation types, with different number of tnc substitutions and/or within different genomic regions. generally, strains possessing different tnc mutation types were not clustered together, but scattered across the tree. based on current evidence, there was no direct phylogenetic association between these different mutation types. this once again revealed that these various tnc mutation types could arise independently. however, some of the tnc substitutions were indeed shared by a few strains, which were clustered together in the tree. thus, the transmission hypothesis cannot be fully rejected yet [ ] . this study is the first showing that linked and coincident mutations observed in ebov could arise independently during human infections. it can be rationally proposed that the "t strains" are the dominant circulating ebovs during the epidemic ( figure ). most of the cases are infected via contact with the patients during the early stages of acute infection. during the late stages of the infection, the "c strains" emerge, although the virus titer has become lower during this period. transmission may also occur rarely between close contacts at this stage of disease. this may explain why the "c strains" could be found in such few patients in the acute infection phase. meanwhile, the tnc mutation happened at different time points of the patients after ebov infection, which may be related with disease progress or prognosis. for patient , the corresponding sites for these tnc were still t, while the tnc substitution was completed on day of the infection of patient . the fever duration and the viremia duration of patient were longer than . however, the relationship of the occurrence time of these mutations with disease progress or prognosis should be further investigated. overall, our data indicate that short stretches of tnc substitutions are part of the linked and convergent evolution during the infection process of evd patients, shedding light on the dynamic intra-host adaptation of ebov during the - ebov epidemic. supplementary data to this article can be found online at https://doi. org/ . /j.bsheal. . . . genetic diversity and evolutionary dynamics of ebola virus in sierra leone evolution and spread of ebola virus in liberia ebola virus epidemiology, transmission, and evolution during seven months in sierra leone genomic surveillance elucidates ebola virus origin and transmission during the outbreak mutation rate and genotype variation of ebola virus from mali case sequences diseases usamrioi, national institutes of h, integrated research facility-frederick ebola response t, monitoring of ebola virus makona evolution through establishment of advanced genomic capability in liberia distinct lineages of ebola virus in guinea during the west african epidemic ebola virus disease in west africa-the first months of the epidemic and forward projections emergence of zaire ebola virus disease in guinea the evolution of ebola virus: insights from the - epidemic non-coding regions of the ebola virus genome contain indispensable phylogenetic and evolutionary information intra-host dynamics of ebola virus during enhancement of replication of rna viruses by adar via rna editing and inhibition of rna-activated protein kinase genotypic anomaly in ebola virus strains circulating in magazine wharf area high-resolution genomic surveillance of ebolavirus using shared subclonal variants human adaptation of ebola virus during the west african outbreak ebola virus glycoprotein with increased infectivity dominated the - epidemic functional characterization of adaptive mutations during the west african ebola virus outbreak preliminary evaluation of the effect of investigational ebola virus disease treatments on viral genome sequences on the ground in sierra leone detection and analysis of ebola virus in sierra leone-china friendship biosafety laboratory from on the ground in western africa: from the outbreak to the elapse of ebola ebola viral load at diagnosis associates with patient outcome and outbreak evolution hundreds of chinese and sierra leonean staff on-site took part in the sample collection, transportation, detection, and data analysis. we especially acknowledge the excellent work of four previous detection teams of chinacdc (mobile and fixed labs) and the essential support (materials and reagents) of the national institute for viral disease control and prevention, china cdc. we also express our deep condolences to the family and associates of dr. abdul kamara of the ministry of health and sanitation, sierra leone, who contributed a lot to this project but succumbed after the successful control of ebola epidemic in sierra leone. the authors declare that there are no conflicts of interest. key: cord- -mbrw og authors: flego, michela; frau, aldo; accardi, luisa; mallano, alessandra; ascione, alessandro; gellini, mara; fanunza, elisa; vella, stefano; di bonito, paola; tramontano, enzo title: intracellular human antibody fragments recognizing the vp protein of zaire ebola filovirus inhibit the protein activity date: - - journal: bmc biotechnol doi: . /s - - - sha: doc_id: cord_uid: mbrw og background: ebola hemorrhagic fever is caused by the ebola filovirus (ebov), which is one of the most aggressive infectious agents known worldwide. the ebov pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. the multifunctional viral vp protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (ifn-α/β) response, and represents a suitable target for the development of strategies to control ebov infection. phage display technology permits to select antibodies as single chain fragment variable (scfv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. scfv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scfv is expressed as intracellular antibody (intrabody) or delivered into the cells. results: monoclonal antibodies (mab) in scfv format specific for the ebov vp were isolated from the eth- library of human recombinant antibodies by phage display technology. five different clones were identified by sequencing, produced in e.coli and expressed in cho mammalian cells to be characterized in vitro. all the selected scfvs were able to react with recombinant vp protein in elisa, one of the scfvs being also able to react in western blot assay (wb). in addition, all scfvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in a cells showed that two of the scfvs can significantly hamper the inhibition of the ifn-β-induced rig-i signaling cascade mediated by ebov vp . conclusion: five antibodies in scfv format recognize an active form of ebov vp in elisa, while one antibody also recognizes vp in wb. two of these scfvs were also able to interfere with the intracellular activity of vp in a cell system in vitro. these findings suggest that such antibodies in scfv format might be employed to develop therapeutic molecules able to hamper ebov infections. ebola hemorrhagic fever caused by the ebov is one of the most aggressive zoonoses affecting humans, leading to death within a few days of the exposure [ ] . six species of ebov are known to date, named after the geographical region in which they were first isolated: bundibugyo, reston, sudan, taï forest (formerly côte d'ivoire ebov), zaire and bombali ebov. sudan, taï forest, and zaire ebov are responsible for outbreaks in humans, whereas reston ebov infects non-human primates, and bombali virus was recently discovered in bats [ , ] . fatal ebov infections are characterized by rapid viral replication combined with an inadequate antiviral response. hallmarks of fatal cases are immune suppression with t cells levels below the normal level, no cd t cell activation, delay of antibody response in the blood, and high viremia ( genome copies/ml serum). the ebov pathogenesis starts with the subversion of both innate and adaptive immune response and the consequent induction of harmful inflammatory responses and tissue necrosis due to disseminated infections [ , ] . ebov has a linear, single-stranded, negative rna genome about , nucleotides in length. it is composed of seven genes coding for eight proteins in this order: np (encoding the nucleoprotein), vp , vp , gp (encoding the glycoproteins), vp , vp , l (encoding the polymerase). the gp gene codes for the two molecular forms gp and gp , generated by rna editing [ ] . vp is a conserved multifunctional protein which is a cofactor of the viral rna polymerase complex along with the np, vp , and l protein. its activity starts at an early stage of the ebov infection; it is also a doublestranded rna-binding protein shown to be implicated in hampering the innate immune response [ ] by blocking the ifn-mediated antiviral activity through multiple inhibitory effects which include disruption of the rig- pathway by preventing irf- phosphorylation [ , ] , and inhibition of activation of the ifn-inducible dsrna and dicer-dependent protein kinase r [ ] . antibody phage display technology makes it possible to select from an artificial immune system human antibodies in scfv format capable of specifically recognizing an antigen of interest. scfvs, consisting of the vh and vl chain regions of a whole immunoglobulin (ig), are the smaller portion still retaining the binding properties of the parental ig. this format is ideal for genetic manipulation in order to obtain antibody constructs potentially useful for diagnostic and therapeutic applications [ ] . furthermore, scfv antibodies can be expressed inside the cell as intrabodies so as to bind to their intracellular target antigen [ ] . the two main mechanisms underlying the efficacy of intrabodies are: ) knockdown of the activity of cytosolic antigens through cytosolic intrabodies [ ] [ ] [ ] ; ) diverting of antigens from their natural intracellular compartment by scfv binding due to an extra-signal for intracellular localization [ ] [ ] [ ] . intrabodies can be used to reveal the function of proteins by interfering with their function, although the possibility of targeting intracellular antigens gives them a therapeutic potential for a number of viral infections [ , ] , neurological diseases [ , ] and cancers [ , , , ] . this study reports the selection by phage display and characterization of different human scfv antibodies binding to an active form of the zaire ebov vp [ , ] . the ability of these scfvs, expressed as cytosolic intrabodies, to reverse the inhibition of type i ifn induction by intracellular expression of vp was tested by a luciferase reporter gene inhibition assay in a cells treated with dsrnas [ ] . in order to isolate antibodies specific for the recombinant vp expressed in e.coli and purified in an active form [ , ] , an approach based on the phage display technology was used. in the eth- library which we used, the diversity (about clones) has been introduced in the complementary-determining region (cdr ) of both the variable heavy chain (vh) and variable light chain (vl) domains [ ] . to recover antigen-specific antibody phages, an aliquot of the eth- antibody library containing cfu phage was used for the panning procedure as described elsewhere [ , ] . in fig. soluble scfvs derived from iptg-induced colonies were screened by elisa to find those specific for the vp protein. all the e. coli colonies corresponding to the clones exhibiting an od λ value in elisa higher than . , were grown and subjected to dna extraction and sequence analysis. several clones had identical nucleotide sequences and five different clones, namely b , a , e , f and h , were identified; the amino acid composition of the complete sequences of the vh and vl domains is shown in fig. along with the schematic representation of a scfv gene in the phagemid cassette. the scfv reactivity towards vp was further characterized by elisa (fig. , panel a) and wb (fig. , panel b) using vp recombinant antigen. the protein glucose oxidase (go) and an anti-go scfv for detection were used as negative controls. the anti-vp reactivity in elisa was confirmed for all b , a , e , f and h scfvs, while in wb the positivity was only observed for scfv a , which reacted with a kda protein identified as the recombinant his-tagged vp protein also detected by the anti-his mab. the scfvs b , e , f , h recognized their antigen in elisa but showed no reactivity in denaturing conditions of wb, suggesting that they probably recognize conformational epitopes. for its part, a is still reactive in wb and probably recognizes a linear epitope. the specificity of the anti-vp reactivity of b , a , e , f and h scfvs is confirmed by the observation that they did not react with the irrelevant go antigen either in elisa or in wb (fig. ) . in order to use the scfvs in the ebov vp luciferase reporter gene inhibition assay, the scfv genes selected were pcr amplified with opportune oligonucleotides and cloned into the ptarget vector for expression in the eukaryotic system. in view of the cytoplasmic vp localization, it was not necessary to provide the scfvs with signal sequences for expression in specific cell compartments. transfection experiments were performed in the cho cells to evaluate the functionality of the scfv ptarget constructs as described in methods. the a , h , b , f and e constructs were all able to express scfvs of the expected molecular mass in eukaryotic cells (fig. , panel a) . evaluation of the anti-ebov vp scfvs ability to restore the ifn-β activity by a luciferase reporter gene inhibition assay to evaluate the capability of the scfvs b , a , e , f and h to block the vp activity, the cell-based miniaturized luciferase reporter gene inhibition assay, previously described [ ] , was used. the assay measures the capacity of the vp expression to inhibit the ifn-β induced by dsrna treatment, in a cells. before performing the ebov vp luciferase reporter gene inhibition assay using the scfvs, it was crucial to exclude that the expression of an irrelevant scfv could influence the ifn-β induction, either in the absence or in the presence of vp expression. to this end, a cells, transfected with the pgl ifn-β luc and the pcdna -ebov-vp expression plasmid, were co-transfected with the irrelevant anti-go scfv expression plasmid. further, to exclude any non-specific effect due to the transfection procedure, the cells were co-transfected in parallel with the pgl ifn-β luc expression vector and an empty pcdna vector. the luciferase signal emitted in the case of pgl ifn-β luc and anti-go scfv concurrent expression, was comparable to that obtained with the ifn-β positive controls. also, in the case of ebov vp and anti-go scfv simultaneous expression, the measured ifn-β signal was comparable to that obtained with the ebov vp expressed alone (fig. , panel b) . the results confirmed that ifn-β induction was affected by neither the transfection procedure nor the irrelevant scfv either in the presence or in the absence of vp expression. next, we tested all the scfv ptarget constructs in the dsrna rig-i-mediated luciferase reporter gene inhibition assay. two of the five scfvs tested, f and e , showed a significant ability (p = . and p = . , respectively) to subvert the inhibition of the ifn-β production generated by dsrna, after the vp expression. by contrast, scfv h , a and b showed no ability to counteract the inhibition of ifn-β induction mediated by ebov vp in the cellular assay (fig. , panel c). competitive elisa using the scfv-expressing phage to verify whether the two different scfvs e and f , able to subvert the inhibition of the ifn-β production, targeted different epitopes, we performed a competitive elisa (fig. ). this relies on detection of the scfv-expressing phage particles that compete with soluble non-phage-fused scfvs for binding to the antigen immobilized on elisa plate. it was not possible to detect the soluble non-phage-fused scfvs using the anti-tag flag ab because the tag is also present on the phage. to determine the best scfv expressing-phage concentration for competition tests, identified as × tu/ml, we first performed a phage elisa experiment in which different scfv-expressing phage concentrations were tested on vp coated plates (data not shown). in a competitive assay, elisa plates coated with vp or with the control antigen go were blocked; they were then incubated with purified scfv-expressing phage both in the absence and in the presence of the competitor soluble non-phage-fused scfv at the maximum concentration of μg/ml. the binding of the scfv-expressing phages was measured. the detection step was via the phage coat protein using an anti-m mab conjugated to hrp. the anti-go scfv-expressing phage clone was assayed against the anti-go soluble nonphage-fused scfv as a positive control for competitive measurement (fig. , panel a) . it was found that μg/ml of soluble non-phage-fused scfv could inhibit the binding of the scfv-expressing phage in a dose-dependent manner. binding in the presence of specific anti-go soluble non-phage-fused scfv was compared: to binding in the presence of e anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the presence of f anti-vp soluble non-phage-fused scfvs (p value = . ); to binding in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. next, we assayed the binding of e scfv-expressing phages both in the absence and in the presence of the competitor soluble non-phage-fused scfv against itself as an intrinsic positive control, against anti-go soluble non-phage-fused scfv as a negative control and against f soluble non-phage-fused scfv for competitive measurement. the inhibiting activity was determined at a single fixed concentration of μg/ml. f soluble nonphage-fused scfv shows a clear competitive effect at the concentration used (fig. , panel b) . binding in the presence of f soluble non-phage-fused scfv was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and in the absence of soluble non-phage-fused scfvs (p value = . ) using student's t-test. as a further confirmation, we performed a one-shot experiment by detecting the competition suffered by the f scfv-expressing phages when co-incubated with e soluble non-phage-fused scfvs and with the control anti-go soluble non-phage-fused scfv at the concentration of μg/ml. also in this case we observed that the control anti-go soluble non-phage-fused scfv has no competitive binding effect. the e soluble non-phage-fused scfv competes with the binding of f scfv expressing-phages. binding in the presence of e soluble non-phage-fused scfvs was compared to the binding in the presence of anti-go soluble non-phage-fused scfv (p value = . ) and to binding in the absence of soluble non-phagefused scfvs (p value = . ) using student's t-test (data not shown). the urgency to find effective counter measures for the ebov disease (evd) was strengthened by the west africa outbreaks occurring in - , which resulted in , cases of ebola with , deaths [ ] , pushing the international scientific community to investigate the widest possible range of defense strategies to counteract the virus. however, the current ebola outbreak, which started in may in democratic republic of the congo (drc) and has caused cases and deaths to date [ ] , received only minor benefits from the use of new diagnostic assays, vaccines and drugs. the reason is that to control ebola outbreaks, transversal coordination between health facilities and communities is essential to achieve rapid isolation of the cases of the disease and to stop the transmission chain. detection of new cases at an early stage by rapid diagnostic tests and ring vaccination strategy with experimental vaccines on volunteers are therefore crucial for controlling ebov in the current drc outbreak [ ] . due to the variable onset of antibody response in the ebola-infected subjects, serology is not used in the acute evd diagnosis. conversely, virus and viral proteins accumulate in blood to detectable levels within a few days from disease onset. molecular tests based on the detection of viral proteins were developed and proved to be effective for diagnosis in acute infection [ ] . currently, in the case of suspected ebov infection, the world health organization (who) recommends a list of validated tests for detection of either viral rna by rt pcr or viral antigens by immunological tests [http://www.who. int/medicines/ebola-treatment/emp_ebola_diagnostics/en/]. most of the antigen-capture tests used in national reference laboratories [ ] utilize mabs generated in mice immunized with the recombinant np [ ] , vp [ ] or gp [ ] ebov proteins. during the recent outbreak, lateral flow immunoassays (lfis) emerged as powerful tools for rapid antibody-mediated antigen-capture practicable at the point of care [ ] . this confirmed the advantages of tests based on antigen-antibody reaction over rt-pcr methodology, which requires significant laboratory infrastructures often lacking in low-income countries. furthermore, in order to control the transmission-chain of the infection and limit virus spread, it is important to have tests that can be easily automated; as such, antigen capture tests meet this requirement. ebov vp is a validated drug target for which only a few small molecules have been reported to be active [ , , ] , albeit no drug has yet been approved. here, we present mabs in scfv format, which are able to react with the zaire ebov vp protein. this is a key viral protein whose action starts at an early stage of the infection and is based on interference with the host immune response by blocking the ifn-mediated antiviral activity. the antibodies presented here enrich the list of available anti-vp antibodies. they could be used individually or in combination to develop novel reagents for ebov vp detection and therapeutics. regarding therapy, pools of neutralizing antibodies have been used in passive immunization of individuals with acute infection [ ] ; nevertheless, antibodies specific for the vp would act with a different mechanism with respect to neutralizing antibodies targeting surface glycoproteins. recent studies showed that targeting vp by either nucleic acid mimics or sirnas, provides protection against the ebov infection in murine [ ] as well as in non-human primate models [ ] . however, the effectiveness of antibodies targeting the vp has not yet been demonstrated in vivo. here, we show that two out of five scfv antibodies selected against the vp significantly hindered the inhibition of the rig-i signaling cascade mediated by vp , in a cellular system. we characterized the binding of these two scfv clones to the recombinant vp antigen by competitive elisa, and found that their epitope is at least partly shared. a more detailed analysis of the binding epitopes could further define whether it is a total or partial sharing and which antigenic region of ebov vp is involved in the inhibition of the interferon pathway. regarding the other anti-vp scfvs selected, we cannot exclude that the observed lack of functionality is due to incorrect scfv folding in the cytoplasmic environment. as antibodies are usually produced in an oxidizing biochemical environment with the help of er-based chaperones, only a fraction of naïve antibodies can be folded correctly in the reducing cytoplasmic environment which prevents the formation of disulfide bridges. however, many examples of successful intrabodymediated proteins knockdown in vitro, obtained using cytosolic intrabodies, have been reported in literature [ ] . scfvs selected in the extracellular environment were previously reported to work intracellularly [ , , ] depending on intrinsic biophysical characteristics such as stability, mainly ascribable to the scaffold. however, several methods have been developed to address the issue of cytosolic intrabodies functioning [ ] . interestingly, anti-vp scfvs able to interfere with vp activity, were recently isolated from a phage library different from the eth- and were linked to a cell-penetrating peptide for intracytoplasmic delivery [ ] . therefore, although we used a different delivery system, our data strengthen the idea that intracellular antibodies in scfv format can be used to counteract ebov vp activity. scfvs against different intracellular ebov targets could be used either to develop a well-defined cocktail of antibodies with different specificities or also in combination with other drug molecules for therapeutic purposes, provided that an appropriate delivery system is developed. furthermore, the vp amino acid sequence is highly conserved among the zaire ebov isolated in several outbreaks (> . % amino acids identity). therefore, it can be hypothesized that the scfvs selected retain a broad-spectrum activity. nevertheless, the efficacy of these new antibodies should be further evaluated either in ebolavirus infected cells or in animal models of ebolavirus infection. five scfv antibodies against an active form of the zaire ebov vp were isolated and characterized. their specific reactivity in elisa and wb suggests the possibility of developing novel reagents for ebov vp detection during the virus life cycle. the two anti-vp scfvs f and e proved to be able to interfere with the vp -depending inhibition of ifn activity in a cell system, so suggesting that such antibodies also represent potential therapeutic agents. further investigations into an ebov infection system in vitro and in animal models are necessary to validate these reagents. the synthetic library of recombinant human antibodies (eth- ) consists of about scfv polypeptides displayed on the surface of the m phage. the library was built by random mutagenesis of the complementarity-determining region (cdr ) of the variable domains of both the heavy (vh) and light (vl) chain of immunoglobulins, using only three antibody germline gene segments (dp for the vh, dpk , and dpl for the vl). in the vh, diversity was created by randomizing four to six positions replacing the pre-existing positions to of the cdr ; in the vl, diversity was obtained by randomizing six positions ( to ) of the cdr [ ] . an aliquot of the eth- library, containing cfu phage, was used to isolate specific human antibodies in scfv format against the recombinant vp protein ( ) . immunotubes (nunc maxisorp; denmark) were coated overnight (on) at room temperature (rt) with purified recombinant vp protein ( μg/ml in pbs). after panning, phages were eluted with ml of mm triethylamine and the solution was immediately neutralized by adding . ml of m tris-hcl ph . . the eluted phages were used to infect an e. coli tg strain in a log phase, and amplified for the next round of selection, as described in flego et al. [ ] . three rounds of panning were performed to recover vp -specific antibody phages from the eth- library. for the preparation of soluble anti-vp scfvs, individual colonies were grown in flat bottomed wells (nunc) for h at °c in μl of . % glucose xyta medium and induced with μl of mm iptg/ xyta medium. the following day, the plates were spun down at g for min, and the supernatants containing soluble scfvs were recovered and tested for specificity with purified vp in elisa and wb, as described in flego et al. [ ] . plasmid dna from individual bacterial colonies clones was extracted using the quiaprep spin miniprep kit and subjected to enzymatic restriction; sequence analysis of the cdr regions was then performed with an automated dna sequencer (biofab, roma, italy) using the fdseq ( ′-gaa ttt tct gta tga gg- ′) and pelbback ( ′-agc cgc tgg att gtt att ac- ′) primers. the scfv gene clones reacting with the recombinant vp in elisa, were pcr amplified using the following primers: eth nco as a forward primer: ′ gcgc acc atg gcc gag gtg cag ctg ′. nhe i stop his as a reverse primer: ′ gcgc gct agc cta atg atg atg atg atg atg tgc ggc cgc gcc tag gac ′ containing the xhis tag sequence. for transient expression in eukaryotic cells, the amplimers were cloned in ptarget (promega) under the strong viral promoter (cytomegalovirus immediate-early enhancer). the clones obtained were sequenced to check for mutations possibly introduced by pcr, and used to transfect cho cells using jetpei dna transfection reagent, according to the manufacturer's instructions. transiently transfected cells were lysed after h with sds-loading buffer ( mm tris-hcl ph . , % sds, % -mercaptoethanol, % glycerol), loaded onto % sds-page, and then transferred to a nitrocellulose membrane using standard procedures. the membrane was blocked in % mpbs on at rt. blotted proteins were incubated for h in % mpbs with μg/ml of tet-ra·his antibody (qiagen), which was the only anti-his mab able to recognize the tag at the scfv cooh terminus, in our experimental conditions. after an additional incubation for h at rt in the presence of goat anti-mouse antibody hrp-conjugate ( μg/ml, dako), the reaction was developed and visualized with a chemiluminescence detection kit (pierce; il, usa). luciferase reporter gene assay ifn-β induction luciferase reporter gene assays a cells ( × per well) were transfected in -well plates with t-pro p-fect transfection reagent (t-pro biotechnology) with the construct pgl ifn-β luc, kindly provided by prof. stephan ludwig (institute of molecular virology, münster, germany). twenty-four hours after transfection, cells were additionally transfected using iav pr vrna and incubated for a further h at °c with % co . cells were harvested with lysis buffer ( mm na-mes ph . , mm tris-hcl ph . , mm dithiothreitol, . % triton x- ). the crude cell lysates were cleared by centrifugation and μl of cleared lysates were added to μl of luciferase assay buffer ( mm na-mes ph . , mm tris-hcl ph . , mm magnesium acetate, . mg/ ml atp) in a white -well plate. immediately after addition of μl of mm d-luciferin into each well, the luminescence was measured in victor luminometer (perkin elmer). the relative light units (rlu) were normalized as the fold activity of the unstimulated control. each assay was carried out in triplicate. the above described luciferase reporter gene assay was also performed for evaluating the ifn-β induction inhibition mediated by ebov vp . twenty-four hours after co-transfection with pgl ifn-β luc and pcdna or pcdna ebov wtvp expression vectors, cells were transfected with the ctrl anti-go scfv ptarget as an irrelevant scfv and with the different scfv p target vectors the next day, cells were additionally transfected with iav vrna. inhibition of luciferase expression was indicated either as the fold activity of the unstimulated control or as a percentage of the induced control. each assay was carried out in triplicate. competitive elisa using scfv-expressing phages for elisa competition assay, we used soluble nonphage-fused scfvs produced and purified as in gellini et al. [ ] . scfv-expressing phages were produced from a monoclonal bacterial culture grown to od = . - . and infected with m k helper phage in a ratio of around : phage/bacteria. one hundred ml of supernatant containing scfv-expressing phages were x concentrated by precipitation with peg and resuspended in pbs. phage titer was determined by plating of bacteria infected with phages at scalar dilutions. coating was performed with vp or go antigen as described in the elisa section. the following day, the plate was blocked with % mpbs for h at rt and washed with tpbs. twenty-five μl of soluble non-phage-fused scfvs at two-fold the desired final concentration were pre-incubated in % mpbs with the antigen for min prior to the addition of μl of the scfv-expressing phage mix at two times the desired final tu/ml in % mpbs, and incubated for h at rt. the plate was washed with tpbs and incubated with anti-m pviii coat protein mab conjugated to hrp (amhersham) diluted : , for h rt. a washing step was conducted followed by the addition of peroxidase substrate as described above. ebola haemorrhagic fever ebola virus disease the discovery of a new ebolavirus, bombali virus, adds further support for bats as hosts of ebolaviruses ebola virus: new insights into disease aetiopathology and possible therapeutic interventions how ebola and marburg viruses battle the immune system strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit the ebola virus vp protein functions as a type i ifn antagonist ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain production technologies for monoclonal antibodies and their fragments expression and targetingof intracellular antibodies in mammalian cells generation and functional characterization of intracellular antibodies interacting with the kinase domain of human egf receptor intracellular antibody capture technology: application to selection of intracellular antibodies recognising the bcr-abl oncogenic protein effects of intrabodies specific for rotavirus nsp during the virus replicative cycle intracellular anti-e human antibodies in single-chain format inhibit proliferation of hpv -positive cervical carcinoma cells in vivo antitumor effect of an intracellular single-chain antibody fragment against the e oncoprotein of human papillomavirus a novel intracellular antibody against the e oncoprotein impairs growth of human papillomavirus -positive tumor cells in mouse models characterization and binding of intracellular antibody fragments to the hepatitis c virus core protein generation and characterization of single-chain anti-body fragments specific against transmembrane envelope glycoprotein gp of maedivisna virus isolation of a human single chain antibody fragment against oligomeric α-synuclein that inhibits aggregation and 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affinity against a marker of angiogenesis eluted from a two-dimensional gel design and use of phage display libraries for the selection of antibodies and enzymes generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (n) sars-cov protein using a phage display approach ebola: anatomy of an epidemic who regional office for africa. ebola virus diseases new ebola outbreak declared in democratic republic of the congo rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome identification of essential outstanding questions for an adequate european laboratory response to ebolavirus zairewest africa detection of ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein development, characterization and use of monoclonal vp -antibodies for the detection of ebola virus production of monoclonal antibodies and development of an antigen capture elisa directed against the envelope glycoprotein gp of ebola virus strategies in ebola virus disease (evd) diagnostics at the point of care antiviral agents against ebola virus infection: repositioning old drugs and finding novel small molecules insights into ebola virus vp and vp interferon inhibitory functions and their initial exploitation as drug targets, infectious disorders -drug targets a randomized, controlled trial of zmapp for ebola virus infection vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study specific in vivo knockdown of protein function by intrabodies human transbodies that interfere with the functions of ebola virus vp protein in genome replication and transcription and innate immune antagonism generation of human single-chain antibody to the cd cell surface determinant specifically recognizing ewing's sarcoma tumor cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank philochem for the generous permission to use of the eth- antibody phage display library. we thank martin bennett for his help in revising the manuscript for grammar and style. authors' contributions mf isolated and characterized the scfvs, participated in the design of the research and drafted the manuscript. aa and mg performed wb analyses and scfv production. am worked on the design and the genetic construction of the anti-vp scfvs in ptarget vector and their expression in cho cells. pdb and la expressed the anti-vp scfvs in the ptarget vector and critically reviewed the manuscript. af and ef produced the recombinant vp , carried out ebov vp luciferase reporter gene inhibition assay and analyzed the data. sv coordinated the study design on the basis of the scfv isolation. pdb and et conceived, coordinated and supervised the study. all authors have read and approved the manuscript. this work was supported by sardinia regional government grants lr / (crp- /f i ), and intramural fund of istituto superiore di sanità. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- -u ts ur authors: furuyama, wakako; reynolds, pierce; haddock, elaine; meade-white, kimberly; quynh le, mai; kawaoka, yoshihiro; feldmann, heinz; marzi, andrea title: a single dose of a vesicular stomatitis virus-based influenza vaccine confers rapid protection against h viruses from different clades date: - - journal: npj vaccines doi: . /s - - -z sha: doc_id: cord_uid: u ts ur the avian influenza virus outbreak in highlighted the potential of the highly pathogenic h n virus to cause severe disease in humans. therefore, effective vaccines against h n viruses are needed to counter the potential threat of a global pandemic. we have previously developed a fast-acting and efficacious vaccine against ebola virus (ebov) using the vesicular stomatitis virus (vsv) platform. in this study, we generated recombinant vsv-based h n influenza virus vectors to demonstrate the feasibility of this platform for a fast-acting pan-h influenza virus vaccine. we chose multiple approaches regarding antigen design and genome location to define a more optimized vaccine approach. after the vsv-based h n influenza virus constructs were recovered and characterized in vitro, mice were vaccinated by a single dose or prime/boost regimen followed by challenge with a lethal dose of the homologous h clade virus. we found that a single dose of vsv vectors expressing full-length hemagglutinin (hafl) were sufficient to provide % protection. the vaccine vectors were fast-acting as demonstrated by uniform protection when administered days prior to lethal challenge. moreover, single vaccination induced cross-protective h -specific antibodies and protected mice against lethal challenge with various h clade viruses, highlighting the potential of the vsv-based hafl as a pan-h influenza virus emergency vaccine. influenza a viruses, which belong to the family orthomyxoviridae, have a single-stranded negative-sense rna genome consisting of eight segments. they are important zoonotic pathogens, with high morbidity in pigs, horses, poultry, and humans. influenza a viruses have two envelope glycoproteins (gps), hemagglutinin (ha) and neuraminidase (na), and are divided into subtypes based on antigenicity. subtypes h - ha and n - na have been isolated from water birds, the natural reservoir of influenza a viruses. , until , avian influenza a viruses were considered unlikely to be transmitted directly to humans because they do not bind the human sialic acid-α , -galactose (saα , gal) receptor with high affinity. however, highly pathogenic avian influenza (hpai) viruses can be transmitted from wild birds upon close contact causing sporadic outbreaks in domestic poultry. this happened for the first time in in hong kong when human cases of respiratory illness, including six fatalities, were caused by hpai subtype h n viruses. [ ] [ ] [ ] since then, human cases, with deaths (~ % case fatality rate), have been reported by the world health organization. furthermore, some reassortant h viruses with different na subtypes (e.g. h n , h n , and h n ) originated from the same ancestral h n virus, and have recently emerged in china and spread to other countries in eurasia and north america. [ ] [ ] [ ] [ ] [ ] [ ] since some hpai viruses are resistant to the currently available treatment options for influenza a virus infections namely oseltamivir, amantadine, and interferon (ifn), , the development of vaccines is an ongoing effort of high priority for public health to be prepared for a potential epidemic or pandemic of hpai. several different vaccination strategies have been developed against influenza a viruses including inactivated whole virus, liveattenuated influenza virus, viral vectors, and dna vaccines. currently, the fda-approved and licensed whole virus and liveattenuated vaccines against human influenza a viruses are mainly produced in embryonated chicken eggs and the manufacturing process can take up to months. [ ] [ ] [ ] unfortunately, the high pathogenicity of hpai viruses for the chicken embryo reduces virus growth complicating efforts to obtain quality allantoic fluid with high virus titers. therefore, hpai viruses are not suitable as seed viruses for inactivated virus-based vaccine production. vesicular stomatitis virus (vsv) is a single-stranded negativesense rna virus in the family rhabdoviridae. although vsv can cause disease in livestock and other animals, it is highly restricted by the human ifn response and generally does not cause any or only very mild disease. the vsv platform used here is based on the attenuated replication-competent vaccine that produces a rapid and robust immune response to foreign antigens after a single immunization and has been shown to protect against numerous pathogens. [ ] [ ] [ ] [ ] [ ] especially, the vsv-based ebola virus (ebov) vaccine, vsv-ebov (also known as rvsv-zebov or ervebo), which expresses the ebov gp instead of the vsv gp, is considered safe and highly immunogenic based on data from multiple clinical trials. , noteworthy, vsv-ebov has shown promising efficacy against ebov in a phase iii clinical trial and is currently being used in the democratic republic of the congo during the ongoing ebov outbreak. the promising safety profile of this liveattenuated vaccine and the favorable immune cell targeting mediated by the ebov gp makes vsv-ebov an interesting platform for vaccine development. the feasibility of this concept has previously been demonstrated in preclinical studies with vaccines for influenza (hpai virus), flavi-(zika virus), and bunyaviruses (andes virus). , [ ] [ ] [ ] in this study, we designed and tested different vsv-ebov-based vaccine vectors expressing different versions of the h n ha (a/vietnam/ / (vn/ )) to demonstrate the feasibility of the platform for a fast-acting pan-h vaccine. mice were vaccinated with a single dose or prime/boost regimen of the different vaccine candidates and challenged with a lethal dose of homologous h n virus. we found that a single vaccination with vsv-vectors expressing the full-length ha (hafl) induced crossreactive h -specific antibodies and conferred complete protection against lethal challenge with various h clade viruses. furthermore, a single dose of these vaccine vectors provided uniform protection in mice against lethal h n challenge within days after vaccination. we generated vsv-based h vaccine vectors by inserting either the full-length open reading frame (orf) of the h n hafl (vn/ ) or a soluble version of this gene lacking the transmembrane and cytoplasmic domains but carrying a mutated single-basic cleavage site to prevent cleavage in the cells and a gcn leucine zipper domain (shazip) for stabilization of the trimeric structure into the vsv-ebov vector , (fig. a) . this shazip antigen has previously been shown to be protective in chickens as a subunit vaccine. we also generated a vsv vector expressing the h n hafl alone without the ebov gp (vsv-hafl; fig. a ) in order to control for the contribution of the ebov gp to vaccine efficacy. expression of the different h antigens from the vsv vectors was confirmed by subjecting the supernatant of infected cells to sds-page and immunoblotting (fig. b, supplementary fig. ). first, we showed the presence of vsv particles by detecting the vsv matrix (m) protein in the supernatant of infected cells (fig. b, supplementary fig. ). the incorporation of ebov gp into vsv particles differed among the vectors and was, as expected, highest for vsv-ebov for which it is the only surface protein and antigen encoded by this vector (fig. b, supplementary fig. ). expression of shazip was verified by detecting the non-cleaved sha precursor likely secreted from infected cells. hafl expression was demonstrated by detecting the furin-cleaved fragment ha in mature spikes on vsv particles. as expected, the incorporation of hafl into recombinant vsv particles was much stronger for vsv-hafl compared to vsv-ebov-hafl, likely because it is the only surface gp and encoded antigen in the vsv vector. next, we performed a series of studies measuring the rate and extent of vaccine virus growth over time. vero e cells were infected in triplicate with each vsv-based vector (multiplicity of infection (moi) . ) and samples were collected from the supernatant at , and h for titration. wild-type vsv (vsvwt) grew more rapidly and to significantly higher titers early post infection compared to any of the other recombinant vsv-based vectors (fig. c) . we did not observe any significant difference in the growth kinetics of vsv-based vectors expressing either one (vsv-ebov, vsv-hafl) or two foreign antigens (vsv-ebov-shazip, vsv-ebov-hafl) with most vectors reaching peak titers between and tcid /ml at h suggesting that the expression of a second antigen did not significantly further attenuate vsv-ebov (fig. c) . ( tcid ) of hpai h n . as expected, control and vsv-ebov vaccinated mice succumbed to the lethal h n challenge within days (fig. ) . single-dose vaccination of the vsv-ebov-shazip showed only . % protection against h n infection with severe weight loss (fig. , left panels). prime/boostvaccination improved the outcome of the vsv-ebov-shazip resulting in mild disease as evidenced by temporary weight loss and moderate disease with % survival for vsv-ebov-shazip (fig. , right panels). in contrast, single and prime/boost vaccination with vsv-ebov-hafl or vsv-hafl protected % of the mice from lethal challenge with no signs of clinical disease (fig. ) . in order to improve the protective efficacy of the vsv-shazip vector, we wanted to increase the antigen expression levels. therefore, the shazip or sha (without trimerization domain) antigens were inserted further upstream into the vsv-ebov backbone resulting in two additional vaccines, vsv-shazip-ebov and vsv-sha-ebov (supplementary fig. a ). these vaccine viruses were recovered and antigen expression was confirmed in the cell supernatant similarly to the previously generated vaccines ( supplementary fig. ). in vitro growth kinetics demonstrated no difference in comparison to the other vsv constructs (fig. b, supplementary fig. b ). next, we analyzed the protective efficacy of these improved vsv-sha-ebov vaccine candidates in mice. single-dose and prime/boost-vaccinations with the vsv-shazip-ebov revealed similar protective efficacies compared to vsv-ebov-shazip (fig. , supplementary fig. c ). interestingly, the h n challenge of vsv-sha-ebov-vaccinated mice demonstrated higher survival rates compared to shazip expressing vsv vectors ( fig. , supplementary fig. d ). taken together, the challenge experiments demonstrated that hafl is the superior antigen to any of the sha versions as a single dose results in uniform protection using the vsv platform. interestingly, sha performed better than shazip. analysis of vsv-based vaccine-mediated antibody responses total anti-ha (h ) immunoglobulin g (igg) and neutralizing antibody responses from all vsv-vaccinated mice were analyzed in serum samples collected directly prior to challenge (day ) and day and day post challenge. enzyme-linked immunosorbent assay (elisa) was performed to determine total anti-ha igg levels in the serum of the mice over time (fig. , supplementary fig. ). in control and vsv-ebov-vaccinated mice, we observed no antibody responses on day , but ha-specific igg was detected on day after h n challenge with all animals succumbing to infection by day (fig. , supplementary fig. ). all haflvaccinated mice responded with ha-specific antibody responses to a single dose on day (fig. , top left panel) that were lower compared to those of the corresponding prime/boost vaccinated mice (fig. , top right panel). the same was observed for sha/ shazip-vaccinated mice ( supplementary fig. ). vsv-hafl prime/ boost vaccination elicited the highest ha-specific igg responses that were significantly higher compared to control mice and the mice vaccinated with vsv-ebov, vsv-shazip-ebov, and vsv-ebov-shazip. in all vaccinated and surviving mice, the h n challenge served as a boost as documented by the increase in haspecific igg titers measured on day post challenge ( fig. , supplementary fig. ). time to immunity of the vsv-based hafl vaccines finally, we determined the minimum time to immunity for the two most promising vaccine candidates, vsv-ebov-hafl and vsv-hafl. groups of eight female balb/c mice were im vaccinated with × pfu of vsv-ebov-hafl or vsv-hafl on days , , or prior to lethal homologous h n challenge. we found that both vaccines resulted in % protection with no or little weight loss when mice were vaccinated at least days prior to challenge, whereas vsv-ebov-vaccinated mice succumbed to infection within days (fig. ) . furthermore, the day − vaccinations resulted in partial survival with . % for vsv-hafl and % for vsv-ebov-hafl (fig. ) . overall, the data demonstrate that both vaccine candidates are equally potent inducers of rapid protection with a slight but not statistically significant benefit of vsv-ebov-hafl over vsv-hafl. cross-protection with a single dose of the vsv-based hafl vaccines due to frequently occurring antigenic changes with influenza viruses, it is important to determine if vaccine candidates elicit antibodies against viruses from different antigenic clades within the same subtype. therefore, we performed hemagglutinin inhibition (hi) tests to examine the ability of the vsv-based h n vaccines to generate cross-neutralizing antibody responses against heterologous h influenza viruses. for this, we used the day mouse serum samples and a panel of nine attenuated candidate vaccine influenza viruses encoding has belonging to different h clades that were isolated from geographically distinct locations (supplementary table ). we found that prime/boost vaccination with vsv-ebov-hafl or vsv-hafl elicited crossneutralizing antibodies against all tested clades (table ) . crossneutralizing antibodies were also detected in the single-dose vaccination group of these two vaccines; however, and similar to the total ha-specific igg, levels were lower and crossneutralization was not detected for all clades (table ). in contrast, all other vaccines expressing the sha/shazip antigen revealed no cross-neutralizing activities after administration of a single-dose and limited cross-neutralizing activity after the prime/boost. these results demonstrated that the vsv-based vaccines expressing hafl induce more potent cross-neutralizing antibodies than the sha/ shazip antigens. in order to support the cross-protective potential of the vsvbased hafl vaccines, we vaccinated groups of mice with a single dose of × in contrast, all mice vaccinated with vsv-ebov-hafl or vsv-hafl were completetly protected aginast lethal challenge (fig. ), but mice challenged with a/indonesia/ / showed minor body weight loss on days - after challenge (fig. c) . taken together, the vsv vectors expressing hafl confer h cross-protection in the mouse model. the outbreak of human h n -caused disease in hong kong was controlled with the depopulation of poultry. however, while this outbreak was contained, hpai h n viruses have been circulating in poultry for almost two decades now and have spread to more than countries. the broad geographic distribution of hpai h n viruses and the risk of transmission to humans causing severe pneumonia with high case fatality rates are a major concern to animal and human health since years. treatment is an option for individual human cases but if these hpais gain transmissibility for humans, vaccines are likely the only public health measure to fight an epidemic or potential pandemic. in this study, we used the well-characterized vsv-ebov vaccine as our starting platform as it has advantages over other vaccine approaches such as ease of genetic modification, efficient and cost-effective manufacturing, proven human safety and immunogenicity profile, and potential favorable immune cell targeting. , to define a more optimized vaccine approach, we generated several different vsv-ebov-based vaccine vectors and compared the protective efficacy against hpai h n virus challenge in the mouse model to a vsv-hafl vector without the ebov gp. despite promising results from previous studies in chickens showing that adjuvanted subunit vaccines consisting of the trimeric h sha (shazip) induced high levels of cross-neutralizing antibodies (clade and . . ), , we could not demonstrate convincing protection with the shazip-expressing vsv vectors in this study (fig. , supplementary fig. c ). in fact, vsv-sha-ebov without the trimerization sequence performed better than the shazipexpressing vectors with complete protection following prime/ boost vaccination (supplementary fig. c) . in contrast to all the sha-based vaccines, single doses of the vsv-ebov-hafl or vsv-hafl vectors were sufficient to provide complete protection from lethal homologous h n challenge in mice (fig. ) . thus, in our study, the vsv vectors expressing hafl are superior over those expressing sha or shazip. it should be noted that vaccine doses in this study were about to times lower than those used in previous vsv-based hpaiv h n vaccine studies. , , this is an important observation, as lower-dose vaccination would likely reduce potential adverse effects of vaccination as has been reported ocassionally from human clinical trials using vsv-ebov vaccination. recently, it has been shown that low-dose vaccination with vsv-ebov does not compromise protective efficacy in nonhuman primates. lower-dose vaccination would also have a beneficial effect on vaccine manufacturing. currently, h hpai viruses have been classified into several clades based on phylogenetic analysis of their ha genes. notably, mainly clade viruses have evolved rapidly and extensively in recent years, and the continued evolution of this particular virus has heightened the concern for a pandemic. thus, here we selected eight viruses from clade and one virus from clade (supplementary table ) to investigate the crossneutralizing nature of the vaccine-induced antibody response by hi test. we found that a prime/boost vaccination with the vsvs expressing hafl elicited cross-neutralizing antibodies against all tested h viruses (table ) suggesting that these vaccine vectors will likely cross-protect. the presence of hi antibodies with titers of : is considered protective as demonstrated in previous animal studies using poxvirus-based vaccination. crossprotection could indeed be demonstrated in mouse challenge experiments utilizing three different h clade isolates (fig. ) highlighting the cross-protective potential of the vaccines. while prime/boost vaccination with vsv-shazip-ebov or vsv-sha-ebov induced cross-neutralizing antibodies, the responses were generally lower in titer and detected in fewer animals. previous studies have shown that the influenza virus ha stem has the potential to induce broad protective immunity and that the removal of the transmembrane domain may affect the native conformation of the ha stem potentially destroying those conformational antibody epitopes. thus, the finding that our vsv vectors expressing sha/shazip did perform worse than those expressing hafl is likely due to the specific design of expressing a soluble antigen that lacks both the transmembrane and cytoplasmic domains. our studies demonstrate that vsv-based vaccines expressing hafl are superior over those expressing modified sha. however, this study did not provide any data supporting an advantage of including vsv-ebov as part of the vector design over just expressing vsv-hafl as both vectors performed similarly well with no statistically significant difference in efficacy following single-dose or prime/boost administration (fig. ) nor in antibody responses (fig. , table ). the lack of differences in ha-specific antibody responses is not necessarily in line with higher expression of hafl following vsv-hafl infection in tissue culture (fig. b) , but replication may be different in vivo. on the other hand, the postulated favorable immune cell targeting through vsv-ebov , - may balance the advantage of higher antigen expression by vsv-hafl. vsv-ebov has been shown to induce rapid protective immune responses in preclinical and clinical studies. , thus, this platform has the potential to be utilized as an emergency vaccine. while we could not demonstrate a significant difference between the vsv-hafl and vsv-hafl-ebov vaccine in regard to fast-acting properties, protection after immunization on day − is marginally better with vsv-ebov-hafl than vsv-hafl (fig. ) . this difference could be due to the favorable immune cell targeting of the ebov gp, , - but further studies with bigger animal group sizes are needed to prove this hypothesis. previous studies demonstrated that vsv-based vaccines provide rapid protection via involvement of the innate immune system combined with an early adaptive response, suggesting that the vsv-ebov-hafl and vsv-hafl vaccines may induce innate immune responses that are able to control the challenge virus, allow for the adaptive immune system to catch up and lead to protection of the mice. nevertheless, the fast-acting feature makes this vaccine extremely valuable for the public health response during an epidemic or pandemic as the vaccine could be strategically administered to more vulnerable populations such as elderly and hospitalized people keeping in mind the replicative nature of the vaccine vectors. vsv-based h influenza virus vaccine candidates have advantages compared to the currently used influenza virus vaccines including the ease of generation of the vectors as well as the vaccine production in cell lines which are already approved for manufacturing of human vaccines. a switch to cell line production would eliminate concerns regarding allergies to egg proteins. the downside of an attenuated, replication-competent vaccine approach such as vsv is adverse reactions to vaccination. however, previous preclinical vaccine work using the vsv platform, including immunization of several immunecompromised animal species, as well as clinical trials with vsv-ebov demonstrated low levels of vaccine-related adverse effects resulting in the general conclusion that the vsv vaccine platform is safe. , in addition, vsv-based replicating vaccines are efficacious at lower doses compared to non-replicating approaches and do not require adjuvants. in conclusion, we have developed two vsv-based vaccine candidates, vsv-ebov-hafl and vsv-hafl, that provide proof-ofconcept for rapid protection against hpai virus infection that are mediating cross-neutralizing responses. if clinical development confirms the promise of being fast-acting and strongly protective, vsv-based vectors might be a promising approach for the development of a pan-h influenza virus emergency vaccine. all infectious work was performed at the required containment level at the integrated research facility, rocky mountain laboratories (rml), division of intramural research (dir), national institute of allergy and infectious disease (niaid), national institutes of health (nih). vaccinations were carried out in bsl settings; h n s were handled exclusively under maximum containment. the animal work was approved by the institutional animal care and use committee (iacuc) and performed according to the guidelines of the association for assessment and accreditation of laboratory animal care, international and the office of laboratory animal welfare. all procedures on animals were carried out by trained and certified personnel following standard operating procedures (sops) approved by the institutional biosafety committee (ibc). humane endpoint criteria in compliance with iacuc-approved scoring parameters were used to determine when animals should be humanely euthanized. african green monkey kidney (vero e ) cells were grown in dulbecco's modified eagle's medium (dmem) (sigma-aldrich) containing % or % fetal bovine serum (fbs), mm l-glutamine, u/ml penicillin, and μg/ ml streptomycin (all from thermo fisher scientific). baby hamster kidney (bhk)-t cells were grown in minimum essential medium (mem) (thermo fisher scientific) containing % tryptose phosphate broth (thermo fisher scientific), % fbs, l-glutamine, penicillin, and streptomycin. madin-darby canine kidney (mdck) cells were grown in eagle's minimum essential medium (emem) containing % fbs, l-glutamine, penicillin, streptomycin, mem non-essential amino acid (thermo fisher scientific), and bicarbonate (thermo fisher scientific). the h n challenge viruses a/ the h hafl orf was constructed using the entire ha cdna sequence of h n a/vietnam/ / . the soluble ha (sha) orf was generated from the hafl orf by deleting the transmembrane domain and replacing the sequences encoding the polybasic cleavage site between ha and ha (pqrerrrkkrg) by one preventing cleavage (pqietrg). the sha with leucine zipper (shazip) was constructed of the sha orf by adding a gcn pll sequence for trimerization. all orfs were cloned into the patx-vsv-ebov plasmid encoding the ebov-mayinga gp. replicationcompetent recombinant vsvs (vsv-ebov-shazip, vsv-shazip-ebov, vsv-sha-ebov, vsv-ebov-hafl, and vsv-hafl) were generated as described previously. the complete sequence of the vsv vaccines was confirmed by sanger sequencing. detailed sequence information can be obtained from the authors upon request. the titer of each virus stock was quantified using standard plaque and tcid assays on vero e cells. the same vaccine virus stock was used for all in vitro and in vivo work. vero e cells were grown to confluency in a -well plate and infected in triplicate with vsvwt, vsv-ebov, vsv-ebov-shazip, vsv-shazip-ebov, vsv-sha-ebov, vsv-ebov-hafl, and vsv-hafl (moi of . ). the inoculum was removed, cells were washed three times with dmem, and covered with dmem containing % fbs, . μg/ml tpck trypsin (thermo fisher scientific), and u/mg na from vibrio cholerae (sigma-aldrich). tpck trypsin and na are required for the propagation of the vsv-hafl vaccine. supernatant samples were collected at , , , and h post-infection and stored at − °c. the titer of the supernatant samples was determined performing tcid assay on vero e cells. samples were generated in parallel from each vaccine virus stocks produced in vero e cells mixed : with sodium dodecyl sulfatepolyacrylamide (sds) gel electrophoresis sample buffer containing % β-mercaptoethanol and heated to °c for min. sds-page with all samples was performed in parallel on tgx criterion pre-cast gels (bio-rad laboratories) (supplementary fig. ). subsequently, proteins were transferred to a trans-blot polyvinylidene difluoride membrane (bio-rad laboratories). the membrane was blocked for h at room temperature in pbs with % powdered milk and . % tween (thermo fisher scientific). protein detection was performed using the following rabbit or mouse primary antibodies: anti-ha : (cat. # -t - , sino biological inc.), anti-ebov gp (zgp / . , μg/ml; kindly provided by ayato takada, hokkaido university, sapporo, japan), and anti-vsv m ( h , : ; kerafast inc.). after horse-raddish peroxidase (hrp)-labeled secondary antibody staining using either anti-mouse igg ( : , ) or antirabbit igg ( : ) (mouse cat. # - - ; rabbit cat. # - - ; both jackson immunoresearch), the blots were imaged using the supersignal west pico chemiluminescent substrate (thermo fisher scientific) and a fluorchem e system (proteinsimple). groups of female balb/c mice (n = ) were vaccinated im with × pfu of the vsv-based vectors in . ml (two sites, . ml each) on day − and − (prime/boost vaccination) or − only (single-dose vaccination). on the day of challenge (day ), four animals in each group were euthanized for serum collection. the remaining animals in each group were challenged intranasally (in) with ld ( tcid ) of hpai h n virus a/vietnam/ / . on day post challenge, four animals in each group were euthanized and samples were collected for serology. the remaining eight mice were monitored until days post challenge when a terminal blood sample was collected prior to euthanasia. for the time to immunity study, groups (n = ) of female balb/c mice were im vaccinated on day − , − , or − with × pfu of the vsv-hafl or vsv-ebov-hafl vaccine in . ml (two sites, . ml each). vsv-ebov was used as a control. all the groups were challenged in with ld ( tcid ) of hpai h n virus a/vietnam/ / . surviving mice were monitored until day post infection. for the h cross-protection study, groups (n = ) of female balb/c mice were im vaccinated on day − with × pfu of the vsv-hafl or vsv-ebov-hafl vaccine in . ml (two sites, . ml each). vsv-ebov was used as a control. all the groups were challenged in with tcid serum samples from h n -infectd mice were inactivated by gammairradiation and used in bsl according to ibc-approved sops. elisa plates were coated with µg/ml ( µl/well) of recombinant influenza ha (h ) (a/vietnam/ / ) antigen (ibt bioservices). after three washes with pbs/tween, plates were blocked with % bsa in pbs for h at room temperature, followed by three additional washes with pbs/tween. the plates were incubated with fourfold serial dilutions of the mouse serum samples for h at °c, and washed three times with pbs/tween. bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-mouse igg (h+l) (jackson immunoresearch) at a : dilution and tmb substrate (kpl). the reaction was measured using the synergy™ htx multi-mode microplate reader (biotek). titers were calculated by a parameter curve fitting model using microsoft excel software. the cutoff value was set as the mean optical density plus three standard deviations of the control samples. hi assay hi assays were performed using eight hemagglutination units/ μl of the different h viruses incubated with μl of the fourfold serial dilutions of each mouse serum sample (day ) in round-bottom -well plates for h at room temperature. then μl of a . % turkey red blood cell solution (innovative research) was added to each well. plates were covered and incubated for min on ice. hemagglutination titers were determined by the reciprocal of the last dilution containing agglutinated turkey red blood cells. hi titers represent the highest serum dilution that completely inhibited hemagglutination. statistical analysis was performed in prism (graphpad). data presented in figs c, (upper panels), (upper panels), (left panels), supplementary fig. c , and supplementary fig. d (upper panels) were examined using two-way anova with tukey's multiple comparison to evaluate statistical significance at all timepoints between all groups. significant differences in the survival curves shown in figs. (lower panels), (lower panels), (right panels), and supplementary fig. d (lower panels) were determined performing log-rank analysis. data presented in fig. and supplementary fig. were analyzed for statistical significance using one-way anova with multiple comparison. statistical significance is indicated as follows: p < . (****), p < . (***), p < . (**) and p < . 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cross-clade immunity in cats vaccinated with a canarypoxvectored avian influenza vaccine towards a universal influenza vaccine: different approaches for one goal enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates ebola haemorrhagic fever vsvdeltag/ebov gp-induced innate protection enhances natural killer cell activity to increase survival in a lethal mouse adapted ebola virus infection cell culture-based influenza vaccines: a necessary and indispensable investment for the future ebola vaccines in clinical trial: the promising candidates properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses we thank the animal care staff of the rocky mountain veterinary branch (niaid, nih) for their support of the animal experiments. we also thank david wentworth, vivien dugan, todd davis, and bin zhou of the virology surveillance and diagnosis branch, influenza division, centers for disease control and prevention for providing the candidate vaccine viruses and the h n viruses utilized in this study. this work was funded by the division of intramural research, niaid, nih. further information on research design is available in the nature research reporting summary linked to this article. the data supporting the findings of this study are available from the corresponding author upon reasonable request. h.f. claims intellectual property regarding the vesicular stomatitis virus-based vaccines for viral hemorrhagic fevers. no other competing interests are to be disclosed. supplementary information is available for this paper at https://doi.org/ . / s - - -z.correspondence and requests for materials should be addressed to a.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. this is a u.s. government work and not under copyright protection in the u.s.; foreign copyright protection may apply key: cord- -nuep nim authors: dewald, lisa evans; johnson, joshua c.; gerhardt, dawn m.; torzewski, lisa m.; postnikova, elena; honko, anna n.; janosko, krisztina; huzella, louis; dowling, william e.; eakin, ann e.; osborn, blaire l.; gahagen, janet; tang, liang; green, carol e.; mirsalis, jon c.; holbrook, michael r.; jahrling, peter b.; dyall, julie; hensley, lisa e. title: in vivo activity of amodiaquine against ebola virus infection date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: nuep nim during the ebola virus disease (evd) epidemic in western africa ( ‒ ), antimalarial treatment was administered to evd patients due to the high coexisting malaria burden in accordance with world health organization guidelines. in an ebola treatment center in liberia, evd patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. as artemether and artesunate are derivatives of artemisinin, the beneficial anti-ebola virus (ebov) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. amodiaquine is a widely used antimalarial in the countries that experience outbreaks of evd and, therefore, holds promise as an approved drug that could be repurposed for treating ebov infections. we investigated the potential anti-ebov effect of amodiaquine in a well-characterized nonhuman primate model of evd. using a similar -day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. however, the treatment regimen did not result in a survival benefit or decrease of disease signs in ebov-infected animals. while amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral. antiviral activity of amodiaquine and artesunate and their metabolites in cell culture. the antimalarial treatment asaq that was administered to evd patients in , is a coformulation of artesunate (as) and amodiaquine (aq). as and aq are rapidly metabolized in the liver to dihydroartemisinin (dha) and desethylamodiaquine (deaq), respectively. prior to in vivo evaluation, the drug asaq, its components, and the metabolites were characterized for their inhibitory effects on ebov (makona variant, ebov/mak) replication in cell culture ( table ). the data confirm previous reports that both aq and the metabolite deaq block ebov replication with similar activity (ic = . to . µm) in huh and vero e cell lines (ic = . to µm). in primary human macrophages, both aq and deaq exhibited elevated cytotoxicity, which precluded any interpretation of antiviral activity. in contrast, activity of as and its metabolite dha was weak or undetectable. combinatorial testing of aq and as or the metabolites, deaq and dha, did not reveal in vitro synergistic effects against ebov replication (data not shown). based on the in vitro data, the decision was made to evaluate aq in the nhp model of ebov infection. nhps with an aq dosing regimen similar to that used for evd patients in ebola treatment centers in . treatment consisted of a -day course of as-aq with the dose determined according to age of the patient. the dose range for aq in humans is . to mg/kg corresponding to a rhesus macaque equivalent dose range of . to . mg/kg based on body surface area . a pharmacokinetic (pk) study in rhesus macaques ( groups of males and females) was performed to monitor plasma concentrations of aq (fig. a ) and the active metabolite deaq (fig. b) . the study evaluated three daily oral doses of aq, using mg/kg or mg/kg. pharmacokinetics were determined by collecting blood samples at , . , , , , , , , , and hours (h) following dosing on day and day ( fig. ) . after days of dosing with mg/kg in healthy male and female macaques, aq was well-tolerated. the higher dose of mg/kg resulted in a variety of clinical observations including hypoactivity, shivering (muscle tremors) and/or diarrhea. extreme hypoactivity was observed in a single male of the mg/kg group that resolved after dosing ceased. animals in both the mg/kg and mg/kg groups had lower blood pressure readings than at predose on both dosing days, typically at and/or h post-dosing. clinical chemistry and hematology parameters for both doses were analyzed and fell within normal ranges with the exception of liver enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) were elevated . -to . -fold on day at h after the rd dose. the increases in alt and ast were seen in both male and female animals at both the and mg/kg dose regimens, though changes were not dose-related. these increases were considered toxicologically significant. aq and the metabolite deaq reached peak concentrations at . to h (t max ) after administration for both day and day dosing (supplementary tables and ). the elimination phase half-life (t / ) varied among the individual animals, ranging from to h for aq and to h for deaq. plasma concentrations of the metabolite deaq were higher than concentrations of the parent drug resulting in auc last , and auc inf values that were . -to -fold higher for deaq than for aq. the mg/kg aq dose resulted in c max for aq ( . - . ng/ml) and deaq ( - ng/ml) that were in a similar range as reported for the mg/kg dose in humans (aq: c max = . ± . ng/ml and deaq: c max = . ± . ng/ml) . based on the results of the pharmacokinetics study, the mg/kg aq dose was chosen for a study to evaluate the in vivo effect of aq in the nhp model of evd (study outline, supplementary fig. ). rhesus macaques were challenged intramuscularly (im) with a target dose of plaque-forming units (pfu) of ebov/mak (measured dose of pfu) on study day . the control group (n = , female, males) received vehicle treatment on days , , , , and postexposure. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , and after exposure to ebov. treatment group (n = , females, males) received a -day course of mg/kg of aq by oral administration on days , , and after exposure to ebov. all animals were euthanized or succumbed to disease by day postexposure (fig. a) . median times to disposition were days for placebo and treatment group , and days for treatment group with no significant difference between the placebo or treatment groups versus the treatment group (p = . and . , respectively). the animals in all groups became febrile by day or postexposure, followed by a marked decrease in body temperature preceding death (fig. b) . no significant difference in febrile illness or weight loss was observed between control and treatment groups (fig. b,c) . nhps had a mild reduction in activity and responsiveness by www.nature.com/scientificreports www.nature.com/scientificreports/ day postexposure with a severe decrease observed for most animals on day postexposure ( table ). loss of appetite and lymphadenopathy began on day to followed by dehydration and rash (petechial, maculopapular) on day postexposure. clinical signs did not differ significantly between the groups. viral loads in plasma were not reduced in treated animals. plasma viremia was quantified by rt-qpcr, and viral titer was determined by plaque assay. viral rna was first detected in the serum of some animals days after exposure, with a rapid and substantial increase by day for both placebo-and aq-treated animals. rna levels at necropsy were in the range of - viral rna copies/ml for all nhps (fig. a) . infectious viral titers followed a similar trend for all groups with a sharp increase in viral titer starting on day postexposure and reaching a peak plateau by day before animal death (fig. b ). viremia correlated with the onset of clinical signs of disease such as loss of appetite, lymphadenopathy, and fever. aq treatment did not reduce plasma viral rna copies or infectious virus titer regardless of when treatment was initiated. no significant difference in viral titers was observed between groups. amodiaquine-treated animals present with abnormal hematology and biochemical profiles and pathological disease that corresponds with ebola virus disease. the animals showed a similar hematological profile regardless of treatment. complete blood counts and serum chemistry analysis were performed at scheduled sampling points, before (days - , - , and ) and after (days , , , and at necropsy) exposure (figs. , ) . the chemistry values for aq-treated nhps followed the same trends as the control animal values. similar hematological and biochemical profiles were observed for all animals regardless of treatment both in aq-and placebo-treated animals. concurrent neutrophilia and thrombocytopenia were observed for aqand placebo-treated animals on day postexposure with ebov (fig. ) . as animals reached endpoint criteria, neutrophil levels decreased (fig. a) . the low-level neutrophil values on day postexposure were from necropsy samples only. nhps from all treatment groups exhibited a concomitant increase in serum creatinine and blood urea nitrogen levels, indicative of renal dysfunction often noted in nhps with concurrent evd (fig. a,d) . other biochemical abnormalities consistent with evd were also observed beginning on day postexposure, including substantial elevation of serum alt, ast, and gamma-glutamyl transpeptidase (ggt) levels that indicate hepatocellular damage (fig. b ,e,f). necropsies were performed on all animals to evaluate pathological disease associated with ebov infection following aq or placebo treatment. the pathologist was blinded to the group identification during necropsy and tissue evaluation. the pathological findings were similar in type and severity between treated and untreated animals, and the gross and histopathologic findings in all animals were consistent with typical evd in rhesus macaques. common gross findings included a cutaneous rash on the face, often extending to other regions of the body, dehydration, discoloration of the liver with increased friability, turgid spleen, and enlarged, often congested kidneys. at the microscopic level, all animals exhibited lymphoid depletion of the axillary lymph nodes, lymphoid depletion and necrosis of the spleen, and hepatocellular degeneration and necrosis. panniculitis and necrosis were observed at the virus challenge site of animals. samples from infected animals collected on days , , , and postexposure and on day of necropsy (days , or ) were analyzed for determination of plasma levels of aq and its metabolite deaq. data indicate negligible levels of aq in all but five plasma samples, each from a different animal and day (fig. a) . as the samples were diluted by a factor of . for decontamination protocol, the concentrations were likely below the limit of detection. plasma levels of the metabolite deaq were higher than aq and detectable in all treated animals from both nhp nhp nhp nhp nhp table . clinical scores for responsiveness of animals. responsiveness of unanesthetized animals was scored using the following criteria: (white) = alert, responsive, normal activity, free of disease signs or exhibits only resolved/resolving disease signs; = slightly diminished general activity, subdued but responds normally to external stimuli; = withdrawn, may have head down, fetal or hunched posture, or reduced response to external stimuli; = recumbent but able to rise if stimulated, or moderate to dramatically reduced response to external stimuli; = persistently recumbent, severely or completely unresponsive, or may have signs of respiratory distress. www.nature.com/scientificreports www.nature.com/scientificreports/ groups and (fig. b) . deaq was evident through day postexposure and/or necropsy in of animals in group and in all animals in group . plasma levels of deaq in ebov-infected nhps were compared at different time points (fig. ) . animals that were treated on days , and (group , fig. a ), had plasma deaq levels ranging from to ng/ml on days , , and postexposure. the highest deaq levels were detected in necropsy samples on day ( and ng/ ml). for animals treated on days , and postexposure (group , fig. b ), plasma concentrations ranged from - ng/ml on day . higher concentrations were detected on day ( - ng/ml) and day postexposure ( - ng/ml). plasma levels of deaq in infected nhps and healthy nhps were compared at select time points that were identical in the pk and the efficacy study. plasma samples from infected group animals on day correspond to pk-plasma samples taken at h after the rd dose. healthy animals had higher plasma levels of deaq ( - ng/ml) than infected animals ( - ng/ml) (fig. a) . similarly, plasma samples on day from infected group animals were in a lower range (n = , - ng/ml) than those of healthy animals at the corresponding time point ( h after rd dose, n = , - ng/ml) (fig. b) . on day , h after the rd dose, deaq levels in the plasma of two animals from group were in a similar range (n = , - ng/ml) as found in healthy animals (n = , - ng/ml) (fig. c,d) . repurposing drugs continues to be of interest to the healthcare professional community for the treatment of emerging and re-emerging hemorrhagic fever viruses such as ebola, marburg, and lassa viruses. extensive efforts to screen approved and established drugs for antiviral activity led to a panel of drugs with broad-spectrum antiviral activity profiles that are available, affordable, have well-characterized pk/safety profiles and could be used under the emergency use authorization (eua) mechanism , , , . while a number of fda-approved compounds have proven to be efficacious against ebov in vitro or in murine models of disease, clinical evaluation or evaluation in more relevant disease models, such as nhps, has been limited. aq is a well-known antimalarial drug with wide usage in the countries that experience outbreaks of evd. the drug has activity against several human pathogens of other virus families including corona-, flavi-, and bunyaviruses [ ] [ ] [ ] [ ] . a recent report showed that entry of lassa-gp pseudotyped virus was blocked by aq, indicating that the drug may also have value for the treatment of other viral hemorrhagic fevers . aq is known to have in vitro antiviral activity against ebov - . multiple mechanisms for antiviral activity have been proposed. aq may block ebov entry, which depends on the acidification of endosomes. as a cationic amphiphilic drug, aq can accumulate in the late endo/lysosome and, as a weak base, neutralize the acidic environment, thereby inhibiting cathepsin b activation required for fusion of the viral and endosomal membrane . aq has also been reported to bind and inhibit cathepsin b directly . in silico binding studies revealed that aq may also bind to the viral protein vp . we found that aq inhibits ebov replication in multiple cell types, including huh and vero e cells and primary human macrophages, with ic 's ranging from . - . um depending on experiment and the cell type (table ) . aq is rapidly metabolized to the active metabolite, deaq, following oral administration. in vitro studies confirmed that the active metabolite also has anti-ebov properties in huh and vero e cells, and primary human macrophages, with ic 's ranging from . - . µm µm depending on experiment and the cell type. in contrast, as and its metabolite dha demonstrated minimal to no in vitro antiviral activity against ebov when tested side-by-side to aq using our assay parameters. when used in combination, as and aq did not have a synergistic effect on ebov replication in vitro. whereas as could possibly act with aq to impact the whole body-system in response to ebov in a way that cannot be replicated in the in vitro assays, this study assumed that aq was the main contributing factor of the drug combination resulting in anti-ebov activity. www.nature.com/scientificreports www.nature.com/scientificreports/ the goal of the study was to treat animals with aq using a similar dosing strategy as for human patients, with a target blood concentration range of the parent compound aq of . ± . ng/ml . indeed, we found that a mg/kg aq dose using the -day treatment regimen resulted in correlating plasma levels of . - . ng/ ml in healthy nhps. however, this treatment regimen did not result in a survival benefit or decrease of disease symptoms in ebov-infected animals. these results are disappointing and highlight the importance of utilizing relevant animal models of evd to evaluate potential antivirals, including detailed characterization of pharmacokinetics and tolerability in the context of evd. whereas aq treatment did not lead to detectable beneficial effects on evd progression in nhps, there may be value in investigating novel aq derivatives that have been improved for anti-ebov potency and/or improved tolerability . a possible impact from aq side effects needs to be considered. rare instances of acute liver injury usually after prolonged treatment have been reported , . for this reason, aq and the combination drug asaq are recommended for use as a malaria treatment in endemic areas, but not for prophylaxis against malaria. the onset of hepatic injury is often associated with agranulocytosis. elevated liver enzymes, alt and ast, were observed in the healthy nhps during the pharmacokinetics study. given that the evd patients in were given a co-formulation of aq and as, it would have been of interest to test the combined regimen rather than just aq to evaluate potential implications of the two drugs on disease progression. whereas as has no measurable anti-ebov effect when tested in combination with aq in vitro, the drug could have an in vivo effect that is not captured in cell culture assays. as and artemether are derivatives of artemisinin and are both metabolized to dha, the active metabolite for the treatment of malaria. however, as www.nature.com/scientificreports www.nature.com/scientificreports/ is water soluble, and reaches peak concentration more rapidly with higher plasma c max than artemether, which could impact in vivo activity against ebov. another consideration is that as and aq may affect each other in terms of pk, drug metabolism, or disposition. for example, as co-administration with aq has been reported to reduce exposure to aq . questions remain on the possible contribution of as to the beneficial effect of the asaq treatment observed in evd patients . therefore, testing the co-formulation would have value, but without testing each drug singly as well, attribution of any observed effect to a specific component will be challenging. other confounding factors not measured in the gignoux study could have accounted for the beneficial effect of asaq . the potential impact of concurrent malaria and the type of antimalarial used in evd patients was not addressed in our study. in conclusion, treatment with aq did not have a beneficial effect on survival or the symptoms of evd in rhesus macaques under the conditions tested. whereas aq on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of aq when used in combination with another antiviral. www.nature.com/scientificreports www.nature.com/scientificreports/ cells and virus. vero e (atcc crl- , manassas, va), hela (atcc ccl- ), and huh (human hepatocellular carcinoma) cells were maintained following recommended protocols. human monocyte-derived macrophages (mdms) were generated as previously described . ebola virus/h.sapiens-tc/gin/ /makona-c (ebov/mak, genbank accession no. kx . ) was propagated in vero e cells (bei resources, niaid, nih: vero c (e ), african green monkey kidney, working bank #nr- ) as previously described . virus stock and challenge inoculum titers were determined by plaque assay on vero e cells as previously described . drugs and treatment preparation. in vitro studies. the antimalarial artesunate-amodiaquine (asaq winthrop) (sanofi-aventis, gentilly cedex, france) was solubilized by crushing the tablets and resuspending in dimethyl sulfoxide to prepare a solution with the aq component at a concentration of mm. the compounds, amodiaquine dihydrochloride dihydrate (#a ), artesunate (#a ), and dihydroartemisinin (#d ) were purchased from sigma-aldrich (saint louis, mo). n-desethylamodiaquine hydrochloride (#sc- ) was obtained from santa cruz biotechnology (dallas, tx). drugs were prepared as mm stocks in dimethyl sulfoxide. animal studies. source, formulation, and preparation of amodiaquine hydrochloride was the same for the pk and the efficacy studies. amodiaquine hydrochloride was obtained from us pharmacopeia (rockville, md; cat. ; lot j ) and a mg/ml aq (free base) solution was prepared in sterile water for injection (usp). cell-based testing of ebov antiviral agents. the cell-based ebov drug screen and cytotoxicity assays were performed as previously described . briefly, vero e and huh cells were seeded at × cells/well, and mdms at × cells/well in -well plates. after h, cells were treated with compounds at -fold dilutions starting from µm. the starting concentration of the asaq tablet suspension corresponded to µm of the aq base component. cells were infected with ebov/mak h after the addition of the drugs in biosafety level (bsl ) containment at multiplicity of infection (moi) of . - . . after h, plates were fixed with % neutral buffered formalin (richard-allan scientific), and ebov/mak was detected with a mouse antibody specific for ebov vp protein (#b-md -bd -ae , usamriid) followed by staining with alexa fluor ® goat anti-mouse igg (heavy + light chain) antibody (life technologies, grand island, ny) or with anti-mouse igg-peroxidase labeled antibody (kpl# - ). fluorescence or luminescence was quantified on a plate reader (infinite ® m pro, tecan us, morrisville, nc). the signal of treated infected wells was normalized to uninfected control wells and measured (in percent) relative to untreated infected wells. non-linear regression analysis was performed, and the % inhibitory concentrations (ic s) were calculated from fitted curves (log [agonist] versus response [variable slope] with constraint to remain above %) (graphpad software, la jolla, ca). the ebov drug screen assay was performed with three replicates for each drug concentration, and the assay was repeated at least twice for confirmation. to evaluate cytotoxicity, cells were treated with compounds as described above in absence of virus. at h after drug addition, cell viability was quantified using the celltiter glo luminescent cell viability assay kit (promega, madison, wi). www.nature.com/scientificreports www.nature.com/scientificreports/ pharmacokinetic analysis of amodiaquine in nonhuman primates. rhesus macaques (macaca mulatta) were obtained from covance research products (princeton, nj). two groups (n = , females and males per group) were dosed with or mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ) orally via nasogastric intubation once daily for days. nhps were housed in stainless steel primary enclosures singly or in pairs if compatible. nhps were provided teklad certified global % protein primate diet (# c) and purified water ad libitum. clinical observations were conducted for days including the day of dosing. blood (maximal μl) was collected from cephalic vessels on day and day . on day , blood was collected pre-dose, . , , , , , , , and h post-dose (immediately prior to dosing on day ). on day , blood was collected h after dose (or h after dose ), immediately prior to dosing on day , and . , , , , , , , and h after dose . drug concentrations of aq and its metabolite, deaq, were determined in collected plasma samples using liquid chromatography with tandem mass spectrometry (lc-ms/ms). plasma samples (volume μl) were prepared by adding ml of methyl t-butyl ether. each tube was vortexed for approximately minutes (min) at maximal speed and centrifuged for min at , g to facilitate separation of the liquid phases. upper layers ( μl) were transferred to new tubes, and the solvent was removed under vacuum in a centrifugal evaporator. the dried residues were reconstituted with μl of internal standard solution ( ng/ml risperidone and ng/ml chloroquine in : [v:v] acetonitrile:water). the tubes were vortexed min and clarified by centrifugation ( , g) for min. the clarified extracts were transferred to high performance liquid chromatography vials containing glass inserts for subsequent lc-ms/ms analysis. lc-ms/ms was performed using a lc- ad pump system (shimadzu, columbia, md) and a qtrap mass spectrometer (sciex, concord, ontario, canada) in multiple reaction monitoring mode and a polaris c -a column ( × . mm, μm; agilent, ca), using gradient elution with . % formic acid in water and . % formic acid in acetonitrile as the mobile phase. the lower limit of quantitation for aq and deaq of the method was ng/ml. the following parameters and constants were determined: maximal plasma concentration (cmax), time to maximum plasma concentration (tmax), area under the plasma concentration-time curve to the last time point and extrapolated to infinity (auc last and auc inf ), terminal elimination half-life (t / ), apparent volume of distribution (v/f), and total clearance (cl/f) after oral administration. pk parameters were analyzed using phoenix ® winnonlin ® software (v . ; certara, princeton, nj) to perform noncompartmental data analysis for extravascular administration. for clinical pathology (serum chemistry, hematology), blood ( ml) was collected prior to dosing on day and approximately h after the final day dose. treatment and challenge of nonhuman primates. rhesus macaques of chinese origin (n = , adult, < years of age) were obtained from charles river laboratories (frederick, md). animals were singly housed in stainless steel primary enclosures and provided chow and purified (reverse osmosis) water ad libitum. the vehicle control group (group ; n = ) was treated with sterile water. two treatment groups were treated with aq orally once daily as a consecutive -day course of mg/kg free base ( mg/kg amodiaquine hydrochloride (us pharmacopeia, rockville, md; cat. ; lot j ). treatment began on the day of ebov exposure (group ; n = , females and males), or on day postexposure (group ; n = , females and males). all groups were challenged im with a target dose of pfu) of ebov/mak (actual dose = pfu). the plaque assay back-titration of the challenge material was initiated on the day of preparation (study day ) on vero e cells, and plaques were fixed with % formalin and stained with crystal violet days later. animals were observed and weighed daily. the vehicle control group (group , n = ) received an equivalent volume of sterile water (gibco, cat. a , lot ). weight was recorded on day - and day - , then daily starting one day before challenge until primates succumbed to disease. responsiveness of the animals was monitored following -point range, in which a score of met primary endpoint criteria and initiated secondary criteria evaluation (table ) . when nhps scored a for primary euthanasia criteria and their temperature was above °c, secondary euthanasia criteria were reviewed. an nhp had to meet at least two secondary criteria, blood urea nitrogen ≥ mg/dl, calcium ≤ . mg/dl, ggt ≥ u/dl, and/or creatinine ≥ . mg/dl, for required euthanasia. two rhesus macaques were euthanized based on their secondary criteria. six of the fifteen rhesus macaques were euthanized based on veterinary discretion, not based on their clinical score or secondary criteria. hematology and serum chemistry. complete blood count with leukocyte differential was performed from peripherally collected blood samples using vacuette k ethylenediaminetetraacetic acid (edta) tubes and a sysmex xt- iv hematology instrument using a preprogrammed monkey species profile (sysmex america, ny). plasma and serum were prepared by separation for min at ambient temperature with centrifuge set to rcf. serum chemistries were performed using the piccolo xpress analyzer and general chemistry discs (abaxis, abbott point of care, nj) from vacuette z serum clot activator tubes (greiner bio-one, monroe, nc). pathology and histology. all animals were humanely euthanatized in accordance with defined experimental endpoints, and gross necropsy was performed. tissue samples were inactivated by fixing for h in % neutral buffered formalin. following fixation and removal from the bsl- laboratory in accordance with standard operating procedures, tissue samples were routinely processed in a tissue-tek vip- automated vacuum infiltration processor (sakura finetek usa, torrance, california, usa) followed by paraffin embedding with a tissue-tek model tec unit (sakura finetek usa, torrance, ca, usa). using a standard semiautomated rotary microtome and lighted water flotation bath (leica biosystems, wetzlar, germany), tissue sections were cut at a thickness of µm and mounted on positively charged uncoated glass slides (thermofisher, waltham, ma, usa), air-dried at room temperature, stained with hematoxylin and eosin (h&e), and coverslipped for microscopic evaluation by the pathologist. plasma samples from rhesus macaques that had received a dose of aq at integrated research facility/ niaid (fort detrick, md) were analyzed for concentrations of aq and the metabolite deaq. prior to shipment to sri, the samples were diluted : . and irradiated at mrad using a cobalt- source to destroy any pathogens that may have been present in the plasma samples. a pilot study with spiked quality control standards confirmed that irradiation does not appreciably alter the level of aq in the plasma (data not shown). a liquid:liquid extraction method with methyl t-butyl ether was used to separate aq and deaq from rhesus macaque plasma. samples were analyzed by lc-ms/ms with a lower limit of quantitation (lloq) of ng/ml ( . ng/ml × . dilution factor) in plasma for both aq and deaq. aq and deaq concentrations were adjusted for the dilution factor of . for analysis in figs. and . the virus working stock ebov/makona used for exposure (genbank accession no. kx . , biosample: samn ). clinical management of patients with viral haemorrhagic fever effect of artesunate-amodiaquine on mortality related to ebola virus disease effect of mass artesunate-amodiaquine distribution on mortality of patients with ebola virus disease during west african outbreak the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry identification of compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs a systematic screen of fda-approved drugs for inhibitors of biological threat agents evaluation of ebola virus inhibitors for drug repurposing identification of agents effective against multiple toxins and 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that inhibit lassa and ebola viruses antiviral activity of cationic amphiphilic drugs a common feature pharmacophore for fda-approved drugs inhibiting the ebola virus novel amodiaquine derivatives potently inhibit ebola virus infection amodiaquine induced agranulocytosis and liver damage tolerability and safety of artesunate-amodiaquine and artemether-lumefantrine fixed dose combinations for the treatment of uncomplicated plasmodium falciparum malaria: two open-label, randomized trials in nimba county evaluation of the activity of lamivudine and zidovudine against ebola virus high dose sertraline monotherapy fails to protect rhesus macaques from lethal challenge with ebola virus makona pathology of experimental ebola-zaire (mayinga) virus infection transmitted to guinea pigs by oral, conjunctival and tonsillar routes the findings and conclusions in this report do not necessarily reflect the views or policies of the us department of health and human services or of the institutions and companies affiliated with the authors. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.d.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ewvnkdr authors: steeds, kimberley; hall, yper; slack, gillian s.; longet, stephanie; strecker, thomas; fehling, sarah katharina; wright, edward; bore, joseph akoi; koundouno, fara raymond; konde, mandy kader; hewson, roger; hiscox, julian a.; pollakis, georgios; carroll, miles w. title: pseudotyping of vsv with ebola virus glycoprotein is superior to hiv- for the assessment of neutralising antibodies date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ewvnkdr ebola virus (ebov) is an enveloped, single-stranded rna virus that can cause ebola virus disease (evd). it is thought that evd survivors are protected against subsequent infection with ebov and that neutralising antibodies to the viral surface glycoprotein (gp) are potential correlates of protection. serological studies are vital to assess neutralising antibodies targeted to ebov gp; however, handling of ebov is limited to containment level laboratories. pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. however, neutralisation capacity can differ among pseudotyped virus platforms. we evaluated the suitability of ebov gp pseudotyped human immunodeficiency virus type (hiv- ) and vesicular stomatitis virus (vsv) to measure the neutralising ability of plasma from evd survivors, when compared to results from a live ebov neutralisation assay. the sensitivity, specificity and correlation with live ebov neutralisation were greater for the vsv-based pseudotyped virus system, which is particularly important when evaluating ebov vaccine responses and immuno-therapeutics. therefore, the ebov gp pseudotyped vsv neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against ebov. www.nature.com/scientificreports/ and time required for plaque development, which can take approximately nine days, makes it time-consuming and restricts high-throughput sample processing. development of novel serological assays that utilise genetically modified recombinant or chimeric viruses with attenuated pathogenicity have enabled more widespread investigation of neutralising antibodies against highly pathogenic viruses including ebov , . pseudotyped viruses are replication-defective chimeric virions that comprise the structural and enzymatic core of one virus, bearing the envelope protein or glycoprotein of another, and encode a quantifiable reporter gene. retroviruses, including lentiviruses and gammaretroviruses such as human immunodeficiency virus (hiv) and murine leukaemia virus (mlv), respectively, and rhabdoviruses, such as vesicular stomatitis virus (vsv), have been used extensively as cores for pseudotyped viruses , , including for ebov , . a number of ebov gp pseudotyped virus neutralisation assays have been developed to investigate immune responses to ebov infection and vaccination [ ] [ ] [ ] , as well as for evaluation of monoclonal antibody (mab) therapies [ ] [ ] [ ] . there are many factors that need to be considered when developing and optimising pseudotyped virus neutralisation assays, to assess experimental parameters that can affect assay performance and to ensure accuracy and reproducibility. these include, choice of core virus and reporter gene, determination of target cell line and amount of pseudotyped virus input, as well as correlation with live virus neutralisation . the aim of this study was to assess the suitability of ebov gp pseudotyped hiv- and vsv systems to measure neutralisation by evd survivor plasma, in comparison with results from a live ebov neutralisation assay. cell tropism of ebov gp pseudotyped viruses. pseudotyped hiv- and vsv bearing the envelope gp from ebov (mayinga) were generated and quantified by measuring luminescence in a range of target cell lines, in order to determine the optimum cell line to use in neutralisation assays. cells only controls were used to determine background levels of luminescence ( supplementary fig. s ). reporter activity was detected in all cell lines infected with ebov gp pseudotyped hiv- and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed (fig. a,b) . for ebov gp pseudotyped hiv- , highest tcid /ml values were observed in t/ cells, followed by huh- cells (fig. c) . titres generated by infection of t/ cells were approximately , and times greater than those produced by infection of huh- , hela and vero e cells, respectively. for ebov gp pseudotyped vsv, highest titres were obtained in during the initial stages of assay development, it is important to evaluate neutralisation of pseudotyped viruses using well characterised antibodies in order to demonstrate the validity and accuracy of the assay. the ebov gp pseudotyped viruses were assessed for neutralisation by the human anti-ebov gp mab, kz . kz is an antibody isolated from a human survivor of the outbreak in kikwit that neutralises ebov in vitro and recognises a conformational epitope at the base of the gp [ ] [ ] [ ] . human anti-ebov gp mab, kz was unable to neutralise the ebov gp pseudotyped hiv- (fig. a) within the range tested, however it was able to neutralise the ebov gp pseudotyped vsv (fig. b) , suggesting that vsv-based pseudotyped viruses are more sensitive to neutralisation then lentiviral-based, possibly the density of ebov gp on the pseudotyped hiv- may differ from that on the pseudotyped vsv or live ebov. to determine the optimal pseudotyped virus input to use in the hiv-and vsv-based assays, neutralisation of different amounts of the ebov gp pseudotyped viruses by plasma from a guinean evd survivor donor or human anti-ebov gp mab kz was assessed. kz was selected as it is commercially available and there is accompanying information regarding its neutralisation activity against ebov gp pseudotyped vsv expressing luciferase. however, as the ebov gp pseudotyped hiv- was not neutralised by kz (fig. a) in the range tested, plasma from an evd survivor was used to assess the effect of pseudotyped hiv- input on neutralisation instead. survivor plasma sample cs was chosen as it displayed strong neutralising ability against live ebov neutralisation, with a geometric mean titre (gmt) of , . percentage infectivity was determined relative to infectivity of cells by the ebov gp pseudotyped viruses alone (fig. a ,b) and % inhibitory concentration (ic ) of pseudotyped virus neutralisation were estimated by model of nonlinear regression dose-response curves (fig. c,d) . plasma from evd survivor cs displayed neutralising activity against all amounts of ebov gp pseudotyped hiv- tested (fig. a) . lower pseudotyped virus input resulted in larger variability and less curve fitting. therefore, an ebov gp pseudotyped hiv- input of at least . × rlu/well, with a target input of . × rlu/well, was used in subsequent neutralisation assays. kz neutralised all dilutions of ebov gp pseudotyped vsv tested (fig. b) and ic values decreased with decreasing amounts of pseudotyped virus input (fig. d , supplementary table s ). when using . × rlu/well of ebov gp pseudotyped vsv, ic of virus neutralisation ( . µg/ ml) was similar to that expected according to the manufacturer's product data sheet ( . µg/ml). therefore, a target input of approximately . × rlu/well was used in subsequent ebov gp pseudotyped vsv neutralisation assays. table s ). neutralisation of ebov gp pseudotyped hiv- and vsv by positive (evd survivor) and negative (uk donor) control plasma was assessed in several independent assays ( supplementary fig. s ). the background level of neutralisation was determined using plasma from a uk negative control donor. for the hiv- -based assay this was calculated as ic . reciprocal dilution. the negative control plasma displayed no neutralising activity against ebov gp pseudotyped vsv, and therefore the background level of neutralisation was assigned the low- www.nature.com/scientificreports/ est dilution of sample tested in the assay ( / ). in the hiv- -based assay, dose-response curves were unable to be fitted for three of the evd survivor samples and six of the samples were deemed below the background level of neutralisation. in contrast, a dose-response curve was unable to be fitted for only one of the evd survivor samples tested in the vsv-based neutralisation assay. in the hiv- -based assay, three of the negative plasma samples tested were above the background level of neutralisation, whereas only one of the negative samples tested was above the background level of neutralisation in the vsv-based assay. although some differences in the discriminatory power of positive and negative samples between the assays were observed, a statistically significant difference in neutralisation titres was detected between the evd survivors and negative plasma samples in the hiv- -based assay (mann-whitney, p = . ) (fig. a ) and in the vsv-based assay (mann-whitney, p < . ) (fig. b , supplementary table s ). remarkably, this difference was more significant and the separation of the positive and negative plasma was better in the vsv-based assay (fig. b) . the sum of these results clearly show that the vsv-based ebov gp neutralisation assay displayed better reliability, specificity and sensitivity compared to the hiv- -based assay. correlation with live ebov neutralisation. the neutralising capacity of the individual plasma samples against authentic ebov was assessed by a live virus neutralisation assay (supplementary table s , supplementary fig. s ). when ic values of ebov gp pseudotyped hiv- neutralisation of the evd survivor and negative plasma samples were compared with gmt values for the live ebov neutralisation assay, a positive correlation (r s = . ) was determined using the nonparametric spearman correlation coefficient (fig. a) and this was statistically significant (p = . ). remarkably, a stronger statistically significant (p < . ) positive correlation (r s = . ) was observed when ic values of ebov gp pseudotyped vsv neutralisation were compared with gmt values for the live ebov neutralisation assay (fig. b) . the correlation coefficients for ebov gp hiv- and vsv ic compared with live ebov gmt without the negative controls were . (p = . ) and . (p < . ), respectively. therefore, the vsv-based ebov gp pseudotyped virus neutralisation assay correlated better with live ebov neutralisation than the hiv- -based neutralisation assay. pseudotyped viruses can be used as alternatives to infectious virus in serological assays to measure neutralising antibodies to viral envelope glycoproteins . pseudotyped virus assays used to profile neutralising antibody responses against severe acute respiratory syndrome-associated coronavirus (sars-cov) , influenza (h n and h n ) [ ] [ ] [ ] , rabies , and chikungunya virus , for example, found that results correlated well with those from replication-competent or live virus assays. a high degree of correlation has been demonstrated between ebov cl prnt and an ebov pseudotyped vsv cl fluorescence reduction neutralisation test (frnt) . however, pseudotyped virus assays may not always accurately determine neutralisation , . live ebov and ebov gp pseudotyped neutralisation assays have previously been shown to yield variable results , , which could be due to differing experimental conditions and viral systems. it is therefore important to optimise pseudotyped virus neutralisation assays in context of the particular viral gp being studied in order to obtain reliable specificity and sensitivity. the aim of this study was to assess the suitability of ebov gp pseudotyped hiv- and vsv systems to measure the neutralising ability of plasma from evd survivors, when compared to live ebov neutralisation. reporter activity was detected in all cell lines ( t/ , huh- , hela and vero e ) infected with ebov gp (mayinga) pseudotyped hiv- and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed. this may reflect general defects in viral entry in different cells. a relatively lower level of ebov gp pseudotyped hiv- transduction was exhibited by vero e cells, which might be due to an intrinsic restriction factor, trim α, which restricts retroviral infection by specifically recognising the hiv- capsid and promoting its rapid, premature disassembly . highest tcid values were obtained following ebov gp pseudotyped hiv- and vsv infection of t/ and vero e cells, respectively. there seemed to be large variability of the luminescent measurement for the vsv-based platform, which may be caused by the www.nature.com/scientificreports/ sensitive nature of the luciferase signal detection. this highlights the importance of titrating each pseudotyped virus batch before use in neutralisation assays, and the inclusion of multiple replicates. the ebov gp pseudotyped viruses were used to assess the neutralising activity of a human anti-ebov gp mab, kz . kz has been shown previously to neutralise ebov pseudotyped viruses , , . however, within the range tested here, kz did not display neutralisation against ebov gp pseudotyped hiv- , suggesting that the ebov gp on the pseudotyped hiv- might be at higher levels, thereby reducing assay sensitivity, and neutralisation may be observed using a higher concentration of kz . in contrast, kz was able to neutralise the ebov gp pseudotyped vsv. to assess the effects of differing amounts of pseudotyped virus input on neutralisation, plasma from an evd survivor of the - ebov outbreak and kz were screened against different amounts of the ebov gp pseudotyped hiv- and vsv, respectively. decreasing quantities of pseudotyped hiv- led to more variable and unreliable results, and the kz ic of pseudotyped virus neutralisation decreased with decreasing amounts of ebov gp pseudotyped vsv input. the variability in neutralisation observed between different amounts of pseudotyped virus input highlights the importance of including standards or reference material with a known activity or potency when comparing neutralising activity, allowing calibration of results . both pseudotyped virus systems were able to measure neutralising antibodies in plasma from evd convalescent patients, and results correlated positively with a live ebov neutralisation assay. however, the discriminatory power of the hiv- -based assay with regards to differing antibody titres appeared to be low. some of the samples tested, which showed neutralising activity against live ebov, did not display neutralisation against the pseudotyped virus and vice versa, therefore raising questions on the sensitivity and specificity of the pseudotyped hiv- assay. in the current study, human embryonic kidney ( t/ ) cells were used for the pseudotyped hiv- neutralisation assays, whereas african green monkey kidney (vero) cells were used in the vsv-based assay and also the live ebov assay. therefore, this could account for some of the differences in results observed between the two assays and for the better performance of the vsv-based assay in relation to live ebov neutralisation. also, the hiv- -and vsv-based pseudotyped virus systems assessed in the current study utilise different transfection methods, which could have implications on the composition of the pseudotyped viruses, density and/or glycosylation of the viral envelope protein on the surface, and consequently neutralisation results. this highlights the importance of assessing experimental conditions and methodology when developing and optimising pseudotyped virus neutralisation assays. a limitation to this study was that the level of ebov gp incorporation per pseudotyped virus type could not be assessed. also, for the vsv-based pseudotyped virus system, traces of vsv-g from the rvsv-Δg-luc-vsv-g virus could be recycled into newly pseudotyped virions . therefore, the use of anti-vsv-g hybridoma cell culture supernatant could give rise to pseudotyped virions covered by anti-vsv-g antibodies, but are still infectious due to ebola gp. this could potentially induce plasma specific reactivity of virions due to bound anti-vsv-g antibodies more than ebov gp specific reactivity. there are several differences between ebov gp pseudotyped and live ebov neutralisation assays that could affect their results . due to their non-replicating nature, such pseudotype systems do not recapitulate all steps in the viral life cycle that may potentially be targeted by neutralising antibodies . in addition, the round, spherical shape of ebov gp pseudotyped hiv- or bullet shape of ebov gp pseudotyped vsv compared to the filamentous shape of authentic ebov could affect their susceptibility to neutralisation. also, the density of gp on the surface of the pseudotyped virus may not be the same as that found on live ebov and may result in the loss or masking of quaternary epitopes , . furthermore, gp maturation and assembly in live ebov could be different in the generation of an ebov pseudotyped virus and may result in different targets and/or conformational epitopes when using whole live ebov as opposed to ebov gp alone in a pseudotyped virus. the presence of shed gp or secreted gp (sgp) in the live ebov assay compared to absence in the ebov gp pseudotyped virus assays could also have an effect on neutralisation. in the live ebov assay, shed gp and sgp could reduce neutralisation of circulating virus by cross-reactive antibodies to surface gp. however, in the current study, weaker relative neutralisation was observed in the hiv- based pseudotyped virus assay. therefore, it is possible that cell debris or free gp generated during ebov gp pseudotyped hiv- production by polyethylenimine (pei) transfection could be interfering with neutralisation. finally, detection of infected cells via measurement of luminescence in the ebov gp pseudotyped virus neutralisation assay compared to plaque formation in the live ebov neutralisation assay could affect neutralisation readout. ebov gp pseudotyped virus neutralisation assays have value for vaccine evaluation and assessment of convalescent blood products and mabs for use as immunotherapeutics. however, pseudotyped virus assays may not always accurately determine neutralisation when compared with neutralisation against live virus. in this study, both ebov gp pseudotyped hiv- and vsv assays were able to detect neutralisation of plasma from evd survivors and correlated positively with live ebov neutralisation. however, the vsv-based assay performed better than the hiv- -based assay in relation to specificity, sensitivity, and correlation with the live ebov neutralisation assay. this research has highlighted the importance of optimising pseudotyped virus neutralisation assays in context of the particular viral gp being studied, especially when evaluating vaccine responses and therapeutics, and could provide a better understanding of the correlates of protection against ebov. and from negative control blood donors in the uk and guinea, who were not knowingly exposed to persons with evd and did not attend high risk events such as funerals, were heat inactivated at °c for min. the samples were obtained from a pre-existing biobank, for which live ebov neutralisation production of pseudotyped viruses. the generation of hiv- pseudotyped viruses was performed as detailed previously , , . twenty-four hours prior to transfection, approximately × t/ cells were seeded into sterile, -well cell culture plates (corning, ewloe, uk) and incubated at °c, % co and % humidity until - % confluence. the hiv gag-pol plasmid, p . , and the firefly luciferase reporter construct, pcsflw, were transfected simultaneously with the ebov (mayinga) gp expression vector at a ratio of . : . : . µg (core:reporter:envelope) using µl of µg/ml polyethylenimine (pei) (sigma-aldrich) per µg dna in opti-mem medium (gibco). following overnight transfection, the cells were incubated with fresh medium and incubated at °c, % co . pseudotyped virus supernatants were harvested at and h posttransfection, passed through a . µm pore filter (millex, millipore, watford, uk) and stored at − °c. ebov gp pseudotyped vsvs were prepared using recombinant vsv, in which the vsv-g gene had been deleted (rvsv-Δg) and replaced with a luciferase reporter gene (rvsv-Δg-luc) by a method similar to that described previously . twenty-four hours prior to transfection, approximately . × t/ cells were seeded into sterile, mm cell culture dishes (corning) and incubated at °c, % co and % humidity until - % confluence. the cells were transfected with the ebov gp expression vectors using transit-lt transfection reagent (mirus bio, madison, wisconsin (wi), usa) as per the manufacturer's instructions. following overnight transfection, the medium was removed and the cells were infected with rvsv-Δg-luc that was pseudotyped with the vsv glycoprotein (rvsv-Δg-luc-vsv-g) (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a multiplicity of infection (moi) of in opti-mem medium and incubated at °c, % co . after h, the inoculum was removed, cells were washed twice with dulbecco's phosphate buffered saline (dpbs) (gibco) and fresh medium was added. pseudotyped virus supernatants were harvested at - h post-infection, clarified twice by centrifugation at xg for min at °c and stored at − °c. prior to use, the pseudotyped viruses were incubated with anti-vsv-g hybridoma cell culture supernatant (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a : dilution for h at °c to reduce background infection mediated by residual virus possessing vsv-g, which can be carried over during preparation . all experiments involving pseudotyped viruses were performed in a cl facility at public health england (phe), porton down, uk. pseudotyped virus titration and neutralisation assays. titration and neutralisation assays were performed in -well solid white flat bottom polystyrene tc-treated microplates (corning) and were based upon previously described protocols , , . for pseudotyped hiv- titration assays, five-fold serial dilutions of pseudotyped virus at a starting dilution of : were prepared in quadruplicate in opti-mem medium at a final volume of µl/well. µl of approximately × t/ , huh- or vero e cells, or × hela cells were then added to each well and incubated at °c, % co for h. the medium was removed and µl of a : mix of bright-glo luciferase assay reagent (promega, southampton, uk):fresh medium was added to each well and incubated for at least min at room temperature to allow complete cell lysis. luminescence was measured using a glomax-multi + detection system luminometer (promega) and relative luminescence units per ml (rlu/ml) were determined. the negative cut-off was set at . times the average rlus of the cells only control wells. % tissue culture infectious dose (tcid )/ml values were determined using the reed-muench method . for the pseudotyped hiv- neutralisation assay, two or threefold serial dilutions of plasma samples at a starting dilution of : or : , respectively, were prepared in duplicate in opti-mem medium at a final volume of µl/well and incubated with µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for h at °c. µl of approximately × t/ cells were then added to each well and incubated for h at °c, % co , prior to taking a chemiluminescent readout as described above. infectivity was calculated using the formula: percentage (%) infectivity = [(rlu with sample)/(rlu without sample)] × . www.nature.com/scientificreports/ for pseudotyped vsv titration assays, h prior, approximately . × t/ or × huh- , hela cells or vero e cells were seeded in -well microplates and incubated at °c, % co and % humidity. the medium was removed and two-fold serial dilutions of pseudotyped virus in opti-mem medium, starting with neat pseudotyped virus were added to each well in quadruplicate at a final volume of µl/well. after h, a chemiluminescent readout was taken and tcid /ml values were determined as described above. twenty-four hours prior to pseudotyped vsv neutralisation, approximately × vero e cells were seeded and incubated as for titration above. twofold serial dilutions of plasma samples at a starting dilution of : were prepared in duplicate in opti-mem medium at a final volume of µl/well in -well microplates, and incubated with µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for h at °c. the medium was removed from the cells, µl of the plasma-pseudotyped virus mixtures were added to each well in quadruplicate at incubated at °c, % co . after h, µl of fresh medium was added to each well. luminescence was measured after h and infectivity was calculated as described above. statistical analysis. pseudotyped virus neutralisation assay raw data were normalised as percentage (%) infection relative to mean values for pseudotyped virus only controls (equivalent to % infection), then ic of pseudotyped virus neutralisation were estimated by model of nonlinear regression fit with settings for log (inhibitor) vs. normalised response curves using graphpad prism v (san diego, california (ca), usa). statistical comparison between two unpaired groups was performed using the mann-whitney test (graph-pad prism v ). correlation between two variables was quantified using spearman nonparametric correlation (graphpad prism v ). www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. ebola haemorrhagic fever emergence of zaire ebola virus disease in guinea world health organization. situation report - biochemical analysis of the secreted and virion glycoproteins of ebola virus a mutation in the ebola virus envelope glycoprotein restricts viral entry in a host species-and cell-type-specific manner characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms systematic analysis of monoclonal antibodies against ebola virus gp defines features that contribute to protection role of antibodies in protection against ebola virus in nonhuman primates immunized with three vaccine platforms the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination current progress with serological assays for exotic emerging/re-emerging viruses construction and use of a human immunodeficiency virus vector for analysis of virus infectivity a system for functional analysis of ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps specific neutralizing response in plasma from convalescent patients of ebola virus disease against the west africa makona variant of ebola virus ebola virus neutralizing antibodies detectable in survivors of the yambuku, zaire outbreak years after infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody potent neutralizing monoclonal antibodies against ebola virus infection technical considerations for the generation of novel pseudotyped viruses ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody structure of the ebola virus glycoprotein bound to an antibody from a human survivor longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h n influenza viruses characterization of lentiviral pseudotypes with influenza h n hemagglutinin and their performance in neutralization assays safe pseudovirus-based assay for neutralization antibodies against influenza a(h n ) virus a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection high degree of correlation between ebola virus bsl- neutralization assays and pseudotyped vsv bsl- fluorescence reduction neutralization test human immunodeficiency virus type env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies broadly neutralizing human monoclonal antibodies to the hepatitis c virus e glycoprotein comparison of platform technologies for assaying antibody to ebola virus specific recognition and accelerated uncoating of retroviral capsids by the trim α restriction factor a shared structural solution for neutralizing ebola viruses ebola virus: pseudotypes, libraries and standards characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins neutralizing antibodies inhibit chikungunya virus budding at the plasma membrane maturation of west nile virus modulates sensitivity to antibody-mediated neutralization multiply attenuated lentiviral vector achieves efficient gene delivery in vivo investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison phase trials of rvsv ebola vaccine in africa and europe a sensitive retroviral pseudotype assay for influenza h n -neutralizing antibodies lyophilisation of influenza, rabies and marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit generation of vsv pseudotypes using recombinant Δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines a simple method of estimating fifty per cent endpoints the authors would like to thank masayuki saijo for providing the rvsv-Δg-luc-vsv-g virus and anti-vsv-g hybridoma cell culture supernatant. we are grateful to eccac for providing vero e and hela cells, and to arvind patel for providing huh- cells. this work was funded by the u.s. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to m.w.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - yhoiv authors: singleton, courtney d.; humby, monica s.; yi, hyun ah; rizzo, robert c.; jacobs, amy title: identification of ebola virus inhibitors targeting gp using principles of molecular mimicry date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: yhoiv a key step in the ebola virus (ebov) replication cycle involves conformational changes in viral glycoprotein (gp ) which facilitate host-viral membrane fusion and subsequent release of the viral genome. ebola gp plays a critical role in virus entry and has similarities in mechanism and structure to the hiv gp protein for which inhibitors have been successfully developed. in this work, a putative binding pocket for the c-terminal heptad repeat in the n-terminal heptad repeat trimer was targeted for identification of small molecules that arrest ebov-host membrane fusion. two computational structure-based virtual screens of ∼ . m compounds were performed (dock program) against a gp five-helix bundle, resulting in commercially available compounds purchased for experimental testing. based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with % inhibitory concentration values in the to μm range. molecular dynamics simulations of the two most potent candidates in their dock-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting ebov. importance the most recent ebola virus disease outbreak, from to , resulted in approximately , individuals becoming infected, which led to over , causalities worldwide. the particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of ebola infection. to limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class i fusion glycoprotein of ebolavirus. in the current work, several promising small-molecule inhibitors that target the glycoprotein gp were identified through systematic application of structure-based computational and experimental drug design procedures. ular modeling tools and experimental characterization. specifically, large-scale virtual screening of ϳ . million small molecules was performed with gp using the program dock ( ) . we hypothesize that small molecules that interact with the gp nhr pocket will interfere with assembly of the hb required for ebov-host membrane fusion (fig. ) . interfering with hb formation is a strategy previously employed successfully against hiv ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) through targeting an analogous pocket on the viral protein gp ( , ) . the computational screening resulted in the prioritization and purchase of compounds for experimental characterization, which led to hits that inhibit viral entry in both ebov-gp-pseudotyped virus and ebov transcription-and replicationcompetent virus-like particle (trvlp) systems. compounds were further evaluated to assess (i) potential activity artifacts using detergent-containing experiments, (ii) specificity for ebov using a vesicular stomatitis virus glycoprotein (vsv-g)-pseudotyped virus particle counterscreen, and (iii) step(s) within the ebov replication cycle where they exerted the majority of inhibitory activity using time-of-addition (toa) analysis. results suggest that of the compounds act to specifically inhibit ebov entry after attachment but prior to virus-host membrane fusion. molecular dynamics (md) simulations in conjunction with genome analysis identified highly conserved residues across different ebola virus strains (e .a, a .a, l .a, f .a, t .c, l .c, and l .c) that contribute a majority of the favorable interactions between the compounds and gp . virtual screening outcomes. the goal of this study was to identify molecules that inhibit ebov infection by interfering with the interactions required for formation of the gp six-helix bundle ( hb). since the conformational change required to produce the postfusion structure is dependent on chr binding the nhr region of gp (fig. ), a virtual screen of approximately . million compounds was conducted to a five-helix bundle model of gp constructed by the removal of one chr from a high-resolution postfusion structure (pdb entry ebo [ ] ) (see materials and methods, below). compound prioritization led to candidates purchased for experimental testing (fig. ) and employed five distinct scoring functions: dce sum (dock cartesian van der waals and electrostatic energy), fps vdw (footprint comparison of the van der waals energy of the reference peptide and selected ligands), fps es (footprint for electrostatic energy), fps sum (footprint for both van der waals and electrostatic energy), and ts (total score; the combination of dce sum and fps sum ). a large number of molecules was prioritized based on their structural and spatial similarity to the reference ligand composed of a segment of the chr that made the most favorable interactions with our model of a gp five-helix bundle. as a rule, all compounds chosen for experimental testing showed good overlap with the reference (fig. a) . however, those selected based on favorable footprint similarity (fps) have somewhat better overlap than those selected based on dce or ts (fig. b) . consistent with visual inspection (fig. ) , molecules in each of the five groups share similar size and flexibility, with a mean molecular weight (mw) distribution of . g/mol and number of rotatable bonds of . (table ) . compounds purchased based on similarity in electrostatic (es) interaction profiles (fps es ) had the overall smallest mw ( . g/mol) and fewer numbers of rotatable bonds ( . ) , while those selected from the ts list were largest ( . g/mol) ( table ) . as expected ( , ) , compounds selected using a specific scoring function (table , scoring function column) generally showed the best average score with regard to that specific chemical or physical property (table , property columns). for example, compounds prioritized using the dce sum function yielded a more favorable (lower) average dce sum energy (Ϫ kcal/ mol) than those obtained using other functions (Ϫ to Ϫ kcal/mol). likewise, molecules selected using fps sum resulted in a more favorable average fps sum score ( . ) than the other groups ( . to . ). for compounds prioritized using fps es and fps vdw footprint components, the scores were the lowest ( . ) and second lowest ( . ), respectively, among their respective fps es and fps vdw groups. for the dce sum -selected group, the favorable scores can be attributed to strong es interactions resulting in an average dce es score of Ϫ . kcal/mol, over -fold greater than the ensemble average (Ϫ . kcal/mol). the overall strength of the dce sum scores, in conjunction with being the second smallest group in terms of mw and number of rotatable bonds ( . ), suggests that the dce sum list compounds are highly polar. in contrast, the ts list interactions are dominated by strong vdw interactions due to their larger size (mw ϭ g/mol) ( table ) . consistent with the fact that fps sum is a part of the ts scoring function, the fps score components are better than those observed using dce sum . however, the overlap is relatively moderate (fps sum ϭ . , fps vdw ϭ . , fps es ϭ . ); therefore, future work could explore increasing the contribution of the fps component of ts. in summary, molecular property analysis confirms that the purchased candidates are similar in size and flexibility but diverse in terms of interaction energy and overlap the reference peptide. nine molecules from the initial in silico screen inhibit ebov-pseudotyped virus entry in vitro. the compounds identified from the aforementioned in silico screen were tested for their ability to inhibit ebov entry and for cytotoxicity at m ( , , ) . ebov (hiv- /ebov)-pseudotyped virus entry into t cells was quantified by luciferase signal normalized by cytotoxicity and dimethyl sulfoxide (dmso) control to yield the infectivity signal per cell as a fraction of the maximum (see materials and methods). encouragingly, nine compounds resulted in a normalized luciferase signal of Յ . (fig. , blue) . additionally, the observed luciferase signal for the nine compounds was approximately . standard deviations below the average infectivity signal for all purchased molecules, . Ϯ . . although the two compounds with the most activity (i and i ) were also the most cytotoxic (fig. , lower, blue), all nine hits with activity were retained and used as starting points for identification of structurally related analogs in a secondary computational screen (see discussion). secondary similarity screen. to identify additional compounds with enhanced activity, a second similarity-based computational screen was conducted to explore the chemical search space around the nine initial hits. each of the hits in turn was used to rescore and rerank the top , docked molecules from the initial screen to identify compounds with similar functionality and three-dimensional ( d) shape using the dock hungarian similarity (hms) scoring function ( ) . the top-scoring molecules from the nine unique lists were further interrogated using five additional functional methods to assess energy score (dce) and similarity to the initial hit (footprint [fps], pharmacophore [fms], volume overlap [vos], and tanimoto). figure compares docked geometries for four of the initial hits (gray) overlaid with two representative compounds each (orange) from the secondary screen. in these examples, with the exception of i , the compounds generally showed strong overlap and made residue-based interaction patterns similar to those of their respective references (fig. ), corresponding to a high average vos score of ϳ . and a low average fps score of ϳ . . despite the overall similarity of ligand scaffolds within each group, the use of different dock functions generally resulted in the selection of chemically diverse molecules at the atomic level. in some cases, however, the same ligand was the top-ranked candidate across the different groups. for example, rank ordering by pharmacophore or volume overlap yielded the same top-scored results for i (fms ϭ . , vos ϭ . ), which suggests high structure and functional similarity with the initial hit (fig. , fms and vos). overall, the secondary virtual screen resulted in the selection of additional candidates, which were subsequently evaluated for inhibition and cytotoxicity at m against ebov-pseudotyped virus. a luciferase signal of Յ . , which was more than standard deviation below the population mean luciferase signal of . Ϯ . , was used to identify additional hits with moderate to low cytotoxicity ( fig. , green, s prefix). dose-response characterization of candidates against hiv/ebov-gp-pseudotyped virus. to further explore the most promising candidates identified from the two in silico screens ( initial plus secondary), in terms of reducing infectivity and their effects on cell viability, the dose-dependent activity for each was measured. of the tested from fig. , compounds exhibited generally well-behaved entry inhibition compared to that of the known control inhibitor, e , seemingly independent of cytotoxicity, especially at the observed % inhibitory concentration (ic ) values, as shown in fig. . the structures of the compounds, with code names, are shown in fig. . encouragingly, of the molecules, exhibited ic values under m, comparable to the results observed for the control inhibitor e (ic ϭ . Ϯ . m) under the same conditions ( fig. and table ). specifically, the ic values for i , i , and s were less than m, and the ic values for s , s , s , and s were less than m (fig. and table ). an accurate cytotoxic concentration that results in % cell death (cc ) could be obtained for of the compounds. for s , s , and e , the computed cc values had large standard deviations, although examination of the cytotoxicity curves suggests minimal impact on cell viability. the two most potent molecules in this assay, i and i , displayed cc values of approximately to m ( table ). all other hits had observed cc values of m or greater. selectivity index (si ϭ cc /ic ) values were also calculated. the higher the si ratio, the more potent and the safer the compound is projected to be in vivo. examination of the data showed a range of si values from to for pseudotyped virus ( table ) . of the compounds with computable si, the two hits with the greatest si were s and s , which have si values around (table ) . to test the effects of the inhibitors in an ebov system that utilizes virus particles of a size and shape similar to that of native ebov ( ), the compounds were assessed for inhibitory effect against the ebov trvlp system at various concentrations ( fig. ) . notably, of the yielded ic s under m (table ) . of particular interest, comparative linear regression analysis between the ic s observed for each candidate against ebov-pseudotyped virus and trvlps yielded an r value of . (n ϭ ), which increased to . (n ϭ ) with the removal of the outliers i and s ( table ) . as expected, based on the good correspondence between the two dose-response assays, i remained the most potent compound, with an ic of . Ϯ . m (fig. and table ). furthermore, s , which exhibited an ic of approximately m in the pseudotyped experiment, was the only compound found to have an ic greater than m. the range of si values from the trvlp experiments was between . and . , with five candidates yielding selectivity indices greater than that of e (table ). of the aforementioned five inhibitors, the two compounds with the largest si values are s ( . ) and s ( . ) ( table ). in summary, the results indicate good reproducibility between pseudotyped virus and trvlp assays, affirming the observed activity of the tested hits. specificity of candidates for ebov-gp. the hits were also examined using computational and experimental methods to ascertain if the observed activity involved nonspecific effects as a result of colloidal aggregation, pan-assay interference compound (pains) liabilities ( - ), or promiscuity. as an initial step to assess whether activity was a result of colloids, the compounds were screened for structural similarity to known aggregators using aggregation advisor (http://advisor.bkslab.org) ( ) . eight candidates exhibited no known similarity to compounds in the current database. the three remaining compounds (i , s , and s ) were found to have %, %, and % structural similarity to a known aggregator. as described by irwin and shoichet ( ), the addition of detergent should lead to a decrease in activity if a compound inhibits exclusively due to colloidal aggregation. thus, activity was also tested in the presence of . % tween (table ) . compounds here were defined as not sensitive to detergent if their ic values with and without detergent were similar, if their ic ranges with and without detergent overlapped, or if their activity increased. based on these criteria, none of the hits appeared to be sensitive, although s was classified as ambiguous due to the absence of a computable error associated with the ic value. the compounds were also subjected to an evaluation for pains alerts using distinct computational filters (cbligand [ ] , fafdrugs [ ] , and swissadme [ ] ). i was the only compound with a pains warning, which occurred for all three programs due to the possibility of mannich reaction ( ) . despite this warning, we opted to retain compound i at this early stage given the fact that multiple fda-approved drugs elicit pains alerts ( ) . finally, pubchem ( ) was searched to assess if any of the compounds fig dose-response infectivity of ebov trvlps with the treatment of hits. the most promising candidates identified from assays using pseudotyped virus were retested against ebov trvlps. molecules from the initial and secondary screens are labeled with the prefixes i and s, respectively. dose-response curves (black) and cytotoxicity results (red) were generated from replicate experiments (n Ն ). ic s are displayed above each graph with the number of biological replicates performed to calculate the viral entry results. were previously reported as being active against multiple targets (i.e., whether or not they were promiscuous inhibitors). results were only available for i , which had been tested in independent studies. in these prior works, i was reported as active in studies to different targets, as an inconclusive inhibitor in experiments, and as a nonspecific inhibitor of steroidogenic acute regulatory protein (bioassay aid ; https://pubchem.ncbi.nlm.nih.gov/bioassay/ ) ( ) . due to its apparent promiscuity, i was not considered further. a counterscreen using vsv (hiv- /vsv-g) was performed to experimentally determine the specificity of the prioritized set of compounds. in a procedure similar to that of the ebov-pseudotyped virus screen (fig. ) , cells were treated with dmso, the ebov inhibitor e , the nonspecific endosome acidification inhibitor bafilomycin a ( ), or the candidates (fig. ). compound i was tested at m due to its low cc (table ) , while the other candidates were tested at m. notably, all compounds showed less inhibitory activity against the vsv-g screen (fig. ) than the initial ebov-gp screen (luciferase signal, Յ . ) (fig. ) . the four compounds with the least average inhibitory activity against vsv-g and, therefore, likely higher specificity for ebov were i , s , s , and s (fig. ) . these hits showed minimal effects on cell viability. based on the aforementioned analysis, although other compounds shown in fig. would also be promising to explore, at this stage only i , s , s , and s were selected for further characterization. candidate compounds exhibited maximal inhibition postattachment and before membrane fusion. to explore the stage in the ebov entry cascade at which the candidates act, time-of-addition (toa) experiments ( , , , ) were performed ( fig. ) for the four compounds showing the most specificity, as suggested by the averaged activity results depicted in fig. . in this toa assay, t cells were treated with the four candidates and the cathepsin inhibitor e d at various time points postinfection. compounds were tested at the concentration required to reach maximum inhibition without a significant effect on cell viability as described by the dose-response curves against pseudotyped virus (fig. and table ). importantly, the four candidate molecules exhibited an activity trend similar to that of the known control e d, where maximum inhibition occurred up until the -min time point and then began to decrease (fig. ). the fact that the compounds track with e d suggests they act after pinocytosis, after cleavage to the npc binding form, but prior to the fusion step, as expected for molecules targeted to disrupt the interaction between the chr and nhr necessary for hb formation. i and s exhibit reproducible pose stability in md simulations. experimental characterization through concentration-dependent analysis, counterscreening, and toa experiments suggested that i , s , s , and s were the most potent, specific inhibitors of the premembrane fusion stage of ebov entry identified from virtual screening. to more fully explore the energetic and geometric compatibility of these inhibitors with gp at the proposed pocket, all atom md simulations of the dockpredicted poses were executed. as previously described ( , , ) , six replica -ns simulations for each candidate-gp complex were performed in explicit solvent, where each replica employed a different random seed. ligand movement was quantified using rmsds (root mean squared deviations) that accounted for translation, rotation, and differences in internal geometry relative to the initial predicted pose. analysis of the trajectories showed that of the four compounds simulated, i and s maintained their dock-predicted poses more closely across all six simulations, as observed by the reproducible average rmsds of . Ϯ . Å and . Ϯ . Å, respectively (fig. ) . since the average rmsds of i and s were less than or equal to . Å, which is close to the typical benchmark ( . Å) commonly used in redocking validation tests ( ) , additional characterization for these two compounds was performed as described further below. in contrast, s and s adopted a wider variety of ligand poses during md simulations, resulting in a larger range of rmsds (fig. ) . visual inspection showed s adopted two overall geometries during its md simulations, one closer to the original dock pose, which contributed to its bimodal rmsd histogram (fig. ) . in general, compound s showed a much larger overall spread in rmsds (mean of Ͼ . Å) as a result of larger changes in internal geometry and/or movement in the pocket. footprint interaction analysis. as a step toward understanding the hypothesized mechanism of action inhibiting six-helix bundle formation, the interactions of i and s with gp were characterized. to determine which residues had the greatest contribution to the ligand-receptor interactions across both hits, footprint interaction profiles were generated for each compound from the energies obtained over the md trajectories (fig. ) . overall, the footprints showed striking similarity to the reference, especially in terms of the vdw profile (fig. ) , suggesting good molecular mimicry of the chr region. moreover, i and s maintained strong contacts to a similar degree with the same residues, consistent with their overlap in the binding site and structural similarity. the residues with the most favorable interactions across the two candidates, which resulted in combined average energies greater than Ϫ . (fig. ) . regarding the es energies, the reference profile contains two es peaks corresponding to e .a and q .a; however, e .a was the only consensus residue with a combined average energy (Ϫ . Ϯ . kcal/ mol) of less than Ϫ . kcal/mol (fig. ) . notably, s also had a considerable interaction with q .a (Ϫ . Ϯ . kcal/mol) (fig. ). further inspection of the individual footprint profiles of i and s showed that s interacted slightly more favorably with the ebov five-helix bundle than i across multiple residues in addition to q .a. for instance, s had stronger predicted interactions with e .a in both the vdw (Ϫ . Ϯ . kcal/mol) and es (Ϫ . Ϯ . kcal/mol) plots than i (vdw, Ϫ . Ϯ . kcal/mol; es, Ϫ . Ϯ . kcal/mol). although simulation of s resulted in slightly greater energies over of the key residues (fig. ) , the energies of the candidates are within one standard deviation from the means and therefore are insignificantly different, highlighting e .a, a .a, l .a, f .a, t .c, l .c, and l .c as the key gp residues that interact with the reference ligand, i , and s . of the corresponding residues, notable favorable vdw interactions were visualized at f .a and t .c. specifically, f .a was involved in strong nonspecific vdw interactions with the -methyloxy, -carboxylphenyl substituent of i and the phenyl substituent of s (fig. ) . additionally, although both hits interact with t .c, i was the only compound to exhibit a vdw interaction with t .c throughout approximately . % of the simulations. regarding es interactions, the two inhibitors established and maintained strong es contacts with e .a across one main substituent throughout the majority of their md simulations. for instance, the protonated nitrogen of the methylpiperidine substituent of i maintained water-mediated hydrogenbonding interactions (ϳ %) with the backbone and sidechain of e .a and direct hydrogen-bonding interactions with the sidechain of e .a about % of the time (fig. ) . on the other hand, s retained water-mediated interaction with e .a through approximately % of the simulations and direct hydrogen-bonding interactions for a total of approximately % of the simulations (fig. ). in summary, results suggest that i and s have the potential to establish and retain strong vdw and es interactions with the predicted gp binding site. sequence conservation across the key residues. to assess whether the inhibitors have the potential to interact favorably with other ebolavirus species and related filoviridae viruses, a comprehensive sequence alignment study was conducted. specifically, human sample sequences containing the complete gp genome for the five known ebolavirus species, zaire, bundibugyo, reston, sudan, and tai forest, were selected via the virus pathogen resource (vipr) database (www.viprbrc.org; nih). an additional virus sequences were selected using blast ( ) based on similarity to the core gp sequence (pdb entry ebo_a) used to conduct the virtual screens. multiple-sequence alignment was then performed using cobalt ( ) to align the above-mentioned , gp -containing sequences to the full-genome sequence of gp (zaire ebolavirus strain mayinga- ; genbank accession number ahc ). ultimately, sequences seen in humans and nonhuman primates were retained with fragmented or complete gp sequences, which were used for sequence comparison analysis (fig. ) . consistent with previous studies ( , , ) , there is high sequence identity for the core region of gp (g -f ), with the exception of an intentional cys ala mutation introduced into ebo_a to facilitate crystallization ( ) . comparison of genomes to a complete gp sequence (residues to ) exhibited approximately % conservation (fig. , bottom) . notably, the zaire sequences, which are the most common and pathogenic ( ) ( ) ( ) , showed % sequence similarity. the seven key residues identified from the md-based footprint analysis of i and s with gp (e .a, t .c, a .a, l .c, l .a, f .a, and l .c) showed greater than % conservation across all sequences, and for zaire in particular there was ϳ % conservation. overall, the high sequence conservation among the subset of surveyed genomes for the five ebolavirus species, for which a representative example is shown in fig. , suggests that i and s have the potential to interact with the seven key residues in analogous gp binding sites (fig. , shaded bars) and thereby inhibit sequence variants of zaire ebolavirus and different ebolavirus species. however, experimental testing would be required to characterize the activity of the small molecules against the different viruses. ebov particles enter the cell through macropinocytosis ( ) , where they are later trafficked to the endosome and a conformation change is induced in the viral envelope protein gp that leads to membrane fusion ( ) ( ) ( ) ( ) . during this conformational change, the three chr regions bind to the nhr trimer, forming a six-helix bundle ( hb) and mediating host-virus membrane fusion ( ) . due to the current lack of fdaapproved therapeutics available to treat evd and the key involvement of gp in virus entry, this study focused on the identification of small-molecule leads to inhibit the formation of the hb necessary for virus entry by targeting gp at the interface where the chr interacts with the nhr. it is important, however, to note that our gp docking model is only an approximation of the ebov prehairpin and, thus, is not likely to reflect all of the subtleties inherent in the actual biological system. nevertheless, as the approach was successfully used by our group in prior work ( , , ) and led to the identification of entry inhibitors targeting hiv gp , we believe that adapting the methods to target ebola is a reasonable strategy. in this work, an initial virtual screen followed by a second similarity screen were performed to prioritize molecules with energetically favorable interactions with the gp nhr pocket. this led to a total of compounds for experimental testing, of which appeared promising in an ebov-pseudotyped virus entry assay. subsequent doseresponse analyses narrowed down the group to inhibitors with low to moderate cytotoxicity. to further validate activity, the hits were tested against ebov trvlps, which are more similar in shape and size to the native virus. the trvlp results correspond well with those obtained using pseudotyped virus, affirming the hits are promising ebov inhibitors. to probe specificity, the hits were also tested using vsv-g-pseudotyped virus-like particles (fig. ) . at this stage, four compounds (i , s , s , and s ) were prioritized for additional analysis given their strong inhibition, low cytotoxicity, and apparent specificity for ebov. in the time-of-addition assay, the control curve for e d showed the maximal level of inhibitory activity occurring between time and up to the -min time point at which inhibition starts to decrease (fig. ) . this is consistent with other studies ( ) that have a lag in the ebov entry pathway compared to that of influenza virus due to trafficking to the late endosome/lysosome. the timing of loss of inhibition of e d and the experimental compounds, as reported in mingo et al. ( ) , occurred with full restoration of infectivity by the -h time point. in contrast, full infectivity was not restored in our system until approximately h. this could be due to differences in vlps, cell types, or the readout assay. importantly, all four hits (i , s , s , and s ) exhibited a time-of-addition trend similar to that of e d, suggesting that they are acting late in entry at a step that is after npc binding. although the hits are hypothesized to prevent the collapse of the metastable intermediate into the stable hb, it is possible they interact with an earlier gp /gp prefusion conformation. they could also disrupt interactions with other partner proteins, the lipid bilayer, or bilayer components or disrupt the putative e d-sensitive cleavage step ( ) . additional mechanistic investigation, such as site-directed mutagen-esis and structural studies, will be required to confirm our hypothesis that the hits are inhibiting hb formation. further study of the candidate compounds and future work on analogs and additional target sites could uncover important details about the fusion trigger. as an initial step, to help validate that the inhibitors prevent hb formation, the steric and energetic compatibility of the hits were explored via md simulations. for the two hits with the most reproducible ligand poses (lower rmsds), the md analysis identified seven key gp residues (e .a, a .a, l .a, f .a, t .c, l .c, and l .c) engaged in significant favorable protein-ligand interactions (fig. ) . notably, these residues are highly conserved (fig. ) across different ebolavirus species, suggesting the hits have the ability to inhibit different types of evd-causing viruses. the compounds identified in this work have efficacy similar to that of other reported inhibitors of virus entry. specifically, we identified compounds with ic values of less than m, and three of the hits had ic values of less than m. previously reported inhibitors include zmapp, which is a combination of three antibodies, two of which appear to prevent conformational changes in the npc- -primed gp that are necessary for progression to late-stage entry ( ) . the estimated ic value for zmapp is to m (estimated from literature values reported by holtsberg et al. [ ] of . to . g/ml). other examples include c-peptide inhibitors ( ) designed on the concept of the successful hiv peptides t (enfuvirtide) and c , which prevent hb collapse ( ) . in contrast to hiv c-peptides, ebov c-peptides showed weak or insignificant antiviral activity due to their inability to access the endosomal compartment ( ) . however, inhibition was significantly improved when researchers added the hiv tat protein transduction domain (ptd), for which the resulting ebo-tat hybrid showed % inhibition at m ( ). other peptide-based inhibitors include prehairpin intermediate mimics reported by clinton et al. ( ) , which showed mid-nanomolar inhibition in a pseudotype assay and a series of cyclopeptides ( ) with ic values ranging from . to . m. in terms of small molecules, basu et al. ( ) reported a benzodiazepine derivative hypothesized to bind in a pocket observed in a prefusion conformation of gp /gp that inhibited entry with an ic of . m. another study identified that the g protein-coupled receptor (gpcr) antagonist benztropine inhibited ebov with an ic of . m. subsequent crystallographic studies by stuart and coworkers ( , ) showed that benztropine and other compounds, including bepridil, paroxetine, sertraline, toremifene, and, interestingly, ibuprofen, bound to the gp /gp site and are thought to destabilize the protein complex ( ) . in contrast, the present compounds are hypothesized to stabilize a gp fusion intermediate, which prevents conformational changes required for formation of the hb. notably, an investigation of drug synergy reported by dyall et al. ( ) using fda-approved drugs showed that the majority of pairs identified as synergistic inhibitors of ebola virus included an entry inhibitor. this suggests it is worthwhile to determine if there is synergy between the entry inhibitors identified in this work and other compounds. in summary, this study has demonstrated the utility of computer-aided modeling, in conjunction with experimental testing, to identify four compounds (i , s , s , and s ) that appear to be specific inhibitors of ebov entry. we targeted a previously unexploited site on ebov gp in a conformation representative of a prehairpin intermediate and utilized protein mimicry to select for small-molecule gp mimics. the identified inhibitors, hypothesized to prevent formation of the critical hb, serve as proof of principle for this technique and as a starting point for further gp -targeted studies. computational methods. in this work, several computational methods were employed to target gp , which can be arranged into five distinct protocols: (i) gp binding site and reference ligand designation through hot-spot identification, (ii) receptor and reference preparation, (iii) dock receptor setup, (iv) dock virtual screening protocols and compound prioritization, and (v) md simulations. the work employed several software packages, including antechamber, tleap, cpptraj ( ) , sander, and pmemd from the amber suite of programs (university of california san francisco) and dms, grid ( ) , and sphgen ( ) , which are part of the dock suite of programs (university of california san francisco). divided into chunks of at most , molecules. compounds were flexibly (flx) ( ) docked to the gp five-helix bundle in parallel, using the mpi version of dock . (university of california san francisco). for each docked compound, the best scoring pose was retained, which was then energy minimized using the standard dock cartesian energy (dce) function to further fine-tune the interactions between the receptor and candidate ligands and permit footprint similarity scoring ( , ) , where the similarity in vdw and es interaction profiles between the reference and screened molecules was quantified using euclidean distance. key descriptors were computed with the program moe for the , top-scoring molecules based on dce score, including the number of lipinski violations, number of chiral centers, and logp, to aid in compound prioritization. the moe maccs clustering method was concurrently employed, using a best-first approach, to group compounds into structurally related families with the best dce scored compound per family to serve as a clusterhead. to promote diversity in compound selection, the top-scored clusterheads were rank ordered using five distinct scoring criteria: (i) the sum of the van der waals and electrostatic dock cartesian energy score (dce sum ), (ii) the van der waals fps score (fps vdw ), (iii) the electrostatic fps score (fps es ), (iv) the sum of the fps vdw and fps es scores (fps sum ), and (v) the combined dce sum and fps sum scores (total score, or ts) ( ) . following d visual inspection of the top-scoring members from each of the five lists, compounds, referred to with the prefix i (initial screen), were purchased for experimental testing. a second set of ligands, referred to with the prefix s (secondary screen), was purchased based on similarity comparisons to hits identified in the initial screen. similarity was computed using the following dock scoring functions: hungarian similarity ( ), footprint similarity ( ), pharmacophore similarity ( ) , and volume overlap. for both screens, additional ligand properties considered included central location in the pocket, number of chiral centers (less than ), formal charge between Ϫ and ϩ , favorable overall score with respect to the particular rank-order method, and favorable electrostatic score. md simulations and analysis. for the most promising candidates, md simulations were performed to assess geometric and energetic stability. the amber accessory programs antechamber and tleap were used to protonate, solvate, assemble, and assign force-field parameters for the protein receptor (ff sb) ( ) , solvent (tip p) ( ) , and ligand (gaff) ( ) . ligand partial charges were obtained from those preassigned by the zinc database ( ) . the five-helix bundle was capped where the n terminus was capped with ace and the c terminus was capped with nme. as previously described ( ), a nine-step protocol was used to equilibrate each solvated ligandprotein complex. briefly, all simulations were performed using the cuda-accelerated version of pmemd ( ) ( ) ( ) in amber . in short, first the solvent and protein-ligand hydrogens were minimized with a restraint weight of . kcal mol Ϫ Å Ϫ on all complex heavy atoms for , cycles. second, the restraint was lifted and the entire complex was minimized for , cycles. third, over ps, the system was heated from to k. fourth, a short md simulation of ps, with an all-atoms restraint weight of . kcal mol Ϫ Å Ϫ , was performed to optimize the water box density to . lastly, each complex underwent five equilibration steps, each ps in length, with lessening restraint weights of all protein and ligand heavy atoms. for the protein, the restraint weights were (i) visualization of md trajectories was conducted using vmd ( ) and chimera ( ) . the amber accessory program cpptraj ( ) and in-house protocols were utilized to extract vdw and es energies (with distance-dependent dielectric) and compute molecular footprints, rmsds (root mean squared deviations), and hydrogen-bonding interactions of each compound throughout its md trajectories ( , frames for each simulation). as previously described ( , ) , predicted interaction energies from all six replica md trajectories were used to calculate the mean vdw and es energies between the small molecule and each residue of the five-helix bundle. residues with energies of less than Ϫ . kcal/mol for the reference ligand and experimentally verified gp entry inhibitors were used to select key gp residues involved in an interaction energy. to compute ligand rmsds, a two-step protocol was executed ( ) . first, the protein-ligand complex in each frame of the trajectory was aligned using cpptraj so that the protein's alpha carbons overlapped. second, atomic-level small-molecule translation and rotation compared to that of the docked pose was quantified. for interpretation, rmsds were binned based on frequency using cpptraj and plotted using python (python software foundation). the amber accessory program cpptraj was used to extract the direct and water-mediated hydrogen-bonding interactions from each trajectory and provide a frequency, location, and frame. experimental methods. the experimental methods to characterize the inhibitory activity of the small molecules identified from in silico screening are described below. three different assays were employed: (i) pseudotyped hiv- /ebov-gp was utilized to assess viral entry, (ii) pseudotyped hiv- /vsv-g was utilized to assess inhibitor specificity, and (iii) ebov trvlp was utilized as a second confirmatory assay of viral entry. cell lines and plasmids. the following reagents were obtained through the aids reagent program, division of aids, niaid, nih: tzm-bl cells (number ; from j. c. kappes and x. wu) ( ) and replication-defective hiv vector pnl - .luc.r-e-(number ; from n. landau) ( ) . the following reagent was obtained through bei resources, niaid, nih: vector pcdna . containing zaire ebolavirus glycoprotein nr- ( ) . plasmid pcmv-vsv-g was a gift from e. freed (nci-frederick). the ebov trvlp transfection plasmids pcaggs-vp , pcaggs-np, pcaggs-vp , pcaggs-l, pcaggs-t , p cis-vrna-rluc, and pcaggs-tim were a gift from h. feldmann (nih) ( ) . t cells (atcc crl- ) and tzm-bl cells were cultured in dulbecco's modified eagle's medium (dmem; corning) supplemented with % heat-inactivated fetal bovine serum (fbs; gemini bio-products) containing g/ml of streptomycin and u/ml of penicillin (dmem-ps- % fbs) in a °c incubator with % co atmosphere. replicationincompetent pseudotyped virus containing the replication machinery of hiv- and the outer glycoproteins of either ebola (hiv- /ebov-gp) or vesicular stomatitis (hiv- /vsv-g) virus were prepared by a standard transfection method using polyethylenimine (pei) max (polysciences) ( , ) . specifically, h prior to transfection, ϫ cells of t cells were seeded per -mm dish. the cells were cotransfected with equal amounts ( . g) of hiv- core plasmid (pnl - .luc.r-e-) and envelope protein plasmid using g pei transfection reagent per plate. twenty-four h posttransfection the medium was replaced, and pseudotyped virus was harvested from the supernatant at and h posttransfection. the supernatant was clarified by low-speed centrifugation followed by filtration with a . -m-pore-size filter (millipore). the filtered supernatant was centrifuged ( , rpm) at °c for h, and the pellet was resuspended in dulbecco's phosphate-buffered saline (dpbs) and stored at Ϫ °c until needed ( ) . infectious titers of virus stocks were quantified by -bromo- -chloro- -indolyl-␤-d-galactopyranoside staining in tzm-bl cells ( , ) . ebov trvlp preparation. a transient-transfection-based transcription-and replication-competent system that models the entire replication cycle at biosafety level was utilized to confirm inhibition. this system is more physiologically relevant than pseudotyped virus due to the native size and shape of the ebov particles. preparations of ebov trvlps were prepared as previously described ( , ) . briefly, t cells were seeded in ml in a -well plate at ϳ % confluence. twenty-four h postseeding, the cells were transfected with the following plasmids per well: ng pcaggs-vp , ng pcaggs-np, ng pcaggs-t , ng pcaggas-vp , g pcaggs-l, and ng p cis-vrna-rluc, using . g pei transfection reagent. twenty-four h posttransfection, medium was replaced with ml dmem-ps- % fbs. seventy-two h posttransfection, the supernatant containing the trvlps was pooled, clarified by lowspeed centrifugation, and stored at °c. screening of in silico-selected compounds in viral entry assays. viral entry was measured using a luciferase reporter. testing of selected compounds and controls against all three types of virus particles, ebov (hiv- /ebov-gp) pseudotyped, vsv (hiv- /vsv-g) pseudotyped, and ebov trvlp, was performed in a similar procedure. t cells were seeded at ϫ cells/well in -well tissue culture-treated white-bottom plates (greiner) that were precoated with g/ml linear pei (sigma). for ebov trvlp infection, helper ribonucleoprotein (rnp) components must be provided in trans through expression plasmid transfection h postseeding (amounts of helper rnp plasmids per well were . ng pcaggs-vp , . ng pcaggs-np, . ng pcaggs-vp , . ng pcaggs-l, and . ng pcaggs-tim , with . ng pei transfection reagent). twenty-four h postseeding (pseudotyped virus particles) or posttransfection (trvlps), t cells were pretreated with selected compounds or controls for h at °c. the medium then was removed and the cells were infected with virus particles that had also been pretreated for h at °c. after h the inoculum was removed, the cells were washed briefly with pbs, and fresh medium was added. plates were incubated for h, and viral entry was measured using the luciferase reporter. the experiment was also performed in the absence of virus to determine the toxicity of the selected compounds and controls. viral entry and cell viability were measured using one-glo ϩ tox luciferase reporter and cell viability assay (promega) according to the manufacturer's protocol using a spectra max m plate reader (molecular devices). luciferase signal was normalized to the cell viability and then further normalized to the luciferase signal in the dmso-treated samples ( ) . compounds with infectivity signal per cell as a fraction of the maximum below . were considered active hits in the initial screening. additionally, for the dose-response assays, % inhibitory concentration (ic ), % cytotoxicity concentration (cc ), and % confidence intervals (ci ) were computed, and ic was plotted using prism . c (graphpad software, la jolla california usa). cc s were reported if a standard deviation within -fold of the cc could be calculated. as previously described ( ), selected controls were dissolved in dmso. cathepsin inhibitor e (millipore) is a cysteine protease inhibitor that prevents cleavage events that are necessary specifically for ebov fusion with the endosomal membrane. it is used as a positive control for inhibition in hiv/ebov-gp and ebov trvlp assays and as a negative control in vsv-g assays, as it does not inhibit vsv-g fusion. e d has the same action as e but is cell permeable. bafilomycin a (calbiochem) is a vacuolar atpase inhibitor that prevents both ebov and vsv entry by alkalinizing the endosome and is used as a positive control for inhibition in both assays. cells were infected with either ebov-or vsv-g-pseudotyped virus at a multiplicity of infection (moi) of . or with l of ebov trvlps. where indicated, . % tween (sigma) was also added to the assay to test for colloidal aggregation. time-of-addition assay. t cells were seeded at ϫ cells/well in pei-precoated -well tissue culture-treated white-bottom plates. the next day, ebov-pseudotyped virus was added to the cells at an moi of . . the plates were centrifuged for h at °c at , ϫ g to allow the virus to attach to the cells and to synchronize the infection. the plates were washed with pbs to remove unbound virus. the plates were then moved to °c to allow for viral entry ( h). small molecules i ( m), s ( m), s ( m), and s ( m) and the e d control ( m; millipore) were added to the plates at various time points as indicated. cell viability and viral entry were measured and analyzed h postinfection as described above. report of an international commission fifteen countries are at risk of ebola outbreak, says who clinical management of ebola virus disease in the united states and europe characteristics and survival of patients with ebola virus infection, malaria, or both in sierra leone: a retrospective cohort study chemical and structural aspects of ebola virus entry inhibitors identification of a small-molecule entry inhibitor for filoviruses a new player in the puzzle of filovirus entry basic clinical and laboratory features of filoviral hemorrhagic fever ebola virus disease in west africa-clinical manifestations and management emergence of zaire ebola virus disease in guinea bioterrorism: management of major biological agents ebola (ebola virus disease) treatments filoviridae: marburg and ebola viruses biochemical and functional characterization of the ebola virus vp protein: implications for a role in virus assembly and budding host factors in ebola infection ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis endosomal proteolysis of the ebola virus glycoprotein is necessary for infection proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection structural insights into the niemann-pick c (npc )-mediated cholesterol transfer and ebola infection ebolavirus glycoprotein structure and mechanism of entry core structure of the envelope glycoprotein gp from ebola virus at . -Å resolution designed protein mimics of the ebola virus glycoprotein gp ␣-helical bundle: stability and ph effects c-peptide inhibitors of ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking design and characterization of ebolavirus gp prehairpin intermediate mimics as drug targets mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies novel small molecule entry inhibitors of ebola virus inhibition of ebola and marburg viral entry by g protein-coupled receptor antagonists toremifene interacts with and destabilizes the ebola virus glycoprotein potent neutralizing monoclonal antibodies against ebola virus infection antibody treatment of ebola and sudan virus infection via a uniquely exposed epitope within the glycoprotein receptor-binding site target identification and mode of action of four chemically divergent drugs against ebola virus infection identification of diaryl-quinoline compounds as entry inhibitors of ebola virus identification of ellagic acid from plant rhodiola rosea l. as an anti-ebola virus entry inhibitor molecular docking based screening of predicted potential inhibitors for vp from ebola virus cysteine cathepsin inhibitors as anti-ebola agents inhibition of heat-shock protein reduces ebola virus replication enfuvirtide dock : impact of new features and current docking performance development of peptide and small-molecule hiv- fusion inhibitors that target gp computer-aided approaches for targeting hivgp small molecule inhibitors of hivgp n-heptad repeat trimer formation footprint-based identification of viral entry inhibitors targeting hivgp development of indole compounds as small molecule fusion inhibitors targeting hiv- glycoprotein- structure-based identification of small molecule antiviral compounds targeted to the gp core structure of the human immunodeficiency virus type development of hiv entry inhibitors targeted to the coiled-coil regions of gp n-substituted pyrrole derivatives as novel human immunodeficiency virus type entry inhibitors that interfere with the gp six-helix bundle formation and block virus fusion design, synthesis, and structureactivity relationship of a novel series of -aryl -( -oxo- -phenethyl- -thioxothiazolidinylidenemethyl)furans as hiv- entry inhibitors development of smallmolecule hiv entry inhibitors specifically targeting gp or gp evidence that a prominent cavity in the coiled coil of hiv type gp is an attractive drug target inhibition of human immunodeficiency virus type infectivity by the gp core: role of a conserved hydrophobic cavity in membrane fusion identification of small molecule inhibitors of botulinum neurotoxin serotype e via footprint similarity implementation of the hungarian algorithm to account for ligand symmetry and similarity in structure-based design a novel life cycle modeling system for ebola virus shows a genome length-dependent role of vp in virus infectivity the ecstasy and agony of assay interference compounds phantom pains: problems with the utility of alerts for pan-assay interference compounds docking screens for novel ligands conferring new biology an aggregation advisor for ligand discovery new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays faf-drugs : a web server for compound property calculation and small-molecule inhibitors of ebola virus entry journal of virology chemical library design swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules pubchem substance and compound databases pubchem bioassay: update vesicular stomatitis virus g protein acquires ph-independent fusion activity during transport in a polarized endometrial cell line ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc ϩ endolysosomes is a rate-defining step ph-remd simulations indicate that the catalytic aspartates of hiv- protease exist primarily in a monoprotonated state basic local alignment search tool cobalt: constraint-based alignment tool for multiple protein sequences in silico prediction of ebola zaire gp , immuno-dominant epitopes for the balb/c mouse structure-based identification of inhibitors targeting obstruction of the hivgp n-heptad repeat trimer mapping of ebolavirus neutralization by monoclonal antibodies in the zmapp cocktail using cryo-electron tomography and studies of cellular entry pan-ebolavirus and panfilovirus mouse monoclonal antibodies: protection against ebola and sudan viruses inhibition of ebola virus entry by a c-peptide targeted to endosomes inhibition of hiv entry by targeting the envelope transmembrane subunit gp novel cyclo-peptides inhibit ebola pseudotyped virus entry by targeting primed gp protein identification of combinations of approved drugs with synergistic activity against ebola virus in cell cultures ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data automated docking with gridbased energy evaluation using shape complementarity as an initial screen in designing ligands for a receptor binding site of known threedimensional structure grid-based molecular footprint comparison method for docking and de novo design: application to hivgp implementation and evaluation of a docking-rescoring method using molecular footprint comparisons comparison of multiple amber force fields and development of improved protein backbone parameters strategies for lead discovery: application of footprint similarity targeting hivgp docking validation resources: protein family and ligand flexibility experiments ucsf chimera-a visualization system for exploratory research and analysis fast, efficient generation of high-quality atomic charges. am -bcc model. i. method fast, efficient generation of high-quality atomic charges. am -bcc model. ii. parameterization and validation development and testing of a general amber force field zinc-a free database of commercially available compounds for virtual screening pharmacophore-based similarity scoring for dock ff sb: improving the accuracy of protein side chain and backbone parameters from ff sb comparison of simple potential functions for simulating liquid water routine microsecond molecular dynamics simulations with amber on gpus. . generalized born routine microsecond molecular dynamics simulations with amber on gpus. . explicit solvent particle mesh ewald spfp: speed without compromise-a mixed precision model for gpu accelerated molecular dynamics simulations vmd: visual molecular dynamics effects of ccr and cd cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type human immunodeficiency virus type viral protein r (vpr) arrests cells in the g phase of the cell cycle by inhibiting p cdc activity the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles optimization of lentiviral vector production using polyethylenimine-mediated transfection transient mammalian cell transfection with polyethylenimine (pei) production, concentration and titration of pseudotyped hiv- -based lentiviral vectors detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene permanent inhibition of viral entry by covalent entrapment of hiv gp on the virus surface clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications. viruses :e development of therapeutics for treatment of ebola virus infection ebola virus entry: a curious and complex series of events key: cord- -w mbvdw authors: volchkov, viktor; klenk, hans dieter title: proteolytic processing of filovirus glycoproteins date: - - journal: activation of viruses by host proteases doi: . / - - - - _ sha: doc_id: cord_uid: w mbvdw filoviruses (marburg virus and ebola virus) have a single envelope glycoprotein (gp) that initiates infection. gp is a class i fusion protein that forms trimeric spikes composed of heterodimers of the subunits gp and gp . gp and gp are derived from the precursor pre-gp by furin cleavage during exocytosis. gp contains a receptor-binding core topped by a glycan cap and a heavily glycosylated mucin-like domain, while gp contains a fusion loop and a membrane anchor. after entering cells by macropinocytosis, the glycan cap and the mucin-like domain are removed from gp by endosomal cathepsins b and l exposing the binding site for the niemann-pick c receptor. it appears that there is no strict requirement for specific proteases involved in gp processing. thus, furin is not indispensible for gp - cleavage, and gp may be trimmed not only by cathepsins b and l but also by other endosomal proteases. two soluble glycoproteins of ebola virus are also processed by host proteases. a significant amount of gp , is cleaved by the metalloprotease tace and shed from the surface of infected cells (gp , delta). the secreted protein sgp is derived from the precursor pre-sgp by furin cleavage. filoviruses comprising marburg virus (marv) and ebola virus (ebov) species (zaire, sudan, reston, bundibugyo, and tai forest virus) cause fulminant hemorrhagic fevers in man and nonhuman primates. marv and ebov have a zoonotic background and, except for reston virus, are endemic in sub-saharan africa. since the discovery of marv in and ebov in , the viruses re-emerged with increasing frequency. most of the outbreaks were dramatic but confined to relatively short time periods and small geographic areas. between and , however, an unprecedented ebov outbreak occurred in west africa with almost , human infections and more than , deaths. the non-segmented negative-stranded rna genome of filoviruses contains seven genes: np, vp , vp , gp, vp , vp , and l. the gp gene of ebov has two overlapping reading frames from which three glycoproteins are expressed by transcriptional editing: the envelope glycoprotein gp and two nonstructural glycoproteins, sgp and ssgp. in contrast, the envelope glycoprotein of marv is expressed as the only gene product from a single open reading frame (volchkov et al. (volchkov et al. , sanchez et al. ) . gp is a type i membrane glycoprotein that matures during export through the exocytotic transport route to the cell surface. er-associated gp, designated pre-gper, contains oligomannosidic n-glycans and shows sensitivity to endoglycosidase h treatment. oligomerization of gp occurs already within the er early after pre-gper synthesis (v. volchkov, unpublished results) . pre-gper lacks the signal peptide sequence which is co-translationally cleaved by cellular signal peptidase. the second precursor identified, designated pre-gp, represents the golgi-associated form of gp. this precursor contains mature n-glycans and is o-glycosylated. still within the golgi apparatus, pre-gp is processed by proteolytic cleavage into gp , consisting of the amino-terminal fragment gp and the carboxy-terminal fragment gp linked by a disulfide bond (volchkov et al. a; sanchez et al. ) (fig. . ). gp , complexes are present at the surface of ebov-infected cells and build up trimeric spikes on virions. proteolytic processing of the envelope glycoprotein of filoviruses has been unnoticed for a rather long period of time, largely due to the fact that pre-gp, mature gp , , and the gp subunit have similar migration rates on polyacrylamide gels and that gp tends to escape detection because it partly comigrates with the vp protein. we know now, however, that cleavage of gp is remarkably efficient and that unprocessed gp is not present on ebola virions in any significant amount. ebov gp is cleaved into subunits gp and gp by furin at the motif r-t-r-r (volchkov et al. a) . furin cleavage was assessed by the observation that cleavage efficiency was dramatically reduced when gp was expressed in the furin-deficient lovo cell line but was fully restored in these cells by vector-expressed furin. the finding that cleavage was effectively inhibited by peptidyl-chloromethylketone containing a furin motif or by site-directed mutagenesis of the furin site further supported this concept. the surface glycoprotein of marv is proteolytically processed in a similar way as that of ebov; two precursor molecules and mature gp , consisting of the disulfide-linked cleavage products gp and gp were identified in cells expressing marv gp and in marburg virions (volchkov et al. a (volchkov et al. , . interestingly, marv gp contains two sites suitable for furin cleavage: r-r-k-r and r-l-r-r . it appears that the second site is not used for protein processing, possibly due to conformational constraints. site-directed mutagenesis revealed that marv gp is indeed proteolytically processed at the first furin site (volchkov et al. ) . mutations introduced at the multibasic site revealed the consensus sequence recognized by furin or the related proprotein convertase pc / which contains arg at positions − and − as a minimal requirement and arg/lys at position − for cleavage optimization (see chap. ). thus, substitution r l at position − resulted in a dramatic loss of cleavage, whereas mutation k m at position − showed a reduction in cleavage efficiency (volchkov et al. ) . a fraction of ebov gp , that is not incorporated into virions is released from the cell surface after removal of the membrane anchor by the metalloprotease released gp , , designated gp , delta, is present in the trimeric form which, however, is more labile than gp , trimers, indicating that the membrane anchor has a stabilizing function. gp , delta released from virus-infected cells activates non-infected dendritic cells and macrophages causing the massive secretion of pro-and anti-inflammatory cytokines and increased vascular permeability. these activities may be instrumental for the excessive and dysregulated inflammatory host reactions to infection and, thus, contribute to the high pathogenicity of the virus (escudero-pérez et al. ) . there is also evidence that fine-tuning of the levels of ebov gp expressed at the surface of infected cells via gp shedding plays an important role in ebov replication by orchestrating the balance between optimal virion gp content and cytotoxicity caused by gp (dolnik et al. ) . tace, also designated adam , is a member of the adam (a disintegrin and metalloprotease) family, a large group of zinc-dependent cell surface proteases. tace mediates shedding of many membrane proteins and has therefore been proposed to have the function of a common sheddase. most, but not all, substrates are cleaved between two hydrophobic residues, but neither a specific recognition sequence nor a specific secondary structure at the cleavage site appears to be required (althoff et al. ). the secreted glycoprotein (sgp) of ebov is derived from a precursor (pre-sgp) that has a length of amino acids and shares the amino-terminal amino acids with the membrane glycoprotein gp. like pre-gp, pre-sgp undergoes several coand posttranslational processing events, such as signal peptide cleavage, n-and o-glycosylation, oligomerization, and proteolytic cleavage by furin to sgp and a small peptide, designated delta-peptide (volchkova et al. (volchkova et al. , . sgp, like gp , delta (dolnik et al. ) , may have a decoy function by binding ebovspecific neutralizing antibodies (sanchez et al. ; volchkov et al. b ). there is also evidence that the cytotoxicity caused by gp is down-regulated through the expression of sgp (volchkov et al. ). the mature envelope glycoprotein of filoviruses is a class i fusion protein that forms trimeric spikes composed of disulfide-linked gp , heterodimers. the structure of the ebov glycoprotein has been analyzed in detail. early studies gave insight into the post-fusion structure of gp (gallaher ; malashkevich et al. ; weissenhorn et al. a, b; volchkov et al. ). more recently, the structure of gp , trimers in the pre-fusion state has been elucidated (lee et al. ; lee and saphire ) . according to these studies, the trimeric spike is shaped like a chalice. the bowl of the chalice is assembled by the three gp subunits, and the base is formed by the gp subunits that cradle and encircle the gp trimer ( fig. . ) . the bowl which is formed by discontinuous sections of the amino-terminal region of gp (residues - ) contains residues required for binding to an endosomal receptor and is covered by a glycan cap with a cluster of n-linked oligosaccharides (residues - ). between the glycan cap and the carboxy-terminal end of gp stretches a mucin-like domain that is about amino acids long and heavily loaded with o-glycans. the gp subunit contains the hydrophobic fusion loop, two heptad repeats typical for class i fusion proteins, and the membrane anchor ( fig. . ). gp presumably initiates infection by binding to the cell surface. a number of cell surface receptors have been implicated, but none of them proved to be necessary and sufficient for viral entry. it is widely accepted, however, that filoviruses are internalized after surface attachment by macropinocytosis and transported to endosomes (saeed et al. ; nanbo et al. ; aleksandrowicz et al. ). within endosomes, ebov gp , is cleaved by cathepsins b and/or l which is an important step in the infection process (chandran et al. ; kaletsky et al. ; sanchez ; schornberg et al. ) . cathepsin trims ebov gp from its original size (ca. kda) to an initial -kda fragment, followed by further cleavage to an approximately -kda species of gp bound to gp by non-covalent linkages and a disulfide bridge between c and c (jeffers et al. ; volchkova et al. ) (fig. . ) . the crystal structure suggests that the site of the final cathepsin cleavage is a loop reaching from residues to (lee and saphire ). this concept is supported by biochemical studies indicating that the cleavage site is located at amino acid (dube et al. ). thus, the entire glycan cap and the mucin-like domain are removed yielding a glycoprotein called gpcl that contains the receptor-binding site exposed on the truncated gp subunit and the fusion loop on gp (fig. . ) . the endosomal receptor has been identified as the cholesterol transporter niemann-pick c (npc ) (carette et al. ; côté et al. ). npc is a ubiquitously expressed endosomal membrane protein involved in the fusion and fission of endosomes and lysosomes (goldman and krise ) . after cathepsin cleavage and receptor binding, the gp subunit unwinds from its gp clamp and rearranges irreversibly into a six-helix bundle to drive fusion of viral and late endosome, gpcl endosomal membrane (bornholdt et al. ; wang et al. ). it has also been suggested that cathepsins are required for a step in genome delivery following fusion triggering (spence et al. ) . like ebov, marv enters cells by macropinocytosis and endosomal fusion, but there are some differences in the structure and in endosomal processing of the glycoproteins. structural analysis by crystallography and small angle x-ray scattering in solution indicated that the mucin-like domains of ebov gp project upward, whereas with marv gp they have a more equatorial orientation. furthermore, the glycan cap is more flexible with marv gp than with ebov gp. thus, the receptorbinding site appears to be tightly masked on the surface of ebov spikes but more exposed on the surface of marv spikes prior to endosomal cleavage (hashiguchi et al. ) . this study showed also other structural differences, particularly at the putative cleavage site, which may explain previous observations indicating that, unlike ebov, marv does not depend on cathepsin b for endosomal gp processing (gnirss et al. ; misasi et al. ). the data presented so far strongly support the concept that removal of the glycan cap and the mucin-like domain which is essential for filovirus infectivity depends on cleavage of gp at the gp -gp interphase followed by endosomal processing of gp , to gpcl. the nature of the proteases responsible for cleavage, however, has been and still is a matter of debate. the finding that ebov gp is cleaved into gp and gp by furin (volchkov et al. a ) did not come as a surprise, since this protease is responsible for the activation of many viral glycoproteins. the role of furin in the ebov life cycle became a mystery, however, when several groups reported that substitution of all basic amino acids at the furin cleavage site did not significantly affect virus infectivity. initially, these unexpected data were obtained, when pseudotype systems based on murine leukemia virus (wool-lewis and bates ) and vesicular stomatitis virus (ito et al. ) were used which allowed generation of surrogate virions carrying mutated ebov gp. the mutated glycoprotein was shown to be transported to the plasma membrane and to be incorporated into virions, predominantly in the uncleaved form, and the pseudotyped viruses infected a wide range of cell types from diverse origins. subsequently, it was reported that recombinant ebov carrying gp in which the multibasic cleavage site was replaced by nonbasic amino acids was able to replicate in vero e cells (neumann et al. ) and to cause lethal infection in nonhuman primates (neumann et al. ). these findings are frequently used as arguments against an essential function of furin cleavage in ebov replication. there is evidence, however, that does not fully support this conclusion. close inspection of the data obtained with the pseudotypes reveals that small amounts of gp , were present. likewise, a minor, but clearly detectable, fraction of gp was present in the cleaved form in the recombinant ebov (volchkov et al. ) . furthermore, recombinant ebov replicated with significantly reduced growth kinetics, when the furin cleavage site was replaced by nonbasic amino acids (neumann et al. ) . it therefore appears that cleavage of gp into subunits gp and gp is accomplished not only by furin but also, yet with lower efficiency, by other proteases that still have to be identified. it is also conceivable that only a fraction of gp has to be present in cleaved form to allow infection as has been observed with other viruses (see chap. ). in any case, there appears to be a preference for furin cleavage, since this is the most efficient processing form. this concept is underlined by the high conservation of the multibasic cleavage site with filoviruses. the only exception is reston ebov. here, the consensus sequence of a typical furin cleavage site is missing which has been suspected to account, at least in part, for the low human pathogenicity of this virus (volchkov et al. a ). as has been pointed out above, endosomal processing of ebov gp , is mediated by the cysteine proteases cathepsin b and l. there is evidence, however, that, again, both enzymes are not indispensible for this process. it could be shown that zaire ebov entry was reduced in cell culture upon selective inhibition of cathepsin b, but not cathepsin l. interestingly, all other ebov species entered the cells efficiently when cathepsin b and/or l activity was blocked. moreover, cathepsin b and cathepsin l knockout mice were equally susceptible to a lethal dose of mouse-adapted zaire ebov as wild-type animals, with no difference in virus replication and time of death (marzi et al. ) . thus, it appears that, like cleavage of gp into subunits gp and gp , endosomal trimming to gpcl is mediated by an array of proteases. this concept is also supported by the observation that cathepsin can be replaced by thermolysin to convert gp , into structurally and functionally competent gpcl (brecher et al. ) . ebov may therefore not be a very suitable target for therapeutic approaches based on protease inhibitors (marzi et al. ) , quite in contrast to other viruses, such as influenza virus, where this strategy is more promising because of the high specificity of the proteases required for activation (see chaps. , , ) . proteolytic processing of the envelope glycoprotein of filoviruses is complex involving a sequence of cleavage steps at different stages of the viral life cycle. in this respect it resembles proteolytic activation of other envelope proteins, such as the f protein of respiratory syncytial virus (see chap. ) and presumably the s protein of coronaviruses (see chap. ), that are also cleaved first during exocytosis by one and subsequently upon virus entry by another enzyme. although cleavage of pre-gp to gp , and triming of gp , to gpcl play essential roles in the processing of the filovirus envelope protein, it is not clear whether there is a strict requirement for furin and cathepsins, respectively. the specificity of the cleavage reactions and the proteases involved will have to be analysed in more detail in future studies. it is well known that cleavage primes a viral fusion protein for the conformational change required for activity, but it has never been shown before that fusion activity depends on removal of a large carbohydrate shield from the top of the spike as is the case with filoviruses. another unique feature is the high amount of virus-encoded glycoproteins that are secreted or shed by proteolytic cleavage from ebov-infected cells and may play important roles in the course of infection and in pathogenesis. ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins host-primed ebola virus gp exposes a hydrophobic npc receptor-binding pocket, revealing a target for broadly neutralizing antibodies cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change ebola virus entry requires the cholesterol transporter niemann-pick c endosomal proteolysis of the ebola virus glycoprotein is necessary for infection small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ectodomain shedding of the glycoprotein gp of ebola virus shedding of ebola virus surface glycoprotein is a mechanism of self-regulation of cellular cytotoxicity and has a direct effect on virus infectivity the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding shed gp of ebola virus triggers immune activation and increased vascular permeability similar structural models of the transmembrane proteins of ebola and avian sarcoma viruses cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss expression niemann-pick c functions independently of niemann-pick c in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo structural basis for marburg virus neutralization by a cross-reactive human antibody ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies covalent modifications of the ebola virus glycoprotein proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity structure of the ebola virus glycoprotein bound to an antibody from a human survivor ebolavirus glycoprotein structure and mechanism of entry core structure of the envelope glycoprotein gp from ebola virus at . -a resolution cathepsin b & l are not required for ebola virus replication filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences ebolavirus is internalized into host cells via macropinocytosis in a viral glycoproteindependent manner reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture proteolytic processing of the ebola virus glycoprotein is not critical for ebola virus replication in nonhuman primates cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing biochemical analysis of the secreted and virion glycoproteins of ebola virus analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein direct visualization of ebola virus fusion triggering in the endocytic pathway gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases the envelope glycoprotein of ebola virus contains an immunosuppressive-like domain similar to oncogenic retroviruses processing of the ebola virus glycoprotein by the proprotein convertase furin polymorphism of filovirus glycoproteins recovery of infectious ebola virus from complementary dna:rna editing of the gp gene and viral cytotoxicity release of viral glycoproteins during ebola virus infection proteolytic processing of marburg virus glycoprotein the nonstructural small glycoprotein sgp of ebola virus is secreted as an antiparallel-orientated homodimer delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus ebola viral glycoprotein bound to its endosomal receptor niemann-pick c the central structural feature of the membrane fusion protein subunit from the ebola virus glycoprotein is a long triple-stranded coiled coil crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function key: cord- -nrluar e authors: park, eun-mee; park, sun-whan; lee, ye-ji; lee, won-ja; choi, wooyoung title: production of ebola virus-like particles in drosophila melanogaster schneider cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nrluar e in this study, we generated recombinant virus-like particles (vlps) against family filoviridae, genus ebolavirus, species zaire ebolavirus, strain makona (ebov) in drosophila melanogaster schneider (s ) cells using the ebov makona. s cells were cotransfected with four viral plasmids encoding ebov makona proteins and protein expression was analyzed by immunoblotting. we confirmed that ebov makona proteins were successfully expressed in s cells. additionally, we further examined the formation of intracellular and extracellular vlps by electron microscopy. evlps were produced by sucrose gradient ultracentrifugation of s cells transfected with ebov makona genes, and production of vlps was confirmed by immunoblot analysis. collectively, our findings showed that the s cell system could be a promising tool for efficient production of evlps. family filoviridae, genus ebolavirus (ebov) causes severe viral hemorrhagic fever in humans and other primates (feldmann and goisbert, ) . previous outbreaks of ebov in central africa are associated with serious public health problems. indeed, since its identification in during an outbreak in zaire (world health organization, ) , there have been over outbreaks of ebov in africa (del and gaurmer, ) . cases were reported between and and patients died. , of , patients died in ebov outbreak that occurred in (cdc, ) . - % of patients infected with ebov die. the ebola virus variant makona was the causative agent of the recent outbreak of ebola during to in west africa, which was the largest outbreak to date. the recently isolated ebov makona variant, from a clinical case of ebov disease (evd) in makona, has been shown to have a varying case fatality rate (cfr) of - % at later stages of the outbreak (who ebola response team, ) . therefore, in this study, we applied s cells for production of species zaire ebolavirus, strain makona (ebov makona) vlps using the makona outbreak strain. ebov belongs to the filoviridae family and contains single-stranded, negative-sense rna genome of kb. the particles resemble long, stretched filaments and consist of three compartments, including the nucleocapsid (np), matrix space, and envelope. additionally, the particles measure nm in diameter and range from to nm in length. the genome of ebov contains seven genes, which encode gp, np, vp , vp , vp , vp , and l proteins (sanchez et al., ; falasca et al., ) . virus-like particles (vlps) mimic the structure of virions and have nonreplicating, noninfective properties because the particles lack the infectious genome and are only composed of structural or capsid proteins (kushnir et al., ) . vlp-based vaccines are safe and high immunogenic. moreover, vlps stimulate innate and humoral immunity and activate antigen presentation in antigen-presenting cells (pushko et al., ) . the development of vaccine candidates derived from vlps may be a promising approach for efficient antibody production. indeed, several vlp vaccine candidates for a variety of viruses have been developed. vaccines based on vlps for hepatitis b virus (hbv), papillomavirus, and influenza virus a have been evaluated in clinical trials in human or have been approved for clinical use (shirbaghaee and bolhassani, ; pushko et al., ) . vlps are produced in several systems, including bacteria, mammalian cells, yeast, plants, and insect cells. insect system is a promising system for high yield and rapid production of vlps (vicente et al., ) . moreover, insect systems can induce post-translational modifications similar to mammalian cells, facilitating the assembly of vlps (fang et al., ; liu et al., ) . vlp-based vaccines, including hepatitis c virus (hcv), papillomavirus, enterovirus type a (ev ), influenza virus a, and sars-coronavirus (cov), have been developed in insect systems (baumert et al., ; acosta-rivero et al., ; lechmann et al., ; senger et al., ; lopez-macias et al., ; ho et al., ) results in a high yield of hiv- vlps and proper cleavage of envelope precursor protein (yang et al., ) . in a previous study, vp / double-layered rotavirus-like particles containing wa capsid protein were produced in s cells transformed with a bicistronic expression system consisting of encephalomyocarditis virus (emcv)-derived internal ribosomal entry site (ires) element (lee et al., ) . glycoprotein (gp), nucleoprotein (np), minor matrix protein (vp) , and vp originating from ebov makona was artificially synthesized. the amplified inserts were cloned into the bglii and xbai sites of the plasmid pmt/v /his (invitrogen, carlsbad, ca, usa) and were confirmed by sequencing. np, gp, and vp of ebola virus have been shown to facilitate assembly and release of vp evlps (kallstrom et al., ) . therefore, to assess ebov protein expression, drosophila s cells were transfected with ebov plasmids using transfection reagent (tranit- ; mirus, usa); at h before harvesting, cells were treated with μm cuso to induce protein expression. the cells and culture supernatants were harvested, and ebov protein expression was confirmed by immunoblot analysis. the cells were lysed in ripa buffer (invitrogen) for min on ice, and lysates were collected after centrifugation at , rpm for min at °c. the culture supernatants were collected and concentrated using a kda centrifugal filter with ultracel- membrane (millipore, germany). to visualize the released particles, the concentrated supernatants were centrifugated through % sucrose and then negatively stained. the cell lysates and concentrated supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis after heating at °c for min, and the proteins were electrotransferred to polyvinylidene difluoride membranes. the membranes were blocked in tbs/tween solution containing % skim milk for h at room temperature. the membranes were then incubated with primary anti-v (invitrogen), anti-np (alpha diagnostics, usa), anti-vp (cosmo genetech, south korea), and anti-gp antibodies for h at room temperature, washed three times with tbs/tween solution, and incubated with secondary anti-mouse-ap, anti-rabbit-ap, or anti-human-ap antibodies for h at room temperature. finally, membranes were washed three times, and proteins were detected using a bcip/nbt substrate kit (invitrogen). as shown in fig. a , we confirmed that ebola viral proteins were efficiently expressed in ebov makona-transfected s cells. the formation of vlps similar to ebola virus particles was visualized in both ebov makonatransfected s cells and culture supernatant by transmission electron microscopy (tem; fig. b ). as negative control for vlps formation, we used cells which were transfected with gp, np, and vp without vp . as shown in fig. b , filamentous particles of approximately nm in diameter and nm in length were observed in culture supernatant of ebov makona-transfected s cells. next, the harvested cells were disrupted by repeated freezing and thawing three times, and the debris was removed after centrifugation at , ×g for min. cell lysates collected by centrifugation were overlaid on top of a continuous sucrose gradient ( %, %, %, %, and %) and ultracentrifuged at , rpm for h at °c using a sw ti rotor (beckman, usa). the procedure for sucrose gradient ultracentrifugation is illustrated in fig. a . to analyze the production of evlps, fractions were collected from top to bottom, and the expression of ebov proteins was confirmed by immunoblotting. as shown in fig. b , abundant amounts of ebov proteins were detected in fractions and . from this result, we found that evlps were mainly concentrated in fractions and . we then aimed to recover the evlps. to this end, fractions and were overlaid on top of a % sucrose cushion and ultracentrifuged at , rpm for h at °c using a sw ti rotor (beckman). the pelleted evlps were resuspended in pbs and subjected to immunoblot analysis to confirm the expression of evlps. as shown in fig. c , viral proteins were detected in the vlp pellets; analysis of the formation of evlps by tem is ongoing. collectively, these findings showed that evlps were effectively produced in an s cell system and that the s cell system may be a promising strategy for effective production of evlps. vlps have been produced in several expression systems and have been shown to provide protective effects against ebov infection in animal model. vaccination with t cell derived-ebola vlps protects nonhuman primates (nhps) or rodents from ebov infection (warfield et al., , a swenson et al., ) . antibodies against ebov are produced in serum of cynomolgus macaques vaccinated with t cell-derived evlps. high active specific antibodies against ebov are produced in serum of cynomolgus macaques vaccinated with t cellderived evlps. additionally, tumor necrosis factor (tnf)-α production in evlp-vaccinated individuals is strongly increased in cd + t cells (warfield et al., a,b) . immunization with kun-vlps generated by transfection with kun replicon rna containing ebov gp protects makona pigs and nhps from challenge with ebov infection (reynard et al., ; pyankov et al., ) . moreover, in previous works, evlps have been produced in insect systems, resulting in activation of the immune response. vlps produced in sf cells using a baculovirus system stimulated cytokine secretion in dendritic cells, and immunization of mice with the vlps increased igg a antibody production through induction of the th -biased immune response (ye et al., ) . in a previous report, high yields of hiv- vlps were produced in hiv- envelope protein-overexpressing s cells, resulting in proper cleavage of envelope precursor protein (yang et al., ) . therefore, we suggested that s cells may be a promising system for efficient production of evlps. in this study, we used s cells for the production of vlps and examined evlps production following overexpression of ebov proteins. we showed that viral proteins of ebov makona were efficiently expressed in s cells and confirmed the formation of evlps by tem. we purified evlps by sucrose gradient ultracentrifugation. collectively, our findings suggested that the evlps produced by the s cell system in this study may be useful for the development of efficient vlp-based vaccines against ebov. to the best of our knowledge, this is the first successful demonstration of vlp production from the new outbreak strain in the s expression system. further in vivo studies on the vlps of ebov are necessary to characterize immune responses to this ebov strain and to study the protective effects of vlp-based vaccines against ebov infection. we have no conflicts of interest to declare. in vitro assembly into virus-like particles is an intrinsic quality of pichia pastoris derived hcv core hepatitis c virus structural proteins assemble into virus like particles in insect cells outbreaks chronology: ebola virus disease ebola: implications and perspectives molecular mechanisms of ebola virus pathogenesis: focus on cell death differences in the post-translational modifications of human papillomavirus type b major capsid protein expressed from a baculovirus system ebola haemorrhagic fever assembly of human severe acute respiratory syndrome coronavirus-like particles biophysical characterization and conformational stability of ebola and marburg virus-like particles analysis of ebola virus and vlp release using an immunocapture assay virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development vaccine hepatitis c virus-like particle induce virus-specific humoral and cellular immune response synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed drosophila melanogaster use of baculovirus expression system for generation of virus-like particles: success and challenges safety and immunogenicity of a virus-like particle pandemic placebo-controlled trial of adults in mexico development of virus-like particle technology from small highly symmetric to large complex virus-like particle structure a kunjin replicon virus-like particle vaccine provides protection against ebola virus infection in nonhuman primates kunjin virus replicon-based vaccines expression ebola virus glycoprotein gp protect the guinea pig against lethal ebola virus infection sequence analysis of the ebola virus genome: organization, genetic elements, and comparison with the genome of marburg viruses enhanced papillomavirus-like particle production in insect cells different applications of virus-like particles in biology and medicine: vaccination and delivery systems vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infection large-scale production and purification of vlp-based vaccines advances in virus-like particle vaccine for filoviruses induction of humoral and cd + t cell responses are required for protection against lethal ebola virus infection filovirus-like particles produced in insect cells: immunogenicity and protection in rodents ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenges ebola haemorrhagic fever in zaire hiv- virus like particles produced by stably transfected drosophila s cells: a desirable vaccine component ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutrializing antibodies this study was supported by korea centers for disease . key: cord- -mb qcd b authors: seymour, elif; Ünlü, nese lortlar; carter, eric p.; connor, john h.; Ünlü, m. selim title: configurable digital virus counter on robust universal dna chips date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: mb qcd b here, we demonstrate real-time multiplexed virus detection by applying dna-directed antibody immobilization technique to a single-particle interferometric reflectance imaging sensor (sp-iris). in this technique, the biosensor chip surface spotted with different dna sequences is converted to a multiplexed antibody array by flowing antibody-dna conjugates and allowing specific dna-dna hybridization. the resulting antibody array is shown to detect three different recombinant vesicular stomatitis viruses (rvsvs) genetically engineered to express surface glycoproteins of ebola, marburg, and lassa viruses in real-time in a disposable microfluidic cartridge. we also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-dna conjugates with the virus sample in solution phase prior to flowing in the microfluidic cartridge, eliminating the antibody immobilization step. this homogenous approach achieved detection of the model ebola virus, rvsv-ebov, at a concentration of pfu/ml in hour. finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. a concentration of pfu/ml was detectable under minutes for the rvsv-ebola virus. utilizing dna microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. we believe these properties will make sp-iris a versatile and robust platform for point-of-care diagnostics applications. rapid and sensitive detection of viral infections is of significant importance for improving patient care and containing outbreaks that threaten public health. current techniques employed in clinical diagnosis of viral infections include polymerase chain reaction (pcr), enzyme-linked immunosorbent assay (elisa), isothermal nucleic acid amplification techniques (e.g. lamp and rpa), or virus isolation in cell culture. , these tests often require sending patient samples to a central laboratory with necessary equipment and trained personnel, and can take on the order of days, or weeks in the case of virus isolation from cell culture, hampering the fast containment of the virus and delaying the appropriate course of treatment. this situation is exacerbated when there is an excessive number of samples to be tested in an epidemic. epidemics are one of the challenging problems that have caused widespread deaths since the beginning of the known human history. starting from the post-classical era, humankind faced several epidemics such as the plague, viral hemorrhagic fever, cholera, smallpox, measles, poliomyelitis, and influenza. it is estimated that more than million people died from spanish flu (swine flu h n ) from to . the rapid spread of the novel coronavirus, sars-cov- , has reminded us that such pandemics are not historical anecdotes and exposed the major gaps in today's infectious disease diagnostics. in many countries, due to the huge demand in rt-pcr tests, clinical laboratories have faced shortages of trained personnel, test reagents and lab space. the system has been rapidly overwhelmed, causing longer wait times and delaying appropriate isolation procedures. to ease the burden on centralized laboratories and ramp up the testing capacity, rapid point-of-care (poc) tests in lateral assay format have been developed. although these tests can provide fast and simple detection, they lack sensitivity to detect low viral loads at the early stages of infection, when detection is essential for stopping community spread. there is a continuing need for alternative viral diagnostic techniques that can meet high sensitivity requirements in easy-to-use and portable poc platforms without the need for laboratory environment and trained personnel. an ideal poc platform should offer rapid (sample-to-answer in about min) and sensitive detection with minimal sample preparation. moreover, it should have multiplexing capability, which is especially important when it is necessary to distinguish between different viral pathogens that cause similar physical symptoms. it is also desirable that the diagnostic platform is easily configurable to new emerging viral pathogens with highly scalable and long-shelf-life consumables. in this paper, we describe a configurable and multiplexed digital virus counter capable of enumerating virions captured on a universal dna sensor chip and its application as a rapid testing platform utilizing dnaconjugated antibodies and disposable microfluidic cartridges. our group developed a label-free biosensor termed interferometric reflectance imaging sensor (iris) that has been used to quantify biomass accumulation on a microarray chip and was shown to detect virus particles captured onto an antibody-printed chip with a limit-of-detection (lod) of x pfu/ml. , iris utilizes a silicon-silicon dioxide microarray chip and an imaging sensor, consisting of different wavelength leds for illumination and a ccd camera that records reflected light intensities. although, this sensor provided a simple, inexpensive, and high-throughput platform for virus detection, due to the ensemble-based nature of this biosensor, sensitivity of detection was moderate. the iris system was modified to image single virus particles and termed single particle -iris (sp-iris). sp-iris has been shown to individually count and size the nanoparticles bound to capture probes on the sensor surface over a large sensor area. in this technique, high affinity capture probes are immobilized on the surface that can selectively bind to the target virus. when virus particles bind to the surface, scattered light from the particles interfere with the reference beam reflecting from the layered substrate, allowing an enhanced signal from the particle that is detected on the ccd camera. particles captured on the sensor surface appear as bright dots in the resulting image. this technique can also be used for single-particle interferometric (spir) microscopy modality and provide shape and size information allowing detailed morphological characterization of viruses. sp-iris has been utilized for detecting viruses in complex media using antibody microarrays by imaging chips both dry (dried after sample incubation and wash steps) and in liquid using disposable microfluidic cartridges. [ ] [ ] [ ] microfluidic integration of sp-iris provided an enclosed chamber for virus incubation, eliminated wash and drying steps and further improved the sensitivity of virus detection. these recent developments rendered sp-iris highly sensitive, fast and easy-to-use. (see table s - of the supporting information for a comparison of different modalities of iris in terms of key sensor properties.) as we improved sp-iris to develop it as a robust poc diagnostic platform, we focused on optimizing virus capture efficiency of antibody microarray chips, one of the most important factors that affect assay sensitivity in solid-phase immunoassays. a major challenge with antibodybased solid-phase biosensors is the immobilization of capture probes on the sensor surface. the surface attachment chemistry can affect the biological activity of the antibody, its affinity and the background noise, ultimately affecting the sensitivity of the biosensor. [ ] [ ] [ ] moreover, printing of antibodies on the microarray surface can introduce issues such as non-uniform surface coverage and assay-to-assay variability, affecting the assay accuracy and reproducibility. [ ] [ ] [ ] a dna-based site-specific antibody immobilization technique, known as dna-directed immobilization (ddi), has gained interest due to the reproducible production of dna microarrays, compatibility of dna microarrays with the fabrication of integrated microfluidic systems, and stability and robustness of dna chips. in this technique, a universal ssdna chip is first converted to an antibody microarray using antibodies tagged with short dna sequences complementary to the immobilized dna capture probes ( figure ). the resulting ddi-antibody microarray can be used for detection of target using a variety of labeled or label-free biosensing techniques. ddi technique has been shown to improve the antigen binding capacity, , the antibody surface coverage, and assay reproducibility, , compared to directly immobilized antibodies. in a previous study, we applied ddi approach to sp-iris to demonstrate the label-free detection of whole viruses. we showed that ddi elevates the antibodies from the sensor surface and improves the virus capture efficiency, increasing the sensitivity of the sp-iris platform. , here, we extend this approach to show multiplexed detection of three virus pseudotypes genetically engineered to express ebola, marburg, and lassa glycoproteins as a model for ebola, marburg and lassa virus detection. utilizing antibody -dna conjugates to convert a dna chip into a multiplexed antibody array suggests an alternative approach for generation of robust and repeatable diagnostic platforms by making use of stability and highly reproducible nature of dna microarrays. we also demonstrate, for the first time, a homogenous dna-directed virus capture assay where the antibody -dna conjugates and the virus sample are mixed in solution phase before incubating the dna chip. we present the combined utility of this homogeneous assay with a passive flow cartridge as an example of the application of sp-iris platform to a rapid test format which is suitable for poc testing. silicon chips with a patterned thermally grown silicon dioxide were purchased from silicon valley microelectronics inc. an oxide thickness of nm was used since the optimization studies for in-liquid visualization of the viruses showed that this thickness gave the highest level of particle contrast. custom-designed, disposable, active and passive microfluidic cartridges were purchased from aline. monoclonal antibodies (mabs) against ebola virus glycoprotein ( f ), marburg virus glycoprotein (agp - ), and lassa virus glycoprotein ( . f) were provided by mapp biopharmaceutical, prof. ayato takada (hokkaido university), and prof. james robinson (tulane university), respectively. recombinant vesicular stomatitis virus (rvsv) stocks expressing surface glycoproteins of ebola, marburg, and lassa viruses were created as described previously. hplc purified ′-aminated single-stranded dna (ssdna) molecules were purchased from integrated dna technologies. antibody-dna conjugation kit was purchased from innova biosciences. polymer kit for chip surface coating (mcp- ) was purchased from lucidant polymers. sensor chip functionalization. the silicon-silicon dioxide chips were cleaned by sonicating in acetone and then rinsing with methanol and nanopure water. chips were then dried under nitrogen. chips were coated with a -d polymeric coating, copoly(n,n-dimethylacrylamide (dma)acryloyloxysuccinimide (nas) - (trimethoxysilyl)propylmethacrylate (maps)) polymer, that offers a simple, inexpensive, and repeatable coating process and provides high density probe immobilization due to its -d structure. , copoly(dma-nas-maps) has nhs esters for covalent binding of proteins and amine-tagged dna molecules. for coating, the chips were first treated with oxygen plasma and then immersed in × mcp- polymer solution for min. chips were then rinsed extensively with nanopure water and dried with nitrogen. polymer coated chips were baked at °c for min and stored in a desiccator until microarray printing. printing of biomolecules on sensor chips. antibody and dna molecules were printed on the polymer-coated chips using a piezo-driven, non-contact dispensing system, sciflexarrayer s (scienion, germany). ′-aminated ssdna surface probes were spotted at µm in mm sodium phosphate buffer (ph = . ), producing dna spots of ~ μm diameter. for passive cartridge experiment and stability test, mg/ml f antibody in pbs with mm trehalose was spotted on the chip along with ssdna (a´ sequence in table ). antibody spots were ~ μm in diameter. during spotting, humidity was kept at % in the spotter chamber and the spotted chips were kept in the chamber overnight at % humidity. the chips were then washed with mm ethanolamine in × tris -buffered saline ( mm nacl and mm tris -hcl, fisher scientific), ph = . , for min to quench the unreacted nhs groups in the polymer. this step was followed by a min wash with pbst (pbs with . % tween) and a rinse with pbs and nanopure water. the chips were finally dried with nitrogen. antibody-dna conjugation. antibody -dna conjugates were prepared using thunder -link oligo conjugation kit (innova biosciences). each monoclonal antibody ( f , agp - , and . f, mg/ml) was reacted with a specific ′ -aminated mer ssdna ( μm) according to the manufacturer's instructions. the dna concentration used in the conjugation was optimized to yield a dna to antibody ratio between - in the final conjugate. ′-aminated mer dna sequences that are immobilized on the sensor chips are partially complementary to the antibody-conjugated dna strands. length of the dna surface probes were optimized in a previous study that showed mer probes provide optimal elevation of the antibodies from the sensor surface. three antibody-linked dna sequences (a, b, and c) and corresponding surface-immobilized probe sequences (a′, b′, and c′) are given in table components for virus detection assay step step step step step complementary regions between the antibody-linked sequences and the surface probes are underlined. to eliminate the formation of hairpin and self-dimer structures and to prevent cross hybridization between dna sequences. a ′ spacer sequence ( -bp polya) was added to the antibody-linked dna sequences to increase the hybridization efficiency. as given by the bradford assay for the protein part of the conjugate and the absorbance at nm for the dna part, dna-to-ab ratios were measured as . , . , and . , for f , agp - , and . f mab conjugates, respectively. antibody -dna conjugates are designated in the text by adding the letter representing the dna sequence to the antibody name as follows: anti-ebov-dna 'a', anti-marv-dna 'b', and anti-lasv-dna 'c'. optical biosensor setup and data analysis. in-liquid virus detection experiments were performed using sp-iris and spotted biosensor chips mounted into either a multi-layer laminate, disposable, active flow cartridge or a disposable passive flow cartridge that is composed of laminate layers and an absorbent pad ( figure ). for the active flow cell, the flow was controlled with a syringe pump (harvard apparatus, phd ) and a flow rate of μl/min was used. sp-iris setup is composed of a single wavelength led ( nm) for illumination of the substrate, a highmagnification objective ( ×, . na) to obtain a high spatial resolution image, and a ccd camera. an autofocus system (mfc- , applied scientific instrumentation) was used to control the focus during image acquisition. the recorded sp-iris images are analyzed for the bound virus particles for each spot. image analysis is performed using a custom software that identifies the particle-associated intensity peaks in a given image and applies a gaussian filter to eliminate the noise from the background. the morphological features of the antibody spots that become prominent due to the high resolution of the optical system cause a low correlation with a gaussian-type intensity profile, and therefore, the background signal caused by these features is eliminated by adjusting the gaussian filter parameters. sp-iris uses a forward-model to correlate the background normalized intensities of the particles to the particle size, allowing size-based filtering of the images to increase the specificity of detection. to quantify the virus particles in a spot, the diffraction-limited particles in the appropriate size range are detected and counted. the signal is expressed as virus density (number of particles per mm ) for a given spot by dividing the number of the detected particles by the analyzed spot area. for the end-point experiments, the initial particle count is subtracted from the final particle count for each spot to obtain the net number of particles bound to the spot during the experiment. table ) that are partially complementary to the antibody-linked dna sequences were spotted on a polymer coated sp-iris chip at a μm concentration. the chip was mounted in the active microfluidic cartridge via a pressure sensitive adhesive (psa) and the assembled cartridge was placed on the sp-iris stage. first, a mixture of dnaconjugated anti-ebov, anti-marv, and anti-lasv mabs (at μg/ml in pbs with % bsa) was flowed through the channel for min at a rate of μl/min. after a μl wash step with pbs, recombinant vsv models of ebov, marv and lasv were flowed sequentially over the sp-iris chip, by flowing each vsv pseudotype for min. μl pbs was flowed through the channel after each virus incubation to wash the extra virus in the channel and the tubing. the order of the virus incubation was rvsv-ebov, rvsv-marv, and rvsv-lasv, and their titers were , and pfu/ml, respectively, as determined by the plaque assay. the images of the anti-ebov, anti-marv, and anti-lasv spots generated by ddi were acquired every minute, and the virus densities on each spot were calculated over the course of the experiment. lod determination for one-step homogeneous detection of rvsv-ebov. one-step homogeneous assay uses a dna chip and a solution-phase mixture of the virus sample and antibody-dna conjugates, eliminating the antibody-dna conjugate incubation step. to determine the lod for the homogenous, dna-directed rvsv-ebov assay using sp-iris, we performed a dilution experiment with fold dilutions of a pfu/ml rvsv-ebov stock, ranging from pfu/ml to pfu/ml. five sp-iris chips were spotted with replicates of a´ probe and washed as described previously. μl of anti-ebov-dna 'a' conjugate at μg/ml was mixed with . ml of each of the rvsv-ebov dilutions prepared in . × pbs with % bsa. a blank sample was also prepared by mixing the same amount of ab-dna conjugate with . ml . × pbs with % bsa. after waiting for min, μl of each virus dilution and the blank sample was passed over a different sp-iris chip in the active microfluidic cartridge in subsequent experiments. for each sample, the channel was first filled with . × pbs with % bsa and the spots were scanned to obtain the pre-incubation particle counts. then, the sample was flowed for h in the cartridge at a rate of μl/min. after the channel was washed with pbs, the spots were scanned again. the net number of virus particles captured on the a´ spots were counted and the average virus densities were calculated from replicate spots for each chip. combining homogenous dna-directed assay with passive microfluidic cartridge. passive microfluidic cartridge has been designed to simplify the sp-iris platform by eliminating the need for an active syringe pump and to create a fully-contained test platform in order to minimize the sample handling. briefly, the passive microfluidic cartridge consists of a sample reservoir with a vented luer cap and an integrated °c fan shape absorbent pad in the channel placed after the chip (figure ) . the sample to be tested is pipetted into the reservoir and the flow is established by applying a pressure through the closure of the reservoir cap. once the sample flows over the chip and touches to the absorbent pad on the other side, the adhesive sealing tab on the cap is removed to let the fluid migrate under the atmospheric pressure. a stable flow rate (~ μl/min) is established by the °c fan shape of the absorbent pad. to demonstrate the feasibility of using the homogeneous assay in combination with the passive flow cartridge and to compare its performance to the directly immobilized antibody assay, an sp-iris chip was printed with anti-ebov mab, a´ probe, and a negative dna sequence, and washed as described previously. . μl of μg/ml anti-ebov-dna 'a' conjugate was added to μl of pfu/ml rvsv-ebov sample in pbs with % bsa. after min incubation, μl of this mixture was placed in the sample reservoir of the passive microfluidic cartridge. the reservoir cap was closed and tightened until the liquid started touching the absorbent pad. the cartridge was immediately placed on the sp-iris stage and the images of the directly immobilized anti-ebov, a´ probe, and negative dna spots were recorded every min during a min incubation. following the image acquisition, the virus density was calculated for each of the three spot types at every time point to show the real-time binding of the viruses. accelerated stability testing of dried antibody-dna conjugates. µl of μg/ml anti-ebov-dna 'a' was aliquoted into five tubes and placed in a vacuum oven for min at °c for drying the conjugate solution. after drying, of the dried conjugate tubes were placed into the oven at °c for the accelerated stability test. one tube was used for the day measurement on the same day. sp-iris chips, spotted with replicates of anti-ebov antibody and a´ sequence, were also kept in the oven at °c. pfu/ml rvsv-ebov sample was prepared on day and aliquoted into five tubes. four of the virus samples were stored at - °c until the virus detection experiments. on each of the days , , , , and , one dried conjugate tube was reconstituted with µl pbs with % bsa and flowed over the sp-iris chip mounted on the active microfluidic cartridge for min for ddi. the channel was washed with pbs and the spots were imaged with sp-iris for the pre-incubation particle counts. then, pfu/ml rvsv-ebov sample was flowed over the chip for min and the spots were scanned again to obtain the postincubation particle counts. average virus densities on the ddi-antibody spots were calculated from replicate spots for each chip. to show the multiplexed detection of the rvsv models for ebola, marburg, and lassa viruses and specificity of the antibody-dna conjugates, we performed a sequential incubation with these viruses after functionalizing a dna spotted sp-iris chip with three antibody-dna conjugates. first, a mixture of dna conjugated anti-ebov, anti-marv, and anti-lasv mabs was flowed through the active microfluidic cartridge for minutes. this step loaded the antibodies onto the specific complementary ssdna spots on the sensor chip. following the antibody immobilization step, rvsv samples were flowed sequentially in the following order: rvsv-ebov, rvsv-marv, and rvsv-lasv. each virus sample was flowed for minutes followed by a μl pbs wash step. sp-iris image acquisition was done with min intervals. figure shows the virus densities (particle count / mm ) on the three ddi-antibody spots (anti-ebov-dna 'a', anti-marv-dna 'b', and anti-lasv-dna 'c') during the course of the experiment. following the rvsv-ebov sample addition (red band), the signal on the anti-ebov-dna 'a' spot starts to increase whereas the other two spots do not show any virus binding. after the rvsv-marv (green band) is introduced, the virus density on the anti-marv-dna 'b' spot starts to increase showing the specific detection of rvsv-marv. finally, when the rvsv-lasv sample is flowed in the channel (blue band), the virus density on the anti-lasv-dna 'c' spot increases whereas the signal on the other two spots remain constant. overall, these results show that the site-specific self-assembly of the three antibody -dna conjugates on a dna surface was performed successfully to generate a multiplexed antibody microarray, and each antibody-dna conjugate was able to detect its target virus specifically with no cross-reactivity from other viruses. such a programmable dna surface can serve as a universal chip that can be adapted for the detection of different target viruses by using different sets of antibody-dna conjugates based on the need. antibody-dna conjugates in solution-phase. one drawback of the conventional ddi-based detection assay is the time associated with the antibody-dna conjugate immobilization step. to reduce the assay time and make this approach compatible with passive flow platforms where it is not practical to have sequential flows, we explored a homogeneous tagging approach where the virus sample is mixed with the antibody-dna conjugates in solution prior to the incubation of the chip. this approach replaces the -min antibody immobilization step of the conventional ddi technique with a simple and fast mixing step, reducing the number of incubation and wash steps. although the homogenous tagging of the target has been demonstrated for the detection of antigens previously, , our work is the first one, to the best of our knowledge, to show the capture of whole viruses decorated with dna-encoded antibodies on a ssdna microarray surface. one important consideration that needs to be addressed for this approach is the amount of the antibody-dna conjugates to be added to the virus sample. presence of excess antibody-dna conjugates in the solution would cause blocking of the dna surface with antibody-dna conjugates, preventing the binding of the virus particles that are already decorated with antibody-dna conjugates. we found that a concentration of . μg/ml antibody-dna conjugate does not saturate the surface, causing only . nm height increase, as measured by iris (data not shown), compared to about a nm height increase when the surface is fully saturated with the antibody-dna conjugates. (in iris, nm surface height corresponds to a surface antibody density of . ng/mm .) we mixed the anti-ebov-dna 'a' conjugate (at a final concentration of . μg/ml) with the rvsv-ebov samples prepared by -fold dilutions from a pfu/ml stock, ranging between - pfu/ml, and waited for min prior to the flow over the sp-iris chip. virus titer of the rvsv-ebov stock was measured by plaque assay. next, we flowed this mixture over the sp-iris chip in the active microfluidic cartridge for h and determined the captured virus density on the complementary a´ spots. figure shows the average virus densities on the a´ spots obtained from replicate spots for each titer tested. the detection threshold, indicated by solid red line, was calculated as the average virus density of six a´ spots plus three times the standard deviation from the blank chip that was incubated with only anti-ebov-dna 'a' conjugate. average virus density from the blank chip, which is virus count/mm , is also shown in the graph as dashed red line. in our earlier work, we have demonstrated that variance scales inversely with the sensor area (equivalently number of spots averaged). thus, for a large sensor area (or many spots), the lod will depend only on the mean virus density of the blank chip. therefore, the lod for a single spot detection is virus count/mm (solid red line) corresponding to pfu/ml, obtained by the extrapolation of the linear fit based on the data presented in figure . similarly, for a large area sensor where many spots can be averaged to virtually eliminate the variance, the lod is expected to approach virus count/mm , further improving the sensitivity. the lod for the homogeneous assay ( pfu/ml) is comparable to the one obtained from the heterogenous ddi technique, pfu/ml, and therefore, our results indicate that the homogenous approach provides a simpler assay procedure without affecting the sensitivity of the detection. sp-iris platform provides a positive result once the adequate number of virions are counted, and therefore, test time can be significantly reduced for high titer samples. according to figure , the experimental virion count response scales with [virus concentration] ( / ) -a perfect theoretical fit to the sheet density of the virus on the chip surface. in contrast, in an rt-pcr test, the cycle threshold (ct) values are inversely and logarithmically related to the viral rna copy number. ct values reported for sars-cov- in a recent study are . , . , . , and . , corresponding to . × , . × , . × , and . × copies/ml, respectively. while a thousand-fold increase in viral load provides a marginal ( %) reduction in ct values corresponding to a small time saving for rt-pcr (about min), the reduction in test time for sp-iris can be as high as -fold. based on our data presented in this and next section, sp-iris is expected to have significantly reduced test times at median viral loads (a few minutes) allowing for high throughput testing. one other advantage of the homogenous assay is the decreased antibody usage compared to the direct immobilization and the conventional ddi. homogenous assay uses at least three-fold less antibody per test, decreasing the cost of the assay. (see supporting information for the comparison of the antibody usage for three methods.) our lod determination experiment used a high number of antibody-dna conjugates per virus particle. this amount can be further decreased (at least -fold) and still have sufficient ab-dna molecules in the solution for efficient tagging of the virus particles (table s- of the supporting information) . moreover, the use of low antibody concentrations makes testing of the antibodies possible even when they are available at low quantities or concentrations. dilution experiment using single -step homogeneous assay approach. average virus densities are calculated from replicate a´ spots that are complementary to the anti-ebov-dna 'a' conjugate. solid red line shows the detection threshold calculated as the mean virus density from six spots plus three standard deviation of the mean from a blank chip. pfu/ml rvsv-ebov was detectable for a -hour incubation. lod, based on the extrapolation of the linear fit, is pfu/ml. passive microfluidic cartridge test using the homogeneous approach. active flow cartridge-based approach is not practical for field testing due to the complexity of the sample flow process that uses a syringe pump and tubing. this especially brings concerns in the case of lethal virus outbreaks due to the risk associated with the sample fluid handling. therefore, we have designed a passive microfluidic cartridge that would eliminate the need for an active pump and provide a fully contained test platform with minimum sample handling. we combined the homogeneous virus tagging approach with this lateral-flow cartridge to show the utility of sp-iris as a rapid testing platform. for this purpose, we added the anti-ebov-dna 'a' conjugate (final concentration of . μg/ml) to the rvsv-ebov sample ( pfu/ml), mixed, and waited for minutes. then, we applied this mixture to the reservoir and closed the cap. once the flow started, we put the cartridge onto the sp-iris stage and started scanning the dna spots (both complementary and negative dna sequences) and directly immobilized anti-ebov spots with min intervals. figure shows the captured virus density as a function of time for complementary dna spots (a´), negative dna spots and directly immobilized anti-ebov antibody. a positive signal can be observed on the complementary dna spots within the first minute of the incubation. moreover, the signal from the directly immobilized anti-ebov spots is lower than that of dna spots which is consistent with our previous findings. our results suggest that the combination of the homogenous assay with the lateral-flow cartridge can provide a suitable platform for rapid and sensitive virus diagnostics. to test the stability of the dried antibody-dna conjugates, we performed an accelerated stability test by storing the dried anti-ebov-dna 'a' conjugates and the spotted sp-iris chips at °c for weeks and performing inliquid virus detection experiments using ddi on the days , , , and . figure shows how the sp-iris signal changes over time for the dna-conjugated antibodies that were reconstituted for ddi on the test day. at the end of the two weeks, the antibody-dna conjugates captured approximately . times more viruses per mm than the directly immobilized antibody spots on the day chip (shown by the dashed red line), showing the superior performance of ddi technique to the direct spotting. the signal on the directly spotted antibodies showed a faster degradation rate over time and higher variability compared to the dried antibody-dna conjugates (data not shown). by extrapolating the line fit for the data points in figure , the stability of the antibody-dna conjugates is calculated as days at °c. using the q-rule we applied the ddi technique to our virus counter, sp-iris, for generation of a multiplexed antibody array for the detection of ebola, marburg, and lassa viruses. we showed the specific self-assembly of the antibodies on a dna microarray surface and subsequent real-time detection of three different rvsvs in a disposable microfluidic cartridge. we also demonstrated the homogeneous tagging of the viruses with antibody-dna conjugates in solution phase. by introducing this dna-encoded virus tagging approach, antibody immobilization step in conventional ddi can be eliminated, decreasing the assay time and complexity. in addition, homogenous dna-directed assay uses substantially less antibody (three-fold) compared to the conventional ddi and direct spotting. moreover, in-solution binding of antibodies eliminates the problems that affect capture efficiency such as antibody orientation, steric hindrance, and the activity loss due to antibody immobilization. we also demonstrated the combined utility of this homogenous method with a passive microfluidic cartridge. this platform allowed us to detect pfu/ml rvsv-ebov in less than min in a disposable, contained cartridge, showing the feasibility of sp-iris as a rapid and sensitive viral diagnostic platform. dna chips also offer the advantage of being configurable, allowing the use of the same multiplexed dna chips to create the desired virus detection panel. for example, clinicians would greatly benefit from a multiplexed test for sars-cov- and different influenza viruses to differentiate between these viruses that cause similar symptoms and that can happen concurrently. we envision that a multiplexed, dna microarray-based sp-iris system would provide a versatile, robust, and simple diagnostic platform through the use of universal dna chips and specific antibody-dna conjugates that can be synthesized quickly according to the need. comparing antibody usage for direct immobilization and dnadirected assays, molecular and immunological diagnostic tests of covid- : current status and challenges. iscience dengue and dengue hemorrhagic fever the lancet see the following epidemics to eradication: the modern history of poliomyelitis reviewing the history of pandemic influenza: understanding patterns of emergence and transmission fast, portable tests come online to curb coronavirus pandemic recent advances in immobilization methods of antibodies on solid supports technological development of antibody immobilization for optical immunoassays: progress and prospects antibody-based protein multiplex platforms: technical and operational challenges surface chemistry and morphology in single particle optical imaging sars-cov- viral load in upper respiratory specimens of infected patients assessing shelf life using real-time and accelerated stability tests authors would like to thank steven m. scherr for his help with microfluidic cartridge experiments and a. j. devaux for taking cartridge pictures. this work was supported by r ai to j.h.c. and m.s.u. key: cord- -k wfyj b authors: paweska, janusz t.; moolla, naazneen; storm, nadia; msimang, veerle; conteh, ousman; weyer, jacqueline; van vuren, petrus jansen title: evaluation of diagnostic performance of three indirect enzyme-linked immunosorbent assays for the detection of igg antibodies to ebola virus in human sera date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: k wfyj b filovirus serological diagnosis and epidemiological investigations are hampered due to the unavailability of validated immunoassays. diagnostic performance of three indirect enzyme-linked immunosorbent assays (i-elisa) was evaluated for the detection of igg antibody to ebola virus (ebov) in human sera. one i-elisa was based on a whole ebov antigen (wag) and two utilized recombinant nucleocapsid (np) and glycoproteins (gp), respectively. validation data sets derived from individual sera collected in south africa (sa), representing an ebov non-endemic country, and from sera collected during an ebola disease (ebod) outbreak in sierra leone (sl), were categorized according to the compounded results of the three i-elisas and real time reverse-transcription polymerase chain reaction (rt-pcr). at the cut-off values selected at % accuracy level by the two-graph receiver operating characteristic analysis, specificity in the sa ebov negative serum panel (n = ) ranged from . % (gp elisa) to . % (wag elisa). diagnostic specificity in the sl ebov negative panel (n = ) was % by the three elisas. the diagnostic sensitivity in rt-pcr confirmed ebod patients was dependent on the time when the serum was collected after onset of disease. it significantly increased weeks post-onset, reaching % sensitivity by wag and np and . % by gp i-elisa. high-mortality and occurrence of ebola disease (ebod) outbreaks almost each year in the last three decades [ , ] are of great public health concern. the unprecedented and first epidemics of ebod in west africa from to [ ] [ ] [ ] [ ] and the large-scale re-emergence of ebod in the democratic republic of the congo in and [ , ] exemplify the devastating health, humanitarian, and socio-economic impacts and challenges in containing ebod outbreaks in resource-poor and politically conflicted settings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the increasing incidence, severity and size of ebod outbreaks highlight the importance of developing and standardizing diagnostic tools for ebod rapid diagnosis and post-epidemic surveillance. the most devastating ebod outbreak to date, the west african outbreak, prompted determining seroprevalence rates, infection risk population studies, and assessing occurrence of asymptomatic infections [ ] . the purpose of this study was to evaluate and compare the diagnostic performance of ebov igg-indirect elisas based on antigens produced by classical virological and recombinant protein expression methods using human serum panels from ebov non-infected and ebov infected humans. a total of individual banked sera collected in south africa (sa) and sierra leone (sl), were used. sa sera (n = ) were originally submitted for various routine diagnostic testing to the centre for emerging zoonotic and parasitic diseases of the national institute for communicable diseases (nicd), johannesburg. these sera represented specimens collected from individuals in ebov non-endemic country and are regarded as igg ebov negative reference serum panel. ethics clearance for using sa human banked sera in the development and validation of diagnostic assays was obtained from human ethics committee, university of the witwatersrand, johannesburg, sa, clearance certification no. m , february . sl blood specimens were originally submitted for ebov rt-pcr testing to the sa modular high-biosafety field ebola diagnostic laboratory (fedl) established in august near freetown, in international response to the rapidly increasing number of ebod cases in sl [ ] . selected aliquots of processed sera were shipped on dry ice from fedl to the nicd's biosafety level (bsl- ) in total, sera obtained from sl patients suspected of having ebod between august and march [ ] were used. of those sera were from ebov rt-pcr confirmed cases for whom date of disease onset was recorded on ebod case submission form. ribonucleic acid from blood was extracted using the qiaamp viral rna kit (qiagen, hilden, germany) according to the manufacturer's instructions. rt-pcr was performed using the qiagen one-step rt-pcr kit (qiagen, hilden, germany) as per the manufacturer's instructions using previously described primers and probes targeting the ebov l gene [ ] . specimens with ct values below were considered positive for ebov rna [ ] and thus confirming ebov infectious status of a patient. serum specimens from the sl ebov rt-pcr positive patients were regarded as sl reference positive serum panel. the remaining specimens were from ebov rt-pcr negative individuals whose sera tested negative for anti-ebov igg by all igg elisas evaluated in this study using cut-offs derived from sa igg ebov negative reference serum panel. this serum panel was regarded as sl reference negative serum panel. the source of positive control serum (c++) was the imported ebod case from gabon to sa [ , ] . the case was a gabonese physician who had been brought from libreville, gabon on october and admitted to a private hospital in johannesburg where a nurse died after being infected through exposure to his blood. negative control serum (reference no. cezpd svpl / ) was obtained from south african blood service. it tested negative for anti-ebov igg and igm antibodies by in-house elisa using procedures published by ksiazek et al. [ ] . internal quality control (iqc) data were generated as described previously [ ] . upper and lower control iqc limits together with coefficients of variations ≤ % for replicates of positive internal control serum and test sera were applied as an assay acceptance criteria. diagnostic performance of three indirect elisas (i-elisa) for the detection of anti-ebov igg antibody in human sera was evaluated. these elisas were based on antigens prepared from infected cell lysate, or on recombinant antigens produced in a bacterial or mammalian expression system. the ebov whole antigen (wag) was produced as previously described with some modifications [ ] . vero c cells (atcc, manassas, va, usa) were infected with the spu / isolate of ebov ( th passage in vero cells) isolated from the serum of a nurse who contracted a fatal infection from a gabonese physician admitted to a private hospital in south africa in [ ] . after incubation at • c when cytopathic effect was observed, cells and supernatant were collected and snap freeze-thawed at − • c and • c. lysed cells and supernatant were separated by centrifugation ( , × g, • c, min). the collected supernatant was gamma irradiated with , gy to inactivate the virus. saturated ammonium sulphate solution ( %) (sigma aldrich, merck, kenilworth, nj, usa) was slowly added with constant stirring to the irradiated supernatant to a final concentration of %, and the mixture was incubated at • c overnight. the antigen-containing pellet precipitate was collected by centrifugation at , × g for min at • c and resuspended in one tenth of the original supernatant volume of phospate buffered saline (pbs) ph . . the antigen was further dialysed against pbs to remove the ammonium suphate ( - buffer changes). the wag preparation was aliquoted and stored at − • c until use. uninfected vero c cells were prepared in the same way and used as control antigen. the codon optimized nucleotide sequence for ebov nucleocapsid (np) (genbank accession number af . ) was synthesized with a c-terminal glycine linker to a × histidine tag (genscript, piscataway, nj, usa) and subcloned into the ncoi and xhoi restriction sites of the pet- b expression vector (novagen, merck, usa). the sequence verified plasmid (pet- b zebov np) was used to transform competent bl star (de ) e. coli. a starter culture was grown overnight at • c, then diluted fold and allowed to reach exponential-phase growth (od of between . - . ) before protein expression was induced using mm iptg (sigma aldrich, merck, usa) for h at • c with vigorous shaking. cells were harvested by centrifugation, resuspended in sodium phosphate buffer ( mm nah hpo , mm nacl, ph . ) and lysed using a combination of bugbuster and lysonase (novagen, merck, usa) treatment, freeze-thaw cycles and sonication. the recombinant np protein-containing soluble phase was collected by high speed centrifugation ( , × g, min, • c) and loaded onto profinity imac (biorad, hercules, ca, usa) cobalt-charged resin. the protein was left to bind overnight with gentle shaking at • c, using a ratio of ml resin to ml soluble protein fraction. contaminating proteins were removed by washing the resin twice with packed-resin volumes of sodium phosphate buffer containing . mm imidazole (ph . ), using gentle centrifugation ( × g, min, • c). the recombinant np was then eluted overnight with gentle shaking at • c in packed-resin volumes of sodium phosphate buffer containing mm imidazole (ph . ). eluted protein was dialysed into . m carbonate/bicarbonate buffer, ph . (sigma aldrich, merck, usa). to remove any residual contaminating proteins, the purified protein was passed through a sec size exclusion column (biorad, hercules, ca, usa) and the fractions containing the np were collected. collected fractions were pooled and concentrated using amicon ultra filters (millipore, merck, usa) with a kd cut-off. the purified np was quantified using the bradford concentration assay kit (pierce, thermofisher scientific, waltham, ma, usa) and aliquots were stored at − • c for later use. for the control antigen, the same process was followed using an expression vector (pet- b) without an insert. recombinant ebov glycoprotein (gp) antigen expressed in human embryonic kidney t cells was obtained from integrated biotherapeutics (rockville, md, usa). optimal immunoreagents concentrations/working dilutions for i-elisa were determined using standard checkerboard titration procedures [ ] . microtiter -wells plates (maxisorb immunoplates, nunc, roskilde, denmark) for wag, np, and gp i-elisas were respectively coated with wag and corresponding control antigen, np and corresponding control antigen or with gp. for wag and np i-elisas virus and control antigens were added to rows of the top half of the plate (rows a-d: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the bottom half of the plate (rows e-h: - ), respectively. for gp i-elisa all rows (rows a-h: - ) were coated with the commercial gp for which control antigen was not available. the np and corresponding control antigen were diluted : (stock concentration . mg/ml) in carbonate-bicarbonate buffer, ph . . all the other antigens were diluted in phosphate pbs without mg and ca, ph . ; wag and uninfected vero c cell culture antigen was diluted : and the gp was diluted : (stock concentration . mg/ml). each -well plate had four replicates of positive serum control (c++), two replicates of negative serum control (c−) and two replicates of conjugate control (cc). for wag and np i-elisa c++ was added to wells a - and b - coated with virus antigen and to corresponding wells e - and f - coated with a control antigen. accordingly, c− was added to c - and g - , and cc (diluent buffer) was added to d - and h - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and corresponding e and f ; adding of the remaining test sera followed the same layout principle. for gp i-elisa, not having control antigen, internal controls were placed as follows: c++ in wells a - and b - ; c− in wells c - ; cc in wells d - . the remaining wells (a-d - and e-h - ) were used for testing test sera in duplicate with test serum no. added to wells a and b and accordingly test serum no. added to wells g and h . all reagents were added to the immunoplates at a volume of µl/well unless otherwise stated. passive adsorption onto elisa plates was performed at • c overnight and all subsequent incubations (except for substrate addition) were performed at • c in a humidified chamber for h. following coating, plates were washed times with µl of pbs containing . % tween ; the same washing procedure followed each subsequent stage of i-elisas. plates were blocked with µl % non-fat milk powder in pbs. after incubation, plates were washed, and control and test sera diluted : in % non-fat milk powder in pbs (diluent buffer) were added. each test serum, negative control serum and conjugate control was tested in duplicate, and positive control was tested in quadruplicate. following incubation with the sera, the plates were washed and goat anti-human igg hrpo conjugate (invitrogen, thermofisher scientific, usa), diluted : , in diluent buffer, was added to the wells. after incubation, plates were washed and , '-azinodiethylbenzothiazoline sulfonic acid (abts) peroxidase (seracare lifesciences, milford, ma, usa) substrate added to wells. plates were incubated in the dark at room temperature (± • c) for min. reactions were stopped by the addition of % sodium dodecyl sulphate (sds) and the optical densities (od) readings were measured at nm. od readings were converted into a percentage of the positive internal control serum (pp) using the equation as previously described [ ] . briefly, a specific activity of each serum (net od) was calculated by subtracting the non-specific background od in the wells with control antigen from the od in wells with virus antigen (for np and wag i-elisa only). the mean net od readings were converted to pp using the formula: pp = (mean net of test serum/mean net od of positive control) × [ ] . cut-off values were determined as mean + standard deviations (sd) of pp values recorded in sa and sl igg ebov negative serum panels and also selected at % accuracy level by the misclassification cost term (mct) option of the two-graph receiver operating characteristics (tg-roc) analysis available from microsoft excel (redmond, wa, usa) [ , ] using sl reference igg ebov negative and positive serum panels. coefficient of variation (cv) values were calculated to measure relative variability using the formula cv = (sd of replicates/mean of replicates) × . optimisation of cut-off values by mct tg-roc was based on the following equation: to compare the diagnostic performance of the assays and agreements between results of matched test samples, data were analyzed in stata using mcnemar's test and cohen's kappa statistic (κ) [ ] . to evaluate the effect of serum inactivation on the levels of the detectable anti-ebov igg by wag, np and gp i-elisas, laboratory protocols previously shown to completely inactivate ebov virus in human serum were used [ , ] . briefly, the c++ was first diluted : in pbs containing either . % triton x- or . % tween- (sigma-aldrich, taufkichen, germany) and heated at • c for min. then, two-fold log dilutions (from : to : , ; log −log . ) of untreated and treated serum was tested by each of the i-elisas. each dilution was tested in duplicate on separate runs. a serum titer was considered the highest sample dilution at which its pp value was ≥ the i-elisa cut-off for at least % of six replicates. both within and between the runs, the c++ od readings for wag, np and gp i-elisas were within the iqc lower (lcl) and upper (ucl) control limits. within the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa). between the runs, the average cv ranged between . ± . (np i-elisa) and . ± . (gp i-elisa) ( table ) . both within and between the runs, the c− and cc od readings were within the iqc lcl and ucl. for wag, np, and gp i-elisa, the iqc c− od control limits ranged from − . to . , − . to . , . to . , and the iqc cc− od control limits ranged from − . to . , − . to . , and . to . , respectively. the distribution of i-elisa pp values and determination of cut-offs as mean plus three standard deviations of pp values recorded by each test in sa and sl ebov igg negative serum panels are shown in figure . selection and optimization of cut-offs by the mct tg-roc in sl serum panels is shown in figure . mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a ) and . (figure a) for wag i-elisa; . , . ( figure b ) and . ( figure b ) for np i-elisa; . , . ( figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ). mct tr-roc was based on the non-parametric program option due to departure from a normal distribution of data sets analyzed. pp threshold values for each of the assays were similar irrespective of serum panels analyzed and the methods used for the determination of cut-offs: . , . ( figure a figure c ) and . ( figure c ) for gp i-elisa, respectively. the higher cut-off values for gp i-elisa were likely due to higher elisa noise resulting from not including a control antigen in this assay. irrespective of data set analyzed and cut-offs used, all the three assays evaluated in this study had high estimates of d-sp, ranging from . % to . % in sa, and from . % to % in sl negative serum panels. however, d-sp for gp i-elisa was generally lower in the sa negative serum panel ( table ) . individual results yielded by wag, np and gp i-elisas in sera from rt-pcr confirmed ebod cases at different times post disease onset and using different cut-off values are given in table . irrespective of the cut-offs used, the results were similar for the same assay, but the np i-elisa was more sensitive in detecting igg antibody during the first two weeks post disease onset compared to wag and gp i-elisas, the latter being the least sensitive. for example, when using the tg-roc derived threshold, of ebod patients bled during the two weeks post onset, ( . %), ( %), and ( . %) were positive by wag, np, and gp i-elisa, respectively. mc nemar test indicate a disagreement of diagnostic capacity (combined dse and dsp) between np and wag (p = . ) or gp i-elisa (p = . ) respectively using all sierra leone data (n = ). agreement was found between wag and gp i-elisa (p = . ; . % κ = . ± . ). most of the discrepant (non-matching) results were recorded during the first two weeks post disease onset. after two weeks post onset, detection of igg antibody and agreement between assays significantly improved. of sera tested - days post onset, all tested positive by wag and np i-eliss irrespective of the cut-off used, and depending on the cut-off, or were positive by gp i-elisa (table ) . mc nemar test indicate agreement between np, wag and gp i-elisas ranging from . % (κ = . ± . ) to % using data from non-ebod and diseased patients bled on day or later post-onset (n = ), except for gp and wag or np i-elisas with the cut-offs of respectively . , . , . (p < . ). estimates of d-se in sera collected at different times post disease onset are given in table . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. estimates of d-se in sera collected at different times post disease onset are given in table . between - days post onset, the d-se ranged from . % (gp i-elisa) to . (np i-elisa). between - days post onset, the d-se was % for both wag and np i-elisa irrespective of the cut-off used, and ranged from . % to . % for the gp i-elisa. table . diagnostic sensitivity of a whole antigen (wag), nucleocapsid (np), and glycoprotein (gp) i-elisas for the detection of anti-igg ebov antibody in humans. cut-off pp wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . / . ( . - . ) / ( . - ) wag i-elisa . mean levels of igg responses measured by wag, np, and gp i-elisas in ebod rt-pcr confirmed cases bled at different times after disease onset are shown in figure . on average, the first seroconversions were detectable by np on days - , then on days - by np and wag, and from day post onset by all i-elisas. the mean levels of igg measured by all the i-elisas were not significantly different. the different inactivation protocols used did not have an adverse effect on the kinetics and the detectable levels of the anti-ebov igg in c++ by either i-elisa evaluated in this study. the titers ( table ) as well as the kinetics of dose-response curves ( figure ) were similar before and after inactivation in each assay. the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate the diagnostic decision limit or cut-off represents a serological assay test value used to dichotomize negative and positive results, and by inference, to define the infection status of an individual against a specific pathogen of disease. the relevance of data used for the determination of cut-off consequently impacts on estimates of d-se and d-sp and other measures of test performance [ ] . important consideration in determining a serological assay cut-off is to select sera from unequivocally infected individuals and sera from individuals who have never been infected with the agent in question. also, in order to account for the distribution of covariate factors (genetic, nutritional, geographical, and stage of infection) that may influence the estimates of d-se and d-sp, the target population should preferably be sampled using simple random, systematic or stratified sampling methods [ ] . these ideal conditions could not be applied during this study. an assay validation data should preferably be derived not only from testing samples from reference individuals of known history and infection status but also from the country or region in which the test is to be used. traditionally, gold standards for selection of truly infected and uninfected subjects include isolation of the agent or pathognomonic histopathological criteria. because a true gold standard is difficult to accomplish, relative standards of comparison are often necessary, and include results from other serological assays [ ] . in order to ensure ebov true infection status of individuals whose sera were used in our study, rt-pcr negative sera that tested negative for anti-ebov igg by all igg elisas using cut-offs derived from sa igg ebov negative reference serum panel were regarded as sl reference negative serum panel. serum specimens from the sl ebov rt-pcr positive patients with known dates of disease onset were regarded as sl reference positive serum panel. various statistical analyses used in our study for the selection of the cut-off values provided similar results. a cut-off value determined as two or three sd above the mean in uninfected individuals is frequently used for the interpretation of serodiagnostic tests. however, this assumes a normal distribution of the test values in population targeted by an assay, and provides only an estimate of d-sp [ ] . deviations from normality are often recorded in serological data and should be addressed in the selection of threshold values [ ] . therefore, we also used the tg-roc analysis for the selection and optimization of cut-off values to account for parametric versus nonparametric distribution of test values. all i-elisas evaluated in our study had high estimates of d-sp, but the d-se was dependent on the time when the serum was taken post disease onset. high detection rate of anti-ebov igg in rt-pcr ebov confirmed cases was only recorded after two weeks post disease onset. results of previous study in a small number of the ebod patients in kikwit, democratic republic of congo, suggested that many patients do not have antibody early in the course of their illness, and that many may die without developing detectable antibodies to ebov. therefore, measurement of igg antibody is of rather limited use in the diagnosis of acute ebod cases [ ] . i-elisa, represents one of the simplest elisa formats, but can be difficult to validate because of signal amplification of both specific and non-specific components [ ] . for these reasons, to determine the specific binding of antibody, sera should be tested with both a specific viral antigen and its corresponding control or comparison antigen to account for possible non-specific background activity. the gp i-elisa evaluated in our study was based on a commercially available recombinant gp for which a negative control could not be obtained. compared to wag and np i-elisas, which included control antigens, the higher gp i-elisa test values in ebov negative serum panels and consequently the higher pp cut-off values derived for this assay are likely due to not having a control antigen. due to inherent differences amongst assay systems, binding-antibody levels should be expressed in relative rather than absolute terms. one of the advantages of using pp values as a measure of antibody activity in the i-elisa is that this method of od readings conversion does not assume a uniform background activity, and therefore it is also more suitable for inter-laboratory standardization [ ] . the first elisas for the detection of antibodies to ebov were based on whole antigen prepared from infected cell lysate [ ] , and variations of this assay are still widely used in diagnostic and research laboratories [ ] . while whole filovirus antigens can be relatively easily produced in large volumes, their preparation pose health risks, restricting their production to biosafety level (bsl- ) facilities. these facilities are not only very expensive to construct and operate, but also are not easily available or accessible for countries where fatal filoviruses are endemic. in addition, the binding of antibodies to cellular contaminants present in ebov-infected cell lysates may lead to cross-reactivity, resulting in reduced specificity [ ] . high quality filovirus recombinant protein antigens can be safely prepared without the need for high bsl- biocontainment facilities and outside ebov endemic areas. their use in elisa has potential to reduce the risk of false positive results and allows for better standardization [ , ] . testing clinical specimens potentially containing a bsl- viral agent presents a serious biohazard. while a number of inactivation methods were shown to completely inactivate ebov [ , ] , they also markedly alter the protein components in human blood, e.g., enzymes and coagulation factors [ ] . results of viral inactivation protocols evaluated in our study indicate that they do not alter detectable levels of anti ebov-igg, thus together with recombinant antigen based elisas, provide a safe and reliable testing platform. it has previously been reported that the use of single filoviral proteins as antigens is disadvantageous for filovirus serology [ , , ] . sera from patients infected with ebov contain antibodies to several viral proteins and therefore might display reduced activity or later seroconversion in elisas based on a single recombinant antigen [ ] . results of our study indicate that anti-ebov np igg is detectable earlier then anti-ebov gp igg antibody during the first week post disease onset. this is likely due to higher abundance of this highly immunogenic protein in infected cells [ ] . long-lasting persistence of igg antibody in humans after infection with ebov [ , , ] , renders igg detection elisa a suitable tool for epidemiological investigations. evaluation of the efficacy of filovirus vaccines and therapeutics require monitoring of immune responses that correlate with protection and survival using reliable and reproducible serological methods. anti-ebov gp igg elisa was recently shown to reproducibly quantify levels of anti-ebov igg antibodies in sera from ebod survivors and immunized individuals [ ] , thus offering an important laboratory tool for assessing immunogenicity of candidate ebov vaccines. the np igg i-elisa evaluated in this study has a potential to be used for testing 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results. key: cord- -l ld cy authors: wertheim, joel o.; kosakovsky pond, sergei l. title: purifying selection can obscure the ancient age of viral lineages date: - - journal: molecular biology and evolution doi: . /molbev/msr sha: doc_id: cord_uid: l ld cy statistical methods for molecular dating of viral origins have been used extensively to infer the time of most common recent ancestor for many rapidly evolving pathogens. however, there are a number of cases, in which epidemiological, historical, or genomic evidence suggests much older viral origins than those obtained via molecular dating. we demonstrate how pervasive purifying selection can mask the ancient origins of recently sampled pathogens, in part due to the inability of nucleotide-based substitution models to properly account for complex patterns of spatial and temporal variability in selective pressures. we use codon-based substitution models to infer the length of branches in viral phylogenies; these models produce estimates that are often considerably longer than those obtained with traditional nucleotide-based substitution models. correcting the apparent underestimation of branch lengths suggests substantially older origins for measles, ebola, and avian influenza viruses. this work helps to reconcile some of the inconsistencies between molecular dating and other types of evidence concerning the age of viral lineages. one of the most powerful forces shaping the human genome is adaptation to pathogens, and the pathogens that appear to have exerted the strongest influence are viruses (worobey et al. ; emerman and malik ) . genes which bear the mark of some of the most potent selective forces detected in the genomes of humans and our relatives are directly related to combating rna viruses (meyerson and sawyer ) . moreover, the ancient history of this association with rna viruses is evidenced by a diverse array of defective viral remnants incorporated into vertebrate genomes, including mounting support for endogenization of rna viruses other than retroviridae (gifford et al. ; belyi et al. ; gilbert and feschotte ; horie et al. ; taylor et al. ) . however, according to molecular dating analyses, many rna viruses have extraordinarily recent origins (holmes a) . at first glance, a recent introduction of many rna viruses into the human population is unsurprising. epidemiological, historical, and phylogenetic approaches agree that some of the most notable rna viruses (e.g., hiv- , worobey et al. ; influenza a virus, taubenberger et al. ; ebola virus [ebov] zaire, walsh et al. ; and sars coronavirus, hon et al. ) emerged as zoonoses within the last century. as one looks further back in evolutionary time, however, specific inconsistencies arise. for example, defective remnants of ancient integrations of filoviridae, the viral family containing the ebov, are found in mammalian lineages that diverged tens of millions of years ago (belyi et al. ; taylor et al. ) , even though molecular dating suggests a time of most recent common ancestor (tmrca) for filoviridae on the order of only thousands of years ago (suzuki and gojobori ) . another inconsistency can be seen in the zoonotic origin of measles virus (mev) from rinderpest virus (rpv) and peste-des-petits ruminants virus (pprv), two viruses capable of infecting large and small ruminants, respectively. mev required at least two conditions to emerge: ) humans had to live in close proximity to rpv, which became commonplace only after the domestication of cattle over the last , years (perkins ; loftus et al. ; beja-pereira et al. ) and ) humans needed societies with a population size above , - , to sustain the epidemic, which did not exist until about , years ago (black ) . furthermore, the first unambiguous historical account of measles dates back to the ninth century (rāzī ). therefore, historical and epidemiological considerations, which place the tmrca of mev and rpv at thousands of years ago, are at odds with molecular dating analysis, which infers the tmrca to be only hundreds of years ago (furuse et al. ) . the same phylogenetic dating methods that provide convincing and reasonable tmrca estimates for recent zoonotic transfers seem to be dramatically underestimating the age of older viral divergence events. therefore, over longer periods of time, the extent of evolutionary change has been lost. in many viral genes, there is remarkable sequence conservation, indicating that purifying selection is a dominant evolutionary force, acting to maintain evidence of homology by preserving amino acid residues, while allowing nucleotide sequences to continue evolving mbe (holmes b; edwards et al. ; pybus et al. ) . although the effect of purifying selection on mutations in rna viruses is becoming better understood (belshaw et al. ) , the importance of spatial and temporal variation in selection pressures has not been explicitly explored in the context of tmrca estimation. it has been well established that failing to account for site-to-site rate variation can lead to an underestimation of branch lengths due to repeated substitutions at rapidly evolving sites (brown et al. ; sullivan and joyce ) , and the use of models, which permit such variation (e.g., yang ) , have become standard. more recently, suchard and rambaut ( ) observed that synonymous substitutions appeared to saturate faster than could be handled by nucleotide models in mitochondrial dna, which is subject to strong overall purifying selection. over longer periods of evolutionary time (i.e., along long internal branches on a phylogenetic tree), evidence of evolution at the nucleotide level could be lost, which could bias tmrca estimates towards younger dates (i.e., underestimate the length of these branches). if a substitution model can account for this evolution, we hypothesize that a comparative analysis of recent viral isolates could be used to make inference about ancient viral divergence events. our work addresses two questions. first, can purifying selection mask the ancient history of rna viruses? and second, by using evolutionary models that account for selection, can we infer more realistic estimates of the ages of these viruses? we investigate these questions using two groups of viruses thought to be older than current molecular dating evidence would suggest: mev/rpv/pprv and ebov. we also consider avian influenza virus (aiv)another well-characterized viral lineage with a young inferred tmrca and long internal branches between different serotypes holmes , ) . the results presented here demonstrate that not accounting for purifying selection may bias tmrca estimation in rna viruses towards more recent dates, and a degree of correction can be realized by employing more realistic codon-based substitution models, capable of partially accounting for the biasing effect of purifying selection. for mev/rpv/pprv, all available full-length nucleoprotein sequences with known years of isolation were downloaded from genbank. vaccine-associated sequences and mev isolates implicated in subacute sclerosing panencephalitis, which experience hypermutation (woelk et al. (woelk et al. , , were excluded. the curated mev/rpv/pprv virus data set contained sequences sampled between and . alignment was trivial due to a lack of insertions or deletions and was performed by eye. next, ebov full-length glycoprotein sequences with known years of isolation were downloaded from genbank. regions containing multiple reading frames (volchkov et al. ; sanchez et al. ) and the mucin-like region-which evolves extremely rapidly due to relaxed selective constraints (wertheim and worobey b )-were removed. the curated ebov data set contained sequences sampled between and . as before, alignment was trivial and performed by eye. the final alignment, aiv neuraminidase, was provided by chen and holmes ( ) . it contained sequences sampled between and . all three alignments in nexus format can be downloaded from http://www.hyphy.org/wiki/codondating. redundant sequences were excluded from the alignments for branch length comparisons and selection analyses. we used a bayesian markov chain monte carlo (bm-cmc) method implemented in beast v . . for phylogenetic inference and tmrca estimation . for each data set and substitution model, four independent bmcmc runs of or million generations were performed. a codon-based substitution model was implemented in beagle (suchard and rambaut ). the first % of the generations were discarded as burn-in. all bmcmc analyses were performed using an uncorrelated lognormal relaxed molecular clock and a bayesian skyline plot coalescent prior, which places the fewest demographic constraints on the analysis . tracer v . was used to check for convergence and adequate mixing (i.e., estimated sample size > for all relevant parameters). finally, the maximum clade credibility (mcc) phylogeny was identified and annotated using the posterior distribution of trees. substitution rates and tmrcas are reported as mean and % highest posterior density values. an earlier study ) developed a hierarchy of five models-all extensions of the muse-gaut probabilistic model of sequence evolution (muse and gaut ) -of differing complexities that incorporate site-tosite and lineage-to-lineage variation of synonymous (α) and nonsynonymous (β) substitution rates. we investigated the ability of these models to accurately infer branch lengths on our viral data sets. the five models are summarized below, with full details available in the original manuscript. note that in all models, e [α] = to ensure identifiability. constant rates: the baseline model, which extends the original muse-gaut evolutionary model by incorporating general nucleotide substitution biases (mg × rev). α and β rates are constant across sites and lineages. proportional: a direct analog to nucleotide + Γ models; β = ωα and α varies from site to site according to a three-bin general discrete distribution (gdd). nonsynonymous: a standard "selection" model, where α rates are constant and β rates are drawn from a bin gdd. dual: a model, where both α and β rates vary from site to site, based on independent three-bin gdd distributions. lineage+dual: in addition to site-to-site rate variation in α and β, some (or all) lineages are endowed with their own mean e [β]/e [α] ratios, to correct for "lineagespecific" effects of selection. unlike the original implementation, our version of the lineage+dual model treated certain interior lineages differently to better reflect the biological reality of the sample. branches which separated clades containing different viral species (mev/rpv/pprv), subtypes (ebov), and serotypes (aiv) were assigned separate e [β]/e [α] ratio parameters because they represent different timescales and selective regimes compared with terminal branches. longer internal branches are expected to bear the mark of stronger purifying selection (kosakovsky et al. ; pybus et al. ). we considered two variations of this model: either ) all long internal lineages share the same ratio parameter, and the remaining branches share another global ratio parameter (two-rate model) or ) each deep lineage possesses its own ratio parameter, and the remaining branches share a global ratio parameter (multirate model). for each data set, we selected the model with the best akaike information criterion (aic) score for further analysis. note that we use the ratio of the means e is possible under dual and lin-eage+dual models, rendering the mean of the ratio infinite. for each data set, we obtained m = , samples from the approximate joint distribution of model parameter estimates using a modified latin hypercube sampling importance resampling scheme, described in detail elsewhere (kosakovsky et al. ). the approach is meant to quickly obtain an approximate joint distribution of maximum likelihood parameter estimators. first, an area of parameter space to be sampled is defined by constructing a d -dimensional rectangle, in which each dimension represents a single model parameter, the corresponding coordinate interval is centered on the maximum likelihood estimate of the parameter, and the lower and upper bounds are determined by profile likelihood. second, each coordinate interval is partitioned into n = , d subintervals of approximately equal probability, based on the asymptotic normal approximation to the likelihood surface. third, n samples are drawn from the d -dimensional rectangle, using the latin hypercube scheme (i.e., each interval in every coordinate is sampled exactly once). fourth, m n points are resampled based on their importance (normalized likelihood), following the procedure described elsewhere (skare et al. ) . variance in estimated branch lengths was computed using this sample. all codon-based analyses were performed in hyphy v . ). we simulated codon sequences along a single branch using the mg × rev codon substitution model ) with site-specific β and α values inferred using a fixed effects likelihood method on internal branches (ifel) (kosakovsky et al. ) from each of the three viral data sets. in this method, three rates are inferred from each site: α s , β i s , and β t s . subscript s indicates the explicit dependence of substitution rates on the site, from which they are being estimated. α s is the tree-wide synonymous rate, β i s is the nonsynonymous rate shared by all internal branches, and β t s is the nonsynonymous rate shared by all terminal branches. only internal branches were used to generate empirical selection profiles because substitutions along tips in viral phylogenies are frequently deleterious and transient (kosakovsky et al. ; pybus et al. ). furthermore, we were primarily interested in the effects of selection on long internal branches, and the ifel profile (α s and β i s inferred for each site) was meant to recapitulate the evolutionary process along an "average" internal branch. simulations were initialized using ancestral nucleotide sequences inferred with marginal maximum likelihood reconstruction (yang et al. ) in hyphy, using the mg × rev codon substitution model on the mcc phylogeny obtained from bmcmc general time reversible (gtr) + Γ analyses. the marginal ancestral sequence was used to provide a realistic starting point for our simulations. ten thousand replicates were generated for each branch length ranging from . to expected substitutions per nucleotide site and analyzed under the gtr + Γ and dual substitution models. we chose to simulate branches based on expected substitutions per nucleotide site, instead of per unit time, because of the difficulty in standardizing time for variable rate parameters (i.e., α s , β i s , and substitution rate) in a reversible model; the expected number of substitutions divided by the substitution rate can be a proxy for time. using bmcmc analysis, we inferred the substitution rate and root age under a standard nucleotide substitution model (gtr + Γ ) for each of our three viral data sets: mev/rpv/pprv nucleoprotein, ebov glycoprotein, and aiv neuraminidase (table ) . the viral lineages examined here had tmrcas ranging from hundreds to several thousand years before present. all three viruses exhibited rapid substitution rates, on par with previous estimates for related rna viruses (duffy et al. ) . our inferred substitution rate for the mev/rpv/pprv nucleoprotein of . × − ( . × − - . × − ) substitutions/site/year under a gtr + Γ model was over % faster than the rate recently reported by furuse et al. ( ) : . × − ( . × − - . × − ) substitutions/site/year. therefore, we inferred a substantially younger tmrca for mev and rpv: ce ( - ce), instead of ce ( - ce). we consider our rate estimates more reliable for three reasons. first, our values are in close agreement with previous substitution rate estimates in mev: . × − ( . × − - . × − ) substitutions/site/year (pomeroy et al. ) . second, furuse et al. ( ) included both mev and rpv vaccine strains in their bmcmc dating analysis (rota et al. ; baron et al. ) , despite evidence that the inclusion of vaccine strains can lead to bias in rate estimation (bush et al. ; wertheim ). third, furuse et al. ( ) fitted an exponential growth coalescent prior to viruses whose effective population size has been declining-due to eradication efforts against both mev and rpv (moss ; normile ). remarkably, our results are in even greater conflict with the historical documentation of measles (rāzī ), further confirming our conjecture that traditional nucleotide substitution models can underestimate the age of ancient viral divergence events. we then explored whether alternative evolutionary models that, to varying extents, account for codon structure were able to recover the expected older tmrcas for these viruses (table ) . the simplest approach, excluding the third codon position in a gtr + Γ model, has been used to remove synonymous sites that might have experienced saturation (worobey et al. ) . a more sophisticated approach, the srd model (shapiro et al. ) , allows first and second codon positions to have a different transition/transversion ratio and Γ shape parameter from the third position. amino acid substitution models have also been successfully implemented to estimate the age of rna viruses (zlateva et al. ; wertheim et al. ). finally, we evaluated an available codon-based substitution model, gy (goldman and yang ) . generally, these alternate models either produced younger tmrcas (e.g., gtr + Γ excluding third positions and whelan and goldman + Γ ) or tmrcas that were indistinguishable from gtr + Γ (e.g., srd and gy + Γ ). a possible exception was analysis of the ebov data set with gy + Γ , which produced a root tmrca that was % older than the gtr + Γ estimate. finally, as a point of comparison, we also investigated how removing site-to-site rate variation affected dating inference by using a gtr model (tavaré ); ignoring rate variation invariably produced younger tmrcas. we simulated codon sequences along a single branch under a substitution model with site-to-site nonsynonymous rate variation and inferred the length of the branch using gtr + Γ . an increase in the proportion of sites simulated under purifying selection (i.e., β s = ) resulted in shorter branches inferred by a gtr + Γ model ( fig. a ) . sufficiently strong purifying selection could theoretically lead to underestimates of branch lengths by an order of magnitude because purifying selection slows down the rate of evolution relative to the synonymous rate. at the extreme (β s = , branch length → ∞), one obtains perfectly conserved amino acid sequences and completely saturated synonymous substitutions. for sequences with % of sites under strict conservation, the nucleotide estimator approached the asymptote of . substitutions/site, even when the simulated branch length was increased to ; the branch length estimate was accurate for up to substitutions/site. for sequences with no nonsynonymous substitutions (i.e., % conservation), saturation occurred much sooner, around . substitutions/site, and the corresponding asymptote was . substitutions/site. the nucleotide substitution model (gtr + Γ ) underestimated the length of branches simulated under empirical (ifel) selection regimes inferred from the three viral data sets ( fig. b- d) ; branch lengths for sequences simulated under neutral selection (i.e., α s = β s = ) were reliably inferred by gtr + Γ . the proportion of sites under strong negative selection (i.e., β s < α s with an ifel p value . ) differed markedly among the three data sets. mev/rpv/pprv nucleoprotein and ebov glycoprotein genes experienced moderate levels of purifying selection along internal branches: % and % of sites under strong purifying selection, respectively. the aiv neuraminidase gene evolved under much stronger selective constraints, with % of sites under strong purifying selection. we note that the power to detect selection using ifel increases with the size of the alignment (kosakovsky et al. ); hence, we would expect more sites evolving under purifying selection to be correctly detected as such in the larger aiv data set. the simulated codon saturation curves varied from virus to virus, indicating that the particular selectiveregime, amino acid composition, and nucleotide substitution model of each viral gene have a substantial influence on the ability to accurately infer branch lengths. although underestimation is more pronounced along longer branches (e.g., about half the true length for substitution/site), not accounting for selection can lead to underestimation of branch lengths even for relatively short branches (e.g., . - . substitutions/site). we then inferred the length of branches simulated under the empirical selective regimes using the dual codon model, which accounts for variation in site-to-site selective pressures ( fig. ) . although the dual model slightly underestimated lengths for the longest simulated branches, the bias was an order of magnitude less than under gtr + Γ . furthermore, similar behavior was observed for the longest branches simulated under neutrality (α s = β s ) and inferred using gtr + Γ ( fig. b - d ) , suggesting that both models perform equivalently, as expected. clearly, not accounting for purifying selection can lead to dramatic underestimation of branch lengths; this effect is exacerbated for longer branches. it is well known that standard nucleotide models differ in their sensitivity to multiple substitutions at the same site (sullivan and joyce ) . we explored how various methods of modeling rate variation affected branch length inference in our three viral data sets, using a fixed mcc tree from the gtr + Γ bmcmc analyses. first, we examined two extremes among nucleotide substitution models that do not adjust for rate variation across sites: jc (jukes and cantor ) and gtr. jc assumes a single substitution rate and equal base frequencies, whereas gtr allows each class of nucleotide substitution to occur at a unique rate and estimates base frequencies from the data. as expected, failure to account for site-to-site rate variation led to severe underestimation of longer branches in all three viral data sets (jc or gtr vs. gtr + Γ , fig. the importance of modeling site-to-site rate variation to accurate inference. allowing for multiple substitution rates and empirical base frequencies had a negligible impact on branch length estimation ( fig. ) . underestimation of long branches likely explains why the gtr model inferred younger root tmrcas for all all three viral data sets (table ) . we hypothesized that codon substitution models, which explicitly account for the differences between synonymous and nonsynonymous substitutions, would permit a more accurate estimation of sequence divergence well past the point a standard nucleotide model would reach saturation. therefore, it may be possible to use rna virus genes, which evolve extremely rapidly (e.g., . - . substitutions/site/year), to estimate ancient viral divergence events using codon substitution models. a crude approximation of a codon model, srd , produced branch lengths that were essentially the same as those from the gtr + Γ model ( fig. ) , which may explain why bmcmc tmrca inference using these two models was so similar. the inference under the gy + Γ codon model resulted in longer internal branch lengths only for ebov ( fig. ) , which was in agreement with our bmcmc tmrca inference using this model. based on the simulations along a single branch, we anticipated that longer branches would be disproportionately affected by site-to-site variation in selection pressures. therefore, we investigated how extensions of the mg model that incorporate β, α, and lineage-specific rate variation affected branch length inference (table ) . for mev and aiv, the lineage+dual (two-rate) model that allowed longer internal branches to share their own ratio e [β]/e [α] provided the best fit to the data. for ebov, a more complicated (eight-rate) model, in which each intersubtype branch and the long terminal branches leading to côte d'ivoire and bundibugyo had their own rates, was fit because e [β]/e [α] varied dramatically among the intersubtype branches. the inclusion of variation in β and α in the nonsynonymous and dual models resulted in notable increases in the cumulative length of deep internal branches (table ; supplementary fig. s , supplementary material online) . in all three viral data sets, however, the most substantial differences were seen with the lineage+dual model, which produced lengths for long internal branches that were substantially greater than those inferred under gtr + Γ ( fig. ) . importantly, the lengths of the more recent intraspecies/subtype/serotype branches were relatively unchanged between these two models for all three data sets; this observation confirms that when the phylogeny is comprised of relatively short branches, it is appropriate to rely on common approximations, such as the gtr + Γ model. this increase in the length of only deep internal branches was likely due to the dramatically stronger purifying selection which we inferred along these lineages (table ). in aiv, for instance, e [β]/e [α] was two orders of magnitude lower on deep interior branches, compared with the rest of the tree. one outcome of including variation in selection pressures across branches into the evolutionary model was that several branches were inferred to have essentially infinite lengths in the ebov and aiv trees. the inference of an infinite branch length is likely caused by the complete saturation of synonymous substitutions along the branch in question; even after accounting for variation in α s and β s , the true branch length was inestimable. in the ebov phylogeny, complete saturation was observed on the branch leading to ebov sudan and on the branch connecting ebov reston/sudan to ebov zaire/côte d'ivoire/bundibugyo (supplementary fig. s , supplementary material online). in the aiv phylogeny, each of the nine neuraminidase serotypes naturally found in avian hosts was separated by branches that experienced saturation at synonymous sites under the lineage+dual model (supplementary fig. s , supplementary material online) . the latin hypercube resampling scheme suggested relatively narrow variance in the expansion under the lin-eage+dual model. the total increase in mev/rpv/pprv tree relative to gtr + Γ was . (approximate % confidence interval: . - . ). due to the inference of branch lengths that experienced saturation in ebov and aiv, the overall increase in tree length was more dramatic under the lineage+dual model, relative to gtr + Γ : . (approximate % confidence interval: . - . ) for ebov and . (approximate % confidence interval: . - . ) for aiv. it is clear that purifying selection can obscure the ancient evolution of the rna viruses examined here. a comparison of the mev/rpv/pprv phylogeny optimized under gtr + Γ and lineage+dual (two-rate) models showed that the elongation of branches occurred along the deep internal branches, leaving the relationships among recently divergent lineages within viral species relatively unchanged ( fig. ) . the depth of the split between mev and rpv under a gtr + Γ model was . substitutions/site, which, according to the bmcmc analysis, occurred in the year table . goodness of fit for various codon models and the effect of model choice on the estimates of the branch lengths of deep and recent lineages. model ( - ce) . this extrapolation is meant as a rather crude approximation of the age of this divergence event; nevertheless, these dates are more likely to be closer to the true split between mev and rpv than previous estimates and are not inconsistent with recorded history of measles. the degree of synonymous saturation observed along the ebov and aiv phylogenies indicate an inability to reliably infer tmrcas for these viruses. too many sites have sunk beyond the evolutionary horizon. nevertheless, these estimates could provide meaningful minimum bounds for the tmrcas. thus, for both ebov and aiv, we applied the mean substitution rate inferred in the bmcmc gtr + Γ analyses to the lineage+dual phylogenies. this approach suggested that the minimum tmrca estimates for ebov and aiv are approximately , and , years ago, respectively. the actual tmrcas may be much older. our results suggest that the ancient age of rna viruses may be partially masked behind a veil of purifying selection. the same forces of purifying selection that maintain evidence of protein sequence homology over great evolutionary distances also truncate long ancestral branches deep within phylogenetic trees. we observed this pattern mbe in three different groups of rapidly evolving negative-sense rna viruses: mev/rpv/pprv, ebov, and aiv. estimating branch lengths under a codon-based substitution model that accounts for spatial and temporal variation in selection pressures yielded phylogenetic trees that were more than twice as long as those obtained under standard nucleotide models. modeling synonymous, nonsynonymous, and lineage-specific rate variation indicates that current estimates of the age of these, and possibly other, viral lineages may be dramatically underestimated. codon models used in this study do not circumvent the issue of substitutional saturation but merely extend the horizon further back in evolutionary time; the application of more complex and biologically realistic models is likely to extend these branches even further into the past. eventually, however, even the most realistic models are likely to fail and external information, such as biogeography and homology to endogenous viral elements, must be brought into consideration to estimate truly ancient events (katzourakis et al. ; katzourakis and gifford ) . the precise age of measles in the human population remains elusive. the domestication of cattle beginning , years ago provided the necessary exposure to rpv, and the development of agriculture allowed human populations to reach sizes necessary to sustain an epidemic. however, the historical record of measles is ambiguous until the ninth century, when rhazes (a persian physician) outlined the criteria for differentiating between measles and smallpox (rāzī ). rhazes discusses both ailments as immemorial, indicating that both diseases predated him by many generations. although there are historical records of earlier plagues that could be interpreted as measles, notably in eighth century france during the battle of tours (rolleston ) , it is also possible that this plague could have been smallpox or another disease with similar presentation. even after rhazes description, western medicine still confounded measles, smallpox, and scarlet fever until the th century (rolleston ) . in light of this confusion, the lack of a definitive description of measles before rhazes (e.g., galen, an ancient roman physician, described smallpox but not measles) does not establish that the virus was absent (mcneill ) . it is plausible that mev might have not entered the human population until the first millennium of the common era, as our analysis suggests. alternatively, mev could have emerged thousands of years ago, and the evolutionary models employed here are too crude to recover the true age of the virus. regardless of which scenario is correct, accounting for variable selective pressures is an important step in revealing the ancient history of rna viruses. the different timescales affecting mutation rates and substitution rates make it difficult to extrapolate population-based estimates of rates over a long evolutionary time (ho et al. ) ; however, it is unlikely that purifying selection alone can account for this difference between short-and long-term evolutionary rates (woodhams ) . furthermore, short-term estimates of viral substitution rates (inferred from population-based rate estimates at the tips of phylogenies) are often found to be several orders of magnitude faster than long-term estimates of substitution rates (inferred from external calibrations located deeper in the phylogeny). this inconsistency has been reported for hepatitis b virus (zhou and holmes ; gilbert and feschotte ) and simian immunodeficiency virus (wertheim and worobey a; worobey et al. ) , suggesting that long-term substitution rates can be orders of magnitude lower than short-term substitution rates. an alternative, and more parsimonious, explanation is that a single substitution rate (albeit one possibly slower than that inferred using short-term population-based data) predominates throughout the history of these viruses. and our inability to accurately estimate branch lengths creates the appearance of dramatically lower substitution rates deep in the phylogenetic tree. although the methods presented here do not correct for branch lengths on the order needed to reconcile short-term substitution rates with deep calibrations, they provide a glimpse at the missing evolution that most current methods of phylogenetic inference fail to capture. although we are unable to provide a full remedy to the problem of underestimated branch lengths due to purifying selection, our results do point to a couple of guidelines when inferring tmrcas in rna viruses. first, the inference of substantially different selective regimes on longer internal branches compared with shorter shallower branches (e.g., using lineage+dual, , or free ratio, yang , codon models) appears to be a sign that older tmrcas may be underestimated. second, if the synonymous substitution rate approaches saturation along a branch or group of branches, it is likely that the tmrca cannot be reliably inferred. if either of these patterns is encountered, the inferred tmrca estimates should be interpreted with caution. rapid advances in cheap and accessible computational power will undoubtedly move the fields of paleovirology and molecular dating towards increasingly more realistic evolutionary models that account for variation in selective regimes (suchard and rambaut ). our results provide compelling evidence that such a movement should be accelerated. in this study, we demonstrate the need to include relevant biological and evolutionary forces in substitution models. it was not until we permitted different evolutionary regimes for different branches in the tree that we saw the most dramatic changes in branch length estimates. current codon models take a very simplistic mechanistic view of the action of purifying selection, and we expect that incorporating processes such as directional selection (seoighe et al. ) , toggling selection (delport et al. ) , and residue-specific (doron-faigenboim and pupko ) 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attachment g protein we thank the associate editor alexei drummond, jeff thorne, and two anonymous reviewers for their valuable comments. this research was supported in part by the national institutes of health ( the relative importance of these factors in understanding the evolutionary history of other taxa remains to be seen. supplementary figs. s -s are available at molecular biology and evolution online (http://www.mbe. oxfordjournals.org/). key: cord- -bgodmbru authors: whitworth, carrie; mu, yi; houston, hollis; martinez-smith, marla; noble-wang, judith; coulliette-salmond, angela; rose, laura title: persistence of bacteriophage phi on porous and nonporous surfaces and the potential for its use as an ebola virus or coronavirus surrogate date: - - journal: appl environ microbiol doi: . /aem. - sha: doc_id: cord_uid: bgodmbru the infection of health care workers during the to ebola outbreak raised concerns about fomite transmission. in the wake of the coronavirus disease (covid- ) pandemic, investigations are ongoing to determine the role of fomites in coronavirus transmission as well. the bacteriophage phi has a phospholipid envelope and is commonly used in environmental studies as a surrogate for human enveloped viruses. the persistence of phi was evaluated as a surrogate for ebola virus (ebov) and coronaviruses on porous and nonporous hospital surfaces. phi was suspended in a body fluid simulant and inoculated onto -cm( ) coupons of steel, plastic, and two fabric curtain types. the coupons were placed at two controlled absolute humidity (ah) levels: a low ah of . g/m( ) and a high ah of . g/m( ). phi declined at a lower rate on all materials under low-ah conditions, with a decay rate of . -log( ) pfu/day to . -log( ) pfu/day, than under the higher ah conditions, with a decay rate of . -log( ) pfu/h to . -log( ) pfu/day. there was a significant difference in decay rates between porous and nonporous surfaces at both low ah (p < . ) and high ah (p < . ). under these laboratory-simulated conditions, phi was found to be a conservative surrogate for ebov under low-ah conditions in that it persisted longer than ebola virus in similar ah conditions. additionally, some coronaviruses persist longer than phi under similar conditions; therefore, phi may not be a suitable surrogate for coronaviruses. importance understanding the persistence of enveloped viruses helps inform infection control practices and procedures in health care facilities and community settings. these data convey to public health investigators that enveloped viruses can persist and remain infective on surfaces, thus demonstrating a potential risk for transmission. under these laboratory-simulated western indoor hospital conditions, we assessed the suitability of phi as a surrogate for environmental persistence research related to enveloped viruses, including ebov and coronaviruses. deaths as of august (https://www.who.int/news-room/detail/ - - -as -ebola-cases-reach- -in-drc-who-calls-on-partners-to-fulfill-promises-to -communities). the largest and deadliest ebov outbreak on record ( to ) had more than , cases and over , deaths globally ( ) . the outbreak directly impacted the united states when, in , a health care worker in texas tested positive for ebov after caring for an infected patient ( ; https://www.cdc.gov/media/releases/ /s -texas-second-health-care-worker.html). recorded cases from past and present outbreaks show that ebov hospital transmission is a global concern as highlighted in a review of health care worker (hcw) infections by selvaraj et al. ( ) , which details occupational exposure increasing transmission risk to health care workers. coronaviruses are also enveloped rna viruses, belonging to the family coronaviridae. human coronaviruses are responsible for some common colds, but in , severe acute respiratory syndrome coronavirus (sars)-cov- emerged from guangdong province, china, as the first known deadly coronavirus ( ) , resulting in , illnesses and deaths. in , the middle east respiratory syndrome coronavirus (mers-cov) outbreak, believed to have originated from saudi arabia, caused deaths across countries (https://www.who.int/emergencies/mers-cov/en/). coronavirus disease , caused by sars-cov- , emerged from hubei province, china (late ), and spread rapidly around the world, being declared a pandemic by the who in march (https://www.who.int/news-room/detail/ - - -who-timeline---covid- ) . fomites were thought to have played a role in the transmission of sars-cov- ( , ) , and though sars-cov- is thought to be primarily spread by aerosols, investigations are underway to determine if fomites also contribute to transmission. emerging data suggest that sars-cov- can persist for days on surfaces ( ) , though the influence of environmental factors still needs to be explored. the centers for disease control and prevention (cdc) recommends a combination of measures to prevent transmission of evd in hospitals, and these recommendations have been adapted for sars-cov- as well. the recommendations include patient isolation and record keeping, proper personal protection equipment (ppe) and correct use of ppe, dedicated equipment, limited use of sharps, avoiding aerosol-generating procedures, hand hygiene, and monitoring potentially exposed personnel and visitors for signs and symptoms (https://www.cdc.gov/vhf/ebola/clinicians/evd/infection-control.html; https://www.cdc.gov/coronavirus/ -ncov/hcp/infection-control -recommendations.html). further guidance from the cdc covers environmental infection control beyond ppe to include disinfectant use, routine cleaning, how to handle soiled surfaces and textiles, and how to transport or dispose of contaminated items and waste (https://www.cdc.gov/vhf/ebola/clinicians/cleaning/hospitals.html; https://www .cdc.gov/coronavirus/ -ncov/hcp/infection-control-recommendations.html). the who offers similar guidance on ppe (including proper donning and doffing), infection prevention and control, hand hygiene, and management of wastes ( , ; https://www.who.int/en/news-room/fact-sheets/detail/ebola-virus-disease). despite implementation of these best practices, transmission of ebola from patient to health care worker continued, as documented by at least four reported cases in the fall of (http://www.cidrap.umn.edu/news-perspective/ / /drc-ebola-cases-surpass -earlier-outbreak-total-virus-infects- -more-health) in the united states. there has also been documented health care worker transmission of sars-cov- and mers ( ) . these transmission events highlight the need to understand the persistence of ebov and other pathogenic enveloped viruses on fomites and the role of fomites in transmission, especially in the presence of body fluids ( , ) . ebov is a select agent and requires a biosafety level (bsl- ) laboratory and specialized ppe to prevent potential life-threatening exposure. sars-cov- and sars-cov- are also labeled as select agents and require a bsl- laboratory in order to culture and conduct environmental persistence, sampling, or disinfection studies in which working with live virus is required. for safety concerns, researchers have incorporated other methods to avoid handling and propagating this virus. some have opted for the molecular detection of viral rna to demonstrate potential transmission in the hospital environment ( , ) . surrogate viruses have historically been used for transmission studies related to health care practices and can be employed as surrogates for viral persistence. bacteriophage phi , a member of the family cystoviridae, was previously used as a surrogate for ebov, influenza virus, coronavirus (sars- ), venezuelan equine encephalitis virus, and other pathogenic enveloped viruses ( ) ( ) ( ) ( ) ( ) ( ) . casanova et al. ( , ) used phi as an ebola surrogate to demonstrate transference to health care worker hands and scrubs during the ppe doffing procedures. however, data regarding the persistence of ebov or its surrogates in the health care environment are limited. this study evaluated the persistence of phi in the presence of artificial test soil (ats) as a potential surrogate for ebov or coronaviruses at two absolute humidity (ah) conditions on four potential fomites: nonporous stainless steel (ss) and plastic (pl) and two types of porous hospital curtain fabrics. at the lower ah ( . g/m ) on ss, phi persisted Ͼ days with . -log reduction (inoculum of . ϫ plaque-forming units [pfu]), and there was no detectable infective phage by days (table ). phi persisted on pl at the same ah up to days with . -log reduction (inoculum of . ϫ pfu), and there was no detectable phage by days. the statistical model, using a least-squares method in sas v . (cary, nc), projected phi persistence until days on ss and days on pl (fig. ) . the model predicted a decay rate of . log /day for both ss and pl, with r values of . and . , respectively, at ah . g/m ( table ). the projected data have a uniform standard error of Ϯ . -log pfu/ml. there was no difference, by analysis of variance and the f statistic, in decay rates between ss and pl at the low-ah ( . g/m ) conditions. at the higher ah ( . g/m ) on ss, phi persisted Ͼ h with . -log reduction (inoculum of . ϫ pfu), and there was no infective phage by h (table ). phi persisted on pl at the same ah up to h with . -log reduction (inoculum of . ϫ pfu), and there was no detectable infective phage by h. the model projected phi persisting until h on ss and h on pl (fig. ) . the model predicted a decay rate of . log /day (r ϭ . ) for ss and . log /day (r ϭ . ) for pl ( table ). the projected data have a uniform standard error of Ϯ . -log pfu/ml. there was a significant difference in decay rates between ss and pl (p ϭ . ) at the higher ah. overall, phi persisted longer on ss and pl surfaces at the lower ah of . g/m (no detectable phi at days to days) than at the higher ah of . g/m (no detectable phi at h to h). phi persistence on porous treated and untreated curtains (tc and uc) at the low-ah ( . g/m ) conditions was Ͼ days with . -log reduction (inoculum of . ϫ pfu) and . -log reduction (inoculum of . ϫ pfu), respectively, and no phi was detectable at d for either curtain type ( table ). the model projected phi persisting until days in the low-ah conditions (fig. ) . the model of persistence on tc and uc predicted decay rates of . log /day for tc (r ϭ . ) and . log /day (r ϭ . ) for uc ( table ). the projected data have a uniform standard error of Ϯ . -log pfu/ml. there was no significant difference in the decay rates of the curtain types in low-ah conditions (p ϭ . ). phi persistence on porous curtains (tc and uc) at high-ah ( . g/m ) conditions decreased drastically compared to the low-ah results for tc and uc. time to no detection decreased from days to hours, where persistence on tc was h with a . -log reduction (inoculum of . ϫ pfu), and, on uc, it was h with a . -log reduction (inoculum of . ϫ pfu) ( table ). the model projected phi persisting until h on tc and until h on uc in the high-ah conditions (fig. ) . the model predicted decay rates of . log /h for tc (r ϭ . ) and . log /h (r ϭ . ) for uc ( table ). the projected data have a uniform standard error of Ϯ . for tc and Ϯ . -log pfu/ml for uc. the difference between curtain types was not significant (p ϭ . ) under high-ah conditions. overall, bacteriophage phi in body fluid simulant persisted longer when held at (table and ). with respect to decay rates, phi declined more slowly on all materials under low-ah ( . g/m ) conditions ( . log /day to . log /day) than under the higher ah ( . g/m ; . log /h to . log /day; table and ). there were significant differences in decay rates between porous and nonporous surfaces at both low-ah (p Ͻ . ) and high-ah (p Ͻ . ) conditions. phi is an enveloped bacteriophage and was chosen for this study because it has been used as a surrogate for the persistence of other enveloped viruses such as influenza virus, coronavirus, and venezuelan equine encephalitis virus ( , , ) . using a nonpathogenic surrogate removes the need for resources associated with a bsl- or bsl- agent and makes the research procedures accessible to more laboratories. this current work demonstrated that the persistence of phi was similar to the published reports of ebov ( ) ( ) ( ) ( ) ( ) , human respiratory viruses ( , ) , and coronavirus ( , ) in that the phage persisted longer in colder temperatures and at lower relative and absolute humidity. to date, two studies have evaluated ebov persistence and found variability based on temperature, humidity, and substrate ( , ) . persistence was also shown to vary between species of ebov, sudan ebov and zaire ebov, and between variants makona-c and yambuku-mayinga ( , , ) . bausch et al. ( ) found the risk of infection from fomites to be low when working with the sudan ebov, where only of surface swab samples taken daily in an ebola isolation ward in uganda were positive by pcr detection only; no samples were culture positive. in contrast, bibby et al. found that the phi was shown here in the current study to be a conservative surrogate for ebov in a laboratory-simulated western hospital room condition of . g/m ah, persisting longer than the makona-c variant (ah ϭ . g/m ), with decay rates of . log /d and . log /h, respectively (table ). due to different conditions, persistence comparisons between the current phi study and the schuit et al. ebola work at the higher ah were not possible; phi was evaluated at an ah of . g/m , and ebov was table for r values. fig. and and expressed in log . d n/a means that no data were provided or that the experiment was not extended until there was no detection. e ah was not calculated because rh was not provided. evaluated at an ah of g/m or . g/m ( ) . interestingly, contrary to the trend we report with phi and studies seen with mers-cov ( ), schuit et al. demonstrated increased survival of ebola at the higher ah of . g/m ( °c; % rh) when deposited in dried blood compared to his lower-ah conditions tested. a controlled laboratory study of sars-cov- and sars-cov- investigated persistence on steel and plastic and found that both viruses behaved similarly ( ) . when conditions were held at ah . to . ( to °c and % rh), no infective virus was detected by and h, respectively (table ). chin et al. ( ) demonstrated that sars-cov- persisted for days at ah . ( °c; % rh). these conditions are close to our ah of . , in which persistence of phi was observed for only days, suggesting phi may not be a suitable surrogate for sars-cov- . additionally, a model based on testing at several humidity and temperature conditions predicted that sars cov- would persist for . days on steel and plastic, as suspended in artificial saliva, and held at an ah of . ( °c; % rh), one of the same conditions we tested phi , though the model would not extend to the lower-ah condition (https://www.cdc.gov/ coronavirus/ -ncov/hcp/infection-control.html). our work reported phi persistence for days under these conditions, a significantly shorter period, though the matrices were different. this adds evidence to the chin et al. ( ) results that phi may not be a suitable surrogate for sars-cov- when persistence is being studied. mers-cov was less persistent than phi under close but not exactly the same conditions, surviving only to days and at low ( . g/m ) and high ( . g/m ) ah, respectively ( ) . our study showed significantly greater persistence of phi ( to days) at lower ah ( . g/m ) than was tested for mers-cov. at the highest-ah conditions tested, . g/m for phi and . g/m for mers-cov, persistence for phi was h, while mers-cov was h. whether these differences can be explained by environmental test conditions, surface characteristics, or organism structure differences remains to be explored, but little parallel is seen between the studies. other coronaviruses tested at ah levels of . to . g/m on aluminum persisted for even less time; human coronavirus (hcv) persisted for h and hcv oc for h ( ) ( table ) . persistence declines with increasing ah for phi , but the literature does not reveal this trend for coronaviruses. an integral data gap for enveloped viruses is the risk of transmission under various humidity and temperature conditions. the two temperature and humidity combinations applied in this study were upper and lower health care facility extremes. as related to ebov and coronaviruses, the upper extreme might be found in a tropical setting without adequate air conditioning, as seen in liberia and sierra leone during the to epidemic, which was . °c ( ) . the lower extreme was chosen as a setting common in western health care facilities. the environmental temperature, along with relative humidity (rh), was used to calculate the ah frequently referenced in the literature. ah is the measure of the water vapor in the air regardless of temperature, while rh is the ratio of the concentration of water vapor to the maximum possible concentration at a given temperature. research on both phi and influenza virus has shown that ah may be more important than rh in virus infectivity ( , , ) and the role of ah may be linked to changes in the viral envelope ( , ( ) ( ) ( ) . shaman and kohn reported that influenza virus survival is more dependent on the water vapor in the air (ah) than how close the air is to saturation (rh) ( ). prussin et al. came to the same conclusion for phi when using a multiple regression analysis, showing that ah is a better predictor of virus infectivity ( ) . the role of ah in survival and infectivity may be linked to changes in the viral envelope during desiccation ( , ( ) ( ) ( ) and the protective effects of proteins upon concentration ( ) . one study noted a drop in infectivity between and % rh at °and °c (ah, . to g/m ) ( ), and similar findings were seen in an influenza virus persistence study ( ) , though these rh and ah conditions were higher than those tested here. health care environments vary around the world, and more information is needed to understand what influences persistence of enveloped viruses. damage from environmental conditions (e.g., ah, surface type, and matrices) to the viral envelope impact infectivity and persistence. mateu et al. ( ) suggest that repeated disruption of the capsid envelope can cause irreparable damage. casanova et al. ( ) suggest that viral inactivation is due to structural damage during desiccation, occurring at the air-water interface of the viral envelope. persistence and infectivity of ebovs have been shown to be influenced by the suspension liquid (blood, serum, or cell culture media) and surface type, such as plastic and metal (nonporous), as well as curtain material (porous) ( , ) . blood, mucus, stool, and other body fluids can provide protection from envelope structural damage due to desiccation ( , ) . in the hospital environment, bausch et al. ( ) detected ebov by reverse transcription pcr (rt-pcr) only upon visibly contaminated surfaces that were bloodstained. the transmission pathway of infected blood, and the protective nature of the blood matrix as shown by fischer et al. ( ) , led to the use, in the current study, of ats as a blood stimulant, as it contains proteins, hemoglobin, and carbohydrates. though testing only continued for h, wood et al. demonstrated better persistence of phi when suspended in blood diluent than in phosphate-buffered saline (pbs) ( table ) and that the influence of the matrix overshadowed the influence of the fomite material ( ) . the current data show that phi infectivity persists longer in dried ats ( °c, % rh, . g/m ah) than the ebola makona-wpgc strain persisted in blood under similar, but not exact, conditions ( °c, % rh, . g/m ah) ( ) . overall, variations in log reductions have been seen between species and strains of filoviruses ( ) as previously mentioned. however, this comparison to the makona variant in blood under similar conditions as used in this work suggests phi may be a conservative surrogate for ebola at lower-ah conditions (table ) . differences in persistence for both lake victoria marburgvirus (marv) and zaire ebola virus (zebov) were seen between metal ( stainless steel) and plastic by piercy et al. ( ) . both lake victoria marv and zebov suspended in guinea pig sera could not be recovered from metal surfaces at any time point regardless of the temperature and humidity. when placed on plastic, however, persistence improved, with infectivity lasting up to days at °c ( ) . nonporous surfaces appear to be a greater risk in the transmission of ebov in hospital settings than porous surfaces. like ebov and coronavirus, phi did not persist as long on porous surfaces (tc and uc) as it did on nonporous surfaces (ss and pl) ( table and one limitation to this evaluation of phi bacteriophage as a suitable surrogate on porous materials is the difficulty in elucidating if there is a true lack of persistence or if the phage adhered to the fabric, making it difficult to elute. adsorption to the curtains could potentially explain some of the declines in recoverable infective phage, though the adsorption would most likely inhibit touch-transfer as well. viral adsorption to fabrics is influenced by whether a fabric is tightly or loosely knit and the surface charge of the virus and the fabric ( ) . these data inform public health investigators that enveloped viruses are likely to persist longer on surfaces in modern climate-controlled health care facilities than in tropical field stations. additional precautions and disinfection strategies must be taken to prevent transmission when treating infected patients. this work also provides additional evidence that phi may be considered a conserva-tive surrogate for ebov when conducting persistence investigations in that it persisted longer. we also report that phi may not be a suitable surrogate for coronaviruses in all environmental conditions, though there seems to be a wide range of persistence reported in the literature. the model for sars-cov- (https://www.dhs.gov/science-and -technology/sars-calculator) predicted persistence of . days ( . % inactivation), which is considerably more than our experimental finding of h for phi . as reported by aquino de carvalo et al. ( ) , when evaluating phi as a surrogate for persistence in water matrices, phi cannot be considered a universal surrogate for persistence of all enveloped viruses on surfaces. instead, multiple surrogate viruses should be considered if the virus of interest itself cannot be investigated. test materials. four test materials were chosen to simulate surfaces typically found within the u.s. health care environment and cut into -cm coupons. the two nonporous surfaces were stainless steel (ss) ( phi propagation. bacteriophage phi and host pseudomonas syringae were obtained from laboratoire de sylvain moineau in québec, canada. using prepared -h growth of p. syringae (her ) in ml tryptic soy broth (tsb), bacteriophage phi (her ) was propagated from lyophilized stock by reconstituting with ml of prewarmed ( °c) tsb. reconstituted phi ( l) was transferred into ml fresh tsb with l of overnight host p. syringae, followed by gentle agitation using a vortex and incubating with agitation ( to rpm) for h at °c. the phi lysate was then filter sterilized using a . , and used as a body fluid simulant, with ats containing proteins (albumin), hemoglobin, carbohydrates, cellulose, and lipids. the stock phi was diluted in series to obtain a pfu/ml suspension in ats. the coupons were inoculated with l of pfu/ml suspension in ats, resulting in an inoculum of pfu/cm . negative controls consisted of ats ( %) without phi inoculated onto coupons. the test coupons were placed in open petri dishes in triplicate, along with the negative coupons, in a caron model environmental chamber (marietta, oh) at two controlled temperature (t) and relative humidity (rh) levels: °c and % rh (ah ϭ . g/m ) and °c and % rh (ah ϭ . g/m ). three inoculated coupons and a negative control were removed and processed immediately (t ) and at designated time points until phi pfu could no longer be detected (limit of detection from coupons was Յ pfu/cm ). each experimental condition was repeated twice with triplicate coupons for a total of coupons at each time point. one exception is ss, which had a total of samples for each time point for the high-ah ( . g/m ah) environmental condition. recovery. the phage(s) were dislodged from the coupons in ml phosphate-buffered saline with tween (pbst; . %) ( . m pbs [ . to . ph] and tween [ . %]) by alternating between vortex and sonicating bath for s each, repeating the rotation times ( ) . the eluate was diluted in series in sm buffer. one ml of sample was plated with l of host p. syringae in tryptic soy agar (becton, dickinson and company, franklin lakes, nj), using a double agar overlay method ( ) . the overlay plates incubated for to h ( °c) before we counted the pfu. coupon removal, processing, and plating continued until no infective phages (as determined by pfu) were observed. pfu per milliliter were calculated based on dilution factors and log transformed. results were calculated in both rh and ah to compare to other studies. statistical analysis. sas v . (sas, cary, nc) was used to create linear models that assessed the potential relationships between the mean log change of virus concentration on ss, pl, tc, and uc at two environmental conditions ( . g/m , . g/m ). for these analyses, the data points were used individually and not averaged. least-squares methods were used to fit the model and determine the rate of log reductions written as decay rate (log reduction per day or hour) in table and for these data; r was used to assess goodness of fit of the model. analysis of variance and the f statistic were used to test the differences between various materials (i.e., ss versus pl, tc versus uc, and porous versus nonporous) under the same absolute humidity, where significance was set at a p value of Ͻ . . after ebola in west africa-unpredictable risks, preventable epidemics texas healthcare worker is diagnosed with ebola infection rates and risk factors for infection among health workers during ebola and marburg virus outbreaks: a 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sm buffer. cold spring harb protoc statistical assessment of a laboratory method for growing biofilms enumeration of bacteriophages by double agar overlay plaque assay the effects of temperature and relative humidity on the viability of the sars coronavirus the authors acknowledge matthew arduino for his help in the design of this experiment and amanda lyons for her technical assistance. key: cord- -c umbcvn authors: reed, patricia e.; mulangu, sabue; cameron, kenneth n.; ondzie, alain u.; joly, damien; bermejo, magdalena; rouquet, pierre; fabozzi, giulia; bailey, michael; shen, zhimin; keele, brandon f.; hahn, beatrice; karesh, william b.; sullivan, nancy j. title: a new approach for monitoring ebolavirus in wild great apes date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: c umbcvn background: central africa is a “hotspot” for emerging infectious diseases (eids) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. ebolavirus is suspected to have caused recent declines in resident great apes. while ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm ebola virus disease (evd) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure. methodology/principal findings: here we report the first successful noninvasive detection of antibodies against ebola virus (ebov) from wild ape feces. using this method, we have been able to identify gorillas with antibodies to ebov with an overall prevalence rate reaching % on average, demonstrating that ebov exposure or infection is not uniformly lethal in this species. furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to ebov (protected from exposure by rivers as topological barriers of transmission). conclusions/significance: our new approach will contribute to a strategy to protect apes from future ebov infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. finally, since human evd is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where ebov causes case-fatality rates of up to %. emerging infectious disease (eid) epidemics and pandemics arise without warning, even with global efforts aimed at tracking pathogens early and at the source, a fact most recently evidenced by the swift global spread of influenza h n [ , ] and a current outbreak of ebolavirus affecting multiple west african countries simultaneously [ ] . most major human eids are of zoonotic origin and include viral infections of both global (hiv- , hiv- , h n ) and localized significance (ebolavirus, monkeypox, marburgvirus, nipah virus, severe acute respiratory syndrome [sars]-associated coronavirus) [ ] . systematic monitoring of people and wildlife at hotspots of eid is one strategy for preventing human pathogens of animal origin from reaching a pandemic state [ ] . by detecting animal pathogens before or just as they emerge in humans, it may be possible to mitigate against their worldwide spread [ ] . furthermore, in the case of some diseases such as ebola virus disease (evd), the monitoring of wildlife disease serves as a critical component of early warning systems aimed at preventing the transmission of zoonotic diseases to humans [ , ] . evd has repeatedly passed from infected apes to hunters, leading to multiple epidemics and human deaths ( cases) in gabon and the republic of congo (roc) alone since [ , [ ] [ ] [ ] . more significantly, human epidemics are often preceded by observed animal outbreaks, underlining the human health implications of surveillance and control of epizootics [ , ] . the international union for conservation of nature (iucn) currently lists the western lowland gorilla (g. gorilla gorilla) as critically endangered and cites infectious disease as one of the top two threats to this species [ ] . ebolavirus is lethal in humans and nonhuman primates and has been described as a significant threat to the survival of western lowland gorillas and chimpanzees (pan troglodytes) in central africa [ , , ] . data from ecological surveys in central african ape habitats illustrate declines in ape signs (nests, feces, prints) temporally and spatially linked with confirmed human evd outbreaks [ ] [ ] [ ] . mathematical modeling suggests that, between and , gorilla numbers in gabon dropped by more than %, and it is hypothesized that infectious pathogens, including ebolavirus and bacillus anthracis, may contribute to gorilla mortality in africa [ , , ] . despite the significance to both human and wildlife health, direct evidence of great ape exposure to ebolavirus or other pathogens (either by pathogen or immune response detection) is scant, complicating our ability to monitor epizootics. therefore, to fill this gap, there is a need for prospective epidemiologic studies combining ecological data with laboratory screening. most currently available data regarding primate pathology and immune response comes from experimentally infected laboratory macaques [ , ] . in direct response to the challenges associated with collecting blood or tissue from wildlife, non-invasively collected biological samples such as feces have been used for wildlife disease screening [ , ] . primate feces have been screened for the presence of viral nucleic acids due to shedding of simian immunodeficiency virus (siv), circoviruses, enteroviruses and hepatitis viruses [ ] [ ] [ ] [ ] . for siv, feces have also shown the presence of virus-specific antibodies [ ] . we developed a non-invasive immunological assay to detect ebolavirus antibodies in great ape feces, allowing us more insight into wild ape ebolavirus infections and their surveillance, and leading the way to identifying the best approaches for their protection. in addition, this new assay may prove valuable in the development and employment of prospective epidemiological ebolavirus studies in wild great ape populations. eighty gorilla fecal samples were collected in different habitats in the roc. in zone a, gorilla fecal samples were opportunistically collected while following habituated gorillas roughly and years after ebolavirus infection was confirmed in ape carcasses at that site using a combination of rt-pcr, immunohistochemistry and antigen capture [ , ] . in june , samples were collected in zone b during a reconnaissance walk survey (recces) composed of eight , km linear recces radiating every u from a central point with terminal ends of every other pair connected by km recces. this zone is southwest of the mambili river in the southeastern-most area of odzala-kokoua national park (oknp), and samples were collected two years after two gorilla carcasses found in this area tested positive for ebov using rt-pcr and antigen capture assays [ , ] . for these surveys, two teams operated simultaneously, each averaging . km per day over days, and following pre-determined global positioning system (gps) points. a continuous gps track log was maintained and uploaded to a garmin xl gps (www.garmin.com) with a position recorded every km. three missions occurred in zone b . the first occurred from th august to th september when a km closed loop survey was conducted on the northeast side of the mambili river. this search was for evidence that would indicate that the abovedescribed may epizootic southwest of the mambili river had also affected wildlife on the opposite side of the waterway. ten gorilla fecal samples were collected and a continuous gps track log was maintained and uploaded to a garmin xl unit, with points taken every km. also, in , a large-scale ecological and large mammal survey was conducted throughout oknp under the auspices of the wildlife conservation society and the projet espèces phares of the european union [ ] . from september th to th , , five gorilla fecal samples were collected during these missions by means of reconnaissance walk surveys and of a systematic unbiased line transect design aimed to estimate animal abundance derived from the density of animal sign, multipliers decay rate and production, and the area of the survey zone; both designed with and analyzed by the distance software program [ ] [ ] [ ] . lastly, in june , the original km loop described above was repeated during which gorilla fecal samples were collected. in november-december (zone d) and march-april (zone c), reconnaissance walk surveys, similar to the approach applied in zone b , were conducted in great ape habitats that, by the end of the study period, had no reported disease outbreaks. the purpose of these missions was to estimate ape abundance by recording all ape nests. gps points were taken every km and samples were collected. ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in central africa. improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. however, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. here we report the first successful noninvasive detection of ebov immunity in wild great apes, demonstrating survival in this species. this tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of evd outbreaks in human populations. sample collectors wore disposable latex gloves and surgical masks while collecting feces. approximately g of fresh feces was placed in ml of rnalater (qiagen gmbh, hilden, germany) in a ml plastic screw-top vial (corning incorporated, corning, new york, usa), sealed with parafilm (pechiney, menasha, wi, usa), and placed in zip-closure plastic bags and stored at ambient temperature (, uc or uf). samples collected in zone b were placed in liquid nitrogen vapor in a dry shipper (arctic express dual , thermolyne) at the end of each day and maintained in this state until arrival at the analyzing laboratory. feces were determined to be that of gorillas when recovered under one of the following conditions: post-observation collection (after seeing gorillas) or post-audition collection (after hearing gorillas), in association with gorilla nests or in association with gorilla trails [ , ] . genotype studies have demonstrated that feces collected using these methods are accurately classified as gorilla feces % of the time [ ] . in addition, the presence of long tri-lobed sections, ample fiber, and abundant green leafy material further classified these samples as gorilla dung [ , ] . only feces estimated to be less than hours old using published criteria [ ] were collected. the plasmid encoding ebov np is a p derivative [ ] . to purify the recombinant viral protein, plasmid p np was tagged at the c-terminus by site-directed mutagenesis with the quick-change xl site-directed mutagenesis kit (stratagene, la jolla, ca, usa). p np was provided with the hexa-histidine tag. the tagged plasmids were transfected into human embryonic kidney cells (freestyle -f cells, catalog no. r - ) obtained from invitrogen (carlsbad, ca) and grown in a shaking flask at uc under % co with freestyle expression medium (invitrogen). the ebov his-tagged np was purified by nickel-affinity gel, ni sepharose fast flow (ge healthcare, piscataway, nj), and eluted with mm imidazol. the concentration of purified np protein was measured with quick start bradford protein assay reagent (biorad, hercules, ca, usa) and used in the western blot assay. to screen nonhuman primate fecal samples for ebolavirus antibodies, we adapted an existing enhanced chemiluminescent western blot assay [ ] . feces were vigorously mixed in rnalater (ambion life technologies, grand island, ny, usa), . ml of the mixture diluted in . ml of pbs-tween- ( . %), heated at uc for minutes, centrifuged at g for minutes and dialyzed in pbs x with stir bar at uc for to hours to resuspend fecal immunoglobulins that normally precipitate in rnalater. purified or cell lysate np protein was denatured in sample reducing agent (invitrogen nupage), heated at uc for minutes, separated by - % gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) ( . mg per well) (invitrogen np , carlsbad, california, usa), and followed by transfer to nitrocellulose membrane (invitrogen lc , carlsbad, california, usa) which was blocked with % nonfat milk in pbs-tween ( . %) and bovine albumin ( . %). membranes were then cut into strips and incubated overnight in fecal extract on a rocking plate. specific np-bound antibody was detected with goat-anti-human igg peroxidase conjugate and the blot was visualized using an enhanced chemiluminescence detection system. the films were exposed to the immunoblot strips then scanned using an epson perfection photo scanner. to define a cut-off of positivity we used the image j program (imagej, nih, bethesda, maryland, usa) that allowed us to subtract the background in each strip and to compute the integrated density of the band that is the sum of the values of the pixels in the selection in the blot. specimens which showed no visible specific band in the blots were scored as negative whereas those which showed specific band (reactivity with a protein of approximate molecular mass of kda corresponding to ebov nucleoprotein np) were regarded as positive if their integrated density was in excess of the mean of integrated density plus standard deviations of the negative blots. blots with a weak specific visible band and an integrated density below this cutoff were classified as uncertain. a subset of samples collected in (the year of the last evd outbreak in the region) was also screened for the presence of filovirus rna using a nested rt-pcr. fifty nanograms of total rna isolated from rnalater preserved fecal samples were extracted using the rnaqueous pcr kit (ambion life technologies, grand island, ny, usa) and used in a onestep rt-pcr, followed by a nested pcr step. we used degenerate primer pairs in order to amplify a bp fragment of the l polymerase gene from any filovirus. the one step rt-pcr primers are: -atmgraayttttcyttytcatt- and rytataawartcactracatgcat- ; the nested pcr primers are -ttyccwagyaayatgatggt- and -ggdattrdrwartgcatcca- . to assess the quality of the total rna from the fecal sample, we amplified a housekeeping gene for each sample, the ß-glucuronidase gene (gus, accession number af ) using a nested pcr assay. the gus primers used for the one step rt-pcr were -gcttaccacccagtttgag- and -tgggga-tacctggtttcattg- , whereas the nested primers were -tcagagcgagtatggagc- and -gcactttttggttgtctc- . we generated a bp fragment. positive and negative controls were included to ensure that cdna product could be amplified and that no contamination from cdna or previous pcr products occurred. we compared antibody prevalence between sampling locations using a log-likelihood ratio test (g-test) [ ] . a % confidence interval (ci) was constructed for the prevalence. in order to examine ebolavirus exposure in wild great apes we sought to develop a strategy of detection in samples collected by non-invasive methods that would be sensitive and specific enough to detect multiple ebolavirus species with minimal false positive results. it has been shown previously that an enhanced chemiluminescent western immunoblot assay is able to successfully detect specific antibodies in rnalaterpreserved feces from simian immunodeficiency virus-infected chimpanzees (sivcpz) [ ] . the sensitivity and the specificity of sivcpz antibody detection in fecal samples were estimated to be % and %, respectively. viral sivcpz nucleic acid could be amplified in an immunoblot-positive fecal sample, confirming sivcpz infection [ ] . furthermore, a similar approach was used to diagnose simian foamy virus infection in wild chimpanzees (sfvcpz). the sensitivities of sfvcpz antibody and viral nucleic acid detection in fecal samples from captive chimpanzees were % and % respectively, and assay specificities were % [ ] . these studies show the potential of assessing rnalater-preserved fecal samples to document wild apes' exposure to viruses. given the success of this approach, we developed a fecal western blot assay to detect ebolavirus antibodies. we chose purified ebov np as the antigen for antibody detection since it is one of the most abundant structural proteins produced during infection and a major target of the host immune response. this is supported by previous studies showing that humans who have survived natural ebov infection developed strong antibody responses mostly against np [ ] [ ] [ ] . in addition, the np sequence is well conserved among ebolavirus species (figure s ), making it useful for detection of antibodies against multiple ebolavirus species [ , ] and potentially increasing the breadth of this detection method. we first assessed the ability to detect np antibodies in rnalater-preserved fecal samples from captive cynomolgus macaques. fecal specimens were experimentally spiked with different dilutions of positive serum containing polyclonal immunoglobulin from a monkey that was vaccinated with a genetic vaccine encoding np [ ] . serum from this vaccinated monkey displayed antibody reactivity with np by both elisa and western blot analysis (not shown). extracts from these positive serum-spiked feces were then used to incubate immunoblot strips containing immobilized np. anti-np antibodies were detected by enhanced chemiluminescent western blot immunoassay in fecal samples at seropositive nonhuman primate (nhp) plasma dilutions of up to -fold (figure ) , indicating a high sensitivity of the assay for fecal antibody detection. a similar level of sensitivity was observed for detection of anti-siv and anti-hiv antibodies by western immunoblots using plasma samples from sivsm-infected nhp diluted up to and plasma samples from hiv- infected individual diluted up to [ ] . in contrast, fecal extracts from captive and uninfected nonhuman primates (cynomolgus macaque and western lowland gorilla species) treated in the same way showed no reactivity in the np immunoblot, demonstrating low background for the assay and lack of cross-reactivity with serum antibodies directed against irrelevant proteins. these results demonstrated that np antibodies present in primate fecal samples can be extracted and detected by immunoblotting. to evaluate whether wild apes show evidence of previous ebolavirus exposure, we screened fecal samples from gorillas living in the roc for ebolavirus antibodies. fecal samples were opportunistically collected from great ape habitats using one of two survey methodologies. the first method employed a systematic unbiased line transect design aimed to estimate animal abundance or the density or size of wildlife [ ] [ ] [ ] . the second consisted of reconnaissance walks to provide a general overview of large animal distributions and investigate animal trails where animal dung is likely to be encountered [ ] . fecal samples were collected from two regions within or adjacent to oknp in western roc near the border with gabon ( figure ). the first is an evd diagnostically confirmed outbreak (dco) region where human cases were laboratory confirmed between and [ ] , and ape carcasses collected between and tested positive for ebov [ , , ] . the presence of long-term and functioning wildlife disease surveillance programs and gorilla habituation and research studies in the roc allowed for immediate access to the dco region during and after evd epidemics which facilitated the collection of samples from gorillas with a high likelihood of previous exposure to ebov, and samples were collected at this site within - months of confirmed great ape evd cases being found. the second region is an area with no reported outbreaks at that time (nro). here, there were no reported human cases, observable signs of epidemics, ebov-positive animal samples, or significant losses in ape numbers despite repeated visits up until the end of this study in april . routine and systematic reconnaissance missions for table ) . all human evd outbreaks that had previously occurred in our sampling zone study are thought to be the result of handling infected wild animal carcasses, including gorillas [ ] . samples from carcasses were used to document ebov outbreaks in gorillas by rt-pcr, antigen detection elisa and immunohistochemistry [ ] . to explore whether ebolavirus antibodies could be detected in fecal samples obtained from wild apes we focused initially on the dco region (zones a and b ; figure ) in order to maximize the likelihood of obtaining fecal samples from apes that had been exposed to ebov. among fecal samples collected from the dco region, tested positive for np antibodies by immunoblot ( figure , table ). two ebov antibody positive fecal samples out of ( %, ci: - . %) came from zone a where, in late and early , ebov was laboratory confirmed in great ape carcasses at the lossi sanctuary [ , ] (figure ). of samples collected in zone b in , two samples were uncertain (defined in methods) and three were positive for np antibodies ( . %, ci: - . %). samples were collected from zone b during a mission two years after ebolavirus was detected in ape carcasses at the site [ , ] (figure ). we also tested ape fecal samples obtained from the outbreakfree (nro) region to explore whether np antibody detection can be used as a potential surveillance tool. the nro region contains zones b , c and d. zone b is adjacent to b , yet separated from it by the relatively large mambili river. in the fall of , fifteen ape fecal samples were collected in zone b to determine whether a may epizootic had also affected wildlife on the opposite side of the waterway. two years later, in june , the original km closed loop track was repeated to explore any temporal changes in ape density or np seropositivity. three positive fecal samples out of twenty ( %, ci: - . %) were found in zone b (table ) and one sample was uncertain. twenty-five fecal samples were collected in zone c (march and april ) and zone d (november and december ). the zone c mission followed the discovery of one chimpanzee carcass that later tested negative for ebov (e. leroy, personal communication, april , ) . no antibodies were found in the fecal samples from zones c and d, where no outbreaks had been reported. altogether, eighty fecal samples from wild great apes were analyzed by western blot and eight ( %) were found to be np antibody positive (table ) . three samples (one from zone b and from zone b ) had blots with a weak specific visible band and an integrated density below the cutoff, and were thus classified as uncertain (not shown). the remaining fecal samples showed no detectable np-specific antibodies and were classified as antibody negative. roughly half of the samples were collected in the dco region and . % of these samples were found to be antibody positive, whereas a smaller proportion ( . %) of samples collected in the nro region were positive. the difference between the nro and dco regions is not statistically significant (log likelihood ratio statistic (g) = . , x-squared df = , p-value = . ) ( table ) , but overall the data show that anti-np antibodies are present in fecal samples from wild ape populations even in areas with no prior reports of human or wild great ape outbreaks. these data demonstrate that the screening of wild gorilla feces by western blot for the purpose of monitoring ebolavirus exposure was successful in detecting np antibodies. this study represents the first time that ebolavirus antibodies have been detected in wild great ape fecal samples, and carries important implications for the future management and survival of these primates. this is especially relevant because intervention strategies to protect apes against future evd infections are being actively explored, including vaccination since ebolavirus vaccines have been shown to protect laboratory monkeys from disease [ , ] . there have been no studies or observations involving great apes that have described immune response, clinical signs, precise mortality rates or whether survivorship provides long-term immunity, and little is known regarding the overall ebolavirus serological status of apes in central africa. to date, serum samples from gorillas (n = ) and chimpanzees (n = ) in central africa have been screened for antibodies against ebov [ ] . most of these animals were sampled while living in captive settings (pets, rescue centers, primate centers); four subjects were free-ranging and sampled directly from the wild in oknp but were seronegative. obtaining samples from free-ranging wildlife is needed to improve our understanding of infectious agents circulating in the environment. all other health data related to ebolavirus from free-ranging apes comes from necropsies performed during wildlife die-offs and, in those cases, the vast majority of samples collected are too degraded to have diagnostic value [ ] . as expected, there is also nothing known regarding potential co-infections involved in great ape evd which may modify the host immune response, alter pathogenesis, increase mortality or influence the effectiveness of any future prophylactic plans, such as the administration of a vaccine once available. this is due to the difficulty in acquiring diagnostic samples from wild populations. capture and subsequent blood collection for serological screening is costly, time consuming, and carries some risk to the animals while providing information on only a few individuals. in fact, despite the disappearance of a staggering number of great apes in gabon and the roc and years of sustained and active surveillance in these countries during the course of this study, only carcasses were recovered, with confirmed ebov infection in of those individuals [ ] [ ] [ ] ] . moreover, finding animal carcasses in vast tracts of rain forest is difficult; it requires intensive searching and often results in the acquisition of highly degraded samples, which are not suitable for detection of viral antigens or nucleic acid and are more prone to negative results [ ] . this newly developed approach for non-invasive sampling of great apes has allowed the successful detection of anti-ebov antibodies in fecal samples, yielding a seroprevalence rate of % in gorillas. since genetic identification of individual fecal samples was not performed, we cannot rule out the possibility of resampling, so the prevalence rate is an upper limit for this data set. however, recent genetic analysis of gorilla and chimpanzee samples collected during iron-cross recces (the type of surveillance executed in sites b , c, and d) from - suggest a low resampling rate. of samples, three were identified with genetic identity the same as three previously sampled individuals, yielding a % resampling rate for sites in which the same site was revisited with the shortest interval of seven-months apart; the resampling rate in the currently study could be higher because two sites were sampled one-month apart and two locations, a and b , were not iron-cross recces (personal communication, k.j. lee) in addition to estimating ebolavirus exposure in nhp, this technique of screening feces by western blot is in fact a multipurpose tool. it provides the potential to employ serial fecal collections to detect a temporal change in incidence exposure in a given zone. for instance, we saw a trend toward a decrease in ebolavirus fecal antibodies in zone b /b from % in to % in , which can be tested in the future using formal prospective studies. fecal antibody screening can also be used before and after vaccination to demonstrate vaccine-induced immune responses developed in great ape populations, noting that antibody levels in vaccinated non-human primates are an immune correlate of protection [ ] . finally, this approach will facilitate the identification of immunologically naïve populations for largescale vaccination trials, thereby improving cost-effectiveness by identifying communities that could benefit the most from vaccination efforts. along these lines, it provides us with the first real possibility to investigate patterns of evd emergence in wild apes independent of animal mortality and the role natural barriers, such as rivers, may have in mitigating its spread. this ability to map exposure patterns across central africa may also provide insight into how this virus spreads within and between ape populations, a question that has generated two disparate theories: multiple virus introductions and a single spreading outbreak [ , , , ] . key to pandemic prevention is disease surveillance at the human/wildlife interface, especially considering the fact that the majority of emerging infectious diseases events (over %) are of animal origin and that those caused by wildlife pathogens are increasing [ , ] . the strategy described herein will be valuable in providing zoonotic information of public health concern from regions where resources are poor and help counter the emergence of diseases which have potential to become the next pandemic. monitoring diseases in animals using methods such as those we describe here allows for the identification and surveillance of many pathogens, including those with potential to adapt and spread in humans, like hiv and plasmodium parasites [ , , ] . these findings also illustrate the role in situ conservation organizations can play in disease surveillance programs. adapting these tools for use in other wildlife species may provide information regarding the transmission of ebolavirus and other emerging infectious diseases to human populations. recent concerns surround the role pigs play in the emergence of diseases such reston ebolavirus and h n [ , ] . central africa's forests are home to tens of thousands of wild pigs, including the giant forest hog (hylochoerus meinertzhageni) and the red river hog (potamochoerus porcus), and are characterized as emerging disease hotspots [ ] . although no evidence has emerged supporting speculation of ebolavirus-associated wild pig die-offs in africa, employing this assay in these species may address whether pigs are amplifiers, victims or carriers of the virus [ ] . it is noteworthy that in the case of influenza pigs are considered ''mixing vessels'' for viruses and capable of generating new strains transmissible to humans [ ] . the extensive bush meat trade in africa provides ample opportunity for pathogen transmission from pigs to humans, and underlines the importance of disease surveillance in this species. wildlife managers frequently perform wide scale ecological surveys, simultaneously collecting biological samples and data on the density and distribution of wildlife. with the benefit of the new diagnostic capacity and sampling strategies described herein, different fecal sampling approaches can be integrated into these surveys to provide information that has thus far eluded us concerning the distribution, ecology and epidemiology of ebolavirus. for the first time, both the logistical and diagnostic capacities are available to immunologically screen large popula-tions of wild great apes for previous exposure to ebolavirus and even estimate and monitor prevalence rates. figure s ebolavirus nucleoprotein sequences. sequence alignment of the nucleoprotein np from zaire ebolavirus (ebov, accession no. np_ ), tai forest ebolavirus (tafv, accession no. aci ), reston ebolavirus (restv, accession no. bab ), sudan ebolavirus (sudv, accession no. aad ) and bundibugyo ebolavirus (bdbv, accession no. aci ). the numbering of the amino acids is according to their position in the sequence. ''*'', identical residues; '':'' conserved residues; ''.'', semi-conserved residues. 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mcelroy, anita k. title: ebola virus disease in humans: pathophysiology and immunity date: - - journal: marburg- and ebolaviruses doi: . / _ _ sha: doc_id: cord_uid: xinkqs u viruses of the ebolavirus genus cause sporadic epidemics of severe and systemic febrile disease that are fueled by human-to-human transmission. despite the notoriety of ebolaviruses, particularly ebola virus (ebov), as prominent viral hemorrhagic fever agents, and the international concern regarding ebola virus disease (evd) outbreaks, very little is known about the pathophysiology of evd in humans and, in particular, about the human immune correlates of survival and immune memory. this lack of basic knowledge about physiological characteristics of evd is probably attributable to the dearth of clinical and laboratory data gathered from past outbreaks. the unprecedented magnitude of the evd epidemic that occurred in west africa from to has allowed, for the first time, evaluation of clinical, epidemiological, and immunological parameters in a significant number of patients using state-of-the-art laboratory equipment. this review will summarize the data from the literature regarding human pathophysiologic and immunologic responses to filoviral infection. ebola virus (ebov) is the prototypic member of the ebolavirus genus in the filoviridae family of negative-sense, single-stranded rna viruses. discovered in during the first documented outbreak of ebola virus disease (evd) in the town of yambuku in northern zaire (today democratic republic of the congo), ebov has since caused sporadic human disease outbreaks of varying magnitude in equatorial african countries (sanchez et al. a ). in march , an ebov variant later named ebov makona was first detected in guinea. this variant was responsible for a -year-long epidemic that affected tens of thousands of people in several west african countries, collapsing the healthcare systems of three of them. ebov makona rampaged through both rural and urban areas, and underscored previously poorly characterized features of evd, like sexual transmission and virus persistence after recovery rowe et al. ; chughtai et al. ; deen et al. ; fischer et al. ; rodriguez et al. ; varkey et al. ; uyeki et al. a) . the scientific and clinical knowledge of human evd before its appearance in west africa was very limited. the scarcity of human cases and their occurrence in rural areas of equatorial africa limited research, as did confinement of filovirus research to biosafety level containment laboratories. in addition, basic studies on evd pathophysiology have been hampered by the lack of susceptible small animal models with competent immunity. for example, laboratory mice, a commonly used disease model, are completely resistant to nonadapted ebov. before , evd was described as an acute hemorrhagic fever, thus earning its former name ebola hemorrhagic fever (ehf); case fatality rates of up to % had been reported. the disease was characterized by lymphopenia, disseminated intravascular coagulation (dic), immunosuppression, and a systemic inflammatory response resembling septic shock (feldmann and geisbert ) . while many of these observations have been strengthened by findings from the west african evd outbreak, some of the previous hypotheses have been revised. perhaps one of the most surprising findings has been the low overall number of human cases presenting with bleeding (schieffelin et al. ) , as well as the lack of correlation between bleeding and disease severity (schieffelin et al. ; mcelroy et al. a, b) . these findings triggered the change in disease nomenclature from ebola hemorrhagic fever to ebola virus disease. moreover, the finding that evd correlates with robust immune activation rather than immunosuppression mcelroy et al. a) , and the ability of the virus to persist in several body fluids long after recovery (varkey et al. ; uyeki et al. a; sow et al. ; green et al. ; deen et al. ) have changed our current view of evd and have prompted new directions in research and new public health policies. here we will aim to integrate these novel findings within the current human evd model, and will discuss future research directions. several ebolaviruses cause evd, and while differences may exist between the diseases caused by the individual viruses, this review will focus on evd as a disease caused by all known viruses in the ebolavirus genus (ebolaviruses) that are pathogenic for humans. the reader will note that most of the available data come from infections caused by ebov rather than the other pathogenic viruses in this genus: sudan virus (sudv), bundibugyo virus (bdbv), and taï forest virus (tafv). the related marburgviruses, marburg virus (marv) and ravn virus (ravv), will be mentioned where appropriate data are available, but unfortunately, information on marburg virus disease (mvd), which is caused by both of them, is still lacking. epidemiological data collected over the last years indicate that human infection with ebov occurs mainly through close contact with infected body fluids. this probably occurs during both spillover events (e.g., contact with infected blood during butchering of bushmeat) and human-to-human transmission. there is no evidence that direct contact with bats causes ebov spillover into humans (mari saez et al. ; leroy et al. ), but infection with marv and ravv via direct or indirect contact with egyptian rousettes (fruit bats of the species rousettus aegyptiacus) (amman et al. ; schuh et al. ) , has been documented. human visits to caves or mines in which these bats roost have been directly associated with the development of mvd (bausch et al. ; centers for disease and prevention ; adjemian et al. ) , strongly indicating that mucosal or skin contact with bat droppings is sufficient to initiate marv infection in humans. with the exception of the first evd outbreaks in zaire, which were linked to substantial percutaneous needle transmission (ebola haemorrhagic fever in zaire / ), most of the data since the early s suggest that exposing skin and mucosae to ebov while conducting activities like body washing during traditional funerals or caring for sick relatives in the household is sufficient for human-to-human transmission of ebov dowell et al. ; francesconi et al. ) . early data collected from a laboratory exposure to sudv even suggest that skin abrasions may not be necessary to allow ebolavirus entry through the skin (emond et al. ) . these findings raise questions regarding how ebolavirus infection takes place in skin and mucosae, and which cells are involved in the primary amplification of the virus. antigen-presenting cells are a putative initial target of ebov infection and previous research in animal models of disease has indicated that dendritic cells (dcs) and macrophages are early and preferred targets of ebov and support virus replication (geisbert et al. a) . both dcs and macrophages can also be productively infected by ebov in vitro (gupta et al. (gupta et al. , mahanty et al. ; bosio et al. ) , and ebov prevents activation of in vitro-derived dcs, mainly through the action of vp and vp (yen et al. ; jin et al. ; ilinykh et al. ) . to further complicate things, a great deal of research over the last decade has been devoted to defining the ontogeny and specific function of dc subsets in mice and humans. the emerging picture is that several cell subsets exist with overlapping and nonoverlapping functions, and these subsets can be roughly classified into classical, plasmacytoid, and inflammatory dcs in humans (see (haniffa et al. ) for an excellent review). whether ebov can equally infect different dc subsets is not known, but some of the existing evidence suggest that it cannot (leung et al. ) . for example, a number of cellular receptors have been involved in the attachment of ebov virions to target cells. these receptors include several c-type lectins present on the surface of dcs, such as dendritic cell-specific icam- -grabbing non-integrin (dc-sign) (simmons et al. ) and liver/lymph node sign (l-sign) . dcs of the epidermis and mucosal epithelium do not express these molecules, but langerhans cells in the skin and cd + dcs in mucosal epithelium do express the c-type lectin langerin (merad et al. ) . in fact, studies in monkeys and pigs have indicated that dc-sign + cells are scarce in the dermis and the lamina propria or submucosa in the steady state (schwartz et al. ; huang et al. ), suggesting that other cell types may be targets for early ebov replication. initiating ebov infection may depend on attachment to target cells via tim- and tim- , which are highly expressed in mucosal epithelia (rhein et al. ; kondratowicz et al. ) . initial virus amplification could then lead to inflammation and infiltration of a high number of myeloid cells expressing dc-sign and other described ebov attachment factors, like triggering receptor expressed on myeloid cells (trem- ) expressed by neutrophils (mohamadzadeh et al. ) , and human macrophage c-type lectin specific for galactose/n-acetylgalactosamine (hmgl) expressed by macrophages (takada et al. ) . the elucidation of the initial steps by which ebov establishes productive infection in a host organism is highly needed to understand the mechanisms by which the virus disseminates from the initial site of entry to the body, and perhaps to design medical countermeasures aimed at preventing virus spread. as mentioned above, dcs and macrophages are early targets of ebov infection. due to the migratory potential of dcs, these immune cells may participate in disseminating ebov from the initial points of entry to the draining lymph nodes (geisbert et al. a ). this strategy is commonly used by other viruses, including sars coronavirus , toscana virus (cusi et al. ) , and measles virus (mesman et al. ) , for dissemination in the host. however, perhaps due to the lack of suitable in vivo models for kinetic studies of ebov, the involvement of dcs in ebov dissemination has not been experimentally addressed. in any case, the specific subsets of cells responsible for ebov dissemination remain to be identified. it is plausible that tissue-resident dcs or inflammatory dcs derived from infiltrating monocytes are important for ebov dissemination. both myeloid dc populations are migratory and can transport a variety of antigens from inflamed tissues to the draining lymph nodes (leon et al. ; ersland et al. ) . conversely, macrophages and neutrophils are less likely to participate in ebov dissemination due to their low mobility and nonproductive infection, respectively (mohamadzadeh et al. ) . recent studies have demonstrated that human evd is associated with loss of peripheral blood monocytes, in particular nonclassical cd + monocytes ) which have been proposed as the main antiviral monocyte subset (cros et al. ). even though this study did not demonstrate direct infection of cd + by ebov, it raised the possibility that this cell subset could be involved in virus dissemination. in fact, cd + monocytes are also called patrolling monocytes due to their ability to attach to endothelial cells in a lfa- -dependent manner and to extravasate into inflamed tissues where they differentiate into inflammatory dcs and macrophages (cros et al. ; auffray et al. ). this hypothesis is also substantiated by a previous study that demonstrated that ebov particles can attach to monocytes and enter these cells only when the monocyte differentiation program has started, that is, during their differentiation into dcs and macrophages in inflamed tissues (martinez et al. ) . the identification of the dc subsets specifically involved in filovirus dissemination is a highly relevant topic of study, because the function of dcs can be enhanced or inhibited, ex vivo or in vivo, by antigen delivery or use of molecules and antibodies. therefore, dcs are putative immunotherapeutic targets for postexposure evd treatment (klechevsky and banchereau ) . for example, the ligand of the dc co-stimulatory molecule cd (scd l) is commonly used to enhance dc-mediated antigen presentation (kornbluth and bot ) , and previous studies have demonstrated a correlation between circulating levels of scd l and survival after sudv infection (mcelroy et al. a, b) . in addition, poor activation profiles of circulating antigen-presenting cells have been correlated with severe evd . these findings provide a rationale for the use of dc enhancers as immunotherapy candidates in filovirus disease. another key driver of ebov dissemination may be the cytokine microenvironment, since these innate immune signaling molecules play an important role in recruiting myeloid cells, which are putative ebov targets, to sites of inflammation. a considerable body of research exists on cytokine and chemokine responses during evd. despite some conflicts, in general, these data correlate fatal outcomes during evd with high concentrations of pro-inflammatory cytokines (e.g., il- ), pro-inflammatory chemokines (e.g., ip- ), and anti-inflammatory cytokines (il-ra and il- ), overall suggesting a general dysregulation in the expression of these key immune signaling molecules (hutchinson and rollin ; gupta et al. ; wauquier et al. ; baize et al. ; villinger et al. ). an inability to control viral replication is likely leading to continued innate immune stimulation. data from asymptomatic human cases have shown an even greater magnitude of cytokine and chemokine upregulation, followed by rapid downregulation of this response in association with control of viral replication (leroy et al. (leroy et al. , , suggesting that cytokine/chemokine dysregulation is a consequence of uncontrolled viral replication rather than a primary mediator of pathogenesis. type i interferons (ifn-i) are key antiviral cytokines, and perhaps one of the more conflicting aspects of comparing ebov experimental and clinical human data is the role of ifn-i in ebov immunity and pathogenesis. importantly, ifn-i not only induces an antiviral state in infected and bystander cells during early virus infection, but also is a key modulator in the transition between innate and adaptive immunity. by enhancing natural killer cell function, antigen presentation by dcs, and expansion of effector t cells, ifn-i bridges natural and acquired antiviral immunity (see (mcnab et al. ) for a review), so elucidating its functions during human evd is highly relevant to understanding disease pathogenesis. ifn is critical in protecting laboratory mice from ebov (brannan et al. ; bray et al. ) , but data are somewhat conflicting in nonhuman primates (nhps) and humans smith et al. ; villinger et al. ; yen et al. ) . higher levels of ifn-alpha were associated with fatal evd cases (villinger et al. ), but higher ifn-beta was associated with less severe evd ) and ifn-beta administration prolonged survival in nonhuman primates (smith et al. ) . however, as ifn responses are highly dynamic, drawing conclusions regarding human pathogenesis is difficult. for example, evd survivors may mount early and robust ifn responses that keep viral replication at bay, while patients who succumb to evd may display higher ifn levels later on due to increased viral replication and inflammation. interestingly, recent studies have shown that patients who survive and patients who succumb to evd both show robust t cell activation (mcelroy et al. a; ruibal et al. ) . since dcs are the only antigen-presenting cells capable of priming naïve t cells (banchereau and steinman ) , these results suggest at least two possibilities. on one hand, infected dcs may retain their capacity to initiate t cell-specific responses, as has been shown in other viral infections (wahid et al. ; rivera and mcguire ; kvale et al. ). on the other hand, some dc subsets may be spared from infection and thus able to prime ebov-specific t cells. the generation of this ebov-specific adaptive immunity is the topic of the next two sections. while innate immune responses may play a chief role in controlling early ebov replication in humans, the current model identifies the character (though not necessarily the magnitude) of adaptive immunity as the main factor driving viral clearance and recovery. both humoral and cellular immunity seem to be required for ebov clearance in humans, a hypothesis strengthened by the finding that evd patients mount robust adaptive immune responses (mcelroy et al. a; ruibal et al. ) with high numbers of circulating plasmablasts and ebov-specific t cells. the more difficult task is assessing whether adaptive immune responses mark substantial differences between fatal and surviving patients. initial studies supported the idea that early development of igm and isotype switching to igg correlated with positive outcome. indeed, a high percentage of patients with fatal outcomes do not seem to develop igm . these field studies are also in agreement with findings in patients evacuated into europe or the us for medical treatment during the recent west african evd outbreak. surviving evd patients mounted early igm responses and showed upregulation of serum igg over the course of the disease, which was correlated with viral clearance (kreuels et al. ; wolf et al. ) . conversely, deficient or diminished igm and igg responses have been reported in fatal cases of both evd and mvd (van paassen et al. ; baize et al. ) . however, limited field data also indicate survivors who did not develop igg, as well as patients who died after developing detectable circulating anti-ebov antibodies (onyango et al. ). in addition, limited clinical data obtained from the sudv-caused evd outbreak in gulu, uganda, did not reveal significant differences between the humoral responses in fatal and nonfatal evd, with very late expression of igg in both groups that was unrelated with viral clearance . as in many other aspects of evd immunology, the kinetics of antibody responses in a statistically relevant cohort of acute-stage patients with defined outcomes must be studied. antibodies play many roles during the immune response to pathogens, including neutralization and antibody-dependent killing of virus-infected cells by targeting them to fc receptor bearing cells (adcc) or complement (cmc). neutralizing antibodies (n ab ) probably play a small role in recovery from acute evd, since in many survivors n ab are not detectable until weeks or even months after recovery (luczkowiak et al. ; sobarzo et al. ). this is a puzzling and as yet unexplained finding. one of the plausible hypotheses is that disrupting lymphoid architecture during acute evd infection may compromise germinal center formation and b cell affinity maturation, a feature that has been observed during lassa fever (carrion et al. ). however, this hypothesis does not reconcile easily with the levels of circulating plasmablasts in patients in the acute stage of illness (mcelroy et al. a) , or with the limited focal necrosis observed in human biopsy samples of lymphoid tissues (martines et al. ) . one interesting possibility is that the long filament shape of filovirus virions may require a highly diverse repertoire of n ab for effective neutralization. in fact, studies have found greater b cell clonality in evd survivors than in individuals with b cell memory against hiv- or influenza a virus (bornholdt et al. b ). still, this cannot be the whole story, as similar delays in n ab production have been described in other viral hemorrhagic fevers (e.g., lassa fever). perhaps more importantly, delayed n ab production strongly suggests long-term virus or antigen persistence, which is in agreement with duration of post-evd sequelae (see below). nevertheless, long-term survivors develop effective n ab , mainly directed against several epitopes of the ebolaviral gp , (bornholdt et al. a, b; misasi et al. ; corti et al. ). many of the described n ab isolated from survivors are directed against the gp , glycan cap as well as against the region bridging gp and gp , which seem to be epitopes amenable for antibody-based therapeutics like zmapp (murin et al. ). importantly, a number of studies in surviving patients have highlighted the presence of naturally occurring n ab with cross-reactivity against other ebolaviruses and even against marv (olal et al. ; misasi et al. ; bornholdt et al. a ). these findings strongly suggest that antibody-mediated immune memory may provide long-term protection against secondary infection with filoviruses, and may have important implications for public health measures (e.g., recruiting survivors as caregivers in future outbreaks). currently, the relative importance of n ab versus other antibody-mediated mechanisms, such as adcc and cmc, is unclear, even though most protective antibodies probably act through both neutralization and adcc/cmc activation (schmaljohn and lewis ) . antibodies with both neutralizing and adcc capacity have been detected in evd survivors more than a decade after recovery , and adcc is probably an important mechanistic feature of zmapp (olinger et al. ). in addition to dissecting whether or not neutralization and adcc specifically contribute to evd immunity, it is important to determine the kinetics of antibody-mediated immunity from acute infection to long-term recovery. lack of information on evd antibody kinetics and ebov-specific quantitative activity is probably largely responsible for the lack of protective effect demonstrated by convalescent plasma therapy (van griensven et al. ). this therapeutic strategy has putative applicability for field outbreak conditions, but requires characterization of the virus specific activity in the product as well as optimization to ensure transfer of sufficient quantities of protective antibodies. t cells, particularly cd t cells, are essential for clearance of acute viral infections. naïve t cells react to stimulation with pathogen-specific peptides by massively expanding, differentiating into effector cells and migrating to peripheral infection sites for elimination of infected cells (see (zhang and bevan ) for a review). because naïve cd t cells can be activated only by dcs (banchereau and steinman ) , the initial assumption was that ebov-induced dc inactivation would in turn result in poor t cell priming and overall inability of the host to eliminate infection. this hypothesis was substantiated by early studies demonstrating that, despite being spared from infection, many t cells underwent apoptosis during human evd (baize et al. ; wauquier et al. ) . while this observation is still valid, data gathered mainly during the recent west african outbreak have suggested that lymphocytes in general display very dynamic kinetics during evd, which may include early proliferation followed by lymphopenia (kreuels et al. ; wolf et al. ) . similar dynamics occur during other systemic viral infections and during pro-inflammatory disorders such as sepsis (luan et al. ) , suggesting that perhaps lymphopenia is not a differential characteristic of evd. a substantial difference between earlier studies and those carried out in the context of the recent outbreak has been the application of multiparametric flow cytometry, which has allowed for the first time collection of phenotypic and functional information from single cells in unprecedented detail. these studies have revealed that, in fact, evd is characterized by massive t cell activation rather than inhibition in both surviving and fatal cases. co-expression of activation markers such as cd and hla-dr, as well as proliferation markers like ki- , were detected in a significant percentage of cd and cd t cells in evd patients (mcelroy et al. a; ruibal et al. ) and were comparable with the magnitude of activation observed in other acute infections or after vaccination (lindgren et al. ; miller et al. ) . of note, since co-expression of cd and hla-dr is correlated with engagement of the t cell receptor (appay et al. ) , these findings strongly suggest that proper t cell priming by antigen-presenting cells occurs during evd in humans. additionally, these findings were comparable between patients receiving experimental therapy (mcelroy et al. a ) and those who received supportive care in the field , indicating that robust t cell activation is a characteristic of evd unrelated to treatment. a paramount question, therefore, is why robust t cell activation does not lead to viral clearance during evd. to some extent, this lack of t cell effectiveness may be related to defects in negative immune checkpoints, namely the molecular mechanisms that control the transition from activation to immune homeostasis and that are essential for autoimmune control (buchbinder and desai ) . two such mechanisms are triggered by the t cell co-inhibitor molecules, programmed cell death- (pd- ) and cytotoxic t-lymphocyte-associated protein (ctla- ). an earlier review already hypothesized that t cell dysfunction during filovirus infection could be related to high expression of pd- and ctla- in t cells (mohamadzadeh et al. ) , which leads to a nonfunctional but reversible status termed t cell exhaustion (wherry ) . studies from the recent west african evd outbreak found that peripheral blood t cells from evd patients expressed high levels of pd- and ctla- (mcelroy et al. a; ruibal et al. ) , which were significantly higher in fatal cases . as a follow-up to these observational studies, determining the correlation between high expression of t cell inhibitory molecules and t cell function and apoptosis will be important. determining this correlation will most likely require relevant in vivo models that can reproduce this t cell phenotype. utilizing immunotherapeutic approaches to block pd- and ctla- function during postexposure filovirus infection treatment may provide an interesting opportunity. several therapeutic products are licensed extensively to block pd- and ctla- in several types of cancer, thereby restoring t cell function (see (sharma and allison ) for a review). another important and related question is whether broad and polyfunctional t cell responses lead to increased disease manifestations, like in hantavirus (cardio) pulmonary syndrome (terajima and ennis ), or to decreased susceptibility, as in dengue virus - infections (weiskopf et al. ). to answer this question, an exhaustive analysis of ebov t cell immunodominance in humans must be performed, which is still not available. previous evidence shows that the viral nucleoprotein (np) drives most of the cd t cell response (mcelroy et al. a; sundar et al. ; wilson and hart ) . this finding is consistent with the observation that hla alleles recognizing conserved filovirus np epitopes provide protection against sudv infection (sanchez et al. b) . additional studies of hla association with evd outcomes in a statistically significant cohort of patients are highly needed to strengthen these initial observations. the finding that np drives most of the cd t cell response also has significant implications for vaccine design and may explain, at least to some extent, why most gp , -based vaccines induce poor t cell immunity (agnandji et al. ; ewer et al. ; zhu et al. ) . the degree to which electrolyte abnormalities contribute to evd pathogenesis was not appreciated in earlier outbreak responses because real-time serum electrolyte data were not available. the ability to acquire these measurements in patients during the west african evd outbreak, as well as the degree of profuse watery diarrhea that was reported, have brought to the forefront the severity of electrolyte imbalances in evd and the impact electrolytes could have on patient outcome. such data were first collected during the sudv outbreak in gulu, uganda, in - , during which elevated bun/cre levels and hypocalcemia were associated with severe disease and fatal outcomes ). data from african cohorts (hunt et al. ) combined with data from repatriated patients who were cared for in developing nations (uyeki et al. b ) have revealed potassium abnormalities, hyponatremia, hypomagnesemia, and hypocalcemia. some of these alterations may be related to acute renal injury that is also common among severely ill patients; others might be related to volume and electrolyte imbalance secondary to profuse watery diarrhea. the clinical consequences of electrolyte imbalances could include cardiac arrhythmias, seizures, or coma. indeed, % of the repatriated patients exhibited arrhythmia or electrocardiographic changes, one patient had seizures, and three were in a coma (uyeki et al. b) . electrolyte levels are easily measured blood chemistry parameters that can be corrected with electrolyte and fluid administration. such measures may have contributed to improved outcomes during the recent outbreak, as one evd treatment center that incorporated these data into patient management had a case fatality rate of only %, significantly lower than the and % rates reported from two other treatment centers in sierra leone (hunt et al. ; schieffelin et al. ; lanini et al. ) . multiple lines of evidence have suggested that the endothelium is dysfunctional during evd. while endothelial cells are directly infected, they do not show significant cytopathic effect, and endothelial infection is thought to occur during the terminal phase of the illness (martines et al. ; geisbert et al. c) . the overall dysfunction of the endothelium is thought to be an indirect effect of pro-inflammatory cytokines like tnf-alpha (villinger et al. ; feldmann et al. ) , or other molecules, like nitric oxide, that increase the permeability of the endothelium during inflammation . increased levels of several pro-inflammatory cytokines and chemokines are associated with evd-related deaths (baize et al. ; wauquier et al. ; gupta et al. ; hutchinson and rollin ; mcelroy et al. a, b) . infected antigen-presenting cells, such as macrophages, dcs, or monocytes are the presumed source of these cytokines (feldmann et al. ; gupta et al. ) , and these cytokines lead to endothelial activation. increased vascular permeability due to loosening of the endothelial barrier is a normal and necessary physiologic function that allows cells and biomolecules to reach sites of inflammation, but widespread activation in many inflammatory diseases results in fluid movement that can be detrimental to the host. clinical and laboratory findings in evd, including tachypnea (with or without pulmonary edema), hypotension, oliguria, tachycardia, impaired distal perfusion, hypoalbuminemia, and hemoconcentration, are consistent with fluid extravasation into extravascular spaces secondary to increased vascular permeability uyeki et al. b; hunt et al. ; chertow et al. ). this constellation of clinical findings is thought ultimately to lead to hypovolemic shock in fatal cases. in recent years, additional evidence that dysfunctional endothelia contribute to the disease process include the findings of increased levels of sicam, thrombomodulin, pe-cam, and p-selectin in patients with severe or fatal disease (mcelroy et al. a, b) . all of these biomarkers, when released into the plasma, indicate an activated endothelium and/or breakdown of endothelial intercellular junctions. an activated endothelium is both pro-inflammatory and pro-coagulant, and likely contributes both to the ongoing inflammatory response that characterizes severe evd and to the coagulopathy that has been observed in some patients (discussed in more detail below). also noteworthy is the sometimes conflicting body of evidence implicating the viral glycoprotein (gp , ) in endothelial dysfunction. the ebov gp gene coding region produces two proteins based on a transcriptional editing site, the soluble gp (sgp) and the full-length structural gp , (sanchez et al. ) . the full-length gp , produced by pseudotyped retrovirus or virion-like particles (vlps) can bind to and activate endothelial cells, leading to increased endothelial permeability (wahl-jensen et al. ; yang et al. ). sgp has been detected in the plasma of infected individuals , and, in fact, inhibits tnf-mediated increases in vascular permeability in vitro, perhaps suggesting a compensatory mechanism to control virus-induced inflammation. a third form of the protein, known as shed gp, is shed from the surface of infected cells in vitro and increases permeability of cultured endothelial cells (escudero-perez et al. ) . while shed gp was detected in infected guinea pigs, it has not yet been detected in vivo in humans. finally, overexpression of gp , in explanted human, porcine, or nhp blood vessels leads to increased endothelial permeability and endothelial cytotoxicity mediated by the mucin domain of the protein (yang et al. ) . the relevance of this finding to evd is unclear, since endothelial cells are infected long after endothelial function has already been compromised, and do not show cytopathic effects when infected in vitro (geisbert et al. c) . taken together, these data suggest that the various forms of ebov gp may modulate endothelial function, but the precise role of the protein in human evd pathogenesis is unclear. the moniker "viral hemorrhagic fever" was applied to ebov evd during the first outbreak identified in , and was appropriate because % of fatal cases had hemorrhagic manifestations, mostly melena (ebola haemorrhagic fever in zaire zaire / . this outbreak was unique, because the route of virus transmission was via injection in approximately one-third of the patients, and this mode of entry could have contributed to the manifestations and severity of disease (the authors note that all patients who were infected by injection died). notably, a concurrent outbreak of sudv-caused evd also had high frequencies ( %) of hemorrhagic manifestations. however, in several of the larger subsequent outbreaks where appropriate data were available, significantly fewer patients had hemorrhagic manifestations of disease: % (ebov ), % (sudv ) , and % (bdbv ) (okware et al. ; macneil et al. ; bwaka et al. ) . additionally, in these three outbreaks, no association was observed between bleeding and death, arguing against the commonly held belief that hemorrhage equates to a fatal outcome. furthermore, in the western african outbreak, hemorrhagic manifestations were rarely reported; fewer than % of all patients from liberia and sierra leone had any bleeding symptom recorded (chertow et al. ; schieffelin et al. ; lado et al. ; yan et al. ; dallatomasina et al. ; li et al. ; qin et al. ) , but two reports from a single center in guinea reported bleeding in and % of patients (barry et al. ; bah et al. ) . perhaps reported differences in hemorrhage frequency are related to genetic or nutritional factors that cannot be controlled for in observational reports. regardless, hemorrhaging can occur during evd, but is not the most prominent feature. in contrast, hemorrhage does seem to be a common ( - % of patients) feature of mvd, based upon limited data from the two largest outbreaks to date (colebunders et al. ; roddy et al. ) . hemorrhaging is a clinical sign that can be secondary to multiple types of hematologic disorders. in the simplest terms, two general categories of hematologic disorders manifest clinically as bleeding: low levels of platelets and coagulation factor deficiencies (hunt ) . platelet counts have not been routinely measured in patients with evd, but in one study of patients with evd during the west african outbreak, platelet counts were not especially low, ranging -  / l (normal range is - ) (hunt et al. ) . interestingly, the same phenomenon was noted years ago in the nhp model, and while absolute platelet counts were not very low, platelet function was severely affected as a result of in vivo activation and degranulation (fisher-hoch et al. ) . no measurements of platelet function have been reported to date in humans. however, elevated levels of scd l were observed in surviving patients with evd caused by sudv (mcelroy et al. a, b) . since platelets are the major source of scd l in the bloodstream (henn et al. ) , this finding suggests platelet activation in humans during evd. these data suggest a process that consumes platelet functional activity in severe or fatal evd; this process would be consistent with the finding that in lethal nhp studies of evd, scd l levels are elevated initially, but decline to undetectable at the time of death (ebihara et al. ) . marv might be a bit different, since in the original report of the first outbreak in europe in , most patients had severe thrombocytopenia, sometimes less than  /l (martini ) , coincident with significant hemorrhage in about half of the patients. finally, the type of bleeding often described in evd (and mvd) patients-epistaxis, conjunctival hemorrhages, bleeding into the gi tract and from the oral cavity-is mostly mucosal in nature, consistent with loss of platelet numbers or function. the second general category of hematologic disorders that manifest as bleeding is deficiency in coagulation factors. coagulation factors are quantitated clinically by measuring partial thromboplastin time (ptt) and prothrombin time (pt) to evaluate the intrinsic and extrinsic coagulation pathways. unfortunately, these measurements have only been reported in case studies, and provide no consensus regarding the levels of pt and ptt during evd. this is a clear information gap that needs to be addressed. an early report on marv states that ptt and pt were measured in patients, but the values obtained did not explain the observed severity of the hemorrhaging (martini ) . dic is often seen in critically ill patients, especially those with sepsis, and involves both low platelet counts and coagulation factor deficiencies. the bleeding seen in patients with evd is often reported as due to dic, although whether the criteria for dic are met is unknown because the necessary laboratory tests are not routinely available. dic laboratory features include thrombocytopenia, elevated fibrin split products, prolonged pt, and consumption of fibrinogen (levi et al. ). as noted above, the level of thrombocytopenia seen in evd patients rarely meets the criteria to assign a dic score, but elevated fibrin split products (such as d-dimer) have been measured retrospectively, are elevated in evd patients, and are associated with fatal outcomes . pt measurements are normal in the few available case reports (sueblinvong et al. ) , and fibrinogen levels were not associated with outcome or hemorrhagic manifestations (mcelroy et al. a (mcelroy et al. , b, . measurement of dic markers is clearly an area that requires additional study for clarification. less conventional evaluations of factors involved in coagulation pathways have also been conducted. thrombomodulin, a protein expressed on endothelial cells, has anticoagulant properties in the microenvironment of the cell surface. when present in the plasma, thrombomodulin can act more globally, as shown in one family with a genetic deficiency that results in elevated levels of free plasma thrombomodulin in association with a bleeding disorder (langdown et al. ) . endothelial cells also release thrombomodulin when they become activated. elevated plasma levels of thrombomodulin were associated with both hemorrhage and death in sudv patients, and with more severe disease in a cohort of evd patients (mcelroy et al. a (mcelroy et al. , b, , suggesting that loss of this protein from the endothelial surface exacerbates both endothelial dysfunction and coagulopathy during evd. additionally, tissue factor, which is implicated in coagulopathy observed in nhps (geisbert et al. b) , was also elevated in patients with severe evd . von willebrand factor (vwf), a protein that is present in both platelets and endothelial cells and mediates interactions between platelets and the damaged endothelium, was elevated both in sudv-infected patients with hemorrhage and in pediatric sudv-infected patients with fatal outcomes. it was also elevated in ebov-infected patients with severe disease (mcelroy et al. b . a complex interplay of activated endothelial cells, activated platelets, inflammation, and coagulopathy is clearly at work during evd. how intervening in any one aspect of the network impacts human disease is still unknown. it would be invaluable to determine the effects on evd outcome of readily available clinical products that affect aspects of these processes. some compounds of interest are statins, which stabilize the endothelium; soluble gpiba, which inhibits the interaction between platelets and vwf; and scd l, which appears to be consumed during severe disease. one key and largely unaddressed question is the role of co-infections and co-morbidities in evd pathogenesis. especially relevant to patient populations in the affected african nations are the possible contributions of malnutrition and malarial co-infection in the disease process. malnutrition is prevalent in the regions affected by evd (wirth et al. ) , and long-standing malnutrition leads to defects in both innate and adaptive cellular immune responses (schaible and kaufmann ) . malnutrition may contribute to the high case fatality rates observed during evd outbreaks in africa as compared to filoviral infections in patients repatriated to the us and europe (uyeki et al. b; martini ) . malaria co-infection is likely to increase evd-related mortality, although this has not been rigorously evaluated, a study of the effects of various antimalarial drugs has been conducted during the west african outbreak. antimalarial drugs were routinely given to evd-positive patients at the etc in foya, liberia; during a time of artesunate-lumefantrine shortage, artesunate-amodiaquine was prescribed. while the amodiaquine preparation was associated with improved survival in malaria-negative patients, interestingly suggesting a direct antiviral effect, this effect was lost in the malaria-positive patients, suggesting that malaria and evd co-infection lead to worse outcomes even when malaria is treated (gignoux et al. ) . also of potential consequence are co-infections with hiv or other hepatotropic viruses. hiv co-infection has only been examined in one study; during the sudv outbreak in gulu, % of the tested patients were hiv- -positive by antibody testing. no differences in evd outcome were observed based upon hiv status in this study (mcelroy et al. a, b) , but no cd counts were obtained, so it is possible that all cases were newly acquired and the patients were not yet immune-compromised enough for hiv- infection to influence evd outcome. one study evaluated publicly available next-generation sequencing data, and using a cohort of patients, posited that co-infection with gb virus c (a common, clinically innocuous pegivirus infection) results in improved outcomes during evd infection (lauck et al. ) . the results were somewhat confounded by the fact that age is a major determinant of both gb virus c infection and outcome during evd. a special mention must be made that early data regarding the effect of age on ebov susceptibility (dowell ) and outcome (mupere et al. ; mcelroy et al. a, b) have been repeatedly observed in large cohorts during the west african outbreak (team et al. ; faye et al. ; li et al. ; schieffelin et al. ; bower et al. ). case fatality rates are high in children under , lowest in school-aged children, reaching a nadir around puberty, and increase again to peak in the elderly. this phenomenon has been seen in other infectious diseases in children, and suggests perhaps that school-aged children are in the perfect immunologic window of life, with a fully mature and functioning immune system without the alterations that occur secondary to the influence of sex hormones. to date, only one study has examined pediatric patients for laboratory evidence of this protective effect in evd; this study demonstrated that pediatric patients have viral loads similar to adult patients (mcelroy et al. a, b) and thus do not appear to control the viremia better. however, higher levels of rantes, a t cell chemokine, were associated with pediatric survival, an association not seen in adults. thus, stronger immune responses in pediatric patients might contribute to better outcomes, but this remains to be proven definitively and will require additional research efforts. perhaps one of the most striking findings during the western african evd outbreak has been the identification of severe sequelae in evd survivors long after recovery. these sequelae have important implications both for medical treatment and for public health. in , one year before the identification of ebov in zaire, marv was successfully isolated from the ocular fluid of a convalescent patient with uveitis (gear et al. ) . a later follow-up study described arthralgia, myalgia, and abdominal pain as common sequelae in evd survivors of the kikwit outbreak . similar findings in individuals long after recovery from bdbv infection (clark et al. ) suggest that post-recovery sequelae may be common in filovirus infections. to date, ebov rna has been detected in semen, ocular fluid, cerebrospinal fluid, breast milk, and other body fluids in evd survivors for several weeks or even months after discharge (green et al. ; chughtai et al. ) . moreover, infectious virus has been isolated from semen (uyeki et al. a) , ocular fluid (varkey et al. ) , saliva, and breast milk , and epidemiological evidence of sexual ebov transmission has been established (mate et al. ) . an important task will be determining the pathogenic potential of virus isolates from semen compared to those of blood from the same patient. because sexual transmission seems to be uncommon (based on the large numbers of male survivors and few sexual transmission events), virus isolates from semen may be less infectious, either due to attenuating mutations or inactivation by ebov-specific antibodies secreted at the mucosal surface. in general, the pathogenic features of evd sequelae and their putative physiological mechanisms are poorly understood. importantly, many of the symptoms reported by evd survivors, as well as some of the observed signs like uveitis and skin desquamation, suggest an inflammatory syndrome. indeed, immune activation persists after the acute phase of evd (mcelroy et al. a; rowe et al. ) , strongly suggesting continuous immune stimulation and postinfection autoimmunity. these hypotheses still need to be experimentally tested, but are consistent with virus persistence in immunoprivileged sites. alternatively, sustained inflammation could be due to deposition of immune complexes in the joints or to viral antigen persistence, as seen commonly in alphavirus infections and influenza a virus infection respectively (hoarau et al. ; tamburini et al. ; tappe et al. ) . since some, but not all, evd survivors suffer sequelae, the factors leading to post-evd syndrome must be determined. co-morbidities and co-infections are likely contributing to sequelae development, particularly those involving immune phenomena like bystander t cell activation (fujinami et al. ) . also, a positive correlation has been described between viremia levels during the acute phase of evd and increased risk of sequelae (mattia et al. ) . these findings suggest that during infection, ebov may be confined to immunoprivileged sites by treatment or by the host immune response, leading to viral persistence, 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immunogenicity of a novel recombinant adenovirus type- vector-based ebola vaccine in healthy adults in china: preliminary report of a randomised, double-blind, placebo-controlled, phase trial key: cord- -bswndfvk authors: lalle, eleonora; biava, mirella; nicastri, emanuele; colavita, francesca; di caro, antonino; vairo, francesco; lanini, simone; castilletti, concetta; langer, martin; zumla, alimuddin; kobinger, gary; capobianchi, maria r.; ippolito, giuseppe title: pulmonary involvement during the ebola virus disease date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: bswndfvk filoviruses have become a worldwide public health concern, especially during the – western africa ebola virus disease (evd) outbreak—the largest outbreak, both by number of cases and geographical extension, recorded so far in medical history. evd is associated with pathologies in several organs, including the liver, kidney, and lung. during the – western africa outbreak, ebola virus (ebov) was detected in the lung of infected patients suggesting a role in lung pathogenesis. however, little is known about lung pathogenesis and the controversial issue of aerosol transmission in evd. this review highlights the pulmonary involvement in evd, with a special focus on the new data emerging from the – ebola outbreak. ebolavirus is part of the filoviridae family, which consists of three genera: marbugvirus, cuevavirus, and ebolavirus. there are currently six known, genetically distinct, species of ebolavirus-ebola virus (ebov), sudan ebolaviurs (sudv), tai forest ebolavirus (tafv), bundibugyo ebolavirus (bdbv), reston ebolavirus (restv), and bombali ebolavirus (bomv) [ , ] . no virus has triggered fear in the general population more than the filovirus ebolavirus [ ] . ebov is categorized among the deadliest viruses, with mortality rates up to %. the zoonotic origin of outbreaks are often the result of transmission from primates, although the suspected natural reservoir for ebov, bats, is still being questioned. since it was first identified in in zaire (the actual democratic republic of congo), confirmed outbreaks, mainly in the central part of africa, have occurred, and each outbreak was accompanied by high case fatality rates up to %, including the new declared outbreak ongoing in the north kivu province of the democratic republic of the congo [ ] [ ] [ ] . the - ebola outbreak is the largest (both by number of cases and geographical extension) ebolavirus outbreak ever reported, resulting . tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory upon entrance into the cell, viruses are recognized by the toll-like receptor (tlr) on either cell membrane or in endosomes. tlrs activate interferon regulatory factors (irfs) leading to ifn-α and ifn-β release via the toll/il- receptor domain-containing adaptor (trif). tlr stimulates irf- and nf-κb via myd activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [ ] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [ ] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent - ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [ ] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il- β, il- , il- , mip- a, mip- β, mcp- , and tnf-α [ , ] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il- β, il- , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl , il , mif, spp [ ] [ ] [ ] . in addition, severe inflammatory cytokines/chemokines may spill over into the circulation and result in systemic cytokine storms, which are responsible for multi-organ dysfunction and for the impairment of the vascular system and disseminated intravascular coagulation [ , ] . dendritic cells (dcs) play an essential role in the link between the innate and adaptive immune response, and their maturation is essential for the correct functionality of dcs, such as the migration, processing, and presentation of viral antigens to t-and b-cells for their activation and correct viral clearance [ , ] . ebov infection has been shown to influence these mechanisms through impairment of dcs in upregulating co-stimulatory molecules (cd , cd , and cd ) and major histocompatibility complex (mhc) class ii, as well as soluble chemokines and cytokines [ ] . ebov infection is also able to influence the adaptive immune response: severe lymphopenia and the destruction of lymphoid tissue is one of the hallmarks of ebov infection. fatal cases showed a more marked reduction of nk cells and γδ t-cell frequency, as well as a loss of peripheral blood cd + and cd + t cells [ , ] . moreover, a recent study showed that patients with fatal outcome presented lower, or often absent, levels of both ebov-specific igm and igg, which, when detected, appeared later than in survivors [ ] . overall, the alteration of the innate and adaptive response explains the paralysis of the immune system and its inability to initiate and maintain a protective immune response. at the pulmonary level, many of the pathological changes are, in fact, secondary to systemic alterations, correlating with general pathogenic mechanisms, which are the major causes of severe disease in humans, even at the respiratory level [ , ] . evd is a viral hemorrhagic fever (vhf) characterized by acute systemic manifestations with vascular damage, plasma leakage, severe inflammation, and disruption of the immune system [ ] . evd transmissibility seems to vary depending on the stage of disease [ ] . a high-level of ebov replication, associated with systemic dissemination to multiple cell types, results in a complex pathogenesis, which is linked to an increased risk of infection transmission [ ] . as stated above, these pathogenic mechanisms include detrimental immune suppression and over-activation of the immune response, disordered coagulation, and tissue damage due to direct viral and indirect host-mediated effectors. in the absence of adequate supportive care, these processes commonly result in multiple organ failure and death within about days of symptom onset in humans. it is well recognized that ebov infection is acquired by direct contact with bodily fluids. notably, studies conducted in animal models have instilled doubts about possible airborne/droplet transmission (see section . ). however, this route of infection in humans is still debated. piercy and colleagues evaluated the actual stability of the virus particles in aerosol droplets [ ] . they created ebola-containing aerosol droplets and, according to the decay rates, estimated that ebov and restv can survive in aerosols for roughly and min, respectively, at % to % relative humidity and ± • c [ ] . therefore, a key additional question to ask is whether primary pulmonary infection of ebov could be a potential scenario for the future. a fair amount of studies, based on animal experiments (table ) and clinical evidence collected during the outbreaks ( table ), suggest that pulmonary infection may be a possibility. this possibility will be fully investigated below. after its first discovery in in cynomolgus macaques imported to reston, virginia, restv was detected in domestic swine in the philippines in a co-infection with the porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae, genus arterivirus) and porcine circovirus type (pcv- ; family circoviridae) [ , ] . later on, restv was identified to cause asymptomatic infections with mild respiratory symptoms, which may result in severe mortality in cases of co-infections with other viral pathogens like viruses in the families arteriviridae and circoviridae. the virus was first isolated in lung and lymphoid tissues in the original disease investigation [ ] . however, the massive presence of the virus in the lungs may be due to the fact that restv infection in pigs has been mostly associated with other infections of the respiratory tract, which may contribute to the specific localization of the virus and the respiratory symptoms of the disease. marsh et al. [ ] conducted an experimental study to rule out the effect of other pathogens affecting pigs, using a philippines swine isolate of restv. specifically, five-week-old pigs were exposed (via the oro-nasal or subcutaneous route) to the virus, and the subsequent viral replication in internal organs and shedding of the virus from the nasopharynx was observed. the researchers detected the highest levels of virus replication in lung and lymphoid tissues, confirming previous results [ ] . the detection of restv in domestic swine raised important biosecurity concerns about the potential for the disease's emergence in humans and other livestock, mainly in animals for food consumption [ , ] . the evidence of restv seropositive individuals further increased the concern for human infections and the worries of researchers, farm owners, and the public at large (world health organization. who, , available online: https://www.who.int/csr/resources/publications/hse_ epr_ _ .pdf). interestingly, so far restv has not been seen to result in any human disease, even if there is concern that its passage through swine may allow restv to diverge and shift its potential for pathogenicity [ ] . on the other hand, several studies investigated if other ebola viruses may be transmitted through the aerosol route and may result in primary pulmonary infection [ , , ] . researchers reviewed the different animal models and offered an overview regarding the possibilities of ebola viruses causing aerosol infections in non-human primates (nhps) and other animals. experimental studies analyzed the respiratory tract involvement in filovirus infections when the animals were exposed to the virus through different aerosol routes (artificially aerosolized virus or natural aerosol transmission) [ , , , ] . in these experimental studies conducted on nhps and pigs, ebov was inoculated via the aerosol route, and, following mucosal exposure, ebov replicated, reaching high concentrations, mainly in the respiratory tract, with the development of severe lung pathology. interestingly, weingartl et al. demonstrated that piglets inoculated oro-nasally with ebov and then transferred to a different room housing macaques in an open inaccessible cage system resulted in ebov infection of all macaques, suggesting a need to revise prevention and control measures during outbreaks [ ] . viral replication was observed within alveolar spaces [ , ] , in type i pneumocytes and macrophages [ ] , and in type ii pneumocytes, bronchiolar epithelial cells, and endothelial cells [ ] , supporting the respiratory involvement. the upper and lower respiratory tract, the lymphoid tissues, and the mediastinal lymph nodes showed infection signs, as well [ ] . similarly, in experiments on cynomolgus macaques placed separately in cages with experimentally infected piglets [ ] , and on guinea pigs exposed via aerosols to a guinea pig-adapted ebov strain [ ] , viral antigens were detected within alveolar and septal macrophages, pneumocytes, epithelial cells, endothelial cells, fibroblasts, and other interstitial cells of the respiratory tree [ ] . considering the pathology of the respiratory system, the expression of disease in the lungs and the patterns of lesions seem to be influenced by the exposure routes (aerogenous or hematogenous). broncho-interstitial pneumonia, characterized by injury to both the bronchiolar and the alveolar epithelium, is commonly caused by aerogenous viral infections [ ] . moreover, such pathological features were generally not evidenced following the inoculation of ebov by other routes in nhps and laboratory animals [ , ] . as shown in animal studies, primary pulmonary infections could occur and cause active viral shedding from the respiratory tract, thus potentially setting up a cycle of ongoing respiratory transmission in humans [ , ] . overall, experimental works conducted so far have shown that ebov infection induces respiratory complications, that the virus can be shed via the respiratory secretions, and that it can cause similar pulmonary lesions both in animals exposed to aerosols and in those kept nearby in separate cages with no close contact. the pathophysiological mechanism of pulmonary disease in patients with evd is unknown. notably, autopsies were performed on a limited number of humans (about cases), primarily during the sudv and ebov evd outbreaks and revealed interesting characteristics at microscopic level. during the first known sudv outbreak, chest pain was almost universal ( % of patients), often accompanied by a dry cough. autopsies were further performed on two patients and thickening of the alveolar walls due to proliferative accumulations of alveolar cells was found [ ] . furthermore, a possible pathogenetic role of the virus in the respiratory tract was suggested by the fact that viral inclusions within alveolar macrophages and free viral particles within alveolar space were found in the lungs from fatal evd cases who showed congestion, focal intra-alveolar edema, diffuse alveolar damage, and hemorrhaging. [ , ] . one of the most common symptoms in evd patients is a cough (up to %), especially during the progression of the disease, when viral loads in serum significantly increase, and the virus is copiously emitted in most body fluids, as well as in aerosol particles of various sizes [ , ] . among the reported evd cases in the literature, respiratory symptoms were commonly reported with a wide range of symptoms, such as a cough (from % [ , ] to % [ ] ), dyspnoea or breathless (detected from % [ ] to % [ ] ), and chest pain (from . % [ ] to . % [ ] ). moreover, a who study on the first months of the epidemic in western africa found that nearly % ( out of ) of the patients experienced coughing and . % ( of ) had a bloody cough [ , ] . a study of ebov-positive patients of the - outbreak in western africa, treated in europe and usa, reported that cough and dyspnoea were present at admission in seven ( %) and five ( %) evd patients, respectively. at symptom onset, only a cough was reported in one patient. furthermore, during hospitalization, patients ( %) experienced hypoxemia while they were breathing ambient air, patients ( %) had pulmonary oedema, seven patients ( %) had pneumonia, patients ( %) had respiratory failure, and six patients ( %) had a diagnosis of acute respiratory distress syndrome (ards). of these patients, four patients ( %) received non-invasive mechanical ventilation, and seven patients ( %) received invasive mechanical ventilation [ ] . notably, the first ebov-positive patient treated in italy, mechanically ventilated for respiratory insufficiency for days, had high levels of ebov rna in the lower respiratory tract secretions. the authors concluded that the absence of other identified respiratory pathogens in broncho-alveolar lavage fluids and aspirates supported the hypothesis of a direct contribution to the lung tissues damage by ebov. notably, ebov rna was detected in bronchial aspirate fluids when the ebov rna concentration in the concomitant blood samples was barely detectable. furthermore, the blood ebov rna concentrations in the previous days were significantly lower than the concentrations detected in the bronchial aspirate samples. these findings suggest that this ebov infection is unlikely a spillover from the blood compartment, eventually accompanied by delayed clearance. instead, the most plausible explanation is that the virus actually replicated into the lower respiratory tract [ ] . in the second ebov-patient treated in italy, our group investigated the presence of ebov genetic material in the lungs and blood during the patient's treatment and recovery. the patient showed a persistence of ebov replication markers within the respiratory tract, with a prolonged detection of ebov viral rnas (negative and positive sense rnas: neg-rna and pos-rna, respectively), known to be associated with ebov replication, in the lower respiratory tract for up to five days after the ebov viral load in blood was already undetectable. these results suggest that ebov may replicate in the lungs, although it is possible that the lungs simply provided a protective environment that allowed rna to linger longer than it did in the plasma. nevertheless, the detection of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). viruses , , x for peer review of of pos-rna together with neg-rna in the sputum (until day and of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [ ] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure ). our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [ ] . interestingly, ( . %) of the cases in the sudv outbreak in nzara, sudan, and ( . %) of the cases during the ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [ , ] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the - western africa epidemic, more than health care workers (hcw) were infected, with a case fatality rate of % [ ] , whereas during the current - outbreak in drc, as of july , hcw have been already affected ( . % of total cases) [ ] . currently, full body protection is recommend by who and cdc [ , ] . all hcw involved in the care of evd patients must receive training and demonstrate competency in performing all ebola related infection control practices and procedures, specifically in proper donning and doffing ppe even if using an n mask or a powered air-purifying respirator (papr). the risk of infection via inhalation of contaminated aerosols from exposed individuals has not been documented. however, droplets containing ebov that have become aerosolized (e.g., from coughing sneezing, vomiting, invasive medical or surgical procedures, or surfaces) may have the potential to come into contact with a person's mucous membrane in their nose or mouth or non-intact skin. therefore, respiratory protection may be helpful in providing a barrier to help prevent infectious materials from contacting a wearer's mucous membranes. finally, the epidemiologic and viral evidence of ebov detection and replication in the respiratory tract raise concerns on the need of strict application of cough etiquette for patients and of droplet and/or respiratory precautions for all hcw involved in the clinical management of evd suspected and confirmed cases. acute respiratory tract infections (artis) remain a leading cause of mortality, morbidity, and economic loss, and viruses are one of the main causes of such disease. who estimates that artis cause nearly four million deaths per year, a rate of more than deaths/ , people [ ] . the microbial etiology of aris is varied, with viruses being the most common cause in humans [ ] , leading to a high level of awareness and the necessity to develop countermeasures to control them (table ) . filoviruses are not commonly considered to be viruses responsible for aris, even if respiratory symptoms may be present as a consequence of diffuse systemic alterations. interestingly, evidence collected in animal studies, in the epidemiological analysis of transmission chains, and in the most recent ebola outbreaks suggests that ebov may be able to cause primary pulmonary infection. this evidence highlights the ability of the virus to be shed in the lung, suggesting a role in lung pathogenesis. specifically, the relevant proportion of evd patients without any epidemiologic link to the exposure to contaminated biological samples or fomites, or to any contact with evd patients; the evidence of respiratory signs and symptoms commonly reported all over the clinical course; the abundance of viral antigens in the lungs in animal necropsies; the prolonged persistence of ebov detection and replication within the respiratory tract days after undetectable ebov viral load in plasma; and similar clinical patterns in several other viral respiratory tract infections are all different parameters with consistent evidence of a major role in the pathogenesis of evd in respiratory tissues [ ] . on the other hand, there is no evidence of aerosol transmission in evd. however, different studies addressing this issue have been performed [ , ] , and aerosol transmission was considered a possibility as a consequence of epidemiological observations in past outbreaks, where people showed signs of evd even in the absence of a direct or physical contact with an infected person or known infected dead body [ , ] . this hypothesis was corroborated by other studies, in which the presence of free viral particles in alveoli and within intra-alveolar macrophages demonstrated a pulmonary involvement [ ] . from a clinical point of view, the - ebov outbreak underlined the lung involvement in evd pathogenesis. in fact, only a few patients treated in europe and usa had a cough and difficulty breathing at admission. nevertheless, during the clinical progression, half of the patients experienced hypoxemia while breathing room air, one third had respiratory failure, and one fourth received invasive or non-invasive mechanical ventilation [ ] . in the italian experience at the national institute for infectious diseases "l. spallanzani" (inmi), respiratory symptoms were present in both patients, in the absence of other common respiratory pathogens [ , ] . one case required mechanical ventilation and the other presented ebov replication markers in the lungs even after clearance of the virus from the blood. the inmi experience suggests a direct role of the virus in lung pathogenesis. although lung pathogenesis in evd may be secondary to systemic alterations (correlating with general pathogenic mechanisms) the direct presence of the virus is undisputable in the lung, and its interaction with the immune system, whose hyper-activation may be the most likely explanation of the lung damage, is also indisputable. further research will be 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of the viral hemorrhagic fevers dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of il- and rantes pathogenesis of lassa fever. viruses pathophysiology of hantavirus pulmonary syndrome in rhesus macaques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -o y rn z authors: ng, melinda; ndungo, esther; kaczmarek, maria e; herbert, andrew s; binger, tabea; kuehne, ana i; jangra, rohit k; hawkins, john a; gifford, robert j; biswas, rohan; demogines, ann; james, rebekah m; yu, meng; brummelkamp, thijn r; drosten, christian; wang, lin-fa; kuhn, jens h; müller, marcel a; dye, john m; sawyer, sara l; chandran, kartik title: filovirus receptor npc contributes to species-specific patterns of ebolavirus susceptibility in bats date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: o y rn z biological factors that influence the host range and spillover of ebola virus (ebov) and other filoviruses remain enigmatic. while filoviruses infect diverse mammalian cell lines, we report that cells from african straw-colored fruit bats (eidolon helvum) are refractory to ebov infection. this could be explained by a single amino acid change in the filovirus receptor, npc , which greatly reduces the affinity of ebov-npc interaction. we found signatures of positive selection in bat npc concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls ebov infection in eidolon helvum cells. our work identifies npc as a genetic determinant of filovirus susceptibility in bats, and suggests that some npc variations reflect host adaptations to reduce filovirus replication and virulence. a single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature. doi: http://dx.doi.org/ . /elife. . ebola virus (ebov) and some of its relatives in the family filoviridae (filoviruses) cause sporadic outbreaks of a highly lethal disease. these outbreaks are thought to be initiated by viral spillover from an animal reservoir to a highly susceptible accidental host, such as a human or nonhuman primate (feldmann and geisbert, ; leroy et al., ; towner et al., ) . recent work suggests that some filoviruses infect bats in nature, and that these viruses may be distributed more widely than previously recognized. very short rna fragments corresponding to portions of ebolavirus genomes were detected in several frugivorous bats of the family pteropodidae ('old world fruit bats') in both africa and asia (leroy et al., ; jayme et al., ) , and longer filovirus rna fragments and near-complete rna genomes were isolated from insectivorous schreibers's long-fingered bats in asia and europe, respectively (negredo et al., ; he et al., ) . however, despite considerable efforts, infectious ebolaviruses have never been recovered from bats. by contrast, marburg (marv) and ravn (ravv) viruses were found to circulate in egyptian rousettes (rousettus aegyptiacus), indicating that these bats are susceptible to marv/ravv and encounter them frequently in nature. egyptian rousettes have been proposed as natural hosts for these viruses (amman et al., ; towner et al., ) . this progress notwithstanding, many key questions remain. for example, the biological factors that influence filovirus host range and interspecies transmission are still poorly understood, as are the virus-host relationships that determine which species of bats are susceptible to infection by ebov and other filoviruses. viral entry receptors are key determinants of tissue tropism and host range (radoshitzky et al., ; sheahan et al., ; hueffer et al., ; demogines et al., ) . niemann-pick c (npc ), a highly conserved endo/lysosomal protein involved in cellular cholesterol trafficking, was recently identified to be an essential entry receptor for all known filoviruses (cô té et al., ; carette et al., ; ng et al., ) . in this study, we uncover a pattern of virus and host species specificity in the filovirus susceptibility of bat cells, which can be explained by elife digest ebola virus and other filoviruses can cause devastating diseases in humans and other apes. numerous small outbreaks of ebola virus disease have occurred in africa over the past years. however, in - , the largest outbreak on record took place in three western african nations with no previous history of the disease. human outbreaks of ebola virus disease likely begin when a person encounters an infected wild animal. though it remains unclear precisely which animals harbor ebola virus between outbreaks, and how they transmit the virus to humans or other primates, recent work showed that some filoviruses do infect specific types of bats in nature. ng, ndungo, kaczmarek et al. sought to identify the genes that influence whether or not a type of bat is susceptible to infection by ebola virus and other filoviruses. several filoviruses, including ebola virus, were tested to see if they could infect cells that had been collected from four types of african fruit bats. these bats are all found in areas where outbreaks have occurred in the past. the tests revealed that a small change in the sequence of the npc gene in some bat cells greatly reduced their susceptibility to ebola virus. npc encodes a protein that mammals need in order to move cholesterol within their cells. in humans, the loss of the protein encoded by npc causes a rare but very severe disease called niemann-pick type c disease. this protein also turns out to be a receptor that the filoviruses must bind to before they can infect the cells. further analysis then revealed that npc has evolved rapidly in bats, with changes concentrated in the parts of the receptor that interact with ebola virus. ng, ndungo, kaczmarek et al. went on to discover some changes in the genome sequence of ebola virus that could compensate for the changes in the bat's npc gene. these findings hint at one way that a filovirus could evolve to better infect a host with receptors that were less than optimal. following on from this work, the next challenges will be to expand the investigation to include additional types of bats, other types of mammals, and other host genes that could influence filovirus infection and disease. further studies could also examine the other side of the arms race -that is, the evolution of viral genes in bats. however, such studies would be complicated by the lack of viral sequences that have been collected from bats, because to date most have been isolated from humans and other primates instead. changes in the affinity of the essential interaction between npc and the filovirus entry glycoprotein, gp. crucially, genetic analyses reveal that npc is under positive selection in bats, with a strong signature of selection at precisely the same residue that influences the filovirus-receptor interaction. our findings suggest that amino acid sequence changes in npc at these positively-selected sites represent host adaptations to resist filovirus infection, and reveal one pathway by which a filovirus could escape from receptor control. in sum, our results support the hypothesis that bats and filoviruses have been engaged in a long-term co-evolutionary relationship, one facet of which is a molecular arms race between the viral glycoprotein and its entry receptor, npc . means ± sd (n = - ) from two biological replicates are shown. in panels c and d, the infectivity of each virus was normalized to that obtained in vero grivet monkey cells. means for infection of the different cell lines by each virus were compared by one-way anova (p-value indicated above each group of bars). tukey's post hoc test was used to compare infection means on hypsignathus monstrosus vs eidolon helvum cells (*p < . ; ****p < . ; ns, no statistical significance). doi: . /elife. . the following figure supplements are available for figure : we first explored the possibility that there exist virus-and/or bat species-dependent differences in the cellular host range of filoviruses. kidney fibroblast cell lines derived from three african pteropodids whose ranges overlap the locations of known african filovirus disease outbreaks ( figure a ,b) were exposed to authentic ebov and marv ( figure c ). we observed a large ebov infection defect in african straw-colored fruit bat (eidolon helvum) cells but not in cells from bü ttikofer's epauletted fruit bats (epomops buettikoferi) and egyptian rousettes. by contrast, cells from bats of all three species were similarly susceptible to infection by marv ( figure c) . thus, cells from african straw-colored fruit bats appear to be selectively refractory to ebov infection. an npc -dependent block to cell entry accounts for the ebov infection deficit in african straw-colored fruit bat cells the viral spike glycoprotein, gp , (herein termed gp) mediates all steps of filovirus entry into the cytoplasm of host cells . vesicular stomatitis viruses bearing filovirus gp proteins (vsv pseudotypes) provide a highly validated surrogate system to recapitulate filovirus entry under biosafety level containment (takada et al., ; jangra et al., ) . to assess whether the ebov infection defect in the african straw-colored fruit bat cells occurs at the viral entry step, we exposed an expanded panel of kidney fibroblast cell lines from four african pteropodids to vsv pseudotypes bearing gp spikes (vsv-gp) from seven filoviruses, including two non-african viruses, reston virus (restv) and lloviu virus (llov) ( figure d ). as observed with authentic ebov, vsv-ebov gp infection was substantially reduced in the african straw-colored fruit bat cells; however, this virus could efficiently infect cells derived from the other pteropodids, including those of a proposed ebov host, the hammer-headed fruit bat (hypsignathus monstrosus) (leroy et al., ) . strikingly, only vsvs bearing ebov gp, and to a lesser degree, those bearing bdbv and tafv gp, were deficient at infecting african straw-colored fruit bat fibroblasts. similar strong but ebov-specific reductions in infection were measured in two kidney and lung cell lines derived from additional african straw-colored fruit bats (figure -figure supplement ) . therefore, reduced infection of these bat cells by ebov reflects a virus-and host species-specific restriction at the cell entry step. we surmised that the filovirus receptor, npc , might explain the selective resistance of the african straw-colored fruit bat cells to ebov entry and infection. accordingly, we engineered these cells to stably express human npc (hsnpc ) (figure -figure supplements , ), and then exposed them to ebov ( figure a ). provision of hsnpc substantially enhanced authentic ebov infection in the african straw-colored fruit bat cells. by contrast, we found no evidence that either marv infection in these cells, or ebov/marv infection in permissive bü ttikofer's epauletted fruit bat cells was limited by receptor availability (figure a ). finally, similar results were obtained with vsvs bearing filovirus glycoproteins ( figure b ). taken together, these findings indicate that ebov infection is reduced in african straw-colored fruit bat cells because of a specific molecular incompatibility between the ebov glycoprotein and the filovirus entry receptor. npc -dependent cell entry is reduced, but not completely eliminated, in african straw-colored fruit bat cells although ebov entry and infection in african straw-colored fruit bat cells was consistently reduced to . - % relative to that in cells from the other pteropodids, we noted that infection was not completely blocked. to determine if ebov could inefficiently infect these bat cells via an npc -independent mechanism, we used crispr/cas genome engineering to derive an african straw-colored fruit bat cell line fully deficient in npc . we identified a single cell clone (eidolon helvum npc -# [ehnpc -# ]) in which all npc alleles bore insertions or deletions (indels) at the expected site ( figure a ). these indels were predicted to frameshift the npc open reading frame at amino acid position (homo sapiens hsnpc numbering), generating truncated polypeptides of , , and residues that lacked the majority of the -amino acid npc sequence. ehnpc -# cells were deficient in clearance of lysosomal cholesterol, a well-established cellular function of npc (carstea et al., ) , but could be rescued by ectopic hsnpc expression, confirming that npc had indeed been disrupted in these cells ( figure b ). we next exposed wild-type (wt) and ehnpc -# fibroblasts to vsvs bearing ebov or marv gp. no detectable infection was obtained with either virus in npc -deficient cells, indicating that filovirus entry into these cells is absolutely dependent on the e. helvum npc ortholog ( figure c ). moreover, ebov gp-dependent infection in ehnpc -# cells reconstituted with hsnpc was dramatically enhanced over that observed in wt cells, whereas marv gp-dependent infection was rescued by hsnpc expression to a level resembling that in wt cells ( figure c ). therefore, the low levels of ebov infection in african straw-colored fruit bat cells likely arise from the weak, but nonzero, activity of ehnpc as an ebov entry receptor. filovirus gps must directly engage the second luminal domain of npc , domain c, during cell entry . accordingly, we postulated that the african straw-colored fruit bat npc ortholog is poorly recognized by ebov gp. to test that hypothesis, we generated and sequenced npc cdnas from all four pteropodid cell lines. alignment of their domain c amino acid sequences with that of hsnpc revealed a high degree of conservation (> %), with identical arrangements of cysteine residues and similar predicted secondary structures suggestive of a similar overall fold (figure -figure supplement ) . to examine gp-npc binding, we engineered and expressed soluble forms of the four pteropodid npc domain cs, as described for hsnpc (figure -figure supplement ) . a cleaved form of ebov gp could capture hsnpc domain c in an elisa, as shown previously . ebov gp bound with similar avidity to npc domain cs derived from egyptian rousettes (ranpc ), hammer-headed fruit bats (hmnpc ) and bü ttikofer's epauletted fruit bats (ebnpc ), but poorly or not at all to that of african straw-colored fruit bats (ehnpc ) ( figure a ). like the infection defect in african straw-colored fruit bat cells, this receptor binding defect was selective for ebov gp, since gps derived from marv and the european filovirus, llov (ng et al., ) , bound equivalently to all four pteropodid domain cs ( figure a ). these findings the restriction in ehnpc -ebov gp binding can be mapped to a single amino acid change in ehnpc to define the molecular basis of the defect in interaction between ebov and ehnpc , we generated a panel of npc domain c chimeras comprising sequences from permissive ranpc and nonpermissive ehnpc , and tested them in the gp-binding elisa. a single chimera, ehnpc domain c containing four ehnpc firanpc amino acid residue changes, regained the capacity to efficiently recognize ebov gp ( figure b ). further dissection revealed that only a single amino acid change, f d, in a central region of npc domain c was needed to effect this complete restoration in gp- figure . the incompatibility between ebov gp and eidolon helvum npc reduces, but does not eliminate, ebov entry into african straw-colored fruit bat cells. (a) crispr/cas genome engineering was used to knock out the npc gene in african straw-colored fruit bat kidney fibroblasts. wt npc gene sequence aligned with the sequences of all three alleles in the knockout (npc -# ) cell clone. the grna target sequence is marked in red, and the protospacer adjacent motif (pam) sequence of the grna target site is underlined. (b) the capacity of wt and npc -# cells, and npc -# cells stably expressing hsnpc , to clear lysosomal cholesterol was determined by staining with filipin iii complex from streptomyces filipensis, as described (carette et al., ) . . we conclude that a species-specific defect in virus-receptor interaction, caused by a single amino acid residue change in ehnpc relative to other, permissive african pteropodid npc orthologs, reduces ebov infection in african straw-colored fruit bat cells. moreover, because residues in the npc -binding site are conserved among all available ebov gp sequences (supplementary file ), this restriction is almost certain to be encountered by all known ebov variants and their isolates, including those detected in ebov disease patients during the recent epidemics in western and middle africa gire et al., ; tong et al., ; carroll et al., ; kugelman et al., ) . previous work has led to the hypothesis that bats in equatorial africa and elsewhere harbor filoviruses (reviewed in [wahl-jensen et al., ] ). these results, together with our findings for virusand host species-specific differences in cellular susceptibility to filovirus infection, hinted at the possibility of a deeper co-evolutionary relationship between filoviruses and bats. one hallmark of such a relationship between a virus and its host is the evolution, under selective pressure to resist infection, of host genes encoding proviral and antiviral factors. to evaluate whether the npc gene has evolved under positive selection in bats, we combined the npc sequences obtained in this study with those of bats from six other species (two non-african pteropodids, two phyllostomids, and two vespertilionids) compiled through assembly of publicly available rnaseq data ( a mutation in ehnpc reduces receptor binding to ebov gp and viral infection, a phenotype that could reasonably produce a selective advantage (figure ) . other codons identified in only some of the tests for dn/ds> , or at slightly lower significance levels, may still have functional significance. for example, additional codons were identified in two regions of domain c that may form a part of the recognition surface for ebov gp ( figure c ). our finding that signatures of accelerated sequence evolution localize to structural features in npc that are important for virus binding (domain c and position ) leads us to postulate that mutations at these sites can protect bats from infection or severe disease caused by filoviruses and/or other intracellular microbes. a single mutation at residue in ebov gp enhances viral entry by strengthening its interaction with ehnpc co-evolutionary arms races between hosts and pathogens are thought to be driven by cycles of genetic adaptation and counter-adaptation (meyerson and sawyer, ; daugherty and malik, ; demogines et al., ) . in this context, we postulated that mutation of residue in ehnpc could be countered by viral mutation. to identify such putative compensatory viral changes, figure . a sequence polymorphism in the npc -binding site of filovirus gp influences gp-ehnpc binding and ehnpc -dependent filovirus entry. (a) binding of ebov gp (wt and mutant v a) to soluble npc domain c proteins derived from african pteropodids measured by an elisa. ranpc , egyptian rousette; ebnpc , bü ttikofer's epauletted fruit bat; hmnpc , hammer-headed fruit bat; ehnpc , african straw-colored fruit bat. (b) infection of african straw-colored fruit bat cells with vsv pseudotypes bearing ebov gp (wt or v a). means ± sd (n = - ) from a representative experiment are shown in each panel. means for vsv-ebov gp wt vs v a infection were compared by unpaired two-tailed student's t-test with welch's correction (**p < . ). (c) surface-shaded representation of a single gp -gp monomer (pdb id: csy highlighting key residues in the npc -binding site (yellow) and residue (red). gp , blue. gp , grey. (d) alignments of gp sequences from a panel of filoviruses. v , orange; a , white text on blue shading; other residues divergent from consensus sequence, black text on green shading. (e) infection of african pteropodid cells with vsv pseudotypes bearing sudv gp (wt or a v). means ± sd (n = ) from two biological replicates are shown. means for vsv-sudv gp wt vs a v infection on each cell line were compared by unpaired two-tailed student's t-test with welch's correction (*p < . , **p < . , ****p < . ). doi: . /elife. . we screened a panel of point mutants in the npc -binding site of ebov gp by elisa for enhanced binders to ehnpc domain c. while no single point mutant bound to ehnpc as well as it did to the other pteropodid npc s or to hsnpc , gp(v a) partially restored ehnpc binding ( figure a ). infection by vsv particles bearing ebov gp(v a) was substantially enhanced in african straw-colored fruit bat cells, commensurate with this mutant gp's increased binding affinity for ehnpc ( figure b) . examination of the x-ray crystal structure of ebov gp revealed that v is located at the edge of the putative npc -binding site, where it forms part of a raised rim ( figure c ). the v a mutation likely creates a more sterically favorable (open) npc binding site that can overcome the structural mismatch at the gp-npc binding interface ( figure c ). naturally-occurring sequence variation at residue in gp contributes to virus-and bat species-specific patterns of cellular susceptibility to filoviruses although no known ebov isolate contains the v a mutation, we observed that llov and sudan virus (sudv) gp naturally possess a ( figure d ). because both gp proteins could mediate efficient viral entry into african straw-colored fruit bat cells ( figure d ) and bind to ehnpc ( figure a data not shown for sudv), we postulated that amino acid changes at position in the gp receptor-binding site broadly influence the capacity of filovirus glycoproteins to utilize ehnpc for viral entry. accordingly, we exposed pteropodid kidney fibroblasts to vsv pseudotypes bearing sudv gp(wt) or sudv gp(a v) ( figure e ). consistent with our hypothesis, the a v mutation substantially reduced sudv gp-dependent infection in african straw-colored fruit bat cells. unexpectedly, this mutant virus also infected egyptian rousette cells significantly less well than wt, pointing to the existence of sequence context-dependent effects that selectively affect sudv gp(a v) binding to ranpc ( figure e ). these findings provide evidence that gp residue can influence cellular susceptibility to infection by modulating npc recognition in a manner that depends on the sequences of both proteins. we speculate that sequence variation at residue and potentially other positions in the receptor-binding site of filovirus glycoproteins has been shaped by selective pressure to utilize restrictive npc receptors, with potential consequences for viral host range and virulence. the ongoing, unprecedented ebola virus disease epidemic in western africa highlights the urgent need to uncover the biological and ecological factors that underlie the distribution, evolution, and emergence of filoviruses. while a full answer to this question will require the integration of knowledge across multiple levels of biological organization, from genes to populations to ecosystems, previous work has shown that studies of molecular interactions between viruses and their host cells can contribute important pieces to this puzzle. the essential interactions between viruses and their entry receptors provide particularly cogent examples. a switch in receptor binding from the feline to the canine ortholog of the transferrin receptor drove the emergence of a new virus, canine parvovirus, and fueled a global disease pandemic in dogs (allison et al., ) . analyses of interactions of sars-like coronaviruses with their receptor ace have helped to trace the emergence of sars coronavirus from bats to humans, and its use of civets as intermediate amplifying hosts ge et al., ; ren et al., ) . in this study, we show that interactions between filoviruses and their entry receptor npc can influence the cellular susceptibility of bats to infection. this observation is especially striking in light of previous findings that filoviruses could efficiently infect a broad range of mammalian cells, including some derived from bats (kuhn, ; kuhl et al., ) . indeed, this prior work and the results of experimental infection studies in rodents and bats have led to the hypothesis that interactions between viral components and those of the host innate and adaptive immune systems constitute the primary molecular variables influencing filovirus host range in nature (ebihara et al., ; volchkov et al., ) . here, we propose that npc is also a genetic determinant of filovirus susceptibility in bats. the essential nature of npc for infection in cells derived from mammals of multiple species, including bats (figure ) , and for infection and in vivo pathogenesis in lethal ebov infection mouse models argues against the existence of alternative filovirus entry receptors (carette et al., ; herbert et al., ) . therefore, strong reductions in the affinity of virus-npc recognition are predicted to reduce or eliminate infection in whole bat hosts, as observed in npc deficient mice (carette et al., ; herbert et al., ) , barring viral mutation to enhance this affinity. it is conceivable that even modest defects or delays in viral multiplication through such a mechanism could help determine host range by accelerating viral immune clearance, as recently observed in npc -heterozygous mice (herbert et al., ) , or by synergizing with other host-virus barriers. the highly virus-and host species-specific nature of the virus-receptor mismatch uncovered in this study warrants the determination of more bat npc sequences for inclusion in genetic analyses (see below), and a more comprehensive phenotypic examination of virus-bat pairs. such studies maydiscover additional interesting bat-filovirus dynamics, including incompatibilities between filoviruses and npc or other proviral/antiviral host factors. such discoveries have potential implications for our understanding of the molecular basis of filovirus infection, virulence, and host range. we found that a single amino acid change, at residue , in the african straw-colored fruit bat ortholog of npc (ehnpc ) greatly diminished the susceptibility of cells from multiple tissues and individuals to ebov. these migratory pteropodids are widely distributed across sub-saharan africa ( figure a) , roost in large colonies near human settlements, and host other rna viruses with zoonotic potential peel et al., ) . moreover, they are extensively hunted for bushmeat in western africa (kamins et al., ) , making them ideal candidates to transmit viruses directly to humans. unfortunately, there is little information currently available on the susceptibility of african straw-colored fruit bats to ebov or their potential role as filovirus hosts. serologic surveys have found some evidence for exposure to one or more filovirus; however, neither infectious virus nor coding-complete or full viral genomes-the gold standards-have been successfully obtained from these bats, indicating they may only have been exposed to filoviruses, rather than being productively infected (reviewed in [wahl-jensen et al., ; olival and hayman, ] ). while more extensive wildlife sampling and, if feasible, experimental infections of african straw-colored fruit bats will be required to clarify this picture, we can extrapolate to several possible scenarios. first, these bats are fully resistant to ebov, and therefore cannot be the source of this virus in the -present ebov disease outbreak in western africa or the outbreak in middle africa. second, because african straw-colored fruit bat cells do remain weakly susceptible to ebov ( figure c ), it is conceivable that they support ebov replication at low levels. indeed, this is one hallmark of a sustaining viral reservoir. third, the filoviruses circulating in these bats, whether ebov or otherwise, bear one or more gp mutations (e.g., v a) that circumvent the infection barrier imposed by ehnpc . assessing this last hypothesis and understanding the nature of the selection pressures that drive gp evolution in vivo will require the isolation of ebolavirus gp sequences from bats-there are none currently available. although these results suggest that african straw-colored fruit bats are selectively refractory to ebov, our genetic findings indicate that this is not merely a special relationship between one host and one virus. rather, we used a diverse set of bat npc sequences, only one of which is from african straw-colored fruit bats, to show that a number of codons, including residue , have evolved under recurrent positive selection. this is a process in which resistant npc variants are 'serially replaced' in response to compensating viral mutations that restore susceptibility. we provide evidence that the filovirus gp interaction surface in the second luminal domain of npc , domain c, is a hotspot for such positive selection ( figure ). by contrast, the vast majority of codons in mammalian npc have evolved under purifying selection. we propose that this pattern of selection is the signature of a long-term genetic conflict between filoviruses and npc in bats, superimposed over the normal evolutionary signature of a housekeeping gene with a critical role in cellular cholesterol trafficking. similar signatures of recurrent positive selection have been identified in other housekeeping genes that encode viral receptors, including the transferrin receptor (kaelber, et al., ; demogines et al., ) (tfr; receptor for new world arenaviruses [radoshitzky et al., ] , the betaretrovirus murine mammary tumor virus [ross et al., ] , and parvoviruses [parker et al., ] ), bat angiotensin-converting enzyme- (demogines et al., ) (ace ; receptor for sars-like coronaviruses [li et al., ] ), and mammalian dipeptidyl peptidase- (cui et al., ) (dpp ; receptor for mers-like coronaviruses [raj et al., ] ). in these cases as well, the preponderance of positively-selected residues localize to virus-receptor interfaces. interestingly, the sequence polymorphism at npc residue did not impair cholesterol clearance from lysosomes ( figure ) , and none of the residues under positive selection were found to be mutated in niemann-pick type c disease patients (runz et al., ; vanier and millat, ) . thus, despite being constrained by its housekeeping function, npc appears to retains a sizeable sequence space accessible to adaptive mutation. it is tempting to speculate that sequence variation at residue ( figure ) and potentially other positions in the receptor-binding site of filovirus glycoproteins represents the other half of the genetic arms race, shaped by selective pressure to utilize restrictive npc receptors. although more data, especially filovirus sequences from bats, are needed, our findings raise the tantalizing possibility that filoviruses, including those yet undiscovered, are each adapted to specific bat hosts, with co-evolved virus-receptor interactions constituting one potential biological barrier to interspecies viral transmission. alternatively, it is conceivable that repeated contacts between unknown (non-bat) reservoir hosts carrying specific filoviruses, and bats of particular species, have driven positive selection in bat npc to limit infection (and selection of filoviruses with compensating sequence changes in gp). in this scenario, detection of anti-filovirus antibodies or filovirus genome-derived oligonucleotides may reflect a type of spillover event from the actual filovirus reservoir hosts into bats. our hypothesis that npc in bats has been genetically sculpted by filoviruses (and vice versa) presupposes not only a long-term coevolutionary relationship, but also one in which these viruses have imposed selective pressure on bats to limit or eliminate infection. the discovery of filovirus np-and vp -related endogenous viral elements (eves) in bat genomes is consistent with such a long-term relationship (taylor et al., ; katzourakis and gifford, ) . to further investigate the deeper origins of filoviruses in bats, we screened all available bat genomes for filovirus-related eves. we obtained evidence for synteny between a filovirus nucleoprotein (np)-like eve in the genome of the big brown bat (eptesicus fuscus) and those previously identified in three, more distantly-related, myotis bats (figure and supplementary file ) (taylor et al., ) . this new discovery strongly suggests that all four eves resulted from a single insertion event prior to the divergence of the myotis and eptesicus genera, » million years ago (miller-butterworth et al., ) . therefore, bats may have been exposed to filovirus-like agents for far longer than previously recognized (» million years ago [taylor et al., ]) . available experimental exposure studies, although limited in number and scope, suggest that some filoviruses isolated from humans can replicate in bats without causing substantial host pathology (e.g., marv and ravv in egyptian rousettes jones et al., ; paweska et al., ] ). these observations therefore prompt a key question: what is the origin and nature of the selective pressure that has driven accelerated npc evolution in bats? our scant understanding admits a number of possibilities. first, it is conceivable that some filoviruses do indeed replicate in a manner that is deleterious to their specific bat hosts-we may simply not have identified the viruses and hosts in question. indeed, the filovirus llov, discovered in schreibers's long-fingered bat carcasses in spain and portugal, may exemplify this possibility (negredo et al., ) . alternatively, in some cases (e.g., ebolaviruses and egyptian rousettes), the human viral isolates used in challenge studies may differ from these bat isolates in important respects due to human adaptation (human ebov, bdbv, tafv, restv, and sudv isolates do not infect egyptian rousettes ). second, filoviruses may have been more virulent in bats in the past. thus, the positive selection signatures observed in bat npc , which cannot be accurately dated, may represent fixed alleles that are the consequence of a selective process driven by ancient filoviruses with properties distinct from their modern counterparts. indeed, the lack of virulence observed in some bats may reflect a dé tente that was shaped by precisely these historic genetic conflicts between filoviruses and bats. third, we cannot rule out the (unlikely) possibility that the evolution of npc in bats was driven by an entirely different infectious agent that also utilizes (or utilized) npc to multiply in its hosts. regardless of the mechanisms that genetically shaped npc , we propose that polymorphisms in this gene nevertheless impose host barriers that impede the colonization and spread of present-day filoviruses in bats in africa and elsewhere. our findings set the stage for broader explorations of species-specificity in filovirus interactions with proviral and antiviral host factors, with an eye to uncovering new molecular arms races between filoviruses and bats and new genetic determinants of filovirus host range and host switching. the following immortalized pteropodid fibroblast cell lines were used: roni/ . (kidney; rousettus aegyptiacus), hypni/ . (biesold et al., ) . the species origin of each cell line was confirmed in the publication in which it was first described (kuhl et al., ) . bat cell populations stably expressing human npc (hsnpc ) were generated as described previously (carette et al., ) . briefly, subconfluent monolayers of cells were transduced with a retroviral vector expressing hsnpc modified at the c-terminus with a triple flag epitope tag. transduced cells were selected by puromycin treatment ( mg/ml). licenses for capturing and export of bats, as well as ethical review and clearances of animal handling procedures were obtained from the ghana forestry commission of the ministry of food and agriculture. bat organ samples were obtained as described (drexler et al., ) . bats were caught, anesthetised with ketamine/xylazine and exsanguinated by heart puncture. carcasses were transported on ice to a nearby laboratory facility, and organs were dissected and immediately snap-frozen for long-term storage. animals were typed morphologically and genetically as described previously (kuhl et al., ) . vero african grivet kidney cells and t human embryonic kidney fibroblast cells were obtained from atcc. cell lines were maintained in dulbecco's modified eagle medium (dmem) (life technologies, grand island, ny) and supplemented with % fetal bovine serum (atlanta biologicals, flowery branch, ga), and % penicillin-streptomycin (life technologies). all cell lines were maintained in a humidified ˚c, % co incubator. we knocked out the npc gene in the eidni/ . cell line by crispr-cas -mediated genome editing as described previously (mali et al., ) . a crispr guide rna (grna) sequence to target '-gttgtgatgttcagcagcttcgg- ' in the e. helvum npc mrna was cloned into the grna cloning vector (addgene plasmid # ). eidni/ . cells were co-transfected with plasmid encoding human codon-optimized endonuclease cas (hcas , addgene plasmid # ), grna cloning vector encoding the e. helvum npc -specific grna, a monomeric red fluorescent protein (mrfp ) expression plasmid (to monitor transfection efficiency), and pmx-ires-blasti (confers blasticidin resistance to transfected cells) using lipofectamine (life technologies). at hr post-transfection, transfected cells were selected with mg/ml of blasticidin for hr and then allowed to recover in the absence of the selection agent. total rna was isolated from surviving cells with the rnaeasy mini kit (qiagen, valencia, ca) as per the manufacturer's directions. the e helvum npc mrna sequence flanking the grna target site was amplified with the one-step rt-pcr kit (qiagen) and the following primers: forward: '-at-tctggactaccaaaatctttgcc- ', and reverse: '-acatggcatccaagcccaag- '. thermocycling conditions used for the rt-pcr were: ˚c for min (reverse transcription), followed by ˚c for min (initial pcr activation), then cycles of ˚c for sec, ˚c for sec, ˚c for min, then a final extension of ˚c for min. amplified pcr products were tested for indels at the target site with the surveyor mutation detection kit for standard gel electrophoresis (transgenomic, omaha, ne), as per the manufacturer's instructions. once indels were confirmed, amplified pcr products from single cell clones were cloned into a topo-ta vector (life technologies). multiple clones for each single cell population were sequenced to confirm disruption of npc alleles. recombinant vesicular stomatitis indiana viruses (vsvs) expressing egfp, and ebov, marv, or llov gp in place of vsv g have been described previously wong et al., ; ng et al., ) . vsv pseudotypes bearing glycoproteins derived from vsv, ebov, bdbv, tafv, sudv, and marv were generated essentially as described previously (takada et al., ) . vsv particles containing gp cl were generated by incubating rvsv-gp-ebov with thermolysin ( mg/ml) (sigma-aldrich, st. louis, mo) for hr at ˚c. the protease was inactivated by addition of phosphoramidon ( mm) (sigma-aldrich), and reaction mixtures were used immediately. infectivities of vsv pseudotypes were measured by manual counting of egfp-positive cells using fluorescence microscopy at - hr post-infection, as described (chandran et al., ) . infectivities of rvsvs were measured in a similar manner, except that nh cl ( mm) was added to infected cell cultures at - hr post-infection to block viral spread, and individual egfp-positive cells were manually counted at - hr post-infection. the wild-type filoviruses ebola virus/h.sapiens-tc/cod/ /kikwit- (ebov/kik- ; "ebov-zaire ") and marburg virus/h.sapiens-tc/deu/ /hesse-ci (marv/ci ) used in this study were described previously swenson et al., ) . cells were exposed to virus at an moi of pfu/cell ( figure c ) or pfu/cell ( figure a ) for hr. viral inoculum was then removed, and fresh culture media was added. at hr (figure a ) or hr ( figure c ) post-infection, cells were fixed with formalin and blocked with % bovine serum albumin (bsa). ebov-infected cells and uninfected controls were incubated with ebov gp-specific monoclonal antibody kz (maruyama et al., ) . marv-infected cells and uninfected controls were incubated with marv gp-specific monoclonal antibody g (swenson et al., ) . cells were washed with pbs prior to incubation with either goat anti-mouse igg or goat anti-human igg conjugated to alexa . cells were counterstained with hoechst stain (invitrogen, carlsbad, ca), washed with phosphate-buffered saline (pbs), and stored at ˚c. infected cells were quantitated by fluorescence microscopy and automated image analysis. images were acquired at fields/well with a  objective lens on an operetta high content device (perkinelmer, waltham, ny). operetta images were analyzed with a customized scheme built from image analysis functions available in harmony software. from bats of four species (hypsignathus monstrosus, eidolon helvum, epomops buettikoferi, and, rousettus aegyptiacus), mrna was collected from cell lines (or spleen samples for additional eidolon helvum npc domain c sequences; figure -figure supplement ), cdna libraries were constructed, and the npc transcript was sequenced (see supplementary file for primers). using available rnaseq read data (supplementary file ), we assembled bat transcriptomes and identified npc sequences in bats of six additional species (myotis brandtii, artibeus jamaicensis, cynopterus sphinx, myotis lucifugus, pteropus alecto, and desmodus rotundus) . transcriptome data were cleaned with trimmomatic (bolger et al., ) and assembled using trinity (grabherr et al., ) and trans-abyss (robertson et al., ) . the -species npc alignment (supplementary file ) was analyzed for positive selection using the m codon model in the codeml package in paml (yang et al., ) , rel, and fel (pond and frost, ) , and meme (murrell et al., ) available at http://datamonkey.org/ (delport et al., ) . all evolutionary analyses were done using both the npc gene tree and a species tree ( figure -figure supplement ) . the species tree represents the accepted relationships amongst the bats analyzed (agnarsson et al., ; almeida et al., ) . to identify orthologous filovirus-related eve insertions, we screened bat genomes in silico for eves. a representative set of filovirus protein sequences was obtained from genbank, supplemented by the putative protein sequences of previously identified filovirus eves (taylor et al., ; taylor et al., ; taylor et al., ; katzourakis and gifford, ) . these sequences were used as 'probes' in tblastn screens of whole genome shotgun (wgs) sequence data derived from bats of ten species (eidolon helvum, eptesicus fuscus, myotis brandtii, myotis davidii, myotis lucifugus, pteropus alecto, pteropus vampyrus, megaderma lyra, pteronotus parnellii, and rhinolophus ferrumequinum) . statistically significant matches to filovirus probes were extracted, conceptually translated, and aligned with homologous filovirus proteins. orthologous flanking sequences were identified by blast comparison of eve-containing contigs. an alignment of the identified eves, along with the flanking information in the relevant bat genomes, is shown in supplementary file . a construct engineered to encode hsnpc domain c (residues - ) flanked by sequences that form a stable, antiparallel coiled coil, and fused to a preprotrypsin signal sequence with flag and hexahistidine tags at its n-terminus has been described (deffieu and pfeffer, ; . similar constructs bearing bat npc domain cs were generated by replacing the human domain c sequence with a sequence encoding domain c from each bat npc ortholog. soluble domain c was expressed in human -freestyle cells (invitrogen) and purified from supernatants by nickel affinity chromatography, as described previously . alternatively, cell supernatants containing soluble domain c were used directly for gp-npc binding elisas following calibration for domain c concentration (see below). npc domain c concentrations used in the elisas were normalized as follows. proteins were resolved by sds-page followed by immunoblotting with an anti-flag antibody followed by an antimouse alexa- secondary antibody (invitrogen). blots were visualized using the li-cor odyssey imager, and the domain c band was quantified using the li-cor image studio package (li-cor biosciences, lincoln, ne). thermolysin-cleaved vsv-ebov gp particles were captured onto high-binding -well elisa plates (corning, corning, ny) using kz , a conformation-specific anti-ebov gp monoclonal antibody. plates were blocked with pbs containing % bsa, and serial dilutions of npc domain c protein were then added. bound domain c was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich) and ultra-tmb substrate (thermofisher, grand island, ny). all binding steps were carried out at ˚c for hr or at ˚c overnight. elisas with vsvs bearing llov and marv gp were performed as above, but with the following modifications. vsv-llov gp particles were cleaved by incubation with a reduced concentration of thermolysin ( . mg/ml, ˚c, hr) due to its enhanced protease sensitivity relative to ebolavirus gps, as described (ng et al., ) . the viral envelope was then labeled with biotin using a function-spacer-lipid construct (fslbiotin) (sigma-aldrich), as described previously (ng et al., ) . biotinylated viral particles were captured onto streptavidin-coated elisa plates ( . mg/ml). the remainder of the steps in the elisa were performed as described above for vsv-ebov gp. vsv-marv gp particles were cleaved by incubation with trypsin ( mg/ml, ˚c, min; sigma-aldrich), modified as above using fslbiotin, and captured onto streptavidin-coated magnetic beads (spherotech, lake forest, il). beads were then aliquotted into a -well round-bottomed plate for the remaining elisa steps. pbs containing % nonfat dry milk was used for blocking and washing steps. binding curves were generated by nonlinear regression analysis using prism ( -parameter logistic equation; graphpad software, la jolla, ca). to detect npc in primate or bat kidney fibroblasts, whole cell lysates were prepared as previously described . briefly, cells were washed with pbs and lysed in nte-chaps buffer ( mm tris [ph . ], mm nacl, mm edta, . % vol/vol -[( -cholamidopropyl)dimethylammonio]- -propanesulfonate) (sigma-aldrich) containing a protease inhibitor cocktail (roche, basel, switzerland), and placed on ice for min. to assist in cell lysis, cell suspensions were vortexed every min, and then placed on ice for min. samples were spun at , Âg for min, and supernatants harvested for western blot. in some experiments, proteins were deglycosylated with protein n-glycosidase f (new england biolabs, ipswich, ma) according to the manufacturer's instructions. proteins were resolved in % sodium dodecyl sulfate (sds)-polyacrylamide gels and transferred to nitrocellulose membranes. endogenous npc was detected using an anti-niemann pick c polyclonal antibody ( : , dilution; ab , abcam, cambridge, ma), followed by incubation with a donkey anti-rabbit antibody conjugated to horseradish peroxidase ( : , dilution, santa cruz biotechnology, dallas, tx). endogenous cyclin-dependent kinase (cdk ; loading control) was detected with a rabbit polyclonal antibody ( : , dilution; sc- , santa cruz biotechnology). ectopic expression of hsnpc -flag was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich). bands were visualized by incubation with an enhanced chemiluminescence reagent (thermofisher) followed by exposure to x-ray film. in figure , cells were visualized using an inverted fluorescence microscope under illumination with a x high-numerical aperture oil objective ( figure b ) or a x air objective ( figure c ). images were captured with an axiocam mrm ccd camera using axiovision software (zeiss usa, thornwood, ny), and imported into photoshop (adobe systems, san jose, ca) for processing. images were cropped, inverted ( figure b) , and subjected to linear adjustment for overall brightness and contrast using the levels tool. developed x-ray films were digitized with a flatbed scanner and processed in photoshop as described above. statistical comparison of means among multiple independent groups was carried out by one-way analysis of variance (anova) with tukey's post hoc test for multiple comparisons. in some figures (see figure legends) , an unpaired two-tailed student's t-test with welch's correction for unequal variances (ruxton, ) was used for pairwise comparison of independent groups. all statistical analyses were performed in graphpad prism. the albert einstein college of medicine. opinions, conclusions, interpretations, and recommendations are those of the authors and are not necessarily endorsed by the us department of the army, the us department of defense, or the us department of health and human services. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. author contributions mn, en, mek, jmd, sls, kc, conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article; ash, rjg, acquisition of data, analysis and interpretation of data, drafting or revising the article; tb, aik, rmj, acquisition of data, analysis and interpretation of data; rkj, acquisition of data, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents; jah, my, acquisition of data, analysis and interpretation of data, contributed unpublished essential data or reagents; rb, acquisition of data, contributed unpublished essential data or reagents; ad, analysis and interpretation of data, drafting or revising the article; trb, conception and design, analysis and interpretation of data, drafting or revising the article; cd, mam, drafting or revising the article, contributed unpublished essential data or reagents; lfw, jhk, analysis and interpretation of data, drafting or revising the 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receptor determinants of zoonotic transmission of new world hemorrhagic fever arenaviruses dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin de novo assembly and analysis of rna-seq data mouse transferrin receptor is the cell entry receptor for mouse mammary tumor virus npc-db, a niemann-pick type c disease gene variation database the unequal variance t-test is an underused alternative to student's t-test and the mann-whitney u test pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections generation of marburg virus-like particles by co-expression of glycoprotein and matrix protein a system for functional analysis of ebola virus glycoprotein evidence that ebolaviruses and cuevaviruses have been diverging from marburgviruses since the miocene evolutionary maintenance of filovirus-like genes in bat genomes filoviruses are ancient and integrated into mammalian genomes genetic diversity and evolutionary dynamics of ebola virus in sierra leone isolation of genetically diverse marburg viruses from egyptian fruit bats niemann-pick disease type c molecular characterization of guinea pig-adapted variants of ebola virus roles of rodents and bats in human viral hemorrhagic fevers a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry codon-substitution models for heterogeneous selection pressure at amino acid sites we thank tyler krause and cecelia harold for technical support, and gary crameri, shawn todd, and mary tachedjian for help with sourcing bat cdna for npc gene amplification. we also thank margaret kielian, jack lenz, max nibert, vinayaka prasad, deeann reeder, nancy simmons, and susan tsang for useful discussions. we thank laura bollinger, integrated research facility at fort detrick, for critically editing this manuscript.supported by grants from the us national institutes of health (ai to kc, gm to sls), the us defense threat reduction agency (hdtra - -c- to sls, cb to jmd), eu fp- antigone (grant ) and the ebokon project (to cd and mam). lfw is supported in part by an nrf-crp grant (nrf nrf-crp - ) in singapore. jhk performed this work as an employee of tunnell government services, inc., a subcontractor to battelle memorial institute, under battelle's prime contract with niaid (no. hhs i). sls is a burroughs wellcome fund investigator in the pathogenesis of infectious disease. kc is additionally supported by a harold and muriel block faculty scholarship and an irma t. hirschl/monique weill-caulier research award at key: cord- -hqnem zs authors: ji, ying-jie; duan, xue-zhang; gao, xu-dong; li, lei; li, chen; ji, dong; li, wen-gang; wang, li-fu; meng, yu-hua; yang, xiao; ling, bin-fang; song, xue-ai; gu, mei-lei; jiang, tao; koroma, she-ku m.; bangalie, james; duan, hui-juan title: clinical presentations and outcomes of patients with ebola virus disease in freetown, sierra leone date: - - journal: infect dis poverty doi: . /s - - - sha: doc_id: cord_uid: hqnem zs background: clinical and laboratory data were collected and analysed from patients with ebola virus disease (evd) in jui government hospital in freetown, sierra leone, where patients with evd were received and/or treated from october , to march , during the west africa evd outbreak. methods: the study admitted patients with confirmed evd and followed them up till the endpoint (recovery or death). evd was confirmed by quantitative rt-pcr assays detecting blood ebola virus (ebov). results: among the lab-confirmed evd cases in jui government hospital, recovered and died, with an overall survival rate of . %. patients under the age of years had a lower survival rate ( . %). most non-survivors ( . %) died within days after admission and the mean hospitalization time for non-survivors was . ± . days. more than half survivors ( . %) turned blood ebov negative within weeks after admission and the mean hospitalization time for survivors was . ± . days. high blood viral load (≥ ( ) copies/ml) was found to be predictive of the non-survival outcome as indicated by the receiver operating characteristic (roc) curve analysis. the probability of patients’ survival was less than % when blood viral load was greater than ( ) copies/ml. multivariate analyses showed that blood viral load (p = . ), confusion (p = . ), abdominal pain (p = . ), conjunctivitis (p = . ), and vomiting (p = . ) were factors independently associated with the outcomes of evd patients. conclusions: most death occurred within week after admission, and patients at the age of or younger had a lower survival rate. most surviving patients turned blood ebov negative within – weeks after admission. factors such as high blood viral load, confusion, abdominal pain, vomiting and conjunctivitis were associated with poor prognosis for evd patients. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. please see additional file for translations of the abstract into the six official working languages of the united nations. ebola virus disease (evd), previously known as ebola haemorrhagic fever, is a rare and deadly disease caused by infection with one of the ebola virus strains. a large-scale outbreak of haemorrhagic fever occurred in southern sudan between june and november . it was transmitted by close personal contact and by use of contaminated needles and syringes in hospitals/clinics [ ] . this outbreak led to the further recognition of the disease, which was subsequently named ebola haemorrhagic fever. since then, outbreaks have occurred sporadically in africa. a evd outbreak was the largest in scale in history, affecting multiple countries in west africa. in the outbreak, the first lab-confirmed evd patient was reported in may, in guinea and since then the zaire ebola virus (zebov) has rapidly spread across sierra leone and to other west africa countries. from march to december , ,there were reported evd infections (including confirmed, probable, and suspected) and reported deaths in west africa. a total of confirmed infections and deaths of health care workers were reported in guinea, liberia, and sierra leone [ ] . as of december , , the sierra leone national ebola response center (nerc) reported a cumulative total of confirmed evd cases with deaths (excluding probable and suspected cases) [ ] . situated on the atlantic coast, freetown is the capital and the largest city of sierra leone, and is densely populated with over million people. due to the heavy population, freetown and its surrounding western region was the most affected area in this epidemic. as of december , , confirmed cases had been reported in this region, accounting for . % of the country's total evd reported cases [ ] . evd imposed a significant economic burden on the west african countries affected. some studies suggest that due to evd deaths, life expectancy may have declined in liberia and sierra leone to a new low since - [ ] . this dramatic healthcare crisis, coupled with human rights and global security concerns, underscored the urgent need for developing resilient healthcare systems, and called for the domestic and international aides and investments in these african countries [ ] . the clinicians of the chinese medical team (cmt) managed evd patients in an ebola holding and treatment center in jui government hospital, which is also known as sierra leone-china friendship hospital. being one of the best hospitals in freetown, jui government hospital received suspected evd patients during the period of october , and march , , of whom were confirmed infected with the virus. all of the cmt clinicians were from beijing hospital, the largest specialized hospital in china for infectious disease treatment. the same hospital had successfully managed and controlled the outbreak of severe acute respiratory syndrome (sars), a/h n influenza, and some other public health emergencies in china. in this study, we described the clinical presentations, clinical courses, and the treatment outcomes of all evd patients admitted in jui government hospital for care. we hope this paper provide further understanding and insights into pathophysiology, clinical manifestations and treatment impact of end outcomes of evd. a retrospective, observational study was conducted using data collected from all patients with confirmed evd who were admitted to the holding and treatment center of jui government hospital from october , to march , . diagnosis of evd was made in accordance with the criteria set by the world health organization (who) in the standard operating procedures (sop) for managing evd. because jui government hospital was designated as an ebola holding center (ehc) on october , , but was not approved as an ebola treatment center (etc) until january , , confirmed evd patients received at this hospital between october , and january , were immediately transferred to other treatment centres once confirmed. after january , , all confirmed patients except pregnant women were treated in jui government hospital; the confirmed evd patients who were pregnant women were transferred to a designated etc (p.t.s. ebola treatment center). we carried out follow-up studies and data collections from all confirmed patients (both the patients treated in jui government hospital and those transferred to other hospitals) before we finished our mission in march . data collected included observations such as the duration of hospitalization, the date when blood ebov turned negative, and the endpoint (recovery or death). the determination of recovery was based on the clinical presentations and the interpretation of laboratory findings. discharge from hospitalization was considered when the following criteria were met: ) three or more days without fever or any other significant symptom, ) significant improvement in clinical presentations, ) a relatively good general condition, and ) a negative pcr result for blood ebov on the third day of being asymptomatic. if a patient continued to suffer symptoms or their condition was not improving, but it was suspected to be unrelated to evd, then two blood ebov tests were carried out h apart, with at least one test being done days or more after the onset of symptoms. if both test results were negative, patients were discharged or referred to a normal hospital for further care. the study protocol adhered to the declaration of helsinki, and the ethical clearance was obtained from military hospital medical ethics committee and the sierra leone ethics and scientific review committee, respectively. the patients were routinely evaluated for clinical presentations. data collected included vital signs at admission, medical history, time points when blood ebov turned negative, durations of hospital stay, and outcome. within h upon admission, ebov detection assays were carried out via quantitative rt-pcr using whole blood samples. if the patient died quickly and the blood samples were not collected due to time constraints or poor venous access after death, ebov detection was carried out using oral swab samples from corpses. samples were collected at : o'clock every day and results were generated within h. samples were obtained using jui government hospital's collection and processing protocols, which was described in the emergencyresponse guidelines established by the sierra leone ministry of health and sanitation. diagnosis testing for ebov was performed by china cdc mobile laboratory. total rna was extracted from patient peripheral blood samples or swab samples in bio-safety level (bsl- ) facilities. ebola viral rna was then detected using detection kit for ebola virus subtype zaire rna (puruikang biotech co. ltd, with pcr fluorescence probing) according to the manufacturer's recommendations [ ] . for quantification purposes, the amplicon concentrations were converted to copies of ebov per milliliter (method provided by china cdc mobile laboratory). the care protocols for confirmed ebola cases before and after june , were similar and were in accordance with the sop of who and the ministry of health of sierra leone. the treatment protocol was as follows: all adult patients received mg of vitamin k and mg of sodium artesunate immediately upon admission. after h all adult patients with confirmed evd received g of ceftriaxone every h, mg of metronidazole every h, - ml of ringer's lactate every h, and - ml of dextrose saline ( % and . %, respectively) every h. all the above medications were administered intravenously. adult patients also received a mg zinc sulfate tablet daily, a mg acetaminophen tablet every h, and mg of ondansetron injection intravenously as needed for nausea or vomiting. after the first days, continuing therapy included a mg metronidazole tablet every h for days, a mg cefuroxime tablet every h for days, and a mg acetaminophen tablet every h. the protocol for children was similar but the dosage was adjusted according to their body weight. oral rehydration solution and juice were provided on an as needed basis. we transfused more fluid to the patients with profound gi losses and those unable to take oral fluids or medications. currently, early supportive care, empiric antibiotic therapy, and maintenance of water and electrolyte balance are the basic interventions to treat evd due to the lack of specific anti-ebov drugs. every etc designed their treatments protocol according to the protocols for viral haemorrhagic fever under the urgent interim guidance for case management established by the who and endorsed by the ministry of health, which were similar. statistical analyses were performed with the aid of spss, version . . (spss inc., chicago, il, usa). the overall survival rate was compared using log-rank test. univariate and multivariate analyses were carried out using the logistic regression model. the intergroup comparisons were performed using chi square test. receiver operating characteristic (roc) curve was plotted using the log value of blood ebov viral load and the survival rate. all the statistical tests were two-tailed, and a p-value of less than . was considered statistically significant. a total of suspected evd patients were admitted to jui government hospital, among which were confirmed with evd. all patients with confirmed evd received supportive treatment and were followed up to the endpoint (recovery or death). among the confirmed evd patients, were transferred to other treatment centres and treated in our centre. of the total confirmed, were female and were male, recovered and died, with an overall survival rate of . % (females . %, males . %). no significant difference was found between the survival rates of females and males (p = . ). the average age of the evd patients was . ± . years, and the median age was years, interquartile range were years and years (iqr, - ). the youngest patient was month old, and the oldest years old. the ages were separated by the following groups: patients ( . %) were under the age of , patients ( . %) between and , patients ( . %) between and , and patients ( . %) above the age of . the median age was (iqr, - ) for the non-survivors and (iqr, - ) for the survivors. the mean hospitalization time for survivors was . ± . days, and the median was (iqr, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days. the surviving patients were discharged after they had been asymptomatic for h, and they had been tested negative for blood ebov using rt-pcr assay. the mean hospitalization time for non-survivors was . ± . days, and the median was (iqr, - ) days. we investigated how long it took for the blood ebov to turn negative in the surviving patients. all survivors were followed up from the time of diagnosis (when blood ebov was first detected) to the time of recovery. the median time for a surviving evd patient to become blood ebov negative was . ± . days, and the range was from days to days. of the total number of survivors observed, . % ( cases) turned negative for blood ebov within weeks after the diagnosis, and . % ( cases) turned negative weeks after the diagnosis (fig. ). among the evd patients who received treatment, died (the fatality rate . %). of the nonsurviving patients, . % died within days after admission, . % died during the first week, and more than . % died within weeks (fig. ) . to investigate the survival rate of evd patients in different age groups, the evd patients were divided into three groups (age - , age - , and ≥ years). the results showed that the survival rate for group - was statistically lower than that of group - (p = . ) or group ≥ (p = . ) (fig. ) . but the survival rates for group - and group ≥ were not statistically different (p = . , kaplan-meier estimate) (fig. ). in addition, we categorized the patients using the median age ( years) or years as the cutoffs, and applied the same statistical analysis, but no statistically significant difference was found in the survival rates between groups. we also looked into whether high viral load in the blood was an indicator for high fatality rate. a receiver operating characteristic (roc) curve was plotted using the blood viral values and the survival outcomes of the evd patients. among the patients, died within h of admission. such patients were tested positive using only oral swab samples, and were excluded from this analysis. a total of patients with detectable blood ebov virus were included in the roc curve analysis. the numbers of ebov copies were converted to log values for further analysis. the results showed that the viral loads had a high predictive power of patients' outcome (p < . ). the areas under operator curve (auoc) were . ( % ci: . - . ). from the roc curve, when the log viral value was greater than , the probability of patients' survival was less than %; and when it was greater than , the probability of patients' survival was less than %. these results suggested that the viral loads can be used as a potential prognostic biomarker for evd patients (fig. a) . we selected copies/ml as the cutoff value to divide the patients into two groups (ebov ≥ copies/ ml and < copies/ml), and applied statistical analysis fig. the time lapsed before survivors turned negative for blood ebov, and the time lapsed before non-survivors died. all patients were confirmed with evd in sierra leone (fig. b) . kaplan-meier estimate showed that when the blood ebov titer was greater than copies/ml, the fatality rate was . % -remarkably higher than that of the group with less than copies/ml viral load ( . %) (p = . ). we analysed the clinical presentations of the evd patients at the time of admission, of which of the patients were excluded due to incomplete data, making the total number of patients studied and included in the analysis to . ( . %), pain behind eyes ( . %), confusion ( . %), and skin rash ( . %). a number of variables between survivors and non survivors were shown to be significantly different amongst the evd positive patients with known outcomes including vomiting (p < . ), abdominal pain (p = . ), jaundice (p < . ), conjunctivitis (p = . ), and confusion (p < . ) (fig. ) . to further explore which factors were associated with the survival of evd patients, a number of factors, including a variety of symptoms (absent or present), age (years), gender (male or female), viral load (log copies/ml), and treatment outcomes (survival or death) were included in the univariate and multivariate analyses ( table ). the univariate analysis showed that vomiting, abdominal pain, jaundice, conjunctivitis, confusion, and the viral load (p < . ) were good candidates for the final logistic regression model. the multivariate analyses showed that higher viral load, severe confusion, vomiting, abdominal pain, and conjunctivitis indicated poor prognosis in evd patients (table ) . jui government hospital in freetown, sierra leone was approved as an ehc on oct , to receive suspected evd patients. once the evd cases were confirmed, the patients were transferred to other etcs such as p.t.s. , in the previous published reports, no detailed data was available on how soon the blood ebov turned negative in evd patients. our observation revealed that blood ebov turned negative very slowly-only . % patients turned blood ebov negative in weeks. this partially explained why evd spread so widely and persisted for so long in west africa. the surviving evd patients in our study were discharged after the symptoms remained absent for h, and the blood samples were negative for ebov in two consecutive tests with quantitative rt-pcr assay. the mean hospitalization time for surviving patients was . ± . days. in other studies, the researchers continued to monitor ebov rna levels in sputum, saliva, conjunctiva swabs, stool, urine, and sweat using real-time rt-pcr assay after the blood ebov rna turned negative. they found that urine samples remained positive for ebov rna for as long as days after blood turned negative,and sweat samples remained positive throughout the observation period for an additional days ( days after blood became ebov rna negative [ ] ). another study reported that ebov existed in cerebral spinal fluids and semen for much longer in evd patients [ ] . even varkey and colleagues described a patient who recovered from evd and subsequently showed severe unilateral uveitis during convalescence. in this case, viable zaire ebola virus was detected in aqueous humour weeks after the onset of evd and weeks after the clearance of ebov from the blood [ ] . such findings warrant further investigations in the future. although it was unclear whether or not such residual viruses were pathogenic, these findings suggested that when evd patients are recovered and discharged, they should be put on continued quarantine for a period of time to avoid close contact with other people. the case fatality rates (cfr) of evd was reported to be between % and % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our study, the total mortality rate was . % for confirmed cases, which was similar to that in the previous reports. as a trend observed in this outbreak, the fatality rate was more than % during the early stage, and dropped to %- % at a later stage. this may be attributable to the public's poor awareness of this disease, the delay in seeking treatment, the inadequate measures for diagnosis and treatment, and the limited media coverage when the outbreak erupted. later, as more awareness came from the international medical associations causing more international medical teams to arrive, the public became better educated and the patients started to receive more effective diagnosis and treatment. thus at the later stage of the virus outbreak, early discovery, early diagnosis, and early treatment helped bring the fatality rate down. we also made observations about the length of the hospital stay for the non-survivors. we found that most non-survivors died within days after admission, which was consistent with other reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . only % died after day upon admission. this indicated that evd has high degree of virulence. it also suggested that early diagnosis and early treatment is critical to the patients' survival. just as some scholars stated, the lack of public awareness of the disease as well as the lack of synergy among governments and international organizations contributed to the current epidemic [ ] . the public should be educated and instructed to seek medical attention as early as they notice any symptoms, and the proactive treatment should be deployed as soon as the patients are admitted. the age factor has been noted in the past evd outbreaks and remains an important factor in the current outbreak [ ] [ ] [ ] . the previous reports showed that older age was associated with worse outcomes, and the association between the two was often attributable to increased coexisting conditions in the elderly [ , , , ] . in contrast, we did not find statistically significant differences between the age of non-survivors and survivors in our study (median age: (iqr: - ) vs. (iqr: - ); average age: . ± . vs. . ± . ). in order to determine the association between age and prognosis, we grouped the patients using different cutoffs, including median age ( ), the cutoffs used in other studies (age , , ), and multiple age groups at intervals of years. in our comparison of the different age groups, no association was found between age group and prognosis. one distinction in our study was that the mortality rate for younger patients under the age of was higher than the other age groups. this may be explained as follows: in the beginning of the evd outbreak, adult patients encountered many complications such as diarrhoea-related electrolyte disorders and secondary infections, but soon received sufficient fluid therapy in many etcs, so the mortality rate dropped among those patients. but for young children, since there was a shortage of paediatricians in many centres, the paediatric patients may not always have received sufficient attention or optimized therapy [ ] . the young children were unable to care for themselves and their daily fluid intake was not always guaranteed while solely depending on the limited hospital resources. additionally, poor access to iv vessels in paediatric patients may limit both the amount and the flow rate of fluid administered. we also found that patients who presented the highest viral loads had the worst outcome, as had been the case for other strains of ebola virus [ , , , ] . although the diagnostic value was excellent for postmortem swab samples [ ] , it was unclear how a cycle threshold (ct) value from an oral swab correlates with that from a whole blood sample, therefore the viral load data for the oral swab samples ( / ) were not included in the analysis. the relationship between the viral load (copies of ebov per milliliter) and the fatality rate was investigated. roc curve analysis was plotted and a positive correlation was found between the two. a low viral load was associated with a better survival outcome, whereas a high viral load was an important indicator for fatality rate. our finding was consistent with those of the previous reports [ , , , ] . based on this finding, it is plausible to assign patients with virus load of ≥ copies/ml to dedicated wards, where they can receive enhanced medical support and palliative care if resources allow, given their increased risk of death. it is noteworthy that the cases for which evd was confirmed using oral swab samples were all severely ill patients who died soon after admission. exclusion of these cases may have introduced some bias in our results. evd was formerly named ebola haemorrhagic fever, and bleeding is one of its hallmarks. bleeding was noted among patients in previous outbreaks. however, in this outbreak, the reported bleeding rates ranged from . % [ ] to . % [ ] . in our study, only of patients ( . %) suffered from visible bleeding during their hospitalization, which is a relatively low number when comparing to other reports [ ] . this suggested that bleeding may not be a major characteristic of evd patients in this outbreak. such discrepancy may be associated with the evolvement of the virus's pathogenicity as its genes undergo mutations. finally, we analysed which factors were associated with patients' outcomes. the incidences of various symptoms in evd patients reported here are consistent with those of previous reports, with only minor differences. the most common evd manifestations on admission were fever, weakness, loss of appetite, vomiting, cough, abdominal pain, headache, joint pain, and diarrhoea. in addition to factors such as age and viral load, previous studies described many other important factors associated with fatal outcomes, including hiccups, haemorrhagic signs, fever, weakness, dizziness, diarrhoea, myalgia, difficulty breathing, extreme fatigue, vomiting, mental symptoms, loss of appetite, confusion, and conjunctivitis [ - , , ] . the multivariate analyses in each study may include one or more of the aforementioned presentations. this indicated that: first, none of the clinical manifestations observed among patients in this outbreak was unique; and second, the way the patient history information was collected may vary in each study. in our study, the multivariate analyses showed that ebov viral load, abdominal pain, confusion, conjunctivitis, and vomiting were independently associated with the death outcome of evd patients. it is understandable that high viral load is related to fatality rate, and that digestive discomfort may be associated with patients' prognosis. in our study, the most powerful predictor of mortality in the multivariable regression model was confusion at admission (p value = . ), with an odds ratio of . ( % ci: . - . ) favouring mortality. this finding indicated that the more severe the condition was at the time of admission, the higher the risk of death. we observed that due the lack of peculiar presentations of evd and the less-developed health care capacity in africa, many patients failed to seek medical attention promptly after they experienced some non-specific symptoms such as fever, thus delaying the diagnosis and treatment. some patients died on their way to the hospital, some died upon arrival before the physicians had the opportunity to examine them, and yet some died on day ( / ) or day ( / ) after admission. in these circumstances, it was too late for the patients to receive proper treatment for such a severe disease. therefore, we should raise the public's awareness that early discovery, early diagnosis, and early treatment are essential. early proactive intervention can significantly improve the prognosis of the patients. in our clinical observations, we found that the clinical presentations in this evd outbreak was primarily severe gastro enteric symptoms, such as nausea and lack of appetite accompanied by excessive vomiting and severe diarrhoea. although due to the limitations of the medical equipment, we were unable to carry out laboratory tests to determine the cause of death for each non-survivor, we suspected that the loss of body fluids, abnormal metabolism, electrolytes imbalance, and hypovolemic shock, which were all secondary to the severe digestive tract disorder, may have been the immediate causes in the death of most non-surviving patients. the patients are usually extremely weak after the onset of the disease and cannot eat or drink by themselves; therefore it is essential for the medical staff, while taking precautions to protect themselves, to administer adequate fluids to the patients as early and as quickly as possible. early initiation of proactive fluid therapy, particularly in the first days of hospitalization and even before the ebov test results are received, is vital to saving patients' lives and reducing fatality rate. a few limitations exist in our study. first was the limitation of the data. there were many challenges regarding the collection of accurate clinical and epidemiological data on site in west africa. heavy workload, language barriers, and lack of an information technology infrastructure which would otherwise allow healthcare professionals to record data electronically at the point of patient contact, had all contributed to the incomplete data presented in our study. the second limitation was that no analysis was available for the time period between symptom onset and admission, and in most cases it was difficult to pinpoint the exact date when symptom started. the majority of the patients were not able to recall or describe when the symptoms started; it was especially so for the more severe patients. another drawback is the limitations on the clinical observations. as we described previously, some patients were transferred to other etcs before jui government hospital became a designated etc, and the treatment protocols in the other etcs may not always be consistent with the evd sop. gi losses and electrolyte disturbances may be the major causes of death in many evd patients. laboratory tests that provide comprehensive data on patients' haematological and biochemical indicators can help delineate the cause of death. more importantly, it may also provide instructions for treatment, thus reducing fatality rate. unfortunately, we did not have a crosscontamination-proof testing facility in place while our centre was serving as an ehc or in its early stage of serving as an etc. therefore, we were unable to determine whether the non-survivors died of evd complications, related co-infections, or some other causes. we only evaluated the severity of the illness by the clinical manifestations, and transfused more fluid for the patients with profound gi losses and those unable to take oral fluids or medications. the biochemical testing and the testing for co-infection with malaria were not launched until a later stage due to the limitation of laboratory facilities. even then, such data was only collected from a limited number of patients. while the data was of some help to the treatment, it was not sufficient for a meta-analysis. in addition, we were not able to analyse the association between the treatment protocols and the outcome. during the months of treatment, three different health care teams rotated and the physicians were on - h shifts. the medical staff professionals had different ways of practicing, thus the medical history, symptoms and signs were recorded in different manners. in conclusion, for the patients confirmed with evd, the survival rate was . %. some surviving patients did not become blood ebov negative until weeks after admission or later. most non-surviving patients died within week after admission. patients under the age of years and those with high viral load had a higher fatality rate. patients who presented confusion, vomiting, abdominal pain, and conjunctivitis at the time of admission were at higher risk of death. such patients should be the priority of medical attention and should be put under intensive treatment, particularly during the first week of hospitalization. additional file : multilingual abstracts in the six official working languages of the united nations. (pdf kb) world health organization international study team. ebola hemorrhagic fever in sudan world health organization: ebola situation report - ebola virus disease update-december assessing the direct effects of the ebola outbreak on life expectancy in liberia, sierra leone and guinea indirect costs associated with deaths from the ebola virus disease in west africa ebola virus outbreak investigation a case of severe ebola virus infection complicated by gram-negative septicemia andré-von arnim a. clinical presentation and management of severe ebola virus disease persistence of ebola virus in ocular fluid during convalescence clinical presentation of patients with ebola virus disease in conakry, guinea clinical illness and outcomes in patients with ebola in sierra leone clinical predictors of mortality in patients with ebola virus disease ebola outbreak in conakry, guinea: epidemiological, clinical, and outcome features clinical features of patients with ebola virus disease in sierra leone ebola outbreak in rural west africa: epidemiology, clinical features and outcomes the contribution of ebola viral load at admission and other patient characteristics to mortality in a médecins sans frontières (msf) ebola case management centre (cmc) high survival rates and associated factors among ebola virus disease patients hospitalized at donka national hospital, conakry, guinea ebola wreaks havoc in sierra leone a limited outbreak of ebola haemorrhagic fever in etoumbi ebola hemorrhagic fever, democratic republic of the congo, : determinants of survival proportion of deaths and clinical features in bundibugyo ebola virus infection who ebola response team. ebola virus disease in west africa -the first months of the epidemic and forward projections rapid diagnosis of ebola hemorrhagic fever by reverse transcription-pcr in an outbreak setting and assessment of patient viral load as a predictor of outcome utility of oral swab sampling for ebola virus detection in guinea pig model basic clinical and laboratory features of filoviral hemorrhagic fever clinical features of patients isolated for suspected ebola virus disease at connaught hospital we thank all staffs of beijing hospital and all members of our families for their hard work and emotional support during we fought again ebola. we thank all nurses, cleaners and managers from sierra leone for their positive teamwork. we thank editors and reviewers for their warm works and valuable comments. no.availability of data and materials authors do not wish to share raw data for readers to open access due to privacy or legal concerns. but we can give our main statistical data as an affiliated file to editors and reviews for reproducing and testing research results.authors' contributions yjj, xzd, xdg, and hjd contributed to the design of the study. yjj, xzd, xdg, ll, cl, dj, wgl, lfw, yhm, xy, bfl, xas, mlg, and hjd entered ebola wards, implemented the field work and collected the on-site data in sierra leone. tj helped with the diagnosis test. yjj, xzd, smk and jb collected the follow-up data. yjj, xzd, xdg and hjd wrote the manuscript. all authors read and approved the final draft. the authors declare that they have no competing interests. the study has been approved by the military hospital medical ethics committee and the committee's reference number is d. the written informed consent was waived because of retrospective, observational study. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -f bs r authors: lai, kang yiu; ng, wing yiu george; cheng, fan fanny title: human ebola virus infection in west africa: a review of available therapeutic agents that target different steps of the life cycle of ebola virus date: - - journal: infect dis poverty doi: . / - - - sha: doc_id: cord_uid: f bs r the recent outbreak of the human zaire ebolavirus (ebov) epidemic is spiraling out of control in west africa. human ebov hemorrhagic fever has a case fatality rate of up to %. the ebov is classified as a biosafety level pathogen and is considered a category a agent of bioterrorism by centers for disease control and prevention, with no approved therapies and vaccines available for its treatment apart from supportive care. although several promising therapeutic agents and vaccines against ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced. like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress. we have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. most of the therapeutic agents in this review are directed against non-mutable targets of the host, which is independent of viral mutation. these medications are approved by the food and drug administration (fda) for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. electronic supplementary material: the online version of this article (doi: . / - - - ) contains supplementary material, which is available to authorized users. the recent outbreak of the human zaire ebolavirus (ebov) infection starting in west african countries has resulted in , infected patients, as of th of november . a total of , deaths have been reported in six affected countries (guinea, liberia, mali, sierra leone, spain, and the united states of america) and two previously affected countries (nigeria and senegal) [ ] . apart from supportive care, neither a licensed vaccine nor a specific therapy is available for the treatment of the human ebov infection [ ] . the world health organization (who) has considered that it is ethically acceptable to offer unproven interventions that have shown promising results in laboratory and animal models, but have not yet been evaluated for safety and efficacy in humans as potential sources of treatment or prevention [ ] . several promising therapeutic agents have been identified for the treatment and immunization of the ebov. these may include monoclonal antibody (mabs)-based therapies (e.g. zmapp), anti-sense phosphorodiamidate morpholino oligomers (pmo avi- ), lipid nanoparticle small interfering rna (lnp-sirna: tkm-ebola), and an ebov glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus (rvsv-ebogp) or a chimpanzee adenovirus (rchad-ebogp)-based vector. human trial results of these agents would not be available until next year. moreover, existing supplies of all these experimental medications and vaccines for compassionate use are either extremely limited or exhausted [ ] [ ] [ ] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions that can target different steps in the replication cycle of the ebov should be explored in the management of the human ebov infection as contingency preparation for the international dissemination of the ebov outbreak in west africa. we have reviewed currently available therapeutic agents that have shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. we propose a therapeutic regimen to supplement the current supportive therapy aiming to reduce viral load, the most important factor in the determination of mortality. through viral load suppression, we may be able to prolong a patient's survival in order to provide a better chance for the patient to develop natural immune defense against the ebov. the ebov is an enveloped filamentous rna virus belonging to the family filoviridae. the -kb linear, non-segmented, negative-sense, single-stranded rna genome of the ebov encodes seven structural proteins and two non-structural proteins in the following order within the genome: ′ non-coding region (leader), nucleoprotein (np), virion protein (vp ), vp , glycoproteins (sgp/ssgp/gp , ), vp , vp , rnadependent rna-polymerase protein (l-polymerase), and ′ non-coding region [ ] . the ebov genome encodes one transmembrane protein gp , (gp -gp ) and two secreted non-structural proteins: secretary glycoprotein (sgp) and small soluble glycoprotein (ssgp). a small soluble delta peptide (Δ-peptide) is secreted from ebov-infected cells after the carboxylterminal cleavage of sgp [ ] . gp , is produced through transcriptional rna editing as a precursor for amino acid polyprotein (gp ), which is post-translationally cleaved by furin into two disulfide-linked subunits; a surface subunit, gp ; and a membrane-spanning subunit, gp . gp contains the receptor-binding domain (rbd) for host cell attachment and a mucin-like domain to protect the rbd from humoral and cell-mediated immunity. the rbd responsible for receptor binding, viral entry, and cellular tropism is covered by a heavily glycosylated "glycan cap". the transmembrane gp contains a helical heptad-repeat region, transmembrane anchor, and a -residue cytoplasmic tail. the gp drives fusion of the viral membrane with the endosomal membrane of the target cell. this gp -gp heterodimer then assembles as a trimer on the viral surface. this homotrimeric gp , complex forms the spike on the envelope membrane of the mature viral particles. during processing, gp , are unstable, and an abundant amount of a soluble non-virion form of gp and a scanty amount of gp , are released into the circulation [ ] [ ] [ ] [ ] . the virusassociated gp , and not the other soluble glycoproteins released during the virus infection are responsible for primary target cell activation [ ] . the highly glycosylated mucin-like region of gp is cytotoxic to the host cells [ ] . the shedding of souble gp , -like protein due to cleavage of ebov glycoprotein on the surface of ebov-infected cells by tumor necrosis factor-alpha converting enzyme (tace) can activate non-infected dendritic cells and macrophages to induce cytokine dysregulation and endothelial cell dysfunction [ ] . the gp of the ebov is able to counter the interferon (ifn)-inducible antiviral protein tetherin which restricts the vp -dependent budding of the progeny viral particles from infected cells [ ] [ ] [ ] . the sgp is produced from non-edited mrna species through furin cleavage from a precursor pre-sgp. the sgp shares the n-terminal amino acids with gp , but differs in the carboxyl terminus by amino acids. the sgp is released into the circulation in the form of homodimers in antiparallel orientation [ ] to evade an antibody-associated innate immune response [ , ] . the sgp has an antiinflammatory function and impairs the transmigration and activation of neutrophils [ , ] . while the gp , in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function, sgp protects the endothelial cell against cytokine-induced barrier dysfunction. the sgp constitutes at greater than % of the total gp synthesized during infection. hence, the hypersecretion of the sgp may protect the ebov against host humoral immune defense and the host endothelial cell against cytokine-induced cytotoxicity during the early phase of the ebov infection [ , , ] . Δ-peptide released in ebov-infected cells joins cathepsins and integrins to inhibit further entry of the ebov in a dose-dependent manner to prevent superinfection of ebov-infected cells. Δ-peptide inhibits entry of both marburgviruses and the ebov, indicating that they might interfere with a common pathway used by filoviruses to gain entry into target cells [ ] . the ssgp of a yet undefined function is produced through transcriptional editing and secreted in the form of a disulfide-linked homodimer that is exclusively n-glycosylated. while ssgp appears to share similar structural properties with sgp, it does not appear to have the same anti-inflammatory function as sgp [ , , ] . the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. the entry of the ebov into cells is initiated by interaction of the viral gp with host cell surface t-cell immunoglobulin and mucin domain (tim- ) receptors. upon receptor binding, the ebov is internalized into endosomes primarily via macropinocytosis [ ] [ ] [ ] . within the acidified endosome compartment of the host cell, the heavily glycosylated gp is cleaved to a smaller -kda fusogenic form by the low ph-dependent cellular proteases cathepsin l (catl) and b (catb), exposing residues in the receptor binding site. this allows the binding of gp to cholesterol transporter niemann-pick c (npc ), a step in the late endosome phase essential for virus-host membrane fusion and viral entry [ ] [ ] [ ] [ ] . cells where the npc function has been biochemically disrupted or cells lacking npc showed resistance to the ebov infection. cells from subjects with npc disease were resistant to the ebov because of defects in the npc protein [ ] [ ] [ ] [ ] . after complete fusion of the viral and host endosomal membranes via conformational change in gp , viral rna and its associated proteins are released into the host cell cytoplasm [ ] . once inside the cytoplasm of the host cell, the ebov suppresses the innate immune response via vp and vp proteins [ ] , and hijacks transcription and translation for robust genome replication and the production of new virions. the ribonucleoprotein (rnp) complex that mediates transcription and replication of the ebov genome comprises np, vp , vp , and l protein [ ] [ ] [ ] [ ] . vp is essential in the initiation of the ebov transcription, but is not required for viral replication. however, dynamic phosphorylation of vp is an important mechanism to regulate the balance between the transcription and replication processes in the ebov replication cycle [ ] [ ] [ ] . this unique property of vp allows the development of a genetically stable vp deleted ebov vaccine with protective efficacy in the mice and guinea pig models [ ] . the matrix proteins vp and vp associated with the viral lipid coat are important for virus structure and stability. both matrix proteins vp and vp contribute to the regulation of viral genome replication and transcription [ ] and the budding of the virus [ ] [ ] [ ] , an important step prior to viral egress [ , ] . this distinct replication cycle of the ebov serves as an attractive target for the development of therapeutic agents against the ebov (see figure and table ). human ebov hemorrhagic fever, characterized by uncontrolled viral replication together with immune and vascular dysregulation, has a case fatality rate of up to % [ ] . type i alpha/beta interferons (ifn-α/β), encoded by a single ifn-β and homologous ifn-α genes in humans, represent an essential element of host defense against virus infections, including the ebov [ ] . the human ebov infection is associated with robust ifn-α production-with plasma concentrations of ifn-α that greatly ( -to -fold) exceed those observed in other viral infections-but limited ifn-β production [ ] . the upon receptor binding of ebov gp with host tim- receptor, ebov is internalized into endosome via macropinocytosis. within the acidified endosome compartment of the host cell, under the action of the low ph-dependent cellular proteases cathepsins, the receptor binding site of gp to cholesterol transporter niemann-pick c (npc ) is exposed. this results in conformational change in gp , leading to complete fusion of the viral and host endosomal membranes in the late endosome and the release of viral rna and its associated proteins into the host cell cytoplasm. ebov then hijacks transcription and translation for robust genome replication and viral protein production under the action of ribonucleoprotein polymerase complex (rnp polymerase). the accumulation of gp , in the endoplasmic reticulum leads to endoplasmic reticulum overload response (er-overload) which, in turn, induces cytokine dysregulation via the activation of nuclear factor kappa b (nfκb) through the production of reactive oxygen species (ros). new virions are released through atp-dependent budding and egress from host cell membrane. currently available therapeutic agents that target the different steps of the ebov life cycle are described in table . ebov, protected from the host interferon response by its encoded vp and vp proteins [ , [ ] [ ] [ ] , produced a heavy viral load [ ] [ ] [ ] , cytopathic damages [ , , ] , and cytokine dysregulation in humans [ ] [ ] [ ] [ ] . the efficient productive replication of the ebov inside monocyte and macrophages leads to a massive release of proinflammatory cytokines/chemokines and reactive oxygen species (ros) [ , , , , [ ] [ ] [ ] , which in turn leads to diffuse endothelial cell dysfunction [ ] [ ] [ ] [ ] [ ] , disseminated intravascular coagulation [ ] [ ] [ ] , and vasomotor collapse [ ] [ ] [ ] . the infection of the antigen presenting dendritic cells [ ] [ ] [ ] [ ] and profound bystander apoptosis of lymphocytes [ , [ ] [ ] [ ] impairs the development of adaptive immunity [ , ] and ebov-specific cd + t [ ] [ ] [ ] , as well as cd + t cells [ ] that are important for the clearance of, and protection from, the ebov infection. infected monocyte-derived dendritic cells were impaired in the secretion of pro-inflammatory cytokines, the up-regulation of co-stimulatory molecules, and the stimulation of t cells [ ] . numbers of cd + and cd + t cells are substantially reduced in fatal human and nonhuman primate (nhp) infections before death [ , , ] . immune evasion by the glycoproteins of the ebola virus: implications on passive immunization and vaccine development the ebov is able to counteract both humoral and cellmediated immunity through its gp , and sgp [ , ] . the overexpression of mature gp , on the plasma membrane results in the masking of antigenic epitopes on gp , itself and the shielding of mhc-i and integrin β, leading to evasion of antiviral immunity. steric shielding of surface epitopes by the heavily glycosylated gp impairs the recognition and killing of ebov-infected cells by the natural killer and cytotoxic cd + t cell during an acute viral infection. it may also contribute to the persistent infection in the natural reservoir host to perpetuate the spread of the ebov [ ] [ ] [ ] . the sgp can evade host antibody-mediated response through "antigenic subversion" by eliciting non-neutralizing antibodies that cross-react with gp , . thus, the massive secretion of sgp by the ebov may prevent effective neutralization of the virus during an ebov infection and reduce the effectiveness of vaccines that rely upon neutralizing antibody responses against gp , [ , ] . some of the antibodies against gp may lead to enhancement of infectivity of the ebov via interaction with complement component c q, a phenomenon known as the antibody-dependent enhancement. the ebov initiates infection by binding its gp to its specific human receptor sites on the surface of human cells. the interaction of c q enhances binding between the virus-antibody complex and the c q ligands on the cell surface, promoting interaction between the ebov and its receptor. these infectivity-enhancing antibodies were virus species specific and were primarily correlated with immunoglobulin igg a and igm levels, but not with igg levels [ , ] . the presence of infectivity-enhancing antibodies against gp , in the ebov infection raises concerns about the effectiveness of gp-based ebov vaccines, and the use of passive prophylaxis or treatment with gp-based antibodies [ , ] . antibodies against gp of the ebov can be neutralizing, enhancing, or non-neutralizing and non-enhancing. neutralizing antibodies are produced in infection by the ebov at a relatively low frequency [ ] . some anti-ebov antibodies are known to be neutralizing in vitro but not protective in vivo, whereas other antibodies are known to be protective in animal models in vivo, but not neutralizing in vitro [ ] . investigations of anti-gp antibodies against the ebov showed that non-neutralizing antibodies induce interferon-inducible transmembrane proteins (ifitmp) production to restrict entry of ebov. favipiravir suppress viral rna polymerase. inhibit na + /k + -atpase that are important in the budding and egress of encapsulated ebov. ouabain digoxin digitoxin anti-oxidants suppress ros-dependent nfκb activation and cytokine dysregulation induced by gp , -induced er-overload. high dose n-acetylcysteine infusion chloroquine, amiodarone, dronedarone and toremifene administration is associated with an increased risk of qt prolongation and torsades de pointes. verapamil should be avoided in patient with hypotension. recognized gp epitopes in the sgp or non-essential mucin-like domain of gp , while neutralizing antibodies were specific to rbd in gp or conformation-dependent epitopes at the base of the gp , spike where gp meets gp . two neutralizing antibodies (kz and jp k ) against ebov-that recognize conformation-dependent epitopes comprising residues in gp and gp -were identified to have quite distinct mechanisms of neutralization. kz is a human recombinant igg neutralizing antibody derived from a human survivor of a natural ebov infection during the outbreak in kikwit, democratic republic of congo. kz has impaired recognition for the sgp and binding was dependent on the presence of gp residues which are not present in the sgp. kz is able to inhibit cathepsin cleavage of gp , . jp k , a monkey derived neutralizing monoclonal antibody against ebov, recognized the cleaved, fusion-active form of gp [ ] . f is a mice derived monoclonal igg antibody that neutralizes sudan ebov by preventing the conformational changes in gp , required for membrane fusion. both f and kz recognize gp -gp -bridging epitopes at the base of the gp , trimer, indicating that this overlapping epitope may be one of the key sites for neutralization of the ebov, and is thus a target for immunotherapy and a key goal of vaccine design [ ] . antibody subclass may be another important factor in protection against the ebov. igg isotype may offer more effective protection against ebov [ , ] . although fully protecting guinea pigs from infection, kz fails to slow viral replication and protect nhps from the ebov infection [ ] . in contrast, rvsv-ebogp [ ] [ ] [ ] [ ] and rchad-ebogp [ ] [ ] [ ] [ ] based vaccination have demonstrated both prophylactic and post-exposure protection in nhps [ ] . this was previously attributed to the protective action of ebov-specific cd + and cd + t-cell response induced by these vaccines in limiting infection and the inability of kz to completely block all entries of the ebov into cells and its subsequent explosive replication [ ] . rchad-ebogpbased vaccination is able to generate potent humoral and cell-mediated responses. significant antibody titers are detectable at weeks post vaccination [ , ] . cd + cellmediated immunity has been shown to play a critical role in protection against the ebov infection in nhps in rchad-ebogp-based vaccination [ ] . on the other hand, humoral rather than the cell-mediated response contributes to protection against the ebov infection in nhps in rvsv-ebogp-based vaccination [ , ] . candidate vaccines expressing the ebov gp or np protect rodents and nhps from the lethal ebov infection [ ] [ ] [ ] . humoral and cell-mediated immune responses are working together to provide protection against the lethal ebov infection. either response alone may be able to limit virus replication but both arms of the immune response are required to clear the infection [ , ] . vp proteins (vp , vp , vp , and vp ) are poor inducers of cell-mediated immunity and are inaccessible to the protective effect of vp-induced neutralizing antibodies because they are not found on the surface of virions or infected cells [ ] . however, the genetic sites of these internal proteins are susceptible to sirna and pmo interference. tkm-ebola (a sirna targeting l-polymerase, vp , and vp ) can be administered intravenously or subcutaneously in a lyophilized lipid nanoparticle formulation. tkm-ebola offers post-exposure protection against the ebov infection in nhps. the fda has approved an "expanded access" program for the use of tkm-ebola in patients with confirmed or suspected infections [ , ] . anti-sense phosphorodiamidate morpholino oligomers avi- effectively reduce viral load, diminish virallyinduced pathology, and improve survival of nhps with the ebov infection by targeting vp and vp mrna. through judicious placement of positive charges on the drug backbone, the drug is able to bind to a negative charge on the virus even if binding at one or more drugvirus base pairs are lost through mutation. this integration of dual targeting and charge complementation significantly lowers the likelihood of drug resistance through viral mutagenesis [ , ] . currently available therapeutic agents that are effective in targeting the ebov infection in cell or animal studies may include convalescent plasma, favipiravir, chloroquine, amiodarone, dronedarone, verapamil, clomiphene, toremifene, ifn-β, na + /k + exchangers, na + /k + -atpase pump inhibitors, and antioxidants. except for convalescent plasma and favipiravir, most of the therapeutic agents under review are acting against the non-mutable targets of the host cells which participate in the replication cycle of the ebov. they may also have a complementary role to conventional therapy in the management of the current ebov outbreak in west african countries (see table ). the who issued a consensus statement that the use of whole blood therapies and convalescent blood serum needs to be considered as a matter of priority in the recent ebov outbreak in west african countries [ ] . the development of neutralizing antibodies and t-cell responses are important for recovery from the ebov infection [ , ] . patients who are able to mount an immune response to the ebov will begin to recover in seven to ten days and start a period of prolonged convalescence [ ] . in survivors, early and increasing levels of igg, directed mainly against the np and the vp , were followed by the clearance of circulating viral antigen and activation of cytotoxic t cells. in contrast, fatal infection was characterized by impaired humoral responses, with absent specific igg and barely detectable igm [ ] . convalescent blood has been shown to improve survival of ebov-infected patients during the outbreak in kikwit in [ ] . immunity against ebov gp is sufficient to protect individuals against infection, and several vaccines based on ebov gp are under development including recombinant adenovirus, parainfluenza virus, venezuelan equine encephalitis virus, vesicular stomatitis virus, and virus-like particles [ ] . neutralizing human monoclonal antibodies is able to protect mouse and guinea pigs from lethal ebov. however, the protection was achieved only by treatment shortly before or after viral infection [ ] [ ] [ ] . the ebov can rapidly mutate to produce antibody-escape mutants. hence, antibody therapy may require hyperimmune polyclonal serum or a panel of monoclonal antibodies of different epitope specificities to be successful [ , ] . these studies have laid the foundation for subsequent clinical research on the development of monoclonal antibodies [ ] [ ] [ ] [ ] and utilization of a monoclonal antibody cocktail such as mb- [ ] , zmab [ ] , and zmapp [ ] in the treatment of the ebov infection in nhps. it is interesting to note that all three monoclonal antibody cocktails include one antibody that binds to or close to the glycan cap and that two of the three monoclonal antibody cocktails include at least one antibody that binds the gp / gp interface, indicating that these two regions may be especially important in protection against ebov [ ] . the treatment window of monoclonal antibody therapy can be extended by the co-administration of adenovirus-vectored interferon therapy. in a guinea pig model, monoclonal antibodies combined with adenovirus-vectored interferon given three days after infection resulted in % survival, a significant improvement over either treatment alone [ ] . a subsequent study showed that such a combination therapy is capable of saving % of ebov-infected nhps when initiated after the presence of detectable viremia along with symptoms [ ] . ( ) favipiravir (t- ; -fluoro- -hydroxy- pyrazinecarboxamide) favipiravir is a broad-spectrum inhibitor of viral rna polymerase that is able to inhibit the replication of many rna viruses. it is registered in japan for the treatment of influenza virus infection [ , ] . favipiravir is able to suppress the replication of the ebov in cell culture. favipiravir, initiated at day after ebov infection, induced rapid virus clearance, reduced the biochemical parameters of disease severity, and prevented a lethal outcome in % of mice lacking the type i interferon receptor [ ] . oral favipiravir taken twice daily for days is able to give % protection against an aerosol ebov infection in an immune-deficient mice model [ , ] . the survival benefit was suboptimal in nhps. only one of the six animals tested survived. studies using dosages that are two to five times higher and have duration longer than shown in influenza studies are being conducted for the human ebov infection [ ] . bcx , a synthetic adenosine analogue with a viral rna polymerase inhibitor function, is active against the ebov and marburg virus in rodent and cell culture. bcx completely protects nhps from the marburg virus infection when administered as late as hours after infection [ , ] . the antimalarial drug chloroquine is able to increase endosomal ph. an acidic endosomal environment is important for the ph-dependent activation of cysteine proteases catb and catl, the proteases responsible for the cleavage of ebov gp , essential for endosomal virus-host membrane fusion [ , , [ ] [ ] [ ] . however, proteolytic processing of the ebov glycoprotein has been demonstrated to be not critical for ebov replication in cell culture [ ] or nhps [ ] . a recent study using a catb and catl deficient mouse model for the study of the ebov infection demonstrates that catb and catl activity is not absolutely required for ebov replication. the ebov glycoprotein cleavage seems to be mediated through a broader spectrum of proteases making therapeutic approaches targeting limited proteases unlikely to be beneficial to combat ebov infections [ ] . a broad-spectrum small molecule that targets the catl cleavage of the ebov and inhibits the entry of a wide variety of viruses has recently been identified. it has been examined for the potential to develop into a potent broad-spectrum antiviral medication [ ] . multiple cationic amphiphiles including amiodarone, dronedarone, verapamil, clomiphene, and toremifene have been identified as potent inhibitors of the entry of the ebov in an npc -dependent fashion [ , ] . amiodarone used for the treatment of atrial fibrillation and ventricular cardiac arrhythmia can induce lipidosis with features similar to niemann-pick c disease [ ] . amiodarone and dronedarone, having basic pka and high water solubility at acidic ph, accumulates within late endosomal compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking, and inducing a niemann-pick c-like phenotype. in contrast to the niemann-pick type-c disease, they are not alleviated by cholesterol removal [ , ] . amiodarone, at concentrations that are routinely reached in human serum during anti-arrhythmic therapy ( . - . μg/ml), is a potent inhibitor of filovirus cell entry through late endosomes (ic . μg/ml for ebov), when induced as a niemann-pick c-like phenotype. significant inhibition is observed in most endothelial and epithelial cells (e.g. macrophage, monocyte, vascular endothelial cell), except for primary hepatocyte and fibroblast. the inhibitory effect of amiodarone on the entry of the ebov was dose-dependent and reversible upon removal of the drug. prolonged exposure to amiodarone will not lead to a compensatory change in the host cell. a similar inhibitory property is observed with the amiodarone-related agent dronedarone and the l-type calcium channel blocker verapamil [ , , , ] . both clomiphene and toremifene have anti-ebov activity in both the vero e (interferon-deficient african green monkey kidney epithelial cells) and hepg (human hepatocellular carcinoma) cell lines. the anti-ebov activity of clomiphene and toremifene is dependent not on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a niemann-pick c-like phenotype to inhibit viral entry at late endosome. clomiphene and toremifene do not disrupt the interaction between primed gp and npc , but mediate the entry block indirectly through npc by targeting other endosomal/lysosomal proteins involved in the cholesterol uptake pathway whose functions may be regulated by npc . clomiphene and toremifene at mg/kg every other day have been shown to result in a % and % survival rate, respectively, in ebov-infected mice compared with % mortality in the control group in an in vivo murine ebov infection model. they are effective in both male and female mice [ , ] . however, the therapeutic dose against ebov cannot be achieved with the oral clomiphene dose used for inducing ovulation in humans [ ] [ ] [ ] . the therapeutic dose against ebov with tolerable side effects can be achieved with toremifene at an oral dose used in the human trial for the treatment of advanced carcinoma of the breast [ ] [ ] [ ] [ ] . toremifene is well absorbed and > . % bound to plasma protein. toremifene undergoes extensive liver metabolism and enterohepatic recirculation. the majority of the toremifene dose is excreted as metabolites in feces. the long terminal half-life of oral toremifene may be due to both plasma protein binding and enterohepatic recirculation [ , ] . interferon-induced transmembrane proteins (ifitms) are expressed basally in the absence of ifn induction in both primary tissues and cell lines [ ] . an ifitm is able to inhibit the entry of viruses to the host cell cytoplasm; permit endocytosis, but prevent subsequent viral fusion; and release viral contents into the cytosol. the human ifitm locus is located on chromosome and composed of four functional genes: ifitm , ifitm , ifitm , and ifitm . ifitm p is a pseudogene. viruses that are restricted by ifitm proteins tend to fuse with host cell membranes in a late endosome or lysosome that precedes the induction of type i ifn in infected cells. viral escape from restriction by ifitm proteins could be more challenging than for antagonizing inhibitory factors that function at later stages of the virus life cycle because the opportunity for de novo synthesis of viral inhibitors is not available. all four human ifitm proteins are induced robustly by both type i and type ii ifns. ifitm is active against multiple viruses, including the ebov and hepatitis c viruses [ ] [ ] [ ] . ifnβ is able to induce interferon-inducible transmembrane protein production to restrict entry of the ebov [ ] . early post-exposure treatment with ifn-β significantly increased survival time of rhesus macaques infected with a lethal dose of the ebov, although ifn-β alone failed to alter the mortality rate. ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [ ] . amiloride and its derivatives are used as potassiumsparing diuretics to treat hypertension and congestive heart failure. apart from inhibiting epithelial na + channel and cellular na + /k + exchangers, these drugs could also affect the function of other less well-defined ionexchangers (na + /ca + and na + /mg + ), and disturb the equilibrium of other ions, such as mn + [ ] [ ] [ ] [ ] . the entry of the ebov into host cells is the first step of infection and a crucial determinant of pathogenicity. upon receptor binding between gp and host tim- receptors, the ebov is internalized into endosomes primarily via the macropinocytic pathway. amiloride is able to inhibit the uptake of many viruses that utilize the macropinocytic pathway for host cell entry [ ] [ ] [ ] [ ] . amiloride at non-cytotoxic dosages leads to potent dosedependent inhibition of the entry and infection of the ebov [ , ] . amiloride can lead to dose-dependent inhibition of rna synthesis. this may be due to a direct blockage of a nucleotide entry tunnel or catalytic site, or due to its effect on the equilibrium of mg + and mn + that are essential co-factors for polymerase activity and nucleotide insertion [ , ] . these novel antiviral mechanisms of amiloride may uncover new targets for drug discovery against the ebov. adenosine triphosphate (atp) is essential in multiple steps in the replication cycle of many viruses. na + /k + -atpase pump is located in the plasma membrane of all animal cells to maintain the cell membrane potential. budding of enveloped viruses is a complex phenomenon that requires concerted actions of many viral and host components. atp may affect multiple steps in the budding process [ ] . atp is required for the assembly and maturation of a number of enveloped viruses such as the influenza virus, vaccinia virus, retrovirus, and herpes simplex virus. the na + /k + -atpase pump inhibitors, ouabain, lanatoside c, strophanthidin, and digoxin are able to inhibit the replication of the influenza virus, newcastle disease virus, and vesicular stomatitis virus through an interferonindependent mechanism [ ] . digoxin and lanatoside c have been shown to inhibit vaccinia virus replication at non-cytotoxic doses [ ] . ouabain has shown antiviral activity against the influenza virus [ ] , herpes simplex virus [ ] , sendai virus [ ] , murine leukemic virus [ ] , cytomegalovirus porcine reproductive and respiratory syndrome virus [ ] , and human cytomegalovirus virus [ ] . one common feature shared by these viruses is that they all possess a lipid envelope. the ebov is an enveloped filamentous rna virus. the secondary matrix protein vp -apart from its role in the evasion of host immune response, nucleocapsid formation, and regulation of replication-has an important role in viral budding and egress. na + /k + -atpase atp a is detected to have a close interaction with vp of ebov during replication. ouabain, at a non-cytotoxic concentration of nm, is able to suppress the replication of the ebov in human mrc- cells [ , ] . among the three cardiac glycosides that may include digoxin, digitoxin, and ouabain, only digoxin is commonly used in clinical practice. ouabain, because of its poor oral availability, is used primarily as a research tool. further research should be conducted to investigate whether digoxin and other na + /k + -atpase inhibitors might play a role in the management of the ebov or other enveloped virus infections. the virus-associated glycoprotein gp , is responsible for the activation of human macrophages [ ] . the highly glycosylated mucin-like region of the gp subunit of gp , is cytotoxic to the host cells [ ] . the mucin-like region in gp leads to an accumulation of gp , at the endoplasmic reticulum, induces endoplasmic reticulum stress [ ] , and activates nuclear factor kappa b (nf-κb) [ ] . mutations of the ebov that lead to an enhanced accumulation of gp , in the endoplasmic reticulum were significantly more cytotoxic than wild-type virus [ ] . in human cells, the accumulation of protein in the endoplasmic reticulum will lead to endoplasmic reticulum overload response (eroverload) which activates nf-κb through the production of ros [ ] . as a major transcription factor for antiviral and immune stimulatory activities, nf-κb is thought to play an important role in the induction of pro-inflammatory molecules, such as interleukin- β (il- β), and tumor necrosis factor α (tnf-α), upon cellular responses against a virus infection [ ] . the cytokine dysregulation of the ebov involves massive ros, nf-κb, tnf-α, and il- β activation [ , ] . the effectiveness of antioxidant therapy for the ebov infection indicates the importance of ros in the pathogenesis of the ebov [ ] . the activation of nf-κb by er-overload is ros-dependent [ ] . nf-κb-induced cytokine dysregulation of novel h n pneumonia has been shown to be suppressible by high-dose n-acetylcysteine (nac) antioxidant therapy at mg/kg continuous infusion daily [ ] . given the poor oral availability of nac in the range of % to % in humans [ ] , a therapeutic dose of nac equivalent to the intravenous route can hardly be delivered by oral preparation. nac is a category b drug for pregnancy and is affordable, with a wide therapeutic window. nac has an established safety profile even in high doses and prolonged use in humans [ ] [ ] [ ] . cytokine dysregulation is a common feature in the ebov infection and is associated with an enhanced mortality [ ] [ ] [ ] [ ] . antiviral medications directed against the mutable viral determinants of the ebov cannot directly prevent cytokine dysregulation. the early endothelial vascular damage characteristic of the ebov infection is not a direct effect of virus-induced cytolysis of endothelial cells, but is due to cytokine dysregulation resulting from massive release of proinflammatory cytokines/chemokines and ros by infected macrophage and monocytes [ ] [ ] [ ] . lymphocytes are resistant to the ebov infection. cytokine dysregulation may also contribute to the diffuse bystander apoptosis of lymphocytes [ , [ ] [ ] [ ] . with the safety profile of nac, if the therapeutic efficacy of a high-dose nac antioxidant therapy to manage ebov-induced cytokine dysregulation is confirmed, it may revamp the future management of the ebov infection. there is a desperate need for a viable treatment regimen in africa to engender hope and encourage people with symptoms and their close contacts to seek medical treatment, so as to limit the spread of the disease. this also helps to recruit and maintain adequate medical staff who are at high risk of contracting the disease. a proposed regimen against the human ebov infection based on available medications and information from in vivo animal testing and in vitro cell culture is attached (see tables and ). this regimen contains a cocktail of currently available medications that can target the different steps in the replication cycle of the ebov aiming to suppress viral proliferation. it has been shown that viral load is major contribution to survival in both human and animal studies [ ] [ ] [ ] ] . through viral load suppression, we may be able to prolong a patient's survival in order to allow the development of natural body immune defense against the ebov. the ebov has undergone a rapid mutation during its spread through humans [ ] [ ] [ ] . the ebov is an rna virus the replication of which is highly error prone with nearly one viral mutation occurring during each cycle of replication. this extremely high mutation rate leads to significant genetic and antigenic diversity that allows the ebov population to evolve resistance to antiviral medications and vaccines [ , ] . a combination therapy has been used in the treatment of rna virus infections, such as the human immunodeficiency virus (hiv) [ , ] and hepatitis c [ , ] to minimize the development of drug resistance. given the broad cell tropism and high replication rate of the ebov due to the potent suppression of both innate and adaptive immune responses of the host, patients with the ebov infection have an extremely high viral load. the selective pressure in the presence of the high mutation rate and viral load during the human ebov infection make the evolution of the ebov viral strains resistant to a single drug inevitable. the currently available medications in the proposed regimen-which is a treatment regimen containing a cocktail of antiviral medications targeting the different steps of the ebov replication in order to achieve maximal suppression of viral replication and to prevent the rapid development of resistance to favipiravir, the only drug in the regimen that is directed against a mutable target of the ebov-has been shown to reduce the replication of the ebov. [ ] [ ] [ ] . the current ebov vaccine (rvsv-ebogp and rchad-ebogp) and therapeutic agents (zmapp, tkm-ebola, pmo avi- , and favipiravir) under development are directed against the mutable targets of the ebov, and their effectiveness is limited by viral mutation. the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. most of the therapeutic agents in this current review are directed against nonmutable targets of the host which is independent of viral mutation. these medications are fda-approved for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. the primary target of the ebov is the mononuclear phagocytic system. the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells during the advanced stage of the disease [ , , ] . the ebov may produce a viral load of up to virions per ml serum in terminally ill patients [ ] . oral amiodarone prophylaxis, by inducing a niemann-pick c-like phenotype in the cells of the mononuclear phagocytic system, may prevent viral entry into these cells during needle stick injury. through protection of the mononuclear system by our prophylaxis and cocktail therapy, we hope to offer a better chance of survival to these patients by allowing them to develop a natural body immune defense against the ebov infection. the liver, containing the largest number of fixed tissue macrophages (kupffer cells), as part of the reticuloendothelial immune defense system of the body, is a major target for the ebov infection [ , ] . the ebov replicates to high titer in the liver [ ] . hepatic apoptosis may play a role in the pathogenesis of the ebov infection [ ] . toremifene is added to the treatment regimen for hepatic protection because amiodarone does not exert inhibitory action against the ebov in hepatocyte. however, both amiodarone and toremifene can increase qtc and the risk of torsades de pointes. therefore electrocardiogram should be carefully monitored if both drugs are to be used. amiodarone, favipiravir, and toremifene are available and stockpileable in oral preparations. these properties are advantageous in outbreak situations and contingency planning of a potential ebov epidemic or pandemic. the avoidance of intravenous administration will prevent needle stick injury in healthcare workers caring for the infected patients. ifn-β may have potential as an adjunctive postexposure therapy for high-risk exposure, such as needle stick injury, by inducing ifitm to limit entry of the ebov. post-exposure ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [ ] . the reduction in viral load and cytokine dysregulation coupled with optimal supportive therapy may improve the chance of survival of the host to allow the development of natural immunity to control the underlying ebov infection. ifitm is active against multiple viruses, including the ebov [ , ] and hepatitis c ml of blood may contain to virions in terminally ill patient. prophylactic amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately from ebov during needle stick injury and accidental exposure and allow time for the consideration of ifn-β, toremifene, favipiravir and convalescent blood serum therapy. amiodarone is unable to protect hepatocyte from ebov infection. both amiodarone and toremifene can increase the risk of qt prolongation and torsades de pointes. the recommended dosage for treatment of human ebov infection may be to times higher than influenza studies. please confirm the recommended dose with the drug company. n-acetylcysteine intravenous infusion at mg/kg/day to control cytokine dysregulation (e.g. add g of intravenous preparation of n-acetylcysteine into each liter of intravenous replacement fluid). [ , , , ] . interferon induced ifitm plays an important role in the treatment of human hcv infection by inhibiting entry of hcv into the host cell [ ] . six million international units (miu) of ifn-β intravenous administration is as effective as a three miu twice-daily regimen for treatment of the hcv infection [ ] , but has lesser side effects that require discontinuation of the medication [ , ] . as the aim of ifn-β therapy in our regimen for post needle stick prophylaxis against the ebov infection is to induce ifitm to limit viral entry, the dose of ifn-β for the post needle stick prophylaxis [ , ] or induction therapy [ , ] for hcv infection in humans is chosen. once infection is fully established, ifn-β are replaced by convalescent blood serum and high-dose nac infusion for providing passive humoral immunity and for the control of ros-dependent nf-κb-induced cytokine dysregulation respectively. the ebov is classified as biosafety level pathogen and is classified by centers for disease control and prevention as a category a agent of bioterrorism with no approved therapies and vaccines for its treatment but carrying a high potential for large-scale dissemination. recent political, economic, military, and religious turbulence around the world raises concerns that the ebov might be used as an agent of bioterrorism [ ] [ ] [ ] . the recent ebov epidemic is spiraling out of control in west africa. the containment measures that worked in the past, such as isolating those who are infected and tracing their contacts, have failed due to an exponential rise in infected patients. although the short-term (threeand six-week) probability of international spread outside the african region is small, the risk of the extension of the outbreak to other african countries followed by international dissemination on a longer time scale is not negligible, indicating that this public health emergency has the potential to grow to extraordinarily destructive dimensions [ , ] . although several promising therapeutic agents and vaccines against the ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced [ ] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions capable of managing the enhanced viral replication and cytokine dysregulation of the human ebov infection should be explored and stockpiled as contingency preparation for the worst-case scenario of an impending human ebov pandemic [ ] . like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress which, in turn, lead to cytopathic damage, cytokine dysregulation, and death of the host. these non-mutable key steps inside the host may be novel targets for future therapeutic strategies against these rapidly mutating viruses. if the efficacy of amiloride, digoxin, amiodarone, and high-dose nac antioxidant therapy against the human ebov infection is confirmed, the availability and affordability of these stockpileable oral regimen are for those workers who are already on amiodarone prophylaxis with a loading dose of amiodarone mg p.o. twice daily for days followed by maintenance amiodarone mg p.o. daily. electrocardiogram and thyroid function should be monitored. monitor for side effect of thrombocytopenia and proteinuria. intravenous dosage of ifn-β that are used for human hepatitis c virus infection to induce ifitm to limit viral entry. intravenous regimen is for those workers who have not been on amiodarone prophylaxis and agreed for the insertion of a central venous line for drug administration. intravenous amiodarone should be administered via central venous line to avoid phlebitis. the dosage for treatment of frequently recurring ventricular fibrillation and hemodynamically unstable ventricular tachycardia is recommended because it can achieve therapeutic drug level immediately after the first dose of amiodarone. agents make them ideal medications in pandemic situation and in countries with limited resources. they may have a complementary role to other antiviral medications to prevent the emergence of resistant strains. this may also signify a major breakthrough in future management of the ebov infection. additional file : multilingual abstracts in the six official working languages of the united nations. the authors declare that they have no competing interests. authors' contributions kyl and gwyn contributed to the conception, drafting, and writing of the paper. kyl, gwyn and fc revised the draft paper. all authors read and approved the revised paper. world health organization ebola response roadmap situation reports world health organization statement on the who consultation on potential ebola therapies and vaccines world health organization: ethical considerations for use of unregistered interventions for ebola viral 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• no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord- -w bpeqzg authors: wong, samson sai-yin; wong, sally cheuk-ying title: ebola virus disease in nonendemic countries date: - - journal: journal of the formosan medical association doi: . /j.jfma. . . sha: doc_id: cord_uid: w bpeqzg the west african outbreak of ebola virus disease was unprecedented in its scale and has resulted in transmissions outside endemic countries. clinicians in nonendemic countries will most likely face the disease in returning travelers, either among healthcare workers, expatriates, or visiting friends and relatives. clinical suspicion for the disease must be heightened for travelers or contacts presenting with compatible clinical syndromes, and strict infection control measures must be promptly implemented to minimize the risk of secondary transmission within healthcare settings or in the community. we present a concise review on human filoviral disease with an emphasis on issues that are pertinent to clinicians practicing in nonendemic countries. the largest outbreak of ebola virus disease (evd) in history has renewed interest in filoviruses and has provided an unprecedented impetus to the development of new therapeutics and vaccines for this highly lethal infection. hemorrhagic fevers caused by ebola and marburg virusesdalso collectively known as filoviral hemorrhagic fever (fhf)dpreviously caused dramatic, albeit limited, outbreaks in central africa. their impact on global health was rather small (except in the realm of biological warfare research) because of the high mortality rate, lack of effective antiviral therapies and vaccines, and potential for person-to-person transmission. the west african outbreak of evd proved that these filoviruses should no longer be considered as merely regional problems. a short review of evd and its clinical relevance to the nonendemic countries is presented. the current epidemic is caused by zaïre ebolavirus; however, references will also be made to the related marburgvirus, which shares many virological, clinical, and epidemiological characteristics. conflicts of interest: the authors have no conflicts of interest relevant to this article. the order mononegavirales consists of enveloped, nonsegmented, negative-sense, single-stranded rna viruses. the family filoviridae comprises three genera: cuevavirus, ebolavirus, and marburgvirus. in , the sole species of cuevavirus, lloviu cuevavirus, was described. it was discovered during an investigation of massive die-offs of miniopterus schreibersii bats in france, spain, and portugal in ; and the virus was detected in bat carcasses collected from northern spain. the genus ebolavirus the name "filovirus" describes a unique morphological characteristic of the viruses. the virions are generally filamentous (in latin, filum means "thread") with a diameter of approximately nm and a highly variable length of e , nm. they may also appear as branched filaments, short rods, u-shaped, circular, or hairpin-shaped. , the genomes of ebov and marv are approximately kb and consist of seven genes (from the to end): nucleoprotein (np), vp , vp , glycoprotein (gp), vp , vp , and rna-dependent rna polymerase (l). , ebolavirus expresses an additional protein through transcriptional editing of the gp gene. in addition to gp, a smaller secreted glycoprotein (sgp) is produced and excreted extracellularly. , at c, ebov and marv are stable and resist desiccation, which probably explains their stability in aerosol droplets. , they are however inactivated by heat and common disinfectants such as detergents, phenolics, and hypochlorites. the usual heat treatment of clinical samples at c for minutes may fail to render the specimen noninfectious. thermal inactivation at c for minutes or c for minutes is necessary. e gamma irradiation readily inactivates the filoviruses, although this method may not be readily available in routine clinical or laboratory settings. the genetics and molecular biology of the filoviral genome have been previously reviewed. , in addition to the essential functions for viral replication and assembly, many viral proteins exert their effects on the host immune system and may contribute to the pathogenesis of the infection (table ) . , , e , , , for example, vp and vp inhibit the normal antiviral activities of type i interferons at multiple steps of the pathway, whereas sgp may contribute to immune evasion by absorbing anti-gp antibodies (i.e., antigenic subversion). e because of the essential roles of many viral proteins in replication and assembly, some viral proteins (e.g., vp and vp ) are potential targets for antiviral agent development. , in recent years, the pathogenesis of fhf has been better elucidated. filoviruses are pantropic with the ability to infect different host cell types. the initial cells in which the viruses replicate are likely dendritic cells, macrophages, monocytes, and kupffer cells at the site of entry. a large number of lectins (e.g., dc-sign and l-sign) and immunorecognition receptors [i.e., triggering receptors expressed in myeloid cells (trem)] can serve as receptors for the viruses. after the initial multiplication in the aforementioned cell types, the viruses are transported to the reticuloendothelial system (e.g., lymph nodes, spleen, liver) and other organs where infection of other cell types occur. the resulting massive necrosis and end organ damage are reflected in the histopathology of human and primate infection models with necrosis in the liver, kidneys, lungs, lymphoid tissues, and other organs. , in addition, various mechanisms contribute to the development of coagulopathy and disseminated intravascular coagulation, which is a hallmark of viral hemorrhagic fevers. patients with fhf develop significant platelet dysfunction (which is not merely accountable for by thrombocytopenia), and this is contributed to by platelet activation and decreased vascular endothelium production of prostacyclin. another important target in the pathogenesis of fhf is the endothelium. human and primate endothelial cells are susceptible to infection by ebov, although direct cyopathic effects are not an important factor in the development of vasculopathy and coagulopathy. the release of vasoactive table filovirus genes and their functions. , , e , , , factors (e.g., nitric oxide) or cytokines and chemokines from monocytes and macrophages (e.g., tumor necrosis factor-alpha, interferons, monocyte chemoattactant protein- , interleukin- ) could be important in the genesis of vascular damage. the combined effects of increased secretions of proinflammatory cytokines, activation of the coagulation cascade, consumption and/or reduced production of protein c, thrombocytopenia and platelet dysfunction, hepatic damage with impaired production of coagulation factors, and vascular damage contribute to the development of coagulopathy in fhf. protective humoral and cellular immune responses can be demonstrated in patients who survive, and antibody levels persist for years. in addition to the various immune evasion mechanisms alluded to previously [e.g., binding of anti-gp antibodies by sgp (i.e., immune subversion), suppression of innate immune responses, and inhibition of interferon pathways], other potential virulence and pathogenic mechanisms of filoviruses such as the np and vp proteins have been identified. the generation of antibodies towards gp is critical for the protective efficacy of vaccines. ebolavirus and marv are geographically restricted to and cause outbreaks in sub-saharan africa; however, rebov is found in nonhuman primates and pigs in the philippines (table ) . , , , , , the first filovirus was discovered in after an outbreak of marburg hemorrhagic fever infection in germany. this infection originated from cercopithecus aethiops monkeys that were imported from uganda. in , the first natural outbreak of zebov occurred in northern zaïre (now called the democratic republic of the congo). in , sebov was discovered in an outbreak in nzara in sudan. in e , bebov caused an outbreak in uganda, followed by another outbreak in the democratic republic of the congo in . , in , tafv was discovered in a swiss biologist who acquired the infection in the republic of côte d'ivoire (i.e., the ivory coast). in , rebov was first discovered in monkeys (macaca fascicularis) that were exported from the philippines to reston, virginia, usa the infected monkeys were subsequently exported to italy ( e ) and to texas ( ) . cynomolgus macaques in the philippines are naturally infected, as are pigs. the rebov virus appears to be nonpathogenic to humans. e human filovirus infections are primary zoonotic diseases with a high propensity for interpersonal transmission. the primary animal reservoir of ebov and marv are bats (especially fruit bats). bats support long-term viral replication without developing the disease. virological and serological studies have confirmed filoviral infection in diverse bat species. , , in the philippines, china, and bangladesh, there is also evidence of rebov (and possibly other filoviral) infection in bats. e cases of fhf have been epidemiologically linked to contact with bat carcasses, spelunking among tourists, and outbreaks among gold miners who work in caves. e peak seasons of bat marv infection have been correlated with the incidence of human "spillover"infections. primates are also naturally infected with filoviruses, although they are likely to be infected by natural reservoir hosts (e.g., bats) and are not considered important reservoirs. , primates, nevertheless, remain important vectors for the introduction of the disease into humans in rural africa, and wild animal mortality sometimes precede human outbreaks of evd. other mammals that can be infected by evd include pigs (especially by rebov and zebov) and dogs; however, their role in causing human evd outbreaks is uncertain. e human infections typically occur with a few patterns of transmission. inhabitants of endemic areas, especially individuals dwelling in forests with occupational exposure to wild animals (e.g., hunters and people handling bushmeat), may develop symptomatic or subclinical infections, presumably because of the exposure to animal reservoirs of the virus. in some cases of human infection, a history of direct exposure to animal carcasses (such as primates, bats, duiker antelopes, and porcupines) or indirect exposure to bats in caves has occurred. e , , these single or multiple introductions to human populations may result in short chains of human transmission. when fhf patients are admitted to healthcare facilities where infection control measures are inadequate, a large outbreak may occur with transmission to other patients and healthcare workers. hospitals have been a major amplifying and disseminating factor in many previous outbreaks of fhf in africa. direct person-to-person transmission of ebov and marv occurs via blood or body fluids by percutaneous inoculation or by mucosal exposures. e during an outbreak of sebov, the virus was detected by viral culture or reverse transcription polymerase chain reaction (rt-pcr) in patients' saliva, skin swab, stool, tears, breast milk, and semen, but not in environmental samples. in view of the persistence of the viruses in semen and breast milk at days and days, respectively, after illness onset, , the use of condoms and withholding breastfeeding are recommended during convalescence. more than % of patients in the kitwit outbreak in the democratic republic of the congo had secondary cases in the household; the risk was highest for people with direct physical contact with the body fluids of symptomatic patients and exposure to patients in the late stages of the disease; this finding can be explained by the high viral load at this phase of disease. reusing needles or other medical instruments without adequate sterilization, needle stick injuries, lack of isolation facilities, and inadequate and inappropriate use of personal protective equipment all contribute to explosive hospital outbreaks. african burial customs of washing dead bodies and transporting bodies without barrier precautions further transmit the disease in the community. , , , infected pigs can transmit rebov to nonhuman primates via the airborne route and nonhuman primates can be infected experimentally by the inhalation of droplets (droplet size of . e . mm), although parenterally infected primates do not transmit the infection via the airborne route. e true airborne transmission of ebov between humans has likewise not been documented. up to % of patients in some ebov outbreaks may have no known contact or exposure history, although it remains unclear whether this is because of fomites, true airborne transmission, or inadequate investigation. , , , table outbreaks of human filovirus infection. , , , , , virus the ongoing evd outbreak at the time of this writing is unique in two aspects: ( ) its unprecedented scale and ( ) its origination in west africa (guinea) rather than in central africa. the first case occurred in guinea in february , and subsequently spread to the african countries of liberia, sierra leone, nigeria, senegal, and mali. in october , the world health organization declared the epidemic over in senegal and nigeria. , further epidemiological investigations suggested the index case was probably a year-old child who died on december , in southern guinea. the infection was subsequently transmitted from the child to family members, a village midwife, and a healthcare worker; some of these patients later caused outbreaks in different areas of guinea. ebola virus disease has caused infection in local healthcare workers of whom ( %) workers died (as of january , ); four of these infected healthcare workers were citizens of the united states of america, spain, and the united kingdom who returned to their respective countries for further management. on the other hand, asymptomatic infection was described in , as evidenced by seroconversion in % of close contacts of patients, in two zebov outbreaks in gabon. in % ( / ) of the seroconverted asymptomatic contacts, rt-pcr of the peripheral blood mononuclear cells was positive for the virus, and viremia persisted up to weeks in some contacts. another line of evidence for asymptomatic infections comes from seroprevalence studies in africa, in which . e . % of the surveyed population in central africa was seropositive for ebov with the seroprevalence consistently higher among forest-dwelling populations and hunters. e the incubation period of evd varies from days to days (commonly, e days), but a recent analysis of the cases in the zebov outbreak in kitwit suggested that the mean incubation period was . days, and the maximum incubation period was up to days. a biphasic illness has been described with an apparent remission of e days in between. , , e the disease usually begins abruptly with nonspecific symptoms such as fever ( e % of patients, ut infra), malaise ( e %), headache ( e %), sore throat, odynophagia or dysphagia ( e %), hiccoughs ( e %), and nonproductive cough ( e %). abdominal pain ( e %) or nausea and vomiting ( e %) often precede the onset of diarrhea ( e %), usually days after the onset of illness). the abdomen can be tender on palpation. in the absence of supportive therapy, diarrhea and vomiting may lead to fluid depletion and electrolyte disturbances such as hypokalemia. other symptoms include arthralgia or myalgia ( e %), chest pain ( e %), and conjunctival injection ( e %). a diffuse erythematous rash ( e %) appears towards the end of the st week which will later desquamate. the nonpruritic rash appears first on the trunk, and then spreads to the entire body with sparing of the face. three early symptoms of bilateral conjunctival injection, rash, and sore throat are suggestive of evd in the differential diagnosis. after the appearance of the rash, patients may either gradually recover or, in severe cases, progress over e days to the full-blown hemorrhagic fever syndrome with petechiae ( %), gum bleeding ( e %), melaena ( e %), hemoptysis ( e %), hematemesis ( e %), epistaxis ( e %), hematuria ( e %), menorrhagia, bleeding at venipuncture sites ( e %), and show features of disseminated intravascular coagulation. , the typical hemorrhagic fever picture, however, occurs only in approximately % of the patients (range, e %). , other manifestations in the late stage include evidence of multiorgan failure such as circulatory shock, obtundation, tachypnea, renal shutdown, convulsion, delirium, and coma. fever is often absent at this stage. death often occurs between days and , whereas patients who survive will show improvement around days to . intrauterine death is common in pregnant patients. mortality among pregnant women is substantial but may not be significantly higher than for nonpregnant patients; and pregnant women do not have an increased susceptibility to the infection. , survivors tend to improve from the nd week of illness. they make a slow recovery during which arthralgia (which could be asymmetric and migratory and often involves the large joints), uveitis, conjunctivitis, orchitis, parotitis, hearing loss, and tinnitus may occur. , chronic infection by filoviruses has not been documented, but male patients may shed the virus in the semen for e days after the onset of illness, and the transmission of marv has occurred via sexual intercourse. , , , common laboratory findings include lymphopenia, thrombocytopenia, elevated aminotransferases, hyperproteinemia, proteinuria, and prolonged prothrombin and activated partial thromboplastin time. with the progression of disease, evidence of disseminated intravascular coagulation and renal failure will appear. disease progression is also associated with worsening lymphopenia and rising antigenemia. compared to survivors, patients with fatal evd are more likely to have tachypnea. patients with disease also have a significantly higher level of viremia and a much weaker humoral immune response to the infection. significant differences in a number of cytokine and chemokine levels have also been detected between patients with nonfatal and fatal evd; in particular, the levels of many proinflammatory cytokines and nitric oxide are higher in nonsurvivors (with the possible exception of bebov), whereas the level of t cells and cd þ t cells were lower. , , e a high viral load is of prognostic significance. in sebov fhf, patients with fatal cases had an average of e (up to ) copies of rna/ml of serum, compared to the approximately copies of rna/mll of serum in survivors. patients with marv fhf likewise have high levels of viremia in blood with a median level of .  (range, .  e .  )/ml of serum serum. there are no pathognomonic signs or symptoms in the early stages of evd. the most useful history is epidemiological linkage to possible cases by travel history or by contact with known or suspected cases. laboratory investigations are necessary to confirm the diagnosis. other differential diagnoses (vide infra) should be excluded, as appropriate. all clinical specimens must be handled with great care from their collection to transport and testing in the laboratories. laboratory-acquired infection of ebov has occurred via percutaneous exposures. marburgvirus retains its infectivity in dried blood for at least days. filoviruses can be inactivated by heat [ c for minutes or c for minutes; or c for minutes in the presence of . % (final concentration) sodium dodecyl sulfate or . % (final concentration) tween ] or inactivated by % sodium deoxycholate solution, acetone, diethyl ether, % formalin, methanol, sodium hypochlorite, glutaraldehyde, % peracetic acid, phenolic disinfectants, and osmium tetroxide (used in fixation for electron microscopy). ultraviolet light is an effective means for surface disinfection. depending on the type of specimen and testing methodology, treatment of the sample with either triton x- , tween , sodium dodecyl sulfate, beta-propiolactone, chloramine b, or % acetic acid (ph . ) should be done before routine hematological, biochemical, and serological testing. heat inactivation is recommended for blood sodium, potassium, magnesium, urate, urea, creatinine, bilirubin, glucose, and c-reactive protein; however, other methods of inactivation would be necessary for calcium, phosphate, albumin, transaminases, gammaglutamyltransferase, and creatine kinase determination. standard virological techniques also apply to the diagnosis of fhf. these include viral culture, electron microscopy, serological tests using antigen or antibody detection, and nucleic acid amplification. , filoviruses can be cultivated from clinical specimens (especially blood and liver samples); however, because of the associated biohazards, a viral culture is notdand should not bedroutinely performed, except in facilities that can handle biosafety level agents. electron microscopy, which has excellent specificity because of the unique morphology of filoviruses, is also not routinely performed because of biosafety considerations, limited availability of electron microscope facilities in routine diagnostic settings, and the relatively high viral load necessary for visualization. the detection antibodies [e.g., immunoglobulin m (igm) or immunoglobulin g (igg)] is commonly achieved using immunofluorescent assays and enzyme-linked immunoassay (elisa) against recombinant np, gp, vp , vp , or vp antigens. e the appearance of igm and igg antibodies occurs at approximately days and e days, respectively, after the onset of illness. various antigen detection assays have been developed for the diagnosis of fhf and some have been used in field situations. the techniques include antigencapture elisa, immunofluorescent assay, dot-immunobinding assay, immunofiltration assay with different genus-specific or species-specific reactivity towards common targets such np, gp, and vp proteins. , , a practical limitation of these serological assays is the limited availability of these tests in laboratories in nonendemic countries. nucleic acid amplification is the diagnostic test of choice because of its high sensitivity (especially in the early phase of illness); its ability to differentiate between different agents of viral hemorrhagic fever; and its relatively lower biohazard, if the viruses are appropriately inactivated; and because antigen and antibody assays are often unavailable in laboratories in nonendemic countries. when the viral load is determined by quantitative assays, prognostic information can also be obtained. the most commonly used test is rt-pcr. a reverse transcriptioneloop-mediated isothermal amplification assay has also been developed for ebov and marv. , all diagnostic nucleic acid amplification tests must be adequately validated before being used clinically. if appropriately validated, the use of multiplex pcr/rt-pcr allows simultaneous detection of multiple pathogens that cause hemorrhagic fever. , the provision of nucleic acid amplification tests should preferably be centralized in national or regional reference laboratories to ensure adequate biosafety containment and quality of results. the rna of ebov can be detected in the sera by rt-pcr e hours earlier than by antigen capture; in some studies, it is detectable on day of the illness. , however, the viral load gradually reaches its peak at approximately e days after the onset of the disease. retesting is therefore necessary if rt-pcr is initially negative but clinical suspicion is high, especially when the first sample was obtained within days of the onset of disease. blood is the most commonly used sample for rt-pcr. oral fluid specimens can be a viable alternative to blood samples for rt-pcr in situations in which blood taking may be difficult or infeasible. common targets for nucleic acid amplification include the l, gp, and np genes. , , , , clinical management and vaccine development treatment of fhf is primarily supportive owing to the unavailability of approved, specific antiviral agents. concurrent infections such as malaria or bacterial sepsis should be treated, as appropriate. fluid and electrolyte replacement, blood product transfusion, renal replacement therapy, and ventilatory support such as extracorporeal membrane oxygenation should be administered, as necessary. , various experimental therapeutic approaches have been attempted in experimental animals or clinically; however, no randomized controlled trials prove their efficacy. examples include recombinant inhibitor of factor vii (rnapc ), recombinant human activated protein c, and interferon-beta. e as in cases of other severe viral infections, convalescent plasma from recovered patients has been used to treat fhf. this was deployed with apparent benefits in the evd outbreak in kitwit. the world health organization (who) published a guideline on the collection and preparation of convalescent plasma for use in the outbreak situation; however, the who recognizes the uncertainties in the efficacy of this treatment. cocktails of monoclonal antibodies have similarly been used recently with some success in reducing the mortality of ebov infection in nonhuman primates. these antibodies have been produced in plants and in mice. e based on these studies, an optimized cocktail of plant-derived monoclonal antibodies, called the zmapp, was produced; it protected % of rhesus macaques infected up to days with ebov. these antibodies have been used experimentally for treating human ebov patients in the west african outbreak, although the actual benefit to human evd remains to be confirmed. a second approach to the treatment of fhf lies in the development of specific antiviral agents. a current nucleotide analogue is favipiravir (t- ), which was developed and approved in japan for the treatment of influenza. favipiravir inhibits viral rna-dependent rna polymerase of the influenza virus. it was subsequently found to exhibit in vitro antiviral activities against certain other rna viruses such as bunyaviruses, arenaviruses, flaviviruses, alphaviruses, norovirus, and ebov. e animal studies also demonstrate the efficacy of favipiravir in the treatment of junín virus, arenavirus, and ebov hemorrhagic fevers, and the drug was used to treat human evd in the west african epidemic. e a dosing regimen of favipiravir for use in a clinical trial for the treatment of evd has been published. another nucleotide analogue, brincidofovir (a lipid conjugate of cidofovir), was previously developed to treat infections due to dna viruses such as adenoviruses, herpesviruses, and orthopoxviruses; the drug was granted emergency investigational new drug applications in october by the united states (us) food and drug administration for evaluation in evd treatment, and a clinical trial was started in january in monrovia, liberia. e the nucleoside analogue bcx was recently shown to inhibit rna polymerase of negative-and positive-sense rna viruses via chain termination effects. its in vivo activity against marv was demonstrated in guinea pigs and cynomolgus macaques. the development of bcx for human clinical trials will require a long time. another approach involves the screening of currently available nonantimicrobials for their activities on filoviruses. compounds such as selective estrogen receptor modulators (e.g., clomiphene, toremifene), amiodarone, dronedarone, and verapamil have antifilovirus activity in cell cultures and/or murine models. , in addition, rna interference using small interfering rnas provide postexposure protection of animals infected with ebov and marv. e a third approach to the specific management of filoviral infections explores the potential of postexposure prophylaxis. such regimens would benefit exposed healthcare workers and other social contacts, and laboratory staff experiencing accidental exposures. protective immunity towards filoviruses does exist, as demonstrated in the possible benefits of convalescent plasma and animal studies, in which humoral immunity (i.e., igg) protects against ebov infection. animals studies also confirm that passive immunization by neutralizing monoclonal antibodies is protective in primates. previously examined filovirus vaccine candidates that elicit protective humoral immunity experimentally include ebov-like particles containing gp, np, and vp ; replication-deficient ebov mutants that lack the vp gene; and ebov gp-containing fragment or fusion protein. e a phase clinical trial has tested dna vaccines encoding the glycoproteins of ebov and marv. one of the most promising vaccine candidates for filoviruses is the recombinant vesicular stomatitis virus-based vaccine system that expresses filoviral gp. this system elicits protective immunity against zebov, sebov, bebov, and marv; it also offers substantial postexposure prophylaxis in primate and murine models. e the vaccine was used for postexposure prophylaxis in a laboratory staff person who sustained a needle stick injury with zebov in . a similar vaccine system used replication-defective recombinant adenovirus that expressed ebov gp was protective in animal models. , phase clinical trials of the vesicular stomatitis virus-based and adenovirus-based vaccines began in late . ebola virus disease as a problem in nonendemic countries: issues on prevention and control the containment of fhf outbreaks in endemic countries requires substantial resources in coordination between the public health system and other authorities of the countries, surveillance of the disease, education and engagement of local citizens, isolation and treatment facilities, and laboratory support. , these requirements are often beyond the capability of endemic countries. significant international assistance is usually needed to contain major outbreaks. the discussion on these public health issues is beyond the scope of this article. for health authorities in nonendemic countries, a preparedness plan for emerging infections is an indispensable component of the public health system. the development and adoption of preparedness and response plans for emerging infectious diseases are first fostered in anticipation of pandemic influenza. the outbreaks of severe acute respiratory syndrome (sars), pandemic influenza a (i.e., h n ), middle east respiratory syndrome coronavirus (mers-cov), and, more recently, avian influenza a (i.e., h n ) and evd underscore the importance of such pre-emptive plans in preventing or mitigating the effects of disease transmission. , the details may vary, depending on the nature of the pathogens; however, key components in public health response include risk assessment; communication and education; establishing a rapid response team and management team to coordinate responses between different government ministries; monitoring and surveillance; providing adequate facilities and supplies for quarantine and infection control; developing laboratory diagnostics; and where appropriate, antimicrobial and vaccination policies, and/or stockpiling. , for clinicians in nonendemic countries, fhf will mostly be encountered in travelers returning from endemic areas. the sars epidemic and influenza a (h n ) pandemic demonstrated the efficiency of international traveldespecially air traveldin the global spread of infectious diseases. , as a continent, africa has a relatively small number of international travelers (only oceania has a smaller number of international tourist arrivals), although the exportation of fhf to nonendemic countries is well documented in the present epidemic. it is tempting to consider establishing travel restrictions to limit the spread of evd outside of africa; however, the actual benefit of such policies is limited. at the time of this writing, the who has issued no travel restrictions to the affected countries. however, it is prudent to issue health warnings to potential visitors to affected countries. pretravel education on transmission routes, precautionary measures, self-monitoring of signs and symptoms, and availability of medical care in destination countries should be provided. as in other subspecialties of travel medicine, the visiting friends and relatives (vfrs) are at particularly high risk of contracting travel-related infections. despite the fact that publicity and health alerts are often announced to the general public, vfrs do not always receive the necessary information on the risks because of cultural differences, language barriers, or different perceptions. hence, efforts must be targeted to vfrs (especially african communities in this context) to reduce the risk of disease importation. remote infrared thermal scanners have been used as a means of fever screening at airports in some countries. the practice first gained popularity during the sars epidemic, and was subsequently evaluated in the influenza a (h n ) pandemic. e to a lesser extent, this method has also been used for the screening of other febrile illness such as dengue fever. infrared thermal scanning is relatively popular in asian countries such as taiwan, japan, korea, and hong kong, and it is usually used in conjunction with health questionnaires for self-reported symptoms such as fever and travel history. the tympanic temperature would be measured for suspected cases. the sensitivity, specificity, and positive predictive value of thermal scanning are affected by a variety of factors such as the instruments used, the threshold temperature, the part of the body being measured, the distance of the instruments from the individual, and the previous use of antipyretic agents. patients in the incubation period of an infection obviously will not be detected by this screening method. despite the relatively low sensitivity, specificity, and positive predictive values of infrared thermal scanning, some investigators consider it a useful adjunctive measure for border screening, although it cannot be relied on as the sole method for screening. e contact tracing must be promptly initiated for any potential contacts of returned travelers diagnosed with a communicable infectious disease such as evd. detailed guidance on contact tracing for patients with evd and other forms of viral hemorrhagic fevers have been published. , all suspected contacts must undergo individual risk assessments, based on the travel history, the epidemic situation in the affected countries, and the nature of potential exposure before and during travel. detailed instructions and information must be provided to the contacts, who will then be monitored for fever and the development of symptoms. the monitoring may be performed in the community or within healthcare facilities, during which some limitations indor at least, advice againstdthe freedom to travel within the community or country may be necessary and interference of daily activities may be inevitable. close and empathetic liaison between the contacts and health departments is essential to ensure compliance with monitoring and to minimize psychological impacts. a preparedness plan for contact tracing, disease surveillance, clinical management, isolation precautions, and outbreak management must be in place to manage such incidents in nonendemic countries. hemorrhagic fever remains an uncommon cause of fever in returned travelers, although it is severe clinically and has substantial public health implications. the risk of fhf in returned travelers is low. ten cases of marburg hemorrhagic fever were reported from to , and all patients had a travel history to africa. cases of imported evd (excluding the nonpathogenic rebov) were described in south africa in (zebov) and in switzerland in (tafv). other causes of fever in such settings must be excluded by appropriate laboratory investigations. these include (depending on the itinerary and exposure history) other causes of fever with or without hemorrhagic presentations such as meningococcal infections, severe sepsis due to other bacterial infections (including rare infections such as anthrax and plague as guided by the clinical picture and exposure history), leptospirosis, typhoid and other causes of enteric fever, rickettsioses, malaria, trypanosomiasis, visceral leishmaniasis, dengue fever, and yellow fever. in particular, malaria (which is a very common treatable cause of fever in returned travelers and is endemic in sub-saharan africa) must be excluded by appropriate testing. depending on the travel destination, other causes of viral hemorrhagic fever have to be considered such as lassa fever, hantavirus infection, crimean-congo hemorrhagic fever, and rift valley fever. the travel history should also include human contacts with sick individuals in the community (e.g., attending local funerals) or in healthcare facilities (as in the case of volunteer workers). another important exposure history is interaction with bats and other wild animals (especially primates). for example, a history of spelunking has resulted in fhf among local citizens and foreign visitors. cases of marburg hemorrhagic fever have occurred after exposure of visitors to bats in caves in kenya and uganda. , when fhf is suspected based on travel and/or exposure histories, pre-emptive isolation is essential to minimize the risk of subsequent interpersonal spread, until the diagnosis is excluded. the routes of transmission of filoviruses are well documented. transmission can be interrupted, provided that proper infection control and public health measures are implemented. detailed infection control guidelines on caring for patients with fhf have been published. , e in essence, the principles of isolation and use of personal protective equipment are not significantly different from standard precautions and transmissionbased precautions (i.e., contact, droplet, and airborne) that are universally practiced in healthcare facilities. however, numerous studies and reviews have confirmed that the compliance with the choice and use of personal protective equipment among healthcare workers are almost always suboptimal. e essential factors that ensure the optimal implementation of infection control protocols are adequate training of healthcare workers on isolation procedures and on the appropriate use of personal protective equipment, preferably by interactive training with clear instructions; adequate manpower and organizational support; and the availability of timely and adequate guidance and support. e such training should not be limited to staff working in clinical areas; it must also include other healthcare workers such as laboratory workers and paramedical personnel. the key elements of infection control consist of patient placement (e.g., isolation facilities), strict adherence to hand hygiene (e.g., the who's "five moments for hand hygiene"), proper use of personal protective equipment such as gloves (e.g., double gloves in special circumstances), waterproof gowns, respiratory protection (e.g., surgical masks, respirators during aerosol-generating procedures), eye protection, rubber boots, and safe handling of sharp objects. support staff engaged in environmental disinfection, funerals, and burial services must also be adequately trained on the proper use of personal protective equipment and chemical disinfectants, and on the handling of human remains to ensure adequate disinfection and minimize the risk of accidental exposure to the virus. , , conclusion filoviral hemorrhagic fevers are uncommon, but they pose real clinical and public health threats to countries outside endemic areas. the routes of transmission, clinical manifestations, and infection 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in health-care settings, with focus on ebola personal protective equipment in the context of filovirus disease outbreak response. rapid advice guideline management of hazard group viral haemorrhagic fevers and similar human infectious diseases of high consequence european network for diagnostics of imported viral diseases. management and control of viral haemorrhagic fevers and other highly contagious viral pathogens canadian nosocomial infection surveillance program. are health care workers protected? an observational study of selection and removal of personal protective equipment in canadian acute care hospitals compliance with isolation precautions at a university hospital a review of the evidence for suboptimal compliance of healthcare practitioners to standard/universal infection control precautions sars hospital investigation team. factors associated with critical-care healthcare workers' adherence to recommended barrier precautions during the toronto severe acute respiratory syndrome outbreak behind the mask: determinants of nurse's adherence to facial protective equipment barriers to implementing infection prevention and control guidelines during crises: experiences of health care professionals guidance for safe handling of human remains of ebola patients in u.s. hospitals and mortuaries supplementary guidance for handling of dead bodies of suspected/confirmed ebola virus disease (evd) the ebola virus vp protein functions as a type i ifn antagonist ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling ebola virus disease centers for disease control and prevention. chronology of marburg hemorrhagic fever outbreaks key: cord- -ez lif authors: wada, yoshiko; wada, kennosuke; iwasaki, yuki; kanaya, shigehiko; ikemura, toshimichi title: directional and reoccurring sequence change in zoonotic rna virus genomes visualized by time-series word count date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ez lif ebolavirus, mers coronavirus and influenza virus are zoonotic rna viruses, which mutate very rapidly. viral growth depends on many host factors, but human cells may not provide the ideal growth conditions for viruses invading from nonhuman hosts. the present time-series analyses of short and long oligonucleotide compositions in these genomes showed directional changes in their composition after invasion from a nonhuman host, which are thought to recur after future invasions. in the recent west africa ebola outbreak, directional time-series changes in a wide range of oligonucleotides were observed in common for three geographic areas, and the directional changes were observed also for the recent mers coronavirus epidemics starting in the middle east. in addition, common directional changes in human influenza a viruses were observed for three subtypes, whose epidemics started independently. long oligonucleotides that showed an evident directional change observed in common for the three subtypes corresponded to some of influenza a sirnas, whose activities have been experimentally proven. predicting directional and reoccurring changes in oligonucleotide composition should become important for designing diagnostic rt-pcr primers and therapeutic oligonucleotides with long effectiveness. viruses have always posed significant threats to public health, as highlighted by the recent ebolavirus outbreak in west africa [ ] [ ] [ ] [ ] and the emerging and re-emerging nature of influenza viruses . to face the world-wide threats caused by zoonotic rna viruses, which suddenly cause serious outbreaks by invasion from nonhuman hosts and mutate rapidly, we must understand the molecular evolutionary changes in their genome sequences extensively by innovating various technologies, including those used in big data analyses, e.g., large-scale word count. time-series word count of oligonucleotides in genome sequences can be conducted without specialized assumptions and prior knowledge and is useful for unsupervised data mining. importantly the obtained results are easily understandable, even for those unfamiliar with molecular evolutionary studies. oligonucleotide composition varies significantly among species even with the same genome g+ c%; therefore, the composition, especially of short oligonucleotides, has been used as a phylogenetic parameter "genome signature" to classify microbial genomes, including viral genomes. viruses are inevitably dependent on many host factors for their growth (e.g., nucleotide pools, proteins and rnas) and must escape from host antiviral mechanisms (e.g., interferon-induced systems) [ ] [ ] [ ] . therefore, after the invasion of zoonotic viruses from nonhumans, a certain level of directional change in genomic sequences and thus in mono-and oligonucleotide compositions should occur during human-to-human transmission. in fact, the g+ c% of influenza a virus strains isolated from humans is lower than that of strains isolated from natural hosts, which were avian , and the cg dinucleotide in an a/u context has been preferentially eliminated from classical h n influenza viruses during human-to-human transmission . by using blsom (batch learning self-organizing map) , , we previously found directional compositional changes in a wide range of oligonucleotides , that occurred after the onset of the influenza h n / pandemic of [ ] [ ] [ ] . in this study, we first conducted time-series word counts on changes in mono-to pentanucleotide compositions, occurring after invasion from nonhumans, for ebolavirus, mers coronavirus and influenza viruses. then, scientific reports | : | doi: . /srep as an example of long oligonucleotides, we analyzed -mers. because -mers are composed of (ca. . trillion) variables, the analysis becomes a big data analysis. long oligonucleotides are key elements of diagnostic pcr primers and potentially promising nucleic-acid therapeutics (e.g., antisense rna, sirna and mirna) , [ ] [ ] [ ] [ ] . rna viruses mutate very rapidly, and therefore, their sequence changes should be intensively characterized for designing diagnostic and therapeutic oligonucleotides with long effectiveness. the present time-series analysis on influenza a genomes showed that the -mers exhibiting the most evident directional changes observed in common for three different subtypes corresponded to several influenza a sirnas, which were experimentally validated. time-series analyses can provide information on changes possibly reoccurring after invasions from natural reservoir hosts. spatiotemporal analysis of mononucleotide composition in ebolavirus genomes. because of the recent major threat to public health in west africa, more than genomes of zaire ebolavirus (ebov) strains, isolated from humans from to , have been sequenced, and the sequences are available from the ncbi virus variation database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/ebola/). to study molecular evolutionary changes in ebov genomes in the current epidemic, we obtained strains, for which the isolated dates are given. although we focused on full-length genomes, a minor portion was relatively short in length, or many unidentified "n" bases were included, partly because of the stringent sterilization treatment obligated by the local government . because these will give odd values of mono-and oligonucleotide compositions, we focused on genomes longer than . kb after omitting n bases, which were derived from guinea, liberia and sierra leone strains. to conduct the present spatiotemporal analysis, strains isolated from several other geographic areas were omitted. we first analyzed time-series change in mononucleotide composition for strains isolated in the three areas separately. figure a plots four mononucleotide compositions (%) in each genome according to the isolated day, which started on march , . because mutations occur primarily as random processes, mononucleotide compositions for strains isolated even around the same day clearly differed from each other, which resulted in evident compositional diversity. despite this diversity, which should also be due to sequencing uncertainty, linear regression lines appear to show a time-dependent increasing or decreasing trend, a possible increase for c% and g% and a possible decrease for a% and u%. this is consistent with the excess u-to-c mutations found by two groups , . . data and regression lines separately analyzed for different geographic areas are differentially colored: guinea (blue), liberia (brown) and sierra leone (green). (b) averaged mononucleotide compositions for strains in each month are plotted according to the elapsed months from march . data and regression lines for the elapsed months are colored as described in (a). a separate and additional regression analysis for data without the averaging (a) that can examine time-series changes from the epidemic start for all three areas showed that the null hypothesis (i.e. no correlation) was rejected for all mononucleotides in the three areas ( cases) at the significance level of . , except for t% and g% for guinea; this was rejected for the t% for guinea at the significance level of . . for data after averaging (b), the null hypothesis was rejected at the significance level of . for mononucleotides in the three areas, except for g% and t% in guinea. scientific reports | : | doi: . /srep the present study focused on time-series changes, but not on data variations caused largely by random mutations and sequencing uncertainty, and therefore, average characteristics of strains isolated in a similar period were analyzed, summing up the sequences isolated in one month and calculating an averaged mononucleotide composition for each month (fig. b) . this could be done because the genome sequence data were distributed rather evenly by area and month; the average number of strains per month was . , . and . for guinea (blue), sierra leone (green) and liberia (brown), respectively. we omitted the data for a few months, for which no more than strains were available in the respective area. figure b shows a more conspicuous increasing or decreasing trend than fig. a , and the time-series increasing or decreasing trend revealed by the regression line for the elapsed months was the same among the three areas for all nucleotides, except for an ambiguous case of g% for guinea. in more detail, positive and negative directions of the regression line were primarily common among the three areas, although the slope angle appear to differ; table lists pearson's correlation coefficients calculated separately for the three areas. the highest negative (− . ) or positive ( . ) correlation among mononucleotides was observed for a% or c%, respectively, in liberia, which corresponded to a decrease of . as or an increase of . cs per genome at the final stage of the current outbreak. notably, the increasing or decreasing trend detected by these averaged data for one month (fig. b) is common to the result without averaging (fig. a) . in these regression analyses, the strains that were isolated in the three areas were independently analyzed. a separate regression analysis that could examine the statistical significance of directional changes from the epidemic start for all three areas, was conducted, by including the data of strains isolated at or near the beginning of the current epidemic (i.e., march strains in guinea) in liberia and sierra leone data. the results thus obtained by the latter analysis were consistent with those obtained by the former, and the null hypothesis (i.e. no correlation) was rejected at statistically significant levels in most cases, as described in figure legend short oligonucleotide composition in ebolavirus genomes. next, we analyzed each dinucleotide composition in the genomes grouped for each month. the results presented in fig. a , table and supplementary fig. a show that a common increasing or decreasing trend was observed among the three areas for more than half of the sixteen dinucleotides. correlation coefficients higher than . or less than − . are colored in table . for nine of the ten colored dinucleotides, the coefficient in the other two areas primarily shows the common directional trend. these high correlation coefficients should reflect the evolutionary dependence of the genome sequences. a decrease in uu and au and an increase in cc (fig. a) are predictable, even as a cumulative effect of their constituent mononucleotides (fig. b) , but an increase in ac, cu, gu and ug is not predictable as a simple cumulative effect and may indicate the characteristics of the contiguous sequence itself. we thus calculated the ratio of the observed dinucleotide occurrence to that expected from the mononucleotide composition (obs/exp) and found a decrease for uu and gc and an increase for gu, ug, ac and ua, which was common among the three areas ( fig. b and supplementary fig. a) . the slope direction of the regression line was common among the three areas, but the slope angle appeared to differ among the areas for a wide range of dinucleotides, as well as for tri-and tetranucleotides (data not shown). a spatiotemporal word count can be conducted without specific assumptions or prior knowledge and visualizes viral evolutionary changes in an easily understandable way. two distinct models can explain the common increasing or decreasing trend among the three areas, which was observed for a wide range of mono-and oligonucleotides. [model ] throughout the current ebov epidemic, virus movement between the three areas was extensive; therefore, the time-dependent trend of directional changes in oligonucleotide composition was common among the areas. [model ] even without the extensive mixing of viruses, common directional changes can occur. for example, viruses inevitably depend on many host factors for their growth, but human cells may not provide an ideal growth environment for invading viruses; therefore, common directional changes occur after invasions from nonhumans for supporting more efficient growth in human cells. in this connection, park et al. reported there was no clear evidence for import or export of ebov across national borders after its initial introduction. furthermore, although an increasing or decreasing trend for a wide range of mono-and oligonucleotides is common among the three areas, their actual composition (%) and the slope angle of the directional change often differ among the areas. this observation is consistent with model and the report by park et al. but not with model , as discussed below from various aspects. when considering the cellular environment differences between natural hosts (bats) and humans, not only the low-molecular substances (e.g., nucleotide pool) but also the macromolecules (e.g., proteins) involved in viral growth must be considered. additionally, antiviral mechanisms - should differ between humans and natural hosts. host macromolecules involved in viral growth (either supporting or inhibiting) should primarily recognize oligonucleotides, such as several nucleotides or longer, rather than mononucleotides. hence, we analyzed time-series changes in pentanucleotide ( -mer) occurrences. we first calculated correlation coefficients for all (= ) -mers and sorted them by the averaged correlation coefficients for the three areas. figure a and supplementary fig. b show time-series patterns of -mers with evidently high or low correlation coefficients; or (of types) -mers showed a common decreasing or increasing trend among the areas, respectively. examination of the ratio of the observed occurrences to those expected from the mononucleotide composition (obs/exp) showed that directional changes of many -mers cannot be explained by a cumulative effect of mononucleotide changes (data not shown), as found for various dinucleotides. when further examining the -mers showing the common directional change in more detail, their actual occurrence level and slope of supplementary fig. b) , and the difference often became clearer compared to that for mono-and dinucleotides. this cannot be explained by the aforementioned model . if the time-series directional change in mono-and oligonucleotide composition found for ebov is actually due to the necessity of supporting efficient growth in human cells (model ), such directional changes should be observed for other zoonotic rna viruses. directional change in mers virus genomes. we next analyzed mers coronaviruses isolated in the recent epidemic starting in april in the middle east , . the ebov outbreak analyzed above was initiated by a single virus strain being introduced from a nonhuman animal; this virus was then transmitted among humans during the outbreak. in contrast, mers viruses were repeatedly transmitted from nonhumans, mainly camels, in this epidemic , although its original natural host is thought to be bats . because direct nonhuman hosts for different human isolates differ among isolates, information on camel-derived strains in the current epidemic is important. from the ncbi virus variation database , we found and strains from humans and camels, respectively, with known isolated dates. the total number of mers virus genomes is approximately one tenth of ebov genomes, but their sequence length, even after omitting n bases, is mostly similar to its genome size (ca. kb); only one sequence that was evidently short ( . kb) was omitted from this analysis. we grouped sequences in each month and analyzed the averaged mono-and oligonucleotides (from to -mers) for each month and host. clear time-dependent increasing or decreasing trends were observed for a wide range of mono-and dinucleotides (fig. a,b) , but the increasing or decreasing trend found for actual mono-and oligonucleotides clearly differed from ebov; e.g., c% and g% decrease and u% increases in mers viruses. ebov is a negative-sense single-stranded rna virus, and the sequences registered in the database and analyzed here were complementary to viral genome sequences, but mers virus is a positive-sense single-stranded rna virus and, therefore, genome sequences were analyzed. this difference, however, cannot explain differences in directional changes in monoand oligonucleotide composition of ebov and mers viruses. figure b presents four -mers with a high increasing or decreasing trend; e.g., during this -month epidemic, approximately six uuuuus or four ccacus have been gained or lost per genome, respectively. the time-dependent gain of u (fig. b) clearly differs from its loss for ebov (fig. a) . although the regression lines (blue lines in figs b and ) were calculated only for human strains, occurrence data for camel strains (brown) were primarily positioned around the regression line, thus fitting the previous report that mers viruses were repeatedly transmitted between camels and humans in the present epidemic . because time-series directional changes in mono-and oligonucleotide compositions were observed for both ebov and mers viruses, the directional change should occur for other zoonotic rna viruses. mononucleotide composition in influenza virus genomes. we next analyzed influenza viruses, for which a large number of sequences are available from the influenza research database for different human a subtypes, epidemics of which have started independently at long intervals, such as several decades. importantly, the aforementioned model , in which the common directional change between different viral populations is expected without extensive virus mixing, is testable more rigorously than for the ebov outbreak, which started from a single invasion in guinea and expanded to other countries. furthermore, we may examine the following important issue. if a common directional change is observed, what types of changes likely reoccur in future epidemics caused by invasions from natural reservoir hosts? in addition to human a subtypes, genome sequences of human b type, which can currently infect humans but not birds, and of various avian a subtypes were available. the genomes of influenza a and b are composed of eight segments, and we first selected ca. , strains with a full set of eight sequences, categorized the full genome sequences according to host and serological type, and grouped them in each year. the sequence length cut-off was not included because the genome is composed of eight segments of various lengths and a simple, satisfactory criterion for length cut-off could not be set. alternatively, because times more sequences are available than for ebov, we selected years with at least ten strains of one category to reduce the artefactual effects derived from sequencing uncertainty. then, to analyze time-series changes, we selected human or avian a subtypes with more than five years fulfilling the above threshold (≥ten strains per year); the human b type also fitted these criteria. in the case of h n / [ ] [ ] [ ] (starting from and abbreviated ph n in the database), a sufficient number of sequences fulfilling the above threshold was available even per month and, therefore, ph n sequences in each month were analyzed; importantly, this allowed the study of changes occurring within one outbreak in detail. collectively, we focused on thirteen subtypes (three human and nine avian a subtypes and a human b type), and first analyzed time-series changes in mononucleotide compositions. three human subtypes of influenza a virus (human h n , h n and ph n ) changed their a%, c% and g% with common increasing or decreasing trends among the three subtypes (fig. a) , despite different epidemics having started by independent invasions from nonhumans; the g+ c% increase in human a subtypes was previously reported by rabadan et al. . figure a shows that the a%, c% and g% move time-dependently apart from those of avian a subtypes (sky blue) and toward those of the human b type (violet). because the b type can currently infect humans, but not birds, this type has possibly adapted better to growth in human cells than a subtypes. the directional change of human a subtypes "apart from avian a subtypes and toward the human b type" may visualize their evolutionary journey from each start of human-to-human transmission. in the case of u%, human h n (blue) and h n (brown) have already reached the b's level, and only ph n (green) shows an increasing trend toward b. averaged mono-and dinucleotide compositions (a,b), for strains in each month are plotted according to the elapsed months from april . camel-derived strains are specified by small brown symbols, and regression lines (blue) were calculated only for human-derived strains. the null hypothesis was rejected for all data ( cases) at the significance level of . , except for a% and g%, with or without camel-derived strains; this was rejected for the g% at the significance level of . . scientific reports | : | doi: . /srep a large number of ph n strains isolated from to could provide detailed information on changes occurring within and after one outbreak because the averaged mononucleotide compositions for each month were analyzed. in fact, a horizontally long panel examining three human a subtypes (fig. b) revealed that a% monotonically increased from to but it abruptly lost half of the increase in (arrowed), followed by a possible partial recursion increase in ; c% and g% ( supplementary fig. a ) also showed a monotonic decrease from to , followed by an abrupt backset. we next analyzed the di-to tetranucleotide compositions. concerning correlation coefficients for the elapsed years in the case of h n and h n and for the elapsed months in the case of ph n , three subtypes show a common increasing or decreasing trend for thirteen of sixteen dinucleotides. the top four cases for the absolute value of averaged correlation coefficients for the three subtypes were examined in fig. a . as observed for mononucleotides, all three subtypes appeared to move from avian a subtypes toward the human b type. a decrease in cg dinucleotides in a/u motifs during human-to-human transmission was previously reported . detailed inspection of ph n (fig. b) showed a monotonic increase of aa% from to was followed by an abrupt backset in (arrowed). here, we discuss the backset occurring after one outbreak may relate to the differential slopes of directional changes observed for different subtypes (figs and ). ph n strains, which are primarily derived from one outbreak, show an evidently steeper slope than h n and h n strains derived from multiple outbreaks, indicating a higher rate of ph n genome sequence changes. the first discussion was whether ph n had an evidently higher mutation rate than h n and h n . we propose here that without introducing the higher mutation rate of ph n , its seemingly higher rate of genome sequence changes can be explained by the model that conforms to the abrupt backset that occurred after an outbreak. when considering a viral infection spread, the ratio of persons with or without proper resistance to the viral infection (e.g., antibodies the same colored and smaller symbols than for human subtypes are used for avian a subtypes because differences among avian subtypes were not a concern in the present study. because the b type has moved gradually toward human a subtypes, this type appears not to have reached its hypothesized goal, which may possibly lie between human a and b types, but nearer the latter. (b) a horizontally long panel for a% of three human a subtypes is presented for clarifying detailed changes occurring within and after one outbreak of ph n . data of c% and g% are presented in supplementary fig. a . scientific reports | : | doi: . /srep that are highly effective against the respective strain) becomes important. therefore, the blooming strains (and their close relatives) in a certain outbreak (especially in a pandemic) may not be a very suitable strain for restarting the next outbreak because antibodies highly effective against the blooming strains are held by a large portion of humans. less blooming strains, that are less adapted to humans, may become good starting strains for the next outbreak, resulting in a backset after one outbreak, as observed in figs b and b and supplementary fig. . hence, the evolutionary change focusing on strains belonging to a single outbreak looks higher than those belonging to multiple outbreaks, giving a steeper slope. to illustrate this interpretation, we added hypothetical data of h n strains belonging to one outbreak as a series of small light-brown dots representing the dinucleotide compositions hypothesized for individual months, whose slope angle simply mimics that of ph n . although other various mechanisms must be included to explain the backset after one outbreak, we propose that the steep slope of ph n can be explained without introducing the higher mutation rate of ph n . a wide range of tri-and tetranucleotides also showed decreasing or increasing trends common among the three subtypes (data not shown). the common increase or decrease of mono-and oligonucleotides should reflect the convergent-type evolution occurring in independently evolving viruses, which have invaded independently from nonhumans. we predict that the time-dependent changes required for efficient growth in human cells will repeat with a significant probability after future invasions from natural reservoir hosts and that this view is most likely applicable to a wide range of zoonotic rna viruses. long oligonucleotides in human influenza a genomes. next, a time-series analysis was performed on long oligonucleotide occurrences. long oligonucleotides are key elements of diagnostic rt-pcr primers , and nucleic-acid therapeutics (e.g., sirnas) , [ ] [ ] [ ] . when designing diagnostic and therapeutic oligonucleotides with long effectiveness for highly mutable viruses, we have to understand fundamental features of time-series directional changes of long oligonucleotides and to predict changes that will reoccur with a high probability. the sizes of pcr primers and therapeutic oligonucleotides range primarily from to nucleotides , [ ] [ ] [ ] . thus, we analyzed -mer occurrences in genomes of three human influenza a subtypes, which independently invaded from nonhumans. of a total of (ca. . trillion) types, ca. . million types of -mers were found in these genomes. importantly, a time-series word count can analyze such big data without difficulty. after calculating correlation coefficients for each -mer for each subtype, we first selected all -mers having an evidently high or low correlation coefficient (> . or < − . ) in common for the three subtypes; all five -mers thus selected had negative correlation coefficients and were found to be mutually overlapped; i.e., these were components of one -mer. figure a presents time-series occurrences for one -mer, acagcagagugcuguggaug; other four -mers gave practically the same decreasing pattern. at the beginning stage of three independent epidemics, most strains had one copy of the -mer, but during the course of human-to-human transmission the copy number decreased in the viral population and were almost completely lost at the final stage in all three subtypes; i.e., the strains having this -mer sequence progressively reduced their share in viral populations, regardless of differences in subtypes. we think that this common decrease reflects a certain inconvenience for efficient growth in human cells and will reoccur with a high probability after a future invasion. we next discuss this common decrease, in connection with sirna designs. because of the social importance of influenza viruses, varieties of sirnas have been designed as promising nucleic-acid therapeutics. actually, virsirnadb , which have complied the experimentally validated sirna sequences, contains sirnas for influenza a viruses; and their length ranges from to . when we searched viral sirna sequences that were highly homologous to the -mers with an evident common increasing or decreasing trend with a blast search against virsirnadb, we found that four -mer influenza a sirnas (virsi , , and ), which had the same sequence (cagcagagugcuguggaug) but were experimentally validated in different cell lines, showed a complete match to the -mer sequence harbored by the aforementioned five -mers, which are components of one -mer (aggaacagcagagugcuguggaug). although these five -mers gave practically the same decreasing pattern to that listed in fig. a , the compensatorily increased -mer responding to their decrease differed slightly in changed positions among the subtypes. figure b ,c present patterns of the compensatorily increased -mers, which had nonsynonymous and synonymous changes from the original -mer, respectively (for details, see the legend). this indicates that the sequence ranging over a certain length is possibly inconvenient for efficient growth in human cells ranges and that changes within the range can resolve the possible inconvenience. since both synonymous and nonsynonymous changes were observed, the possible inconvenience should not relate to protein sequence. the information about the time-dependent directional change and the changed base should become important for designing sirnas with long effectiveness. future prospects. finding time-series directional changes in long oligonucleotide occurrences for a large number of influenza a genomes can provide a new refinement strategy for designing therapeutic oligonucleotides and diagnostic rt-pcr primers with long effectiveness. three influenza a subtypes may not be sufficient to judge the reoccurrence of the change, and therefore, our group has started to analyze the time-series changes in experimentally validated sirna sequences, by focusing on both human and avian strains. an important issue is clarification of biological mechanisms responsible for directional changes in oligonucleotide occurrences after host changes. if the mechanism is clarified, reoccurrence in the future should become much clearly defined. systematic analyses on a wide range of oligonucleotides with various lengths for the genomes of human and avian strains may give useful information about the possible mechanisms. judging from the directional changes observed in ebov and ph n outbreaks, genome sequences from strains isolated within several months after a new invasion appear to give reliable information about the directional change. if a large number of viral sequences becomes available, an averaging even per week for strains that are isolated in properly assigned geographic areas may clarify time-dependent directional changes. undoubtedly, past sequences from the same or closely related viral genomes become important for comparison. in the present study, we focused on zoonotic rna viruses whose reservoir hosts are vertebrates, but it is undoubtedly important to analyze other rna viruses spread by bugs (e.g., ticks and mosquitos), which have caused serious emerging infectious diseases, such as dengue fever and zika virus disease , . results obtained with time-series word count are easily understandable even for those unfamiliar with evolutionary biology. when social importance of time-series word count of oligonucleotides becomes clear, experts in various fields will participate actively. huge numbers of genome sequences, including those from disease-causing microbial strains, have accumulated rapidly because of revolutionary development in sequencing technologies and of social importance. in this era of big data accumulation, participation of experts in big data analysis becomes increasingly important for interdisciplinary efforts against big threats presented by infectious microorganisms. genome sequences of human zaire ebolavirus (ebov) and mers coronavirus strains were downloaded from the ncbi virus variation database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/) on dec. and dec. ( ) , respectively. because the number of mers virus sequences is small, compared with the other two viruses analyzed, a time-series analysis was conducted for the months having at least three strains, and when the number of strains for a certain month was less than , these were combined with those of the nearest neighboring month that had the lower number of strains than the other neighbor. from the ncbi influenza virus resource (http://www.ncbi.nlm.nih.gov/genomes/flu/), a total of ca. , segment sequences derived from ca. , influenza a and b virus strains were obtained on sep. ( ) . we calculated mono-and oligonucleotide occurrences in eight genome segments of influenza virus strains and summed up the occurrences for each strain. to prevent possible misassignment from a large number of ph n strains, relatively small numbers of human classical h n strains isolated from were omitted from the present analysis. influenza virus is a negative-sense single-stranded rna virus, and therefore, the sequences analyzed are complementary to viral genome sequences. computer codes are available from k.w. (k_wada@nagahama-i-bio.ac.jp). response team. ebola virus disease in west africa -the first months of the epidemic and forward projections genetic diversity and evolutionary dynamics of ebola virus in sierra leone ebola virus epidemiology, transmission, and evolution during seven months in sierra leone temporal and spatial analysis of the - ebola virus outbreak in west africa emerging viral diseases comparative dna analysis across diverse genomes inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses induction and suppression of rna silencing: insights from viral infections interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures comparison of avian and human influenza a viruses reveals a mutational bias on the viral genomes oligonucleotide motifs that disappear during the evolution of influenza in humans increase ifn-α secretion by plasmacytoid endritic cells analysis of codon usage diversity of bacterial genes with a self-organizing map (som) -characterization of horizontally transferred genes with emphasis on the e. coli o genome informatics for unveiling hidden genome signatures prediction of directional changes of influenza a virus genome sequences with emphasis on pandemic h n / as a model case novel bioinformatics strategies for prediction of directional sequence changes in influenza virus genomes and for surveillance of potentially hazardous strains novel swine-origin influenza a (h n ) virus investigation team. emergence of a novel swine-origin influenza a (h n ) virus in humans emergence and pandemic potential of swine-origin h n influenza virus origins and evolutionary genomics of the swine-origin h n influenza a epidemic progress toward oligonucleotide therapeutics: pharmacodynamic properties broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr nucleic-acid therapeutics: basic principles and recent applications mechanisms of gene silencing by double-stranded rna rna targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform virus variation resource -recent updates and future directions evidence for camel-to-human transmission of mers coronavirus close relative of human middle east respiratory syndrome coronavirus in bat influenza research database: an integrated bioinformatics resource for influenza research and surveillance. influenza and other respir a multiplex rt-pcr for detection of type a influenza virus and differentiation of avian h , h , and h hemagglutinin subtypes virsirnadb: a curated database of experimentally validated viral sirna/shrna the global distribution and burden of dengue history, epidemiology, and clinical manifestations of zika: a systematic review this work was supported by a grant-in-aid for scientific research (c: no. ) from the ministry of education, culture, sports, science and technology, japan. t.i. and s.k. conceived and designed this project. k.w., y.w. and y.i. developed computer programs. t.i. wrote the manuscript. all authors participate in the discussion through the project. key: cord- - qlk y h authors: friedrich, brian m.; trefry, john c.; biggins, julia e.; hensley, lisa e.; honko, anna n.; smith, darci r.; olinger, gene g. title: potential vaccines and post-exposure treatments for filovirus infections date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qlk y h viruses of the family filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. while many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. contemporary methods of supportive care and previous treatment approaches for human patients are also discussed. the family filoviridae includes two accepted genera, ebolavirus and marburgvirus. the genus ebolavirus includes five species (each represented by a single virus): zaire ebolavirus (ebola virus, ebov), sudan ebolavirus (sudan virus, sudv), reston ebolavirus (reston virus, restv), taï forest ebolavirus (tai forest virus, tafv), and bundibugyo ebolavirus (bundibugyo virus, bdbv). the genus marburgvirus includes a single species, marburg marburgvirus, which has two members: marburg virus (marv) and ravn virus (ravv) [ , ] . in , the first cases of filoviral infection were documented in three simultaneous outbreaks in west germany and yugoslavia. the virus responsible for the outbreaks was named "marburg virus" after the german city of marburg in which it was first recognized [ , ] . from documented instances of infection, it seems that the members of the filovirus genera may exist in quite opposite climates of africa with marburgvirus infections occurring more frequently in the dry woodlands, while ebolavirus infections occur more frequently in rain forests [ ] . more than years of effort have focused on the search for the reservoir of these viruses in africa, and while the search is still ongoing, recent evidence indicates that bats may be reservoirs for both marburgviruses and ebolaviruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, the recent outbreak of restv in domestic pigs in the philippines demonstrated the potential for animals other than primates and bats to be infected and potentially spread or amplify outbreaks [ ] . filoviruses are named for their long, filamentous shape which can been seen on the order of micrometers in length, while their width is more narrow (usually around nm) with little fluctuation [ ] . contained within this filamentous virus is a single, -kb negative-sense rna genome that encodes seven proteins [ , ] . the seven filoviral proteins are the glycoprotein (gp), the polymerase (l), the nucleoprotein (np), a secondary matrix protein (vp ), the transcriptional activator (vp ), the polymerase cofactor (vp ), and the matrix protein (vp ) [ , ] . homotrimers of the viral gp cover the surface of the virion, and this viral gp is believed to be the sole host attachment factor for filoviruses [ , ] . candidates for filoviral receptor and co-factors include transferrin, dc-sign, tim- , and npc [ , [ ] [ ] [ ] [ ] . after entry, filoviruses replicate their genomes and viral proteins in the cytoplasm using a rna-dependent rna-polymerase which is carried in with the virus. wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as % [ ] . in humans, filovirus infection is characterized by an abrupt onset of flu-like illness, after an initial incubation period of - days. following this initial illness, signs and symptoms of disease include anorexia, nausea, vomiting, chest pain, cough, edema, postural hypotension, neurologic complications, petechiae, and mucosal hemorrhage. there have also been several observed wild-type filovirus outbreaks among great apes in africa that have demonstrated similarly high mortality rates [ ] . in an effort to create cost and time-effective models of filoviral disease for the development of vaccines and therapeutics, small animals, such as mice and guinea pigs, are often used. however, these animals usually demonstrate significant resistance to wild-type filovirus infection, and only demonstrate mortality rates similar to primates when the filovirus in question has been adapted to the model species [ ] . due to the difficulties in evaluating wild-type filovirus infection in small animals and the generally high level of immune protection correlates derived from non-human primate (nhp) models of infection, therapeutics and vaccines are ultimately evaluated in nhp species for efficacy against filovirus. of the nhp models available for filovirus study, rhesus and cynomolgus macaques have been the most highly characterized and utilized for therapeutic and vaccine development, respectively. however, the starting point of vaccine and therapeutic development remains small animal models due to the cost, ethical, and time-associated benefits [ ] . this review will highlight the current research into filovirus vaccines and therapeutics. the current clinical standard for filoviral infection is supportive care as there are currently no fda-approved treatment strategies. supportive care consists of oral fluid rehydration, oral medication, nutritional supplementation, and psychosocial support [ ] . nasogastric feeding tubes and i.v. administration of both fluids and medication are increasingly considered supportive care where possible during outbreak scenarios to prevent dehydration and facilitate support of blood pressure [ , ] . however, given the limited equipment and laboratory support during outbreaks, care must be taken to prevent overaggressive fluid administration [ ] . fluid replacement was evaluated briefly in rhesus macaques, and while there was no significant benefit to survival, a less severe renal compromise was observed [ ] . while supportive care may (or may not) reduce the overall case fatality rate in humans, the true impact of simple interventions such as fluid management has yet to be fully evaluated and the potential for benefit in combination with direct antiviral measures has yet to be assessed [ ] . treatment of filovirus infection with passive transfer of antibodies is an attractive therapy. while there have been conflicting results in vitro, in animals, and in humans, recent breakthroughs have solidified the potential for this strategy of intervention. in addition, there have been a number of immunotherapies developed for other agents, such as respiratory syncytial virus (rsv), which can provide potential roadmaps or precedence to facilitate the advancement of these products through the necessary regulatory hurdles [ , ] . transfer of immune serum for the treatment of filovirus infection in humans has previously been attempted. however, interpretation of these results has been cautious due to the study conditions as well as the uncertainty of the disease stage at which the individuals were treated [ ] . as a result, much attention has focused on animal studies evaluating candidate products. while many of the early studies in mice and guinea pigs were successful, these successes did not translate to nhp studies and tempered the enthusiasm for further evaluation of candidate products. [ , [ ] [ ] [ ] [ ] . when similar passive transfer strategies were attempted in nhps, viremia onset and outward signs of disease were reduced, but the treatment did not affect survivorship [ ] [ ] [ ] . in addition, the suggestion that antibodies could enhance filovirus infections in vitro caused further concern [ ] [ ] [ ] . however, recently a series of experiments have made researchers, developers, and funding agencies reconsider the potential of this category of products for filoviruses. both polyclonal and monoclonal passive therapies have been shown to be efficacious in rodents for filovirus infection [ ] [ ] [ ] . furthermore, evidence of enhancing antibodies exists in the antibody response to ebov [ ] . more recent studies have demonstrated protection in macaques with polyclonal and monoclonal passive therapy [ ] [ ] [ ] . these sources of monoclonal antibodies have ranged from murine monoclonal antibodies to recombinant-derived cloned human monoclonal antibodies from survivors of filovirus infection [ , ] . development of new antibodies to be used for post-exposure treatment is on-going. in one study, an antibody ( f ) targeting the ebov gp mucin-like domain was generated and subsequently shown to protect % of mice against a lethal ebov challenge when given days post-exposure [ ] . this antibody was then modified to generate h- f , a human recombinant antibody. this human recombinant antibody also significantly protected mice against a lethal challenge of ebov [ ] . in another method, a recombinant vsvΔg/ebovgp was used to generate a total of monoclonal antibodies which were subsequently characterized. all monoclonal antibodies improved survival rate of mice ( %- %) against a high-dose lethal challenge by mouse-adapted ebov [ ] . another antibody, kz , was isolated from the bone marrow of a human survivor of ebov infection and is specific for the complex of gp and gp [ ] . kz neutralized ebov in vitro and offered protection from lethal ebov challenge in a rodent model [ ] , but was non-protective in nhps [ ] . table . . . dna vaccines the first clinical trials involving filovirus vaccines were based off of plasmids expressing ebov np and gp as well as sudv gp [ ] . this strategy proved safe in subjects involved with phase i testing. however, the prime/boost dna vaccine strategy covering four separate plasmid doses administered three times each was ineffective at creating durable immunity as evidenced by the near non-existent antibody titer in these subjects after year [ ] . while clinical trials with this vaccine have halted, it may be possible that this strategy can supplement another vaccine technology in a prime/boost capacity. while no vaccines or therapeutics are currently licensed for use by the fda, phase i clinical trial safety tests have been performed on one particular platform for an ebov vaccine. this vaccine is based on a replication deficient, recombinant adenovirus serotype (rad ) vector genetically engineered to carry the genes for ebov glycoprotein (gp (z)) and sudv glycoprotein (gp (s/g)). as a common human pathogen, this vector vaccine utilized the broad-tropism of the adenovirus vector to infect cells and once inside the inserted ebolavirus glycoproteins are expressed. upon expression of these inserted ebolavirus genes, the host immune system will recognize them as foreign and mount a response against them. the advancement of this vaccine technology to phase i trials manifested from its ability to provide % protection among cynomolgus macaques vaccinated - days prior to challenge and its ability to generate potent humoral and cell-mediated immune responses [ , ] . in the clinical trial participants remained asymptomatic after a single vaccination with either × or × viral particles [ ] . furthermore, for both doses of the vaccine significant antibody titers were observed at weeks post-vaccination with % and % of participants receiving × viral particles being positive for gp (s/g) and gp (z), respectively. significant antibody titers were observed again at weeks post-vaccination and, while decreased from weeks, demonstrated the potential durability of this vaccine over time [ ] . t-cell activation was also examined for these individuals and found to directly correlate with the dose administered, but to a lesser extent than the previously mentioned antibody response. cd + activation was observed with greater frequency than cd + activation in those receiving the vaccine. importantly, these results were obtained in the context of significant pre-existing immunity to ad as pre-entry evaluation of antibody titers revealed that % of participants were positive against ad , showing that pre-existing immunity to ad still resulted in protection against ebov [ ] . while this study shows great promise, further development and additional studies in nhps and humans are needed. table . in addition to the adenovirus platform in clinical trials, additional variations of the rad vaccine are also in development and have been evaluated in nhp models. based on the adenovirus vector platform, the complex adenovirus (cadvax) technology substantially increased the genetic payload capacity of the vector, up to kb. additionally, this strategy involved the blending of four separate vectors expressing the glycoproteins of ebov, sudv, and marv along with the nucleoproteins of ebov and marv. when administered in a prime/boost strategy, this technology offered % protection against ebov, sudv, and marv [ ] . another variation of the cadvax system designed to express modified ebov glycoprotein and sudv glycoprotein was effective in protecting against both parenteral and aerosol challenge when administered in a prime/boost strategy [ ] . both implementations of the cadvax technology demonstrated significant antibody titers. further improvement upon the adenovirus-based ebov vaccine technology is ongoing. richardson et al. reformatted the genetic insert for the vector which included the addition of a cytomegalovirus (cmv)-chicken β-actin hybrid promoter, optimized codons and a consensus kozak sequence [ ] . these improvements led to three-to seven-fold increases in ebov glycoprotein expression. neutralizing antibody titers were found at doses as low as infectious forming units (ifu) with comparable titers requiring ifu of the unmodified vaccine in mice. these modifications demonstrated % protection of mice at doses two orders of magnitude lower than the unmodified vaccine. interestingly, at min post-challenge, the modified ad-cmvzgp/ad-cagoptzgp offered % protection compared to the % protection of mice offered from the original vaccine [ ] . this vector format has also recently showed promise when administered sublingually in mice, therefore eliminating the complexities of parenteral administration such as the necessity for sterile tools, aseptic chemicals, and the risks of potential blood-borne pathogen exposure [ ] . while this adenovirus vector vaccine technology is promising, demonstrations that pre-existing immunity to the ad vector depressed the desired immune response may impede its implementation. in efforts to circumvent issues of pre-existing immunity to ad , geisbert et al. sought out a less prevalent serovariation [ ] . in their study, a heterologous prime/boost strategy with recombinant adenovirus serotypes and carrying gp (z) and gp (s/g) demonstrated complete protection among nhps. each of these vectors was capable of stimulating humoral and cell-mediated immune responses in the context of nhps pre-vaccinated with rad as evidenced by antibody titers reaching an order of magnitude above those achieved in rad vaccinated subjects ( : , compared to : , ), and cd + intracellular cytokine staining was . -fold greater among heterologous prime/boosted subjects ( . % compared to . %) [ ] . rhabdoviruses have recently offered unique vaccine platforms to generate both genus/species specific immunity as well as potential for cross-protective immunity for filoviruses. for example, based on an attenuated recombinant vesicular stomatitis virus (rvsv), the replication-competent virus expresses the glycoprotein of the target filovirus in place of its wild-type membrane glycoprotein. as this virus is primarily an agricultural pathogen, pre-existing immunity interfering with the desired immune response and subsequent protection is unlikely [ ] . several studies have begun to address the safety of the filovirus vsv platforms. evaluation of this platform in immunocompromised nhps has suggested that this technology may be safe among similarly immunocompromised humans [ ] . further encouragement for the safety of this live-attenuated vaccine came recently from mire et al. who showed that ebov and marv rvsv showed no signs of neurovirulence associated with vsv [ ] . the utility of the vsv-based vaccine for protection against filoviral hemorrhagic fever was highlighted by geisbert et al. [ ] . using a blended vaccine consisting of equal amounts of three different vsv vectors each carrying the ebov, sudv or marv glycoprotein, they were able to generate % protection of nhps against challenges with ebov, sudv, tafv, and marv with no observed ill effects from this replication-competent vaccine. of all vaccinated nhps, only one showed signs of viremia as assayed by rt-pcr. each of the vaccinated nhps also demonstrated elevated antibody responses after vaccination, with titers ranging from : to : for all three glycoprotein components of the blended vaccine [ ] . in addition to providing such high levels of protection as a prophylactic vaccine strategy, the vsv-based technology has demonstrated post-exposure protection for both ebov and marv when administered via intramuscular (i.m.) injection [ ] . when marv-rvsv was administered i.m. - min post-challenge with marv, % of nhps survived. in this study viral rna was observed in the blood on day three post-challenge when assayed by rt-pcr, but active virus was unobservable by traditional plaque assay. clinical chemistry results demonstrated that these surviving nhps experienced significant rises in aspartate aminotransferase, gamma-glutamyl transferase, total bilirubin, and blood urea nitrogen indicating that, while protective, the post-exposure treatment did not completely prevent typical pathogenic events associated with marv infection. similar experiments demonstrated sudv-rvsv delivered - min after challenge offered % protection [ ] . post-exposure protection for ebov-rvsv was less effective at - min but still afforded % protection to eight nhps [ ] . as a post-exposure treatment, ebov glycoprotein rvsv was used recently h after a suspected human exposure via needlestick in the laboratory. while there is no direct evidence the laboratory worker was indeed exposed, that person survived the experience with no discernible sequelae from the treatment outside of a transient fever occurring h after an injection of × plaque forming units (pfu) [ ] . also of note for rhabdovirus-based filoviral vaccines was a recent report that generated dual immunity for both ebov and rabies virus infection. ebov gp was efficiently expressed from an attenuated vaccine used for wildlife against rabies virus in place of the wild-type rabies envelope glycoprotein, g [ ] . this vaccine vector was capable of inducing protective immunity to ebov infection as well as to rabies virus infection in both live-attenuated format and β-propriolactone inactivated vaccine. neurovirulence of the recombinant vector was unobserved in suckling mice when compared to the unaltered vaccine [ ] . this versatility offers increased storage options with an inactivated vaccine as well as the opportunity to vaccinate for each disease where they are both endemic. alphavirus particle-based vaccines also provide high levels of protection to nhps against lethal filovirus challenge. these vaccines for ebov, sudv, and marv are composed of venezuelan equine encephalitis virus (veev)-based replicon particles (vrps) that express the viral glycoprotein of interest [ ] . replicon particles are replication-defective, single-cycle vectors which cannot spread from cell-to-cell. the vrps are composed of an attenuated veev replicon that contains veev non-structural genes and the filovirus glycoprotein. vrps are generated when the replicon is co-transfected into cells with helper plasmids containing the veev structural genes. the resulting single-cycle propagation defective particles are then administered to the appropriate animal model for efficacy testing [ , ] . this technology offers several advantages, including high expression levels of heterologous genes, dendritic cell tropism, induction of robust cellular and antibody immune responses, rapid construction into single and multivalent vaccines, and relative resistance to anti-vector immunity [ , ] . in vivo studies with the marv vrp offered the first demonstration of a fully protective filovirus vaccine. nhps exhibited % survival after vaccination with focus forming units (ffu) of vrp in three consecutive doses spaced at day intervals prior to challenge with marv [ ] . this protection was offered when the vrps were constructed to express gp alone or gp + np; however, the nhps vaccinated with np alone all exhibited clinical symptoms of illness and only two out of three survived the challenge. substantial antibody titers were found in each of the vaccinated nhps. additionally, no conspicuous elevations in clinical chemistries were observed in nhps throughout the experiment. experiments performed on mice and guinea pigs supported the ability of vrps expressing gp to mediate complete protection from lethal marv and ebov challenge [ ] . in mice, adoptive transfer of cd + cells, but not cd + cells or passive antibody transfer, from vrp-np-immunized mice was protective, suggesting this vaccine may be most protective by stimulating the host cell-mediated immune response [ ] . additionally, adoptive transfer of cd + t-cells after activation via specific ebov peptides provided mice complete protection indicating a mechanism for vrp-based immunity [ ] . recent studies indicate that a vrp-based vaccine is fully protective in cynomolgus macaques against ebov, sudv, and marv parenteral and aerosol virus challenges (unpublished observations, olinger). paramyxovirus-based vectors for vaccination against filoviral threats have recently demonstrated the capacity to protect nhps from infection and stimulate strong immune responses. paramyxoviruses have a natural tropism for the respiratory tract and, as filoviruses are both emerging diseases and potential weaponized threats, the idea of targeting vaccines to this area is ideal. two candidates for this category of vaccines have been investigated to date: human parainfluenza virus (hpiv- ) and newcastle disease virus (ndv). of these two systems, the hpiv- system has been evaluated in nhp models of ebov infection. combinations of ebov gp alone, ebov gp + np, and ebov gp + human granulocyte macrophage colony stimulating factor (gm-csf) were inserted into the genome of hpiv- . each of these vaccine vectors was used to vaccinate nhps both intranasally (i.n.) and intratracheally (i.t.) as initial studies offered complete protection of guinea pigs vaccinated via the respiratory route [ ] . at least one nhp in all three vaccine groups receiving × tcid (median tissue culture infective dose) of their respective vector displayed clinical signs of illness after challenge during the study. each group held two nhps and out of the three groups only one vaccinated animal from the ebov gp + np succumbed to the disease. immune responses from these subjects, prior to challenge, revealed antibody titers ranging from : to : , [ ] . by manipulating dose and administration strategies, bukreyev et al. were able to achieve complete protection of nhps after two successive doses of × tcid given at day zero and again at day with challenge occurring on day [ ] . the two-dose strategy produced igg titers ranging between : , and : , , much higher than the single dose. each of these experiments highlights the potential of the hpiv- platform for ebov vaccination but the known prevalence of pre-existing immunity to hpiv- in humans could hinder the generation of targeted immunity [ ] . to address these concerns, bukreyev et al. compared the immunogenicity of ebov gp expressing hpiv- vector among naïve and pre-immune nhps [ ] . in these experiments ebov-specific igg levels were substantially decreased among hpiv- pre-immune nhps; however, this hindrance was overcome when the nhps were vaccinated with two doses of recombinant vector which was previously shown to offer complete protection against ebov challenge [ ] . in efforts to diversify the paramyxovirus-based vectors and avoid issues surrounding the pre-existing immunity found for hpiv- , a new vector design based on ndv was established. ndv is an avian paramyxovirus that infects the respiratory tract. this virus has been shown to be highly attenuated in nhps due to natural host restriction processes [ ] . additionally, this vector system has proven successful as a vaccine platform for severe acute respiratory syndrome-associated coronavirus (sars-cov) and influenza h n in nhps [ ] . although this system has yet to be evaluated in the context of nhp models of ebov infection and disease, it was recently shown to be immunogenic in nhps. single vaccination with ndv expressing ebov gp produced lower titers than the hpiv- platform, demonstrating this vector is less immunogenic; however, in a homologous prime/boost vaccination strategy ebov-specific mucosal iga levels reached those similar to the hpiv- homologous prime boost vaccination strategy [ ] . igg specific for ebov did not reach levels comparable to the previous hpiv- platform. these reports support the potential use of paramyxoviruses as vaccine candidates, but further examination of immunostimulatory effects and pre-existing immunity will require investigation. the vlp technology works by generating non-replicating virus particles that do not contain any filoviral genetic material. immune responses generated in response to exposure to vlps are derived from the filoviral protein shell that is the vlp itself. vlps are constructed through the matrix protein vp 's ability to drive the budding process. by simple transfection and expression of vp into target cells, filamentous structures can be generated [ ] . co-expression of additional filoviral proteins, such as gp and np, can dramatically enhance and stabilize the production of vlps from target cells [ ] . when traditional target/production cells were swapped out for insect cells and filoviral protein expression was driven from a baculovirus vector, vlps were generated and found to have filamentous structures resembling wild-type virus. vlps have demonstrated immunogenicity and the ability to protect nhps from lethal challenge. the first such study involving nhps utilized the baculovirusproduced vlps containing ebov gp, np, and vp [ ] . five cynomolgus macaques were vaccinated three times at -day intervals with µg of vlp with ribi adjuvant. after the full vaccination schedule, % survived a lethal ebov challenge, and there was a three-to ten-fold increase in ebov specific antibodies which possessed an % plaque reduction at titers between : and : [ ] . additionally, these antibodies were shown to have both compliment-mediated and antibody-dependent cell-mediated cytotoxic properties [ ] . using this same vlp technology, swenson et al. were able to show a similar effect for marv. three i.m. injections of mg of vlp in . ml of qs- adjuvant spaced days apart offered % protection against marv and ravv; however, one animal challenged with ravv did exhibit slight morbidity [ ] . all animals had no detectable viremia as determined by plaque assay at days , , , , , , and post-exposure. additionally, two injections of the vlps correlated with a peak in homologous antibody titer while three injections correlated to peak heterologous antibody titer [ ] . the ability of vlps to generate protective immunity against filoviral challenge has been demonstrated in guinea pigs as well [ ] . table . immunogenic virus-like particles (vlps) of ebov and marv can be easily generated in mammalian systems. ebov-like particles (vlps) can be efficiently generated in mammalian cells after expression of vp alone, but other filovirus proteins can be co-expressed as well [ ] [ ] [ ] . baculovirus-derived vlps have also been successfully generated in insect cells and stimulated both cellular and antibody immune responses against hepatitis e virus, human papilloma virus, rotavirus, and simian immunodeficiency virus [ ] [ ] [ ] [ ] . several groups have made and tested baculovirus-generated filovirus-vlps as vaccines in small animal models. warfield et al. generated ebola-vlps (evlp) and marburg-vlp (mvlp) containing vp , np, and gp. this vaccine had up to % survival following a lethal infection in mice (vaccine dose-dependent) [ ] . sun et al. produced an evlp containing vp and gp. this vaccine was also up to % effective following a lethal ebov infection in mice (vaccine dose-dependent) [ , ] . many reports document the ability of filoviral gp to act as a potent immunogen, and as such, viral vectors are a popular method of vaccinating through the expression of gp by the host and subsequent immune recognition. however, a recent report sought to utilize gp itself as the vaccination agent. in order to efficiently produce the protein, an expression vector was constructed such that the fc portion of a human igg was fused to ebov-gp [ ] [ ] [ ] . this gp-fc fusion protein induced both cell-mediated and humoral immune responses, and mice vaccinated with zebovgp-fc demonstrated % protection against a lethal ebov challenge. [ ] . while further studies are required, these results show that vaccination with ebovgp-fc alone is effective against a lethal ebov infection, and thus fusion proteins could potentially be used as an effective vaccine against filoviruses [ ] . the use of plants to produce vaccine antigens and antibodies has been demonstrated previously and is attractive for several reasons, including low manufacturing cost, efficient production, minimal risk of contamination with human pathogens or toxins, and the fact that plants have similar secretory pathways and endosomal systems as mammalian cells [ ] [ ] [ ] [ ] . recently, a bean yellow dwarf virus (beydv)-derived replicon system was developed and shown to efficiently produce antibodies against ebov in nicotiana benthamiana leaves [ ] . poolcharoen et al. used this system to develop and fuse ebov-gp to the c-terminus of an igg heavy chain [ ] . these ebov immune complexes (eic) were then used as a vaccine to immunize mice. serum antibody response tests showed that this eic was highly immunogenic in mice and produced antibody levels similar to mice protected from a lethal ebov challenge [ ] . while antibody titers alone do not fully correlated with protection from lethal challenge, there is potential for further development of this vaccine. a replicating vaccine that could spread through the target population after initial inoculation would be an attractive, alternative approach to filovirus development. this approach could provide high coverage with minimal initial vaccinations. a cmv-based vaccine would allow for this type of spread [ ] . cmv, a member of the family betaherpesvirinae, induces a high "effector" memory t-cell (t em ) response before establishing a low-level persistent infection [ ] [ ] [ ] . this high immunogenicity makes cmv a good potential vaccine vector, and the cmv vector has been previously shown to be effective as a vaccine for siv in rhesus macaques [ , ] . tsuda et al. constructed a mouse cmv vector expressing a cd + t cell epitope from the nucleoprotein (np) of ebov (mcmv/ebov-npctl). this vaccine induced high levels of ebov-specific cd + t cells, and subsequently, protected % of mice against a lethal ebov challenge. this shows a proof-ofconcept for cmv as a potential vaccine vector for ebov [ ] . as discussed previously, paramyxovirus-based vectors have demonstrated the capacity to induce strong immune responses and protect animals from infection. as such, a chimeric hpiv was developed where all the hpiv surface markers/receptors were deleted and replaced with ebov-gp [ ] . this chimeric virus can be amplified and recovered easily. additionally, this chimeric virus has -fold greater incorporation of ebov-gp into the virion due to the lack of competition with hpiv surface proteins [ ] . testing in guinea pigs showed that hpiv /Δf-hn/ebogp is highly attenuated, as compared to both hpiv and hpiv /ebogp, and immunogenic, as % developed neutralizing antibodies against ebov. finally, a × pfu i.n. inoculation of hpiv /Δf-hn/ebogp was able to protect % of guinea pigs against a lethal ebov challenge [ ] . table . . . rnapc severe coagulation disorders are one of the most prominent features of filoviral infection. in the event of a breach in vascular integrity a strict balance of pro-and anti-coagulant host factors must be maintained to successfully clot the breach and to prevent too much or too little clot formation. in the instance of filovirus infection, sustained microvascular injury in effected organs results in host coagulation inhibitor depletion which results in disseminated intravascular coagulation (dic). in dic, tissue factor (tf), a clotting factor normally present on cells not exposed to blood, complexes with circulating factor vii leading to clot formation and fibrin deposition through the extrinsic pathway [ , ] . as numerous studies have demonstrated a clear link with dic and resultant organ failure, geisbert et al, sought to eliminate potential tf pathogenesis during filovirus infection by using recombinant nematode anticoagulant protein c (rnapc ) [ ] . they demonstrated that rnapc , an inhibitor of the tf pathway, provided partial post-exposure protection to rhesus macaques during filovirus infection [ ] . previous studies with rnapc have already gone through phase ii trials in orthopedic surgery [ ] and coronary revascularization [ ] . geisbert et al. showed a % survival rate, in addition to a . -day increase in mean time-to-death, for ebov-infected rhesus macaques and a % survival rate, with a . -day increase in mean time-to-death, in marv-infected rhesus macaques, when treated with rnapc post-exposure [ , ] . in a normally % lethal model of filovirus infection, rnapc demonstrated a clear benefit to survival as an increase in the mean time-todeath was observed. rnapc has completed phase ii clinical trials with a good safety record. primates. comparison of current drug candidates at the highest level of development, either in human clinical trials or those that have shown promise in nhps. also listed are the afforded levels of protection in nhps, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. phosphorodiamidate morpholino oligomers (pmos) exert their anti-translation effects through steric hindrance of the translation machinery. this steric hindrance is possible due to a morpholino group, similar to a ribose base in rna, and a methylene phosphorodiamidate linking moiety that physically bind to mrna and prevent the translation machinery from accessing the mrna [ ] . once the antisense pmos bind to their target mrna, they are highly stable and highly soluble which would allow high levels of translation inhibition and predictably low levels of potential cytotoxicity [ ] [ ] [ ] [ ] . pmos have previously demonstrated effective antiviral activity against coronaviruses and flaviviruses [ , ] . swenson et al. initially utilized pmos targeting ebov vp and ebov vp to highly protect mice and guinea pigs against a lethal challenge with ebov and marv [ , ] . subsequently, avi- (a combination of pmos against both ebov vp and vp ) and avi- (a combination of pmos against both marv vp and np) were developed and tested in a nhp post-exposure scenario. these pmos, delivered - min post-exposure, protected > % of rhesus macaques against lethal ebov infection and % of cynomolgus macaques against marv infection [ ] . both the ease of controlled manufacture and their efficacy in nhp models to combat filovirus infection, pmos represent a viable therapeutic strategy [ ] . currently, avi- and avi- are in phase i clinical trials. table . . . recombinant human activated protein c ebolavirus disease (evd) and severe sepsis (or septic shock) share many clinical features including fever, hypotension, increased production of tissue factor, elevated levels of nitric oxide, and elevated levels of d-dimers [ ] [ ] [ ] . in addition, the most prominent and consistent finding in severe sepsis is severe protein c deficiency [ , ] . it was shown that treatment of patients with severe sepsis with recombinant human activated protein c (rhapc) resulted in improved survival [ ] . later experiments in nhps demonstrated that ebov infection results in rapid reduction of circulating protein c levels [ , ] . therefore, it was tested if treatment with rhapc could protect against lethal ebov infection in rhesus macaques. fourteen rhesus macaques were infected with a lethal dose of ebov; eleven were then treated with iv rhapc - min after challenge, continuing for days. all control animals died on day post-exposure; however, of the rhapc-treated animals survived (~ % survival). additionally, the mean time-to-death for rhapc-treated animals was . days, which is significant compared to the . days observed in placebo and historical controls [ ] . this product was pulled as a single post-exposure treatment, but given that this intervention is not directly targeting the virus, there may be additional merit in assessing this product in conjunction with a direct antiviral. rna interference (rnai) represents a powerful, naturally occurring biological strategy for inhibiting gene expression. rnai interferes with the translation of mrna to protein products by either sterically blocking mrna or by triggering rnase h-mediated cleavage of the dna/rna duplex, resulting in inhibition of gene expression [ ] . for many years rnai as demonstrated clear efficacy in preventing viral replication in vitro against a number of viruses, including coxsackieviruses, hepatitis b virus (hbv), hepatitis c virus (hcv), herpesviruses, human immunodeficiency virus (hiv), human papillomavirus, rsv, influenza a virus, lymphocytic choriomeningitis virus, polioviruses, and sars-cov [ ] [ ] [ ] [ ] . fowler et al. demonstrated that small-interfering rna (sirna) downregulation of various marv mrna transcripts was able to significantly decrease viral protein production and subsequent viral release in cell culture [ ] . unfortunately, efficient delivery vehicles providing effective drug targeting and stability have hindered the application of rnai in the clinical setting. however, recent developments in the field of nanotechnology have made nanoparticles the solution to increasing pharmacokinetic profiles for rnai therapies [ ] . additionally, tekmira, inc. developed proprietary lipid encapsulation as a means of improving the pharmacology of sirna targeting the ebola rna polymerase l protein, as demonstrated by geisbert et al. [ , ] . to efficiently deliver the sirna to target cells, a mixture of lipids forming a bilayered liposome, or stable nucleic acid-lipid particles (snalp), was designed. the snalp ensures cell entry by preferential fusing with the endosomal membrane upon exposure to the decreasing ph of the endosome. the snalps were further modified by conjugation with polyethylene glycol (peg) ensuring stability and efficient delivery of the sirna payload by neutralizing surface charges and presenting a hydrophilic exterior. this encapsulation was initially demonstrated to significantly increase the stability, half-life, and effectiveness of sirna directed against hbv [ ] . the snalp-encapsulation of sirna targeting the ebov l protein was initially shown to completely protect guinea pigs when administered shortly after a lethal ebov challenge [ ] . this treatment was then assessed for efficacy in rhesus macaques. snalp-encapsulated sirnas targeting ebov l polymerase, vp , and vp were given to rhesus monkeys either four or seven times following a lethal challenge of ebov. two of the three monkeys given four doses survived lethal infection, while all four monkeys given seven doses survived infection [ ] . the enhanced survivorship among the snalp-treated group highlights the efficacy of this potential therapeutic. additionally, there was little to no evidence of side effects associated with the treatment group, aside from mildly altered liver enzyme levels (which could have been an artifact separate from the challenge course) [ ] . table . innate immunity is often the first line of defense against invading pathogens. one mechanism by which innate immunity functions relies on the identification of pathogen-associated molecular patterns (pamps). pamps consist of unique carbohydrate moieties on the external surfaces of foreign microbes, such as hexose and mannose, which are not expressed on the surfaces of the host cells. these pamps are then recognized by host proteins such as mannose-binding lectins (mbl), which recognize these high hexose and mannose contents [ ] . filoviruses have large amounts of mannose comprising their glycoproteins and thus are a target of mbl [ ] . upon exposure, host mbl targets filoviruses for compliment-dependent virus neutralization through the lectin pathway of the compliment cascade [ ] . when administered in supraphysiological doses before or after lethal challenge with ebov, recombinant mbl treatment protected % of mice [ ] . these studies showed that mbl may be a potential post-exposure prophylactic. table . post-exposure treatments effective in small animal models. comparison of current treatment candidates at the small animal model level of development, specifically mouse models. also listed are the afforded levels of protection, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. high-throughput screening (hts) is a significant tool for novel drug discovery. hts involves screening libraries consisting of thousands to hundreds of thousands of unique molecules against specific targets. available libraries used in hts have included natural compounds [ , ] , peptides [ ] , drugs [ ] , and synthetic compounds [ , ] . recently, compound fgi- was identified during a screen with an ebov-gfp pseudotyped virus and has shown strong antiviral activity in vitro against high doses of ebov and marv. fgi- was subsequently shown to protect mice against lethal challenges of both ebov and marv [ ] . additionally, compound fgi- was initially identified in a similar manner and was shown to exhibit strong antiviral activity in vitro against ebov, rift valley fever virus (rvfv), all four types of dengue virus (denv), hcv, and hiv- . fgi- also protected mice against a lethal challenge of ebov when given postexposure [ ] . taken together, this suggests that fgi- probably acts on a conserved pathway common to these four viruses, and potentially makes for a very intriguing antiviral. a second screen was done using a collection of , small molecule compounds obtained from the nci and the ebov-gfp pseudotyped virus [ ] . as a result, nsc , a reactive oxygen species scavenger, was identified and shown to have high antiviral activity against ebov, marv, lassa virus, rvfv, and veev. in vivo studies demonstrated that this compound protected mice against a lethal challenge of ebov and marv when given either pre-or post-exposure [ ] . metal ion-based therapeutics are a new potential class of drugs because they differ from carbon-based compounds due to the charged central ion which determines the molecular geometry of the compound. through these unique molecular geometries, specific compounds can be isolated that inhibit biological processes, and are unlike traditional carbon-based compounds because of their unique geometry. this difference allows these compounds to form octahedral and square planar molecular geometries. hexamminecobalt (iii) chloride (cohex) is a complex of a cobalt (iii) ion surrounded by six ammonia ligands in a full octahedral coordination. cohex was initially reported to have antiviral activity against adenovirus and sindbis virus, and was subsequently thought to have potential broad-spectrum antiviral activities [ ] . cohex was shown to be well-tolerated in mice with no apparent toxicity. mice were treated with cohex daily and infected with a lethal dose of ebov. cohex-treated mice had a significant increase in mean time-to-death, and the highest concentration treatment group had a % survival rate [ ] . this suggests that cohex has the potential to be an effective treatment against ebov infection. niemann-pick c (npc ), a cholesterol transporter found in endosomes and lysosomes, was recently identified as being required for ebov replication during a gene trap screen in hap cells using a replication-competent vsv bearing the ebov glycoprotein (rvsv-gp-ebov). in these experiments, cells with non-functional npc demonstrated decreased infectivity by rvsv-gp-ebov; however, expression of a functional npc rescued the normal infectivity of these viruses [ ] . npc is known to affect calcium homeostasis as well as endosomal and lysosomal fission and fusion [ ] [ ] [ ] . it has also been shown to be involved in hiv- release [ ] . loss of npc causes a neurological disorder called niemann-pick disease, which is characterized by cholesterol accumulation in lysosomes [ ] . while heterozygous npc knockout mice (npc +/− ) do not show evidence of niemann-pick disease, most npc +/− knockout mice were protected against a lethal challenge of mouse adapted ebov ( % survival) and marv ( % survival) [ ] . additionally, small molecules, such as u a, have been identified that interfere with npc and cause a cellular phenotype similar to npc deficiency [ ] . as such, u a was subsequently shown to block infection of ebov in vitro [ ] . heat-shock protein (hsp ) is a molecular chaperone that aids in the folding, trafficking, and proteolytic processing of many proteins [ , ] . as a result of their many functionalities, hsp inhibitors have been designed to combat diseases such as cancer, and there are currently several drugs now in phase i and ii clinical trials [ , ] additionally, hsp has shown to be important for replication of negative-strand viruses, as well as hcv, hbv, and polio [ ] [ ] [ ] [ ] . the effects of several natural and synthetic hsp inhibitors on ebov replication were tested in vitro. results of this study demonstrated that three hsp inhibitors significantly inhibited the replication of ebov in vero cells and primary human monocytes, suggesting their use as a potential therapeutic [ ] . ebolaviruses express two secreted glycoproteins, soluble gp (sgp) and small soluble gp (ssgp) [ ] . sgp has been associated with stabilization of the endothelial barrier function and reduction of endothelial barrier permeability by opposing the effects of tnf-α. these effects are in direct opposition to the observed roles for gp, which has been associated with endothelial cell destruction [ ] . ssgp has yet to have a clear role during infection [ ] . each of the gp forms contains identical n-termini but differ in the structure of their c-termini. during the differentiation process of the c-termini, the homodimer sgp is cleaved by furin to yield the mature sgp and a Δ-peptide which is retained in cells longer as compared to sgp. when these Δ-peptides from ebov, sudv, and tafv were fused with fc tags and transfected into cells before infection, they were capable of inhibiting both ebov and marv infection in a dose dependent fashion [ ] . these observations indicated that Δ-peptides might play an important role in filovirus pathogenesis, and could be exploited as a novel anti-filoviral therapeutic [ ] . while many events in filovirus entry remain undiscovered, a fusion mechanism similar to hiv and sars-cov is thought to occur such that a conformational rearrangement of glycoproteins on the viral surface results in viral fusion with the cellular membrane. inhibitors of viral fusion have been developed for hiv- and sars-cov. these inhibitors prevent the c-terminal heptad repeat in the envelope protein from interacting with the cellular membrane proteins by directly competing and blocking its interaction with the n-terminal heptad repeat, which normally would result in the formation of the six-helix bundle required for membrane fusion. c-peptides, which are inhibitors of viral envelope fusion, have had limited success against filoviruses in the past, most likely due to the suggestive evidence that filovirus fusion occurs far along in the endosomal maturation process [ ] [ ] [ ] [ ] [ ] . however, when these c-peptides were conjugated with an argenine-rich segment of hiv- 's tat protein (a protein known for its endosomal localization) the conjugated c-peptide exhibited marked antiviral effects, up to % inhibition of ebov and marv in vitro [ ] [ ] [ ] . unfortunately, the concentrations used to generate this inhibition in vitro were not possible in the clinical setting, but this report indicates that future c-peptide research may result in filovirus entry prevention for future therapeutics. a recent report that screened , molecules demonstrated that chlorophyllide was able to decrease the section of hbv dna in a hbv antiviral assay. these results were obtained at compound concentrations which exhibited no cytotoxic effects. this molecule is an alkylated porphyrin containing copper and as such this compound is carries a charge at neutral phs [ ] . during these screens, the chlorin e compound, a metal-free chlorophyllide-like molecule, was found to be the most potent and was subsequently tested against other viruses, including marv. during testing, the chlorine e compound showed significant antiviral activity in vitro against marv. this compound also inhibited junin virus, denv, hcv, and hiv- [ ] . another recent study that examined a library of , compounds yielded candidates capable of generating ≥ % inhibition of a hiv- /ebov-gp pseudotyped virus, while not interfering with the hiv- /vsv-g control virus. from these candidates, compound , a benzodiazepine derivative, showed an ability to inhibit both ebov and marv to a similar degree in vitro [ ] . results from these experiments suggested that compound acts at an early stage of viral entry, preventing infection by an unknown mechanism [ ] . lj , an aryl methyldiene rhodanine derivative, was identified during a high-throughput screening of inhibitors for nipah virus entry in the context of a vsv-pseudotyped vector [ ] . this compound was subsequently shown to inhibit a variety of enveloped viruses, including marv, ebov, nipah, rvfv, hiv- , hcv, wnv, etc. [ ] . however, it did not inhibit nonenveloped viruses such as adenovirus and reovirus. further testing demonstrated that lj binds to the viral membrane and prevents virus-cell fusion. while initial testing in mice did not show antiviral efficacy, further development of this compound to improve pharmacokinetics and potency is distinctly possible [ ] . development of medical countermeasures for ebov and marv remain a high priority and substantial progress has been made over the past decade. we have moved from the inability to protect from infection in various animal models of disease to a realm of medical countermeasures that protect prophylactically and more recently successful treatments that can be employed following known exposure to the viruses. initial efforts, focused on preventing the disease with vaccination strategies, ranged from subunit vaccines to vlps, vectored systems, dna vaccines, and live-attenuated virus systems that express the ebov or marv glycoproteins. to that aim, vaccine efficacy has been achieved by multiple vaccines against parenteral and aerosol routes of exposure. with the success of these new vaccine platforms, the attention of the past years has focused on the ability to treat infected patients. in the animal models, success has been demonstrated with traditional small molecules and antibodies directed against the virus or critical host proteins or pathways associated with pathogenesis. the ability to utilize various rna silencing technologies has been a focus for therapeutics that could be beneficial for filovirus infection, other infectious diseases and cancer therapy. despite these successes, there is much work to do to adequately prepare for this infectious threat. the ability to provide a beneficial therapeutic impact at a point when patients experience clinical symptoms and seek relief from caregivers remains a hurdle for the medical countermeasure development. moreover, the quality of life of patients following infection and treatment may require additional development efforts or the combination of multiple therapeutic approaches. as seen in outbreaks, the clinical sequelae observed in patients that survive infection are severe and life changing. these observations emphasize the need for medical countermeasures that not only provide survival but also decrease morbidity and long-term pathological outcomes following infection. lastly, the funding resources fortitude and the ability to navigate regulatory pathways will be essential to reaching either emergency use authorization (eua) status or licensed drug status for therapeutics and vaccines. however, the field remains optimistic that medical countermeasure solutions for human use are possible. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations virus taxonomy-ninth report of the international committee on taxonomy of viruses a hitherto unknown infectious disease contracted from monkeys filoviridae: a taxonomic home for marburg and ebola viruses? a compendium of years of epidemiological, clinical, and laboratory studies drug targets in infections with ebola and marburg viruses ebolavirus and other filoviruses human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus marburg virus infection detected in a common african bat discovery of swine as a host for the reston ebolavirus differentiation of filoviruses by electron microscopy virion nucleic acid of ebola virus marburg and ebola viruses recent advances in ebolavirus vaccine development filovirus vaccines. hum. vaccin processing of the ebola virus glycoprotein by the proprotein convertase furin c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus recovery potential of a western lowland gorilla population following a major ebola outbreak: results from a ten year study correlates of immunity to filovirus infection filovirus hemorrhagic fever outbreak case management: a review of current and future treatment options the medecins sans frontieres intervention in the marburg hemorrhagic fever epidemic, uige, angola, . i. lessons learned in the hospital dengue and dengue hemorrhagic fever: management issues in an intensive care unit real-time monitoring of cardiovascular function in rhesus macaques infected with zaire ebolavirus progress towards the treatment of ebola haemorrhagic fever a phase , randomized, double-blind safety and pharmacokinetic assessment of respiratory syncytial virus (rsv) prophylaxis with motavizumab and palivizumab administered in the same season antibodies for prevention and treatment of respiratory syncytial virus infections in children ebola virus: from discovery to vaccine treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee the marburg virus outbreak of and subsequent episodes an infectious disease transmitted by cercopithecus aethiops. ("green monkey disease") evaluation of immune globulin and recombinant interferon-alpha b for treatment of experimental ebola virus infections neutralizing antibody fails to impact the course of ebola virus infection in monkeys protective efficacy of neutralizing antibodies against ebola virus infection antibody-dependent enhancement of marburg virus infection epitopes required for antibody-dependent enhancement of ebola virus infection antibody-dependent enhancement of ebola virus infection recombinant human monoclonal antibodies to ebola virus pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody epitopes involved in antibody-mediated protection from ebola virus postexposure antibody prophylaxis protects nonhuman primates from filovirus disease protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies enhanced potency of a fucose-free monoclonal antibody being developed as an ebola virus immunoprotectant characterization of zaire ebolavirus glycoprotein-specific monoclonal antibodies neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps development of a preventive vaccine for ebola virus infection in primates a replication defective recombinant ad vaccine expressing ebola virus gp is safe and immunogenic in healthy adults vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections protection of nonhuman primates against two species of ebola virus infection with a single complex adenovirus vector enhanced protection against ebola virus mediated by an improved adenovirusbased vaccine a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs recombinant adenovirus serotype (ad ) and ad vaccine vectors bypass immunity to ad and protect nonhuman primates against ebolavirus challenge rhabdoviridae: the viruses and their replication vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine recombinant vesicular stomatitis virus vector mediates postexposure protection against sudan ebola hemorrhagic fever in nonhuman primates effective post-exposure treatment of ebola infection inactivated or liveattenuated bivalent vaccines that confer protection against rabies and ebola viruses replicon-helper systems from attenuated venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo recombinant rna replicons derived from attenuated venezuelan equine encephalitis virus protect guinea pigs and mice from ebola hemorrhagic fever virus marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates vaccine potential of ebola virus vp , vp , vp , and vp proteins protective cytotoxic t-cell responses induced by venezuelan equine encephalitis virus replicons expressing ebola virus proteins a single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose ebola virus challenge successful topical respiratory tract immunization of primates against ebola virus parainfluenza viruses mucosal parainfluenza virus-vectored vaccine against ebola virus replicates in the respiratory tract of vector-immune monkeys and is immunogenic recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of h n highly pathogenic avian influenza virus protect against virus challenge in monkeys respiratory tract immunization of non-human primates with a newcastle disease virus-vectored vaccine candidate against ebola virus elicits a neutralizing antibody response ebola virus vp -induced particle formation and association with the lipid bilayer contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple marburg viruses filovirus-like particles produced in insect cells: immunogenicity and protection in rodents ebola virus vp -induced particle formation and association with the lipid bilayer ebola virus vp drives the formation of virus-like filamentous particles along with gp immunogenicity and protective efficacy of rotavirus / -virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice expression and self-assembly of empty virus-like particles of hepatitis e virus enhanced mucosal and systemic immune responses following intravaginal immunization with human papillomavirus l virus-like particle vaccine in thermosensitive mucoadhesive delivery systems intranasal immunization with siv virus-like particles (vlps) elicits systemic and mucosal immunity protection against lethal challenge by ebola virus-like particles produced in insect cells ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies immunogenicity of the outer domain of a hiv- clade c gp increased potency of fc-receptor-targeted antigens cross-reactive hiv- -neutralizing activity of serum igg from a rabbit immunized with gp fused to igg fc: possible role of the prolonged half-life of the immunogen ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice transgenic plants as protein factories monoclonal antibody manufacturing in transgenic plants--myths and realities high-level rapid production of full-size monoclonal antibodies in plants by a single-vector dna replicon system recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor expression of an immunogenic ebola immune complex in nicotiana benthamiana a replicating cytomegalovirus-based vaccine encoding a single ebola virus nucleoprotein ctl epitope confers protection against ebola virus effector memory t cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge broadly targeted human cytomegalovirus-specific cd + and cd + t cells dominate the memory compartments of exposed subjects human cytomegalovirus tropism for endothelial cells: not all endothelial cells are created equal profound early control of highly pathogenic siv by an effector memory t-cell vaccine chimeric human parainfluenza virus bearing the ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against ebola virus challenge disseminated intravascular coagulation (dic) coli septic shock is prevented by blocking tissue factor with monoclonal antibody treatment of ebola virus infection with a recombinant inhibitor of factor viia/tissue factor: a study in rhesus monkeys dose-response study of recombinant factor viia/tissue factor inhibitor recombinant nematode anticoagulant protein c in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement recombinant nematode anticoagulant protein c , an inhibitor of the tissue factor/factor viia complex, in patients undergoing elective coronary angioplasty marburg virus angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c cell penetrating peptide conjugates of steric block oligonucleotides phosphorodiamidate morpholino oligomers: favorable properties for sequencespecific gene inactivation morpholino antisense oligomers: design, preparation, and properties stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice chemical modifications of antisense morpholino oligomers enhance their efficacy against ebola virus infection advanced antisense therapies for postexposure protection against lethal filovirus infections ebola hemorrhagic fever and septic shock recombinant human activated protein c for the postexposure treatment of ebola hemorrhagic fever marburg and ebola hemorrhagic fevers: does the primary course of infection depend on the accessibility of organ-specific macrophages? severe protein c deficiency predicts early death in severe sepsis protein c concentrations in severe sepsis: an early directional change in plasma levels predicts outcome efficacy and safety of recombinant human activated protein c for severe sepsis mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event silencing viruses by rna interference. microbes infect rnai, a new therapeutic strategy against viral infection rna interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus inhibition of sars-cov replication by sirna inhibition of marburg virus protein expression and viral release by rna interference lipidic systems for in vivo sirna delivery postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by rna interference postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study potent and persistent in vivo anti-hbv activity of chemically modified sirnas high-dose mannose-binding lectin therapy for ebola virus infection phase i safety, tolerability, and pharmacokinetic study of recombinant human mannanbinding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dcsign and complement-mediated virus neutralization marine natural product libraries for high-throughput screening and rapid drug discovery identification of upregulators of bmp expression via high-throughput screening of a synthetic and natural compound library in vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes high-content assay to identify inhibitors of dengue virus infection drugs for bad bugs: confronting the challenges of antibacterial discovery antiviral activity of a small-molecule inhibitor of filovirus infection development of a broad-spectrum antiviral with activity against ebola virus development of high-content imaging assays for lethal viral pathogens identification of an antioxidant small-molecule with broad-spectrum antiviral activity hexamminecobalt (iii) chloride as a broad-spectrum antiviral complex niemann-pick c disease gene: homology to mediators of cholesterol homeostasis niemann-pick c functions independently of niemann-pick c in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo niemann-pick disease type c is a sphingosine storage disease that causes deregulation of lysosomal calcium deficiency of niemann-pick type c- protein impairs release of human immunodeficiency virus type and results in gag accumulation in late endosomal/lysosomal compartments cholesterol synthesis inhibitor u a and the role of sterol metabolism and trafficking in numerous pathophysiological processes inhibition of heat-shock protein reduces ebola virus replication regulation of signaling protein function and trafficking by the hsp /hsp -based chaperone machinery phase i trial of -allylamino- -demethoxygeldanamycin in patients with advanced cancer hsp and the chaperoning of cancer antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses evolutionary constraints on chaperonemediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp is important for vaccinia virus growth in cells heat-shock protein is essential for stabilization of the hepatitis c virus nonstructural protein ns ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry effects of ebola virus glycoproteins on endothelial cell activation and barrier function heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type gp are potent inhibitors of virus infection evidence that a prominent cavity in the coiled coil of hiv type gp is an attractive drug target capture of an early fusion-active conformation of hiv- gp inhibition of ebola virus entry by a c-peptide targeted to endosomes tat transduction: the molecular mechanism and therapeutic prospects cellular uptake of unconjugated tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses identification of a small-molecule entry inhibitor for filoviruses a broad-spectrum antiviral targeting entry of enveloped viruses the authors declare no conflict of interest. key: cord- - rhoffx authors: yu, changqing; li, sunan; zhang, xianfeng; khan, ilyas; ahmad, iqbal; zhou, yulong; li, shuo; shi, jing; wang, yu; zheng, yong-hui title: march inhibits ebola virus glycoprotein, human immunodeficiency virus type envelope glycoprotein, and avian influenza virus h n hemagglutinin maturation date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: rhoffx membrane-associated ring-ch-type (march ) strongly blocks human immunodeficiency virus type (hiv- ) envelope glycoprotein (env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. we now report that march also blocks the ebola virus (ebov) glycoprotein (gp) incorporation via surface downregulation. to understand how these viral fusion proteins are downregulated, we investigated the effects of march on ebov gp maturation and externalization via the conventional secretion pathway. march interacted with ebov gp and furin when detected by immunoprecipitation and retained the gp/furin complex in the golgi when their location was tracked by a bimolecular fluorescence complementation (bifc) assay. march did not reduce the gp expression or affect the gp modification by high-mannose n-glycans in the endoplasmic reticulum (er), but it inhibited the formation of complex n-glycans on the gp in the golgi. additionally, the gp o-glycosylation and furin-mediated proteolytic cleavage were also inhibited. moreover, we identified a novel furin cleavage site on ebov gp and found that only those fully glycosylated gps were processed by furin and incorporated into virions. furthermore, the gp shedding and secretion were all blocked by march . march also blocked the furin-mediated cleavage of hiv- env (gp ) and the highly pathogenic avian influenza virus h n hemagglutinin (ha). we conclude that march has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the golgi, which inhibits their transport from the golgi to the plasma membrane and incorporation into virions. required for viral entry ( , ) . ebov also expresses several different mature and soluble gp forms that are secreted or shed extracellularly, which is easily detectable by western blotting (wb) ( ) . thus, we used ebov gp and its mutant bearing mld deletion (Δmld) to study the march activity. march is expressed in terminally differentiated myeloid cells ( ) . we could not detect march expression in commonly used human cell lines including t, vero, thp , huh , hep g , hela, and hela-derived tzm-bi cells (fig. a) . thus, we investigated the march antiviral activity via ectopic expression. initially, we tested how march proteins from human (h), cow (bos taurus, b), and mouse (m) affect ebov entry using pseudotyped hiv- , which is a faithful system for studying the ebov gp function ( ) ( ) ( ) ( ) . all these march proteins restricted the pseudotyped virus replication as effectively as the retroviral restriction factor apobec g (fig. b) . to explore the march antiviral mechanism, ebov virus-like particles (vlps) and hiv- vlps were produced by expressing ebov vp or hiv- gag proteins in the presence of march and gp or gpΔmld. march strongly reduced the gp incorporation into these virions ( fig. c and d) . in contrast, the inactive ring domain mutant w a did not have such in addition, hiv- pseudoviruses containing the full-length ebov gp were produced and purified similarly. the levels of gp expression in these vlps were analyzed by wb using anti-flag, and the levels of vlps were determined by anti-hiv- gag (p ). protein expressions in t cells are shown in fig. d . (d) ebov virus-like particles (vlps) were produced from t cells after expressing ebov vp , human march or its w a mutant, and ebov gp or gpΔmld. after purification via ultracentrifugation, these vlps were analyzed by wb. protein expressions in viral producer cells are shown in fig. c . (e) ebov gpΔmld that has a gfp tag was expressed in hela cells in the presence or absence of human march . after staining with dapi, the gp localization was detected by confocal microscopy. (f) ebov gpΔmld was expressed with increasing amounts of human march in t cells. the gp expression on cell surface was detected by flow cytometry using anti-flag followed by alexa fluor -conjugated goat anti-mouse igg. activity (fig. d ). when gp subcellular localization was determined by confocal microscopy, although gp alone was detected predominantly on the plasma membrane, it was retargeted into the cytoplasm when march was expressed (fig. e) . consistently, gp was strongly downregulated from the cell surface by march in a dose-dependent manner when detected by flow cytometry (fig. f) . march retains ebov gp in the golgi. to understand where gp is targeted by march , we studied gp and march subcellular localization. first, we tested their interactions by immunoprecipitation. gpΔmld could specifically pull down march ( fig. a, lanes to ) , and furin could pull down gpΔmld and march ( fig. a , lanes to ). these results demonstrate that gp, furin, and march interact with each other in cells. second, we set up a bimolecular fluorescence complementation (bifc) assay to track their interactions in live cells ( ) . the c termini of gp, march , and furin were fused to the n-terminal to amino acids (aa) of a green fluorescent protein venus (vn) or its c-terminal to aa (vc). as a control, serine incorporator (serinc ) protein in addition, flag-tagged furin was expressed with ha-tagged human march and gpΔmld in t cells. proteins were immunoprecipitated with anti-flag and analyzed by wb. march , furin, and gp were detected by anti-ha, anti-flag, or anti-ebov gp, respectively. (b) gfp, gp-vc, furin-vn, or human march -vn proteins were individually expressed in hela cells. after staining with dapi, fluorescent signals were observed by confocal microscopy. (c) the march -vn-ha/gp-vc or furin-vn-ha/gp-vc bifc pair was expressed in hela cells. cells were stained with fluorescent anti-ha to detect march or furin. in addition, furin-vn that does not express the ha tag was expressed with gp-vc and ha-tagged march in hela cells. cells were stained with fluorescent anti-ha to detect march . fluorescent signals were observed by confocal microscopy. (d) indicated bifc fusion proteins were expressed individually or pairwise, and the levels of bifc signals were determined by flow cytometry. serinc , serine incorporator . experiments were repeated three times, and identical results were obtained. was also fused to vc. when gp-vc, furin-vn, and march-vn were expressed individually, no green bifc signal was detected by confocal microscopy (fig. b) . when gp-vc was expressed with march -vn or furin-vn, strong green fluorescent signals were produced that overlapped march or furin (fig. c) , confirming the interactions between gp and march , or gp and furin. unlike gp alone that was found on the plasma membrane (fig. e) , the gp-march bifc complex was found in intracellular compartments, confirming that march downregulates gp from the cell surface. in addition, when the furin-vn/gp-vc pair was expressed with march , their colocalization was also detected (fig. c ), confirming that these three molecules form a complex in live cells. when these interactions were quantified by flow cytometry, both march /gp and furin/gp pairs generated ϳ % bifc-positive cells, whereas positive cells produced from the march /serinc pair were only around % (fig. d ). thus, we detected specific march -gp and furin-gp interactions via bifc. third, we determined how march affects the gp and furin subcellular localization via confocal microscopy. the furin-vn/gp-vc bifc pair was expressed with an er marker, calnexin (cnx), or a tgn marker that was fused with mcherry. although the gp-furin complex colocalized with both cnx and tgn, its colocalization with tgn was much stronger than that with cnx when march was not expressed (fig. ). in the presence of march , its colocalization with tgn was strongly increased, whereas that with cnx was decreased. these results suggest that march retains ebov gp in the golgi. march blocks ebov gp proteolytic processing. the full-length gp was expressed with march from different species or its w a mutant, and gp processing was analyzed by comparing gp and gp expression levels via wb. as reported previously ( ) , because of the heavy o-and n-glycosylation of mld in the golgi, gp exhibited a higher molecular weight than gp (fig. a ). when march proteins were expressed, gp proteins were almost undetectable, indicating that march blocks the gp processing. the w a mutant was inactive, indicating that the ring domain is required for this activity. to understand whether this activity is cell type specific, similar experiments were conducted in hela, hep g , and huh cells. again, the gp expression in these cells was selectively inhibited by march (fig. b) . to confirm the specificity of this march effect, huh cells were also transfected with small interfering rnas (sirnas) to silence the march expression. when the march expression was decreased, the gp expression was effectively recovered (fig. b, lanes to ) . thus, the march effect is not cell type specific. to understand whether this march activity was affected by the mucin-like domain, the full-length gp and gpΔmld processing were compared side-by-side in the presence of wild-type (wt) march or its w a mutant (fig. c ), or march proteins from different species (fig. d ). unlike gp from the full-length gp, gp Δmld exhibited a lower molecular weight than gp Δmld. in addition, unlike similar levels of gp and gp from the full-length gp, the gp levels were much higher than gp from gpΔmld, confirming that gpΔmld is more effectively processed than the full-length gp. nevertheless, gp expressions from both full-length gp and gpΔmld were completely inhibited by march proteins but not by the w a mutant ( fig. c and d) . to test whether march destabilizes gp , cells were treated with inhibitors that target different degradation pathways, including mg for proteasomes, nh cl and bafilomycin a (bafa ) for lysosomes, and dbeq for er-associated protein degradation (erad). none of these inhibitors rescued gp expression or increased gp expression, indicating that gp is not subjected to degradation by these two pathways (fig. e) . we also tested how march affects the expression of vesicular stomatitis virus glycoprotein (vsv-g), a class iii fusion protein. the vsv-g expression was strongly reduced by march but not by its w a mutant, and the reduction was blocked by nh cl but not mg (fig. f ), confirming that march triggers vsv-g degradation via the lysosome pathway as reported previously ( ) . human march has amino acids (aa) that create a ring-ch finger in the n-terminal cytoplasmic tail (ct), two tm domains, and a c-terminal ct (fig. a ). the critical w residue is in the ring region and is required for recruitment of ubiquitinconjugating e enzyme ( ) . to uncover other important domains, we created three march c-terminal ct deletion mutants by expressing its -to- -aa ( - ), -to- -aa ( - ), and -to- -aa ( - ) regions. when these mutants were expressed with the full-length gp or gpΔmld, wt and mutants - and - blocked the gp processing, whereas the - mutant did not (fig. a ). these mutants were then fused with a green fluorescent protein (gfp) tag, and their subcellular localizations were tracked and compared by confocal microscopy. both wt and the - mutant displayed a scattered punctate localization in the cytoplasm, whereas the - mutant protein with a ring domain and two tm domains is presented. three march c-terminal ct deletion mutants, - , - , and - , were created and expressed with the full-length gp or gpΔmld. march and gp expression was detected by wb using anti-ha or anti-flag. (b) march and its three deletion mutants that have a c-terminal gfp tag were expressed in hela cells. after staining with dapi, their subcellular localizations were detected by confocal microscopy. (c) a schematic diagram of human furin functional domains is presented. flag-tagged cd, p domain, and cd subdomain-deletion mutants were created and were expressed with gpΔmld. proteins were immunoprecipitated by anti-flag and detected by wb. furin was detected by anti-flag, and ebov gp was detected by anti-ebov gp antibodies. (d) the full-length gp was expressed with increasing amounts of furin in t cells. the gp and furin expression was determined by wb. ® exhibited diffuse localization in the cytoplasm and nucleus (fig. b) . the - mutant diffused partially in the cytoplasm. these results demonstrate that the - region determines march intracellular compartmentalization that is critical for its activity. identification of a novel furin cleavage site on ebov gp. human furin is a -aa type i tm protein with a large lumenal region that has a signal peptide (sp), prodomain (pro), subtilisin-like catalytic domain (cd), p domain, cysteine-rich region (crr), tm domain, and a -aa ct ( ) (fig. c) . the cd and p domain are functionally critical for furin convertase activities. we created five furin dominant negative mutants, including Δcd, Δp, Δcd , Δcd , and Δcd , by deleting cd ( to aa), p domain ( to aa), and the three cd subdomains cd ( to aa), cd ( to aa), and cd ( to aa). when these five mutants were expressed with gp, they all blocked the gp cleavage from gp to gp (fig. c , lanes to ), confirming that ebov gp is processed by furin ( ) . consistently, when their interactions were determined by immunoprecipitation, gp was pulled down by the Δp mutant, but not by the Δcd, Δcd , Δcd , and Δcd mutants (fig. c) , indicating that gp interacts with the furin catalytic domain. to detect furin-cleaved ebov gp products, full-length gp was expressed with increasing amounts of furin. gp and gp were detected even in the absence of ectopic furin, which is produced by endogenous furin (fig. d , lane ). in the presence of ectopic furin, although the levels of gp were not changed, those of gp and gp were decreased or increased in a dose-dependent manner by furin (fig. d , lanes to ). in addition, there appeared another gp at ϳ kda that was also increased by furin. accordingly, a novel furin cleavage site, rkir , was found in gp in front of mld, and this novel -kda product was named gp * (see fig. b and also fig. s in the supplemental material). we conclude that gp * is predominantly processed from gp by furin. analysis of the gp glycosylation becomes complex due to proteolytic processing. to detect gp glycosylation, we blocked gp processing by removal of the furin cleavage site between gp and gp . two furin cleavage site-deficient (Δfr) mutants were created from the full-length gp and gpΔmld, and gp glycosylation was analyzed by treatments with endoglycosidase h (endo h) and peptide-n-glycosidase f (pngase f) that trim off nh-glycans or all n-glycans, respectively. gpΔfr was detected at and kda, and gpΔmldΔfr was detected at and kda (fig. a , lanes and ). all these proteins were sensitive to pngase f, confirming that they are modified by n-glycans (fig. a, lanes and ) . a ϳ -kda, pngase f-resistant protein was detected from gpΔfr after treatment with pngase, confirming that gp but not gpΔmld is modified by o-glycans (fig. a, lane ) . the -kda gpΔfr and -kda gpΔmldΔfr were resistant to endo h (fig. a, lanes and ) , indicating that they are modified by nc-glycans. the -kda gpΔfr and -kda gpΔmldΔfr were sensitive to endo h (fig. a, lanes and ) , indicating that they are modified by nh-glycans. when march was expressed, only the endo h-sensitive -kda gpΔfr and -kda gpΔmldΔfr were detected (fig. a, lanes to and lanes to ) . additional experiments were conducted to verify these results by using the fulllength gp and gpΔmld that have an active furin cleavage site. much more o-glycosylated gp proteins were detected from the full-length gp, which should come from gp (fig. a, lane ) . all gp proteins were sensitive to, whereas all gp proteins were resistant to, endo h (fig. a, lanes and ) , confirming that gp and gp are modified by nh-or nc-glycans, respectively. again, march selectively inhibited the endo-h-resistant and pngase f-resistant gp expressions (fig. a, lanes to and lanes to ) . collectively, these results demonstrate that march blocks gp modifications by nc-and o-glycans, but not by nh-glycans. modification of ebov gp by nc-and o-glycans is required for its proteolytic cleavage by furin and incorporation into virions. because gp is packaged into ebov virions ( ), we addressed how glycosylation affects its incorporation into ebov vlps. gpΔfr and gpΔmldΔfr were expressed with ebov vp and march , and their expressions in vlps were compared. we confirm again that march selectively inhibited the expression of gp with nc-and o-glycans (fig. b, lanes , , , and ) . notably, only gp with these two types of glycans was detected from vlps (fig. b) , indicating that only gp proteins with nc-and o-glycans are selectively incorporated into virions. when march was replaced with furin in this experiment, the expression of gp with nc-and o-glycans was also inhibited, but gp * showed up that was also incorporated into virions (fig. b, lanes and ) . gp * was produced much more from gpΔmldΔfr than gpΔfr, and it was also detected from gpΔmldΔfr in the absence of ectopic furin expression that was also enriched in virions (fig. b, lane ) . collectively, these results demonstrate that furin selectively cleaves gp with nc-and o-glycans. in addition, because gp * is incorporated into vlps (fig. b, lanes , , and ), we confirm that only fully glycosylated gps are incorporated into virions. because gp is modified by nc-and o-glycans, it further confirms that furin selectively processes fully glycosylated gp. march blocks ebov gp shedding and secretion. after maturation, gp /gp trimers on the plasma membrane are further cleaved by the tumor necrosis factor alpha (tnf-␣) converting enzyme (tace) at the gp membrane-proximal external region to release a soluble shed gp ( ) . to verify march 's inhibitory effect on ebov gp march inhibits viral fusion protein maturation ® maturation, we tested how march affects gp shedding. after expression of gp and gpΔmld with march or furin, gp proteins were immunoprecipitated from supernatants and analyzed by wb. march strongly blocked gp shedding, and in contrast, furin strongly increased it (fig. a ). gp * was also detected as soluble proteins. in addition to gp, ebov produces secreted gp (sgp) and small secreted gp (ssgp) via rna editing, all of which share the n-terminal aa (fig. b) . to address how march affects the gp secretion, we expressed sgp with march proteins and measured its secretion. unlike its w a mutant, march retained sgp in cells and effectively reduced its secretion (fig. c, lanes to ) . next, we expressed only gp or gp Δmld that should also be secreted as sgp and tested how march affects their secretion. march proteins from different species strongly increased gp and gp Δmld expression in cells but completely blocked their secretions (fig. c, lanes to ) . march blocks hiv- env and h n ha maturation. unlike hiv- env, most iav has are processed by trypsin-like serine proteases. however, a highly pathogenic avian strain such as h n has gained an additional furin cleavage site to expand its tropism ( ) . thus, we used h n ha as well as hiv- env to confirm the march antiviral activity. when hiv- env and h n ha were expressed with furin and march , furin pulled down gp or ha with march (fig. a) , confirming that march interacts with env/furin or ha/furin complex. in addition, when march was expressed with these fusion proteins, hiv- gp /gp and h n ha /ha expression levels were all decreased (fig. b) , confirming that march also blocks the env and ha cleavage by furin. when the env and ha glycosylations were analyzed after treatments with glycosidases, they were all sensitive to pngase f (fig. c, lanes and ) , confirming that they are modified by n-glycans. hiv- gp was completely sensitive to endo h, indicating that it is modified by nh-glycans (fig. c, lane ) . hiv- gp was only partially sensitive to endo h (fig. c, lane ) , indicating that it is modified by both nh-and nc-glycans. h n ha and ha were completely sensitive or resistant to endo h (fig. c, lane ) , indicating that ha and ha were modified by nh-or nc-glycans, respectively. when march was expressed, the gp expression was reduced likely due to inhibition of the furin activity (fig. c, lane ) . in addition, endo h-sensitive ha expression was increased (fig. c, lane ) . collectively, these results demonstrate that march does not block the env and ha modification by nh-glycans in the er, but rather they suggest that it should target the other glycosylation steps. class i fusion proteins project from the virion surface as highly glycosylated heterotrimeric spikes and initiate infection by binding to receptors on the surface of target cells. although it is clear that glycosylation and proteolytic cleavage are critically important, it remains incompletely understood how these steps are coopted to generate mature spikes that are incorporated from the plasma membrane into virions. we march inhibits viral fusion protein maturation ® now show that march specifically inhibits these processes, which provides new understanding for this poorly defined mechanism. our results highlight the fundamental role of n-glycosylation in the spike formation. first, n-glycosylation is absolutely required for ebov gp maturation. although march does not affect the gp modification by nh-glycans in the er, it inhibits the gp modification by nc-and o-glycans as well as its proteolytic processing in the tgn. these results suggest that march inhibits the conversion of nh-glycans to nc-glycans, likely by blocking the mannose trimming step in the cgn and/or the following glycosylation steps in the medial-golgi and the tgn. in addition, it is possible that o-glycosylation and furin cleavage are both dependent on the mannose trimming. in fact, when gpΔfr and gpΔmldΔfr were expressed with furin, only gp proteins with nc-and o-glycans were processed into gp * (fig. b, lanes and ) . consistently, when gp was expressed with furin, only gp but not gp was effectively processed into gp * (fig. d) . these results strongly suggest that the gp processing by furin is dependent on the completion of the glycosylation processes. second, n-glycosylation is required for ebov gp transport from the tgn to the plasma membrane. march not only blocked gp shedding and secretion (fig. ) , but also downregulated gp expression on the cell surface ( fig. e and f) . these inhibitory effects were likely caused by an inhibition of the gp glycosylation in the golgi by march . in fact, we found that only gp proteins modified with o-and/or nc-glycans were selectively incorporated into virions (fig. b) . thus, glycosylation is required for gp externalization and incorporation into virions. because we detected the gp interaction with march as well as the gp retention in the tgn by march , it is possible that march directly or indirectly retains viral glycoproteins at an early location so they never physically reach the location where they would be glycosylated. in that case all the glycosylation machinery would be intact but the gp would never reach it. in addition to the three class i fusion proteins, march also targets a class iii fusion protein, vsv-g, via a different mechanism. instead of inhibiting maturation, march triggers vsv-g degradation via the lysosomal pathway. although march does not trigger class i fusion protein degradation, its activity still depends on the ring domain, because the w a mutant that has a dead ring domain is inactive. these results demonstrate that the march activity depends on the ubiquitination pathway. we speculate that march might ubiquitinate and degrade a critical cellular transport facilitator, which blocks gp from access to the glycosylation and furin cleavage machinery. however, the degradation of vsv-g by march follows a similar mechanism by which march proteins downregulate immune receptors from the cell surface ( ) . march proteins usually ubiquitinate a lysine residue in the cytoplasmic tail of these immune receptors, which redirects them from the endosomes to the late endosomes and the lysosomes for degradation when they are endocytosed from the cell surface ( ) . thus, march proteins use two different mechanisms to target different proteins. for class i fusion proteins, march blocks their anterograde transport from the tgn to the plasma membrane; for vsv-g and immune receptors, march proteins block their recycling back to the tgn after retrograde transport from the plasma membrane to the endosomes. in addition to these four viral proteins, march proteins can target an amazingly large number of cellular proteins ( ) . most of these targets are immune receptors on the cell surface that play an important role in innate and adaptive immunity, including mhc i, mhc ii, cd , cd , cd , cd , cd , cd , intracellular adhesion molecule (icam)- , nkg d ligand mult , transferrin receptor (tfr), trail receptor , and interleukin- receptor (il rap). thus, the expression of march proteins is tightly regulated, which causes their poor expression in immortalized cell lines and a major difficulty in studying their endogenous function ( ) . thus, we were unable to further confirm the march antiviral activity by silencing its endogenous gene expression. in addition, due to lack of structural data on march proteins, it is difficult to understand how they have so many different targets. the role of tm domains of march proteins in substrate recognition has been suggested ( ) . a gxxxg motif is conserved in march tm domains, and it is also present in the mhc ii tm domain, which may mediate their interactions. in addition, the recognition could be mediated by a common adaptor protein. in addition to march , the maturation of viral fusion proteins is inhibited by guanylate-binding proteins (gbp) and ( ) , and interferon-induced transmembrane (ifitm) protein ( ) . gbp / directly target the cytoplasmic part of furin and inhibit its enzymatic activities, which exert broad antiviral activity by inhibiting furin-mediated cleavage of viral fusion proteins. ifitm interrupts viral glycoprotein processing of murine leukemia virus (mlv) env, hiv- env, and vsv-g, but not ebov gp, suggesting that it does not affect furin function. in addition, it interferes with env trafficking and promotes its degradation in lysosomes. thus, mammalian cells have evolved diverse mechanisms to intercept the production of mature viral fusion proteins for assembly of infectious virus particles. march is naturally expressed in terminally differentiated myeloid cells such as macrophages and dendritic cells (dcs). these cells are the primary targets for hiv- and ebov infection, transmission, and dissemination, and establishment of persistent tissue virus reservoirs in the case of hiv- . these cells can also be infected by h n , although the infection may not necessarily contribute to the significant part of pathogenesis. thus, march should play a role in defending these viruses during natural infection. in fact, when the march expression was silenced in macrophages, hiv- replication was strongly promoted ( ) . although macrophages and dcs express all hiv- receptors, they are much less permissive to hiv- infection than cd ϩ t lymphocytes. the hiv- resistance in these myeloid cells is currently attributed to their high expression levels of the host restriction factor samhd . however, the role of march should not be ignored. in addition, it should be addressed how viruses counteract march to establish productive infection. cell lines, rna oligonucleotides, and transfection reagents. hek t, hela, hep g , huh , vero e , and tzm-bi cells were cultured in dulbecco's modified eagle medium (dmem). thp- cells were cultured in roswell park memorial institute (rpmi) medium. media were supplemented with % fetal bovine serum and % penicillin-streptomycin (gibco), and cells were cultured at °c in a humidified atmosphere in a % co incubator. march sense/antisense rna oligonucleotides =-ccu ucucucgcacuucuautt- =/ =-auagaagugcgagagaaggtt- = and negative-control sense/antisense rna oligonucleotides =-uucuccgaacgugucacgutt- =/ =-acgugacacguucggagaatt- = were ordered from genepharma. t cells were transfected with polyethylenimine (pei) from sigma, huh cells were transfected with lipofectamine from thermo fisher, and hep g cells were transfected by amaxa nucleofector using program t- from lonza. plasmids. the human march expression plasmid pcaggs- xha-hmarch and its w mutant pcaggs- xha-hmarch -w a were provided by kenzo tokunaga ( ) . bovine and murine march cdnas were amplified from bovine peripheral blood mononuclear cells or mouse raw . cells by rt-pcr and cloned into pcaggs with a c-terminal ha tag after ecori/xhoi digestion. the human march c-terminal deletion mutant - , - , and - expression vectors were created by pcr in pcaggs- xha-hmarch vector. ebov gp and ebov vp expression vectors were described previously ( ) . the n-terminal flag-tagged ebov gpΔmld expression vector was provided by shan-lv liu. pcdna . -flag-sgp, pcdna . -flag-gp , and pcdna . -flag-gp Δmld were created by pcr from the ebov-gp and ebov gpΔmld vector and cloned into pcdna . (ϩ) vector by bamhi/ecori digestion. pcmv -furin-flag/myc was purchased from origene (catalogue number rc ). the furin Δsd, Δsd , Δsd , Δsd , and Δp mutants were created in the same furin expression vector by overlapping pcr. pcdna . -hmarch -gfp and pcdna . -gpΔmld-gfp were constructed by inserting march and ebov gpΔmld cdnas into pegfp-n vector (genbank accession no. u ) via ecori/agei digestion. to construct bifc expression vectors, the furin, march , and gp-Δmld cdnas were inserted into pcdna . -vn, pcdna . -vn-ha, or pcdna . -flag-vc (c-terminal vn/vc) vectors by ecori/agei digestion, which generated pcdna . -furin-vn, pcdna . -furin-vn-ha, pcdna . -march -vn-ha, or pcdna . -gpΔmld-flag-vc, respectively. pcdna . -serinc -flag-vc was reported previously ( ) . mcherry-tgnp-n- and mcherry-calnexin-n- expression vectors were obtained from michael davidson via addgene. h n ha expression vector was obtained from gary nabel. confocal microscopy assay. a total of ϫ hela cells were seeded on a glass slide h before transfection. cells were transfected with lipofectamine reagent (thermo fisher). after h, cells were washed and fixed with % paraformaldehyde and permeabilized with . % triton x- . after washing, cells were blocked with % bovine serum albumin. for immunofluorescence assay, cells were incubated with anti-ha primary antibody for h at room temperature (rt). after washing times with phosphate-buffered saline (pbs), cells were stained with alexa fluor- conjugated secondary antibody h at rt. following washing times with pbs, cells were stained with =, -diamidino- -phenylindole (dapi) for min, washed for h at rt, and observed by a confocal microscopy assay (zeiss lsm ). at least random cells per slide were analyzed, and the most representative images from each slide were selected for presentation. flow cytometry analysis. to detect ebov gp expression on the cell surface, a total of ϫ t were seeded in each well of a -well plate h before transfection. cells were transfected with pcdna . -flag-gpΔmld and increasing amounts of pcaggs- xha-hmarch expression vectors. after h, cells were centrifuged at , rpm for min and fixed with % paraformaldehyde for min at rt. after brief centrifugation, cells were blocked with % bovine serum albumin and incubated with anti-flag primary antibody for h at rt. after centrifugation and washing three times with pbs, alexa fluor -conjugated secondary antibody was added for h at rt. after washing times, cells were collected and analyzed by flow cytometry. to detect bifc, t cells in -well plates were transfected with the pcdna . -march -vn-ha/pcdna . -gpΔmld-flag-vc, pcdna . -furin-vn-ha/pcdna . -gpΔmld-flag-vc, or pcdna . -march -vn-ha/pcdna . -serinc -flag-vc pairs similarly and directly analyzed by flow cytometry. western blotting. forty-eight hours posttransfection, cells were lysed with ripa buffer (sigma) and cytosolic fractions were collected. after incubation with loading buffer, samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). some samples were subjected to endo h or pngase f treatment before applying them to the sds-page gel. for endo h (catalogue no. p l; neb) treatment, a total of g of cell lysate was first denatured by heating at °c for min in denaturing buffer. denatured proteins were added to a -l reaction system containing l of glycobuffer and , units of endo h and incubated at °c for h. for pngase f (catalogue no. p l; neb) treatment, the same amount of cell lysate was added to l of pngase f buffer ( ϫ) to make a -l total reaction volume and incubated at °c for min. after cooling down, l of rapid pngase f was added and samples were incubated h at °c. after transferring to polyvinylidene difluoride (pvdf) membrane, % nonfat milk was used to block the membrane for h at rt. the membrane was incubated with primary antibody and horseradish peroxidase (hrp)-conjugated secondary antibody. alternatively, the membrane was incubated with hrp-conjugated primary antibody. membranes were exposed after incubation with enhanced chemiluminescence (ecl) substrate (thermo fisher). mouse anti-march antibody was purchased from proteintech; mouse anti-actin, -ha, and -flag monoclonal antibodies were purchased from sigma; rabbit anti-ebov gp and rabbit anti-iav-ha polyclonal antibodies were purchased from sino biology (china). the mouse anti-hiv gp , anti-hiv gp , anti-hiv gp , and anti-hiv p /p monoclonal antibodies were obtained from the nih aids reagent program. hrp-conjugated anti-human, anti-rabbit, or anti-mouse immunoglobulin g secondary antibodies were purchased from pierce. immunoprecipitation. a total of ϫ t cells were seeded in each well of a -well plate h before transfection. forty-eight hours posttransfection, cells were lysed by ripa buffer. cytosolic fractions were collected and incubated with anti-flag antibody-conjugated magnetic beads (sigma) overnight at °c. the beads were isolated by magnetic shelf, washed several times, and used for wb assay. viral infectivity assay. a total of ϫ t cells were seeded in each well of a -well plate h before transfection. cells were transfected with pnl-luc-Δenv, pcdna . -flag-gpΔmld, and march expression vectors. forty-eight hours posttransfection, virion-containing supernatants were collected and subjected to ultracentrifugation at , ϫ g for h. pellets were collected and analyzed by wb. in addition, -l supernatants were collected for quantification by p gag enzyme-linked immunosorbent assay (elisa). supernatants with equal amounts of p gag were used to infect vero e cells that were seeded in a -well plate. after another h, infected cells were washed times with pbs and lysed with l ripa buffer. twenty-five microliters was collected and mixed with an equal amount of substrate from the bright-glo luciferase assay system (promega), and luminescence activity was determined by a luminometer. supplemental material is available online only. fig s , pdf file, . mb. unconventional protein secretion in animal cells er to golgi-dependent protein secretion: the conventional pathway glycosylation in health and disease arms race between enveloped viruses and the host erad machinery common features of enveloped viruses and implications for immunogen design for next-generation vaccines influenza virus n-linked glycosylation and innate immunity gp on hiv- virions lacks o-linked carbohydrate the secret life of viral entry glycoproteins: moonlighting in immune evasion structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme the membrane associated ring-ch proteins: a family of e ligases with diverse roles through the cell downregulation of cell surface receptors by the k family of viral and cellular ubiquitin e ligases downregulation of major histocompatibility complex class i by human ubiquitin ligases related to viral immune evasion proteins c-mir, a human e ubiquitin ligase, is a functional homolog of herpesvirus proteins mir and mir and has similar activity overview of the membrane-associated ring-ch (march) e ligase family march inhibits hiv- infection by reducing virion incorporation of envelope glycoproteins membrane-associated ring-ch (march) and are march family members that inhibit hiv- infection covalent modifications of the ebola virus glycoprotein mechanistic understanding of n-glycosylation in ebola virus glycoprotein maturation and function ebola virus glycoprotein with increased infectivity dominated the - epidemic human adaptation of ebola virus during the west african outbreak characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines bimolecular fluorescence complementation (bifc) analysis as a probe of protein interactions in living cells solution structure of the kaposi's sarcomaassociated herpesvirus k n-terminal domain reveals a novel e -binding c hc -type ring domain furin at the cutting edge: from protein traffic to embryogenesis and disease processing of the ebola virus glycoprotein by the proprotein convertase furin endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function ectodomain shedding of the glycoprotein gp of ebola virus the proteolytic regulation of virus cell entry by furin and other proprotein convertases . march inhibits viral infection by two different mechanisms guanylate-binding proteins and exert broad antiviral activity by inhibiting furin-mediated processing of viral envelope proteins ifitm reduces retroviral envelope abundance and function and is counteracted by glycogag murine leukemia virus glycosylated gag reduces murine serinc protein expression at steady-state levels via the endosome/lysosome pathway to counteract serinc antiretroviral activity we thank bin wang and wen-bo tan for reagents or technical assistance. we thank ian a. york for reading and very insightful comments on the manuscript. we thank kenzo tokunaga, shan-lv liu, michael davidson, and gary nabel, as well as the nih aids reagent program, for providing various reagents.changqing yu and sunan li are supported by national natural science foundation of china grants and , respectively. key: cord- -rmxin da authors: de clercq, erik title: new nucleoside analogues for the treatment of hemorrhagic fever virus infections date: - - journal: chem asian j doi: . /asia. sha: doc_id: cord_uid: rmxin da eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of ebola virus (ebov) and/or other hemorrhagic fever virus (hfv) infections. they can be considered as either (i) adenine analogues ( ‐deazaneplanocin a, galidesivir, gs‐ and remdesivir) or (ii) guanine analogues containing the carboxamide entity (ribavirin, eicar, pyrazofurin and favipiravir). all eight owe their mechanism of action to hydrogen bonded base pairing with either (i) uracil or (ii) cytosine. four out of the eight compounds (galidesivir, gs‐ , remdesivir and pyrazofurin) are c‐nucleosides, and two of them (gs‐ , remdesivir) also contain a phosphoramidate part. the c‐nucleoside and phosphoramidate (and for the adenine analogues the ′‐cyano group as well) may be considered as essential attributes for their antiviral activity. abstract: eight differentc ompounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of ebola virus (ebov) and/oro ther hemorrhagic fever virus (hfv) infections.t hey can be considered as either (i)adenine analogues ( -deazaneplanocin a, galidesivir,g s- andr emdesivir) or (ii)guanine analogues containing the carboxamide entity (ribavirin, eicar, pyrazofurin and favipiravir). all eight owe their mechanism of action to hydrogen bondedb ase pairing with either (i)uracil or (ii)cytosine. four out of the eight compounds (galidesivir, gs- , remdesivir and pyrazofurin) are c-nucleosides,a nd two of them (gs- , remdesivir) also contain ap hosphoramidate part. the c-nucleoside and phosphoramidate (and for the adenine analogues the '-cyano group as well) may be considered as essential attributes for their antiviral activity. the hemorrhagic fever virus (hfv) infections, including lassa and ebola, that we highlighted as potential targets for antiviral agents in [ ] have essentially remained unchanged,a nd now, years later,w eare still awaiting the first antiviral drug(s)t ob ef ormally approved for the treatment of hfv infections.t he first antiviral compound ever to be reported to be effective in the treatment of any hfv infection, that is, lassa, that could be used at any point in the illness as well as for postexposure prophylaxis, was ribavirin. [ ] ribavirin (virazole, -b-d-ribofuranosyl- , , -triazole- -carboxamide) ( figure ) was the first synthetic nucleoside analogue ever reported (in ) to be active against ab road spectrumo fb oth rna and dna viruses. [ ] in their final remarks, sidwell et al. ( ) stated, rightfully that "the antiviral spectrum of virazole was the broadest ever reported for any syntheticm aterial that does not induce interferon", although the eventual clinicalu se of virazole would eventually be limited to rna virus infections. [ ] one of the structural analogues mentioned in the latter article wase icar ( - eicar had originally been reported as an antileukemic agent in mice. [ ] in its antiviral activity,i ts howed as imilar spectrum as ribavirin, being active against pox-, toga-, arena-, reo-, orthomyxo-and paramyxovirus infections, with ap otency that was -to -fold greater than that of ribavirin. [ ] as originally ascertained for ribavirin, [ ] eicar also provedt ob eapotent inhibitor of imp dehydrogenase, thus blocking the biosynthesis of gmp. [ ] the depletion of the intracellular gtp and dgtp pools, resulting from the decreased biosynthesis of gmp,m ay also explain variouso ther effects of eicar, such as its potentiating effect on the anti-hiv activity of ddi ( ', '-dideoxyinosine) [ ] and its inhibitory effects on the replication of av ariety of viruses such as the double-stranded rna virus, ipnv (infectious pancreatic necrosis virus). [ , ] in the latter publication, yet another antiviralc ompound, pyrazofurin, was mentioned to be as pecific inhibitor or ipnv. [ ] pyrazofurin [ -(b-d-ribofuranosyl)- -hydroxypyrazole- -carboxamide] (figure ) is an inhibitor of omp decarboxylase, a key enzyme in the de novo pyrimidine mononucleotide biosynthesis:i th as provent ob ea ctivea gainst both (+ +)rna viruses [picorna (polio, coxsackieb ), toga (sindbis) and flavi (yellow fever)] and (À)rna viruses [paramyxo( measles, rsv), orthomyxo (influenza), rhabdo( vsv) and arena (junin, tacaribe)]. [ , ] pyrazofurinh as been found to be an exquisitely potent inhibitor of vsv (vesicular stomatitis virus), which belongs to af amily (rhabdoviridae), closely relatedt ot he family of the filoviridae to which ebola virus (ebov) and marburg virus belong. vsv can be handled in conventionals afety conditions, whereas ebov and marburgv irus require biosafety level ; vsv could therefore be recommended as ap aradigm for predicting antiviral activity against ebov. [ ] pyrazofurinh as so far not been evaluated for its potentiala ctivity against ebov;i t should be done so, as has already been done with the neplanocin aa nalogues. these analogues are knownt ob et argeted at the s-adenosylhomocysteine hydrolase. [ ] the prototype of this class of compounds is -deazaneplanocina( figure ). two decades ago, -deazaneplanocin aw as shownt ob ee ffective against al ethal ebov infection in mice. [ , ] the compound induced massively increased interferon-a production in ebov-infected mice, [ ] as tartling observation that was never followed up. when reviewing in possible therapeutics trategiest o block ebov infections, im entioned -deazaneplanocin a, besides bcx andt - (favipiravir). [ ] later added to the list were gs- (remdesivir) and zmapp. [ ] the response to zmapp, am ixture of three monoclonal antibodies directed against the surfaceg lycoprotein of ebov,w as beneficial but did not meet the prespecified statistical threshold for efficacy [death occurred in of patients ( %) who received the current standardo fc are alone as compared to o f p atients ( %) who received the current standard of care plus zmapp]. [ ] apparently the outbreak of ebola in - ended before any incontrovertible evidenceo fa ny intervention could be assessed. in this review iw ill focus on the potentialo ff avipiravir (t- ), bcx (galidesivir) and gs- (remdesivir) on the treatment of ebov and other hfv infections. the imino-c-nucleoside bcx (galidesivir)w as first reported by warrene tal. [ ] to be activea gainst aw ide range of viruses, including filo-, toga-, bunya-, arena-, paramyxo, corona-, flavi-, orthomyxo-andp icornaviruses. it was found to completely protectc ynomolgus macaques from marburg virus infection when administered as late as hours after infection. [ ] it effectively blocked yellow fever virus infection in ah amster model. [ ] it is now under development for the treatmento f ebov infection. [ ] bcx (galidesivir) (figure ) has also been found effective against zika virus in cell culture and in a lethal mousem odel. [ ] antivirala ctivity of bcx has also been reported against west nile virus, at ypical mosquitotransmitted flavivirus, and against tick-bornef laviviruses, kyasanur forest disease virus (kfdv). [ ] it inhibits infection of rift valley fever virus (rvfv), am osquito-borne pathogen that causes severe disease in humansa nd livestock in sub-saharan africa and the arabian peninsula, in syrian golden hamsters. [ ] the phosphoramidatep rodrug of the pyrrolo[ , -f][triazin- amino]a denine c-nucleoside gs- (remdesivir) ( figure ) was first reported to protect %o fe bov-infected rhesus monkeys against al ethal ebov infection. [ ] it wasa lso found active against other emerging viruses such as respiratory syncytial virus (rsv) and hepatitis cv irus (hcv),a nd the presence of the '-cyano group in remdesivir wasf ound to be critical in providing selectivity toward the viral (rna) polymerases. [ ] gs- was then found to be highly inhibitory to various viruses other than filo, that is, pneumo-, paramyxo-and coronaviruses. [ ] that gs- inhibited both epidemic and zoonotic coronaviruses, and might prove effective against emerging coronaviruses in the future was emphasized by sheahane tal. [ ] the viral rna polymerase and the proofreading exoribonuclease were suggested as being responsible fort he coronavirus susceptibility to remdesivir. [ ] the viral rna polymerase has also been identified as the targete nzyme of paramyxoviruses (i.e. nipah virus) for the antiviral activity of gs- . [ ] for both ebov rna-dependentr na polymerase (rdrp) and rsv rdrp, chain termination was delayeda nd predominantly seen at position i + . [ ] the first newborn baby to have survived congenital ebov infection, received remdesivir in addition to zmapp and ab uffy coat transfusion from an ebola survivor. [ ] favipiravir (t- ) ( figure ) is ap yrazine derivative, that is, -fluoro- hydroxy- -pyrazinecarboxamide, sharing ac ommon structural feature, that is ac arboxamide entity,w ith ribavirin, eicar and pyrazofurin, andw hich is also an essential (hydrogen bonding) part of guanine( -hn -c o-). [ , ] as already mentioned, ribavirin and eicar would primarily owe their antiviral activity to interference with the imp dehydrogenase, ak ey enzyme in the biosynthesis of gmp and gtp.y et, eriksson et al. [ ] reported inhibition of influenzav irus rna polymerase by ribavirint riphosphate,a nd when furuta et al. [ ] had revealed the in vitro and in vivo activities of t- against influenza virus, they attributed the mode of action of t- to as pecific inhibition of the influenzavirus rna polymerase by the t- rtp (ribofuranosyl triphosphate. [ ] to this end (scheme ), t- had first to be converted to its ribofuranosyl monophosphate( rmp) by a phosphoribosylt ransferase, and then further processed by kinase(s) to its diphosphate( rdp) and triphosphate (rtp), before the latter would interact in ag tp-competitive manner with the viral rna synthesis. that t- (favipiravir), as av iral rna polymerase inhibitor, offers great potential in the treatment of aw idev ariety of rna virus infections, has been reviewed repeatedly. [ ] [ ] [ ] [ ] sidwell et al. [ ] demonstrated that t- inhibited al ethal avian influenza a( h n )v irus infectioni nm ice. this was confirmed by kiso et al.. [ ] it was ascertainedt hat in the influenzaa(h n ) virus-infected cells t- was metabolized to t- rtp. [ ] the latter is incorporated into the nascent rna strand as ap urine nucleotide analog (reminiscent of gmp) and inhibits strand extension. [ ] the rtp of t- acts as ag tp-competitive inhibitor of the influenza viral rna polymerase. [ ] favipiravir inhibits in vitro replication of aw ide range of influenzav iruses, including those resistantt oc urrently available drugs [ ] andt hose isolated from patients who had previously received favipiravir. [ ] favipiravir has been approved, as avigan, in japan for the treatment of influenza. [ ] lethal mutagenesis hasb een proposed to be ak ey mechanism in the activity of t- (favipiravir) againsti nfluenzaa (h n )v iruses in vitro. [ ] lethalm utagenesis has also been suggested for the activity of favipiravir against norovirus repli-cation [ ] and hepatitis cv irus. [ ] such mechanism of antiviral activity was originally proposed for ribavirin. [ , ] however,t he activity of ribavirin against yellow fever virus could not be explained by an error-prone mechanism. [ ] favipiravir has provene ffective againstv irtually all hemorrhagic fever virus infections:a rena-and bunyaviridae, [ ] pichinde virus (an arenavirus), [ , ] lassa fever virus, [ ] yellow fever virus, [ ] western equine encephalitis virus, [ ] west nile virus, [ ] punta to ro, ap hlebovirus, [ ] the hantavirus maporal, [ ] rift valley fever virus [phlebovirus (bunyaviridae)], [ ] crimean-congo hemorrhagic fever virus [nairovirus (bunyaviridae)], [ ] severef ever with thrombocytopenia syndrome virus (sftsv), ar ecently identified emerging virus, [ ] and norovirus (caliciviridae). [ , ] resistance to t- has never been reported, except for chikungunya virus (alphaviridae) which acquired resistance due to the k r mutation in the rna-dependentr na polymerase (rdrp). [ ] favipiravir proveds uccessful in the treatment of ebov infection in mice. [ , ] in nonhuman primates, treated intravenously with favipiravir,f ive of six animals ( %) survived al ethal marburg virus infection. [ ] in patients with the ebov infection in www.chemasianj.org sierra leone, favipiravir was found to increase the survival rate from . %( / ) to . %( / ). [ ] another study carried out in guinea ended with the statement that favipiravir monotherapy merits furthers tudy in patients with mediumt oh igh viremia but not in those with very high viremia. [ ] guedj et al. [ ] concluded that favipiravir mayh ave ap otentialr ole at high doses in the treatment of ebov infections in humans. ta king into account that rhabdo-and filoviruses are closely related,i ti sn ot surprising that favipiravir has also been advocated for the postexposure prophylaxis of rabiesvirus, as ap otential alternative to rabies immunoglobulin. [ ] the compounds described here can be considered as either adenine derivatives ( -deazaneplanocin a, galidesivir,r emdesivir or guanine derivatives (ribavirin), eicar, pyrazofurin,f avipiravir). this is immediatelyo bvious for the adenine derivatives, which can be expected to interferew ith viral rna synthesis at the level of the rdrp (rna-dependentr na polymerase), where they can serve as competitive inhibitors with respectt o atp( based on the hydrogen bonds formed between adenine and uracil) ( figure ). in addition, the adenine analogues can also serve as s-adenosylhomocysteine (sah) hydrolase inhibitors, thus interfering with the s-adenosylmethionine (sam)-dependentmethylationr eactions. that ribavirin, eicar, pyrazofurin and favipiravir would be able to act as guanined erivatives thus competing with gtp at the rdrp level is less obvious. yet, all c ompounds share a carboxamide group, reminiscent of the carboxamide (-c ( ) o-n ( ) h-) part of guanine, which could explain the hydrogen bondingw ith cytosine ( figure ). in addition to their action at the rdrp level, the carboxamide-containing derivatives could also interfere with the imp dehydrogenase activity,t hus suppressing the biosynthesis of gtp,a sh as been specifically demonstrated for ribavirin and eicar. the exact mechanism of action of favipiravir remains to be assessed;i tm ay well vary from one virus to another.a sf ar as the activity of favipiravir rtp against influenzaavirus polymerase is concerned, this has been ascribed to "ambiguous base-pairing". [ ] in , ip roposed that "c-nucleosidess hould be revisited". [ ] this proposal was prompted by the advent of two new c-nucleosides, bcx (figure ), which has in the meantime, been further pursued (as galidesivir) for the treatment of ebov infections (see supra), and gs- (compound , containing a '-c-methyl group imparting specific activity against hepatitis cv irus (gcv). [ ] gs- (figure ) also contained a '-cyano group, which later on was found to be critical in providing selectivity towardv iral (rna) polymerases. [ ] gs- has only been reported for its potentiala sa na nti-hcv agent. [ ] [ ] [ ] it has not been thoroughly explored for its potential activity against ebov or any other hemorrhagic fever virus (hfv) infections. according to literatured ata, [ ] it would only have weaka ctivity against ebov. remdesivir (gs- ) is at present aleadingd rug candidate for the treatment of ebov infections. its chemical attributes are that it is ac -nucleoside, extendedb yaphosphoramidate and equippedw ith ac ng roup. galidesivir (bcx ) is also acnucleoside, but what would happeni fi tw ould also contain a cn group and would be converted to ap hosphoramidate prodrug?f or favipiravir,i ts n-nucleoside t- is more effective than the free -pyrazinecarboxamide against yellow fever virus infection in hamsters, [ ] but less so against punta to ro virus in mice. [ ] what would happen if t- would be convertedt o its c-nucleoside, and, furthermore, extended by ap hosphora- www.chemasianj.org wiley-vch verlag gmbh &co. kgaa, weinheim midate entity?a lso, the forgotten c-nucleoside, pyrazofurin, [ ] could be converted to its phosphoramidate, and eicar, which containsa ne thynyl, reminiscent of the cyano group, could be first transformed to its c-nucleoside before being converted to its phosphoramidate (scheme ). any of these chemical interventions may provide potential clues in the design of new medicines against ebovand other hfv infections. the (candidate) antiviral compoundsc urrently availablef or the treatment of ebov and other hfv infections are -deazaneplanocin a, galidesivir,g s- , remdesivir,r ibavirin, eicar, pyrazofurin andf avipiravir.they are boxed in (scheme ). in attempts to increase their efficacy and potentially lower their toxicity, they could, where applicable, first be converted to their c-nucleoside, and, subsequently,t ot heir phosphoramidate (scheme ). for gs- and remdesivir,w hich,i na ddition, also contain ac yanogroup, these manipulations have already been achieved. the authordeclares no conflict of interest. keywords: antivirals · ebola · hemorrhagic fever viruses · nucleoside analogues proc. natl. acad.sci.u sa antiviral res currentc hemotherapy:p roceedings of the th international congress of chemotherapy proc. jpn. acad. ser.b proc. natl. acad.s ci proc. natl. acad. sci revised manuscript received key: cord- -qhtj pef authors: dash, raju; das, rasel; junaid, md; akash, md forhad chowdhury; islam, ashekul; hosen, sm zahid title: in silico-based vaccine design against ebola virus glycoprotein date: - - journal: adv appl bioinform chem doi: . /aabc.s sha: doc_id: cord_uid: qhtj pef ebola virus (ebov) is one of the lethal viruses, causing more than epidemic outbreaks to date. despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of ebov infections in humans. disclosing this, the present study described an epitope-based peptide vaccine against ebov, using a combination of b-cell and t-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. here, protein sequences of all glycoproteins of ebov were collected and examined via in silico methods to determine the most immunogenic protein. from the identified antigenic protein, the peptide region ranging from to and the sequence hkegaffly from the positions of – were considered the most potential b-cell and t-cell epitopes, correspondingly. moreover, this peptide (hkegaffly) interacted with hla-a* : with the highest binding energy and stability, and also a good conservancy of . % with maximum population coverage. the results imply that the designed epitopes could manifest vigorous enduring defensive immunity against ebov. ebola virus (ebov) is an antisense-strand rna virus from the filoviridae family, and it is structurally filamentous. although the initial discovery of ebov was in , till now more than epidemics have been reported from africa, mostly with the zaire species (http://who.int/mediacentre/factsheets/fs /en/). [ ] [ ] [ ] the genome of ebov enciphers the seven structural proteins, ie, nucleoprotein (np), viral structural proteins (vp , vp , vp , and vp ), glycoprotein (gp), and rna-dependent rna polymerase (l). among these, three different versions of glycoprotein are transcribed by the gp gene. [ ] [ ] [ ] [ ] both attachment protein (gp ) and entry/fusion protein (gp ) are expressed from the full length of the gp chain, which are synthesized from messenger rnas (mrnas), containing an additional nontemplated adenosine. the soluble gp (sgp) is synthesized from the unedited rna transcript. on the contrary, small soluble gp (ssgp) is translated during this process by adding two additional adenosine residues. the gps are expressed virally on the virion surface, which plays a crucial role in the catalysis of membrane fusion and amalgamation to host cells. as a result, it is considered not only a crucial component for vaccines but also an essential target for developing inhibitors and antibodies of attachment and fusion. [ ] [ ] [ ] protein sequence retrieval, evaluation analysis, and antigenic protein identification all available sequences of the gp of ebov were extracted from the uniprot database. after that, multiple sequence alignment was performed by using the clustalw tool, and a phylogenetic tree was assembled by mega . software. and then, vaxijen v . was used to predict most efficient antigenic protein from the available protein sequences. top scored eiptope subjected to ns md simulation **rmsf **rmsd **hydrogen bond occupency analysis secquence, having highest vaxijen score prediction of b cell epitope, using-**t cell epitope prediction by proteasomal c terminal cleavage, tap transport efficiency and mhc class binding **epitopes with ic value less than for their binding to mhc class molecule from iedb analysis along with binding to highest number of alleles in both analyses were chosen **epitope conservancy analysis **population coverage analysis **kolaskar and tongaonkar antigenicity scale **emini surface accessibility prediction **karplus and schulz flexibility prediction **bepipred linear epitope prediction **chou and fasman beta turn prediction vaxijen analysis with a threshold score of > . secquence, having highest vaxijen score vaxijen analysis with a threshold score of > . in silico-based vaccine design against ebov gp t-cell epitope identification and conservancy analysis t-cell identification was done using the netctl . server, setting thresholds at . , . , and . for sensitivity and accuracy. mhc-i binding of the identified epitopes and epitope conservancy were then calculated using tools from the immune epitope database (iedb). [ ] [ ] [ ] these tools calculate the half maximal inhibitory concentration (ic ) value of epitope binding to human leukocyte antigen (hla) molecules using the stabilized matrix base method. , the restriction for epitope identification was set to mhc-i supertypes. prior to the run, all the alleles were considered, and the length of the peptides was set at . . the population coverage tool from iedb was applied to determine the population coverage for every single epitope by selecting hla alleles of the corresponding epitope. allergenicity of the predicted epitope was calculated using allerhunter, which can predict both nonallergens and allergens with a high level of accuracy , by comparing the input sequence with the sequence of known allergen. molecular simulation analysis of hla allele interaction design of the three-dimensional structure of epitope and hla protein the three-dimensional structures of all the five epitopes were predicted by a pep-fold web-based server. for each sequence, this server predicted the five most provable structures, the best of which, having the lowest energy model, was chosen for further analysis. to validate the binding of identified epitope and hla molecule, we considered the homology modeling as there is no relevant structure available in the protein data bank. we selected homology modeling using the most popular online protein fold recognition server, phyre , to generate the three-dimensional structure of hla-a* : (accession id: am ). then, modrefiner was used to minimize and correct the hypothetical structure. the validation of the predicted structure was done using procheck, verify d, errat, prove, and qmean. molecular docking analysis was performed using autodock vina, by considering hla molecule as a protein and identified epitopes as ligands. first, we used the protein preparation wizard of ucsf chimera to prepare the hypothetical protein for docking analysis by adding hydrogens and gasteiger-marsili charges. the prepared file was then converted into pdbqt format. the parameters used for the docking simulation were set to default. the size of the grid box in autodock vina was kept at . , . , and . , respectively, for x, y, and z. the energy range was kept at , according to the default setting. autodock vina was implemented via the shell script offered by autodock vina developers. docking results were observed by negative score in kcal/mol, as binding affinity of ligands. binding energy estimation and molecular dynamics (md) simulation the binding free energy of hla-epitope complexes were calculated by using mm (charmm) -generalized born surface area (gbsa) and poisson-boltzmann surface area (pbsa) protocols, implemented in accelrys discovery studio . . using implicit solvent models of gbsa and pbsa, the binding free energy (Δg bind ) for each epitope was calculated by maintaining salt concentration of . m. default value was set for conformational entropy and ligand minimization. the distance cutoff value was set to . Å. the binding energy was calculated by using following equation: the entire dynamics simulation study for the hlaepitope complex was accomplished in yasara dynamics software. prior to simulation, the complex was cleaned and optimized the hydrogen bond network. after that, a cubic simulation cell was created with a periodic boundary condition, and the atoms of the complex were typed using the amber force field. the pka (acid dissociation constant) values of protein titratable amino acids were calculated and solvated the simulation box using the transferable intermolecular potential points (tip p) water model (density: . g/l - ). the system consistent with atoms was energy minimized using the steepest gradient approach ( cycles) followed by simulated annealing method. restrained and unrestrained all-atom molecular dynamics simulation were performed in solvent using the pme method to describe long-range electrostatic interactions at a cut off distance of Å at physiological conditions ( k, ph . , . % nacl). a multiple time step algorithm together with a simulation time step interval of . fs was chosen. molecular dynamics simulations of ns long were performed at constant temperature using a berendsen thermostat and constant submit your manuscript | www.dovepress.com dash et al pressure. the md trajectories were saved every ps for analysis. the trajectories generated from the simulation were analyzed for the stability by various evaluative measures viz. rmsd, rmsf (rms fluctuations), and initial and final protein backbone comparisons using yasara structure built in macros and vmd software. to detect b-cell epitope, various tools from iedb were used to identify the b-cell antigenicity, together with the emini surface accessibility prediction, kolaskar and tongaonkar antigenicity scale, karplus and schulz flexibility prediction, and bepipred linear epitope prediction analysis. since antigenic parts of a protein belong to the beta turn regions, the chou and fasman beta turn prediction tool was also used. a total of gp sequences from the different variants of ebov were collected from the uniprotkb database. multiple sequence alignment analysis was then performed, and a phylogenetic tree (figure ) was constructed thereby. using the unweighted pair-group method with arithmetic mean, a phylogram was constructed using the bootstrap with , replications in mega . from the multiple sequence alignment analysis, it is clearly seen that protein sequences that isolated from various strains were having a close relationship. also, from the multiple comparison result, the selected sequences of ebov of the same subtype have %- % similarity. this result also confers the possibilities of mutation in glycoprotein of all strains, which demonstrates a good agreement with the results from veljkovic et al. antigenic protein prediction protein sequences in this study were considered to screen out using vaxijen web server for the identification of potent antigenic protein. as a corollary, uniprotkb id: q ymg was identified as the most potent antigenic protein having a maximum total prediction score of . . here the threshold of . is considered as the potent antigenicity. this sequence was used for further analysis. on the basis of the high combinatorial score, the five best epitopes were predicted by the netctl server from the selected protein sequence in a preselected environment. the identified epitopes are represented in table . in combination with several methods such as proteasomal cleavage/transporter associated with antigen processing (tap)/mhc-i combined predictor, mhc-i processing of the netctl server calculates an overall score for each peptide's intrinsic potential from a protein for the designing of t-cell epitope. peptides with a higher score represent higher processing capabilities. the five t-cell epitopes were subjected to mhc-i binding prediction, using the stabilized matrix base method. the epitopes that elicited higher affinity (ic < nm) were subjected to afterward analysis (table ) . notably, proteins are transformed into peptides by proteasome complex, which cleaved the peptide bonds. by combining with class i mhc molecules, these peptides were deported to the cell membrane, where they were introduced to t helper cells. as shown in table furthermore, this epitope retained the highest conservancy of . %, according to the iedb conservancy analysis, as tabulated in table . as population coverage in vaccine design generally plays a crucial role, it was calculated in this study. the cumulative percentage of population coverage was obtained for the predicted epitope hkegaffly. as shown in table , the population coverage for east africa was found to be . %; in west and north africa, it was . % and . %, respectively; and for central africa it was observed to be . %. the population coverage was recorded at . % for the east asian region, which was a major hotspot for viral infection. for north america, the population coverage was found to be . %. in current vaccine design pipeline, allergenicity is considered the most prominent barrier in vaccine designing, since most vaccines convert the immune system into an "allergic" reaction by inducting type t helper cells and immunoglobulin e. that is why we predicted allergenicity of the selected epitope by the allerhunter web server, where the probability is > . . the epitope hkegaffly was scored . (sensitivity = . %, specificity = . %), and was thus considered a nonallergen, according to the food and agriculture organization/world health organization evaluation system of allergenicity prediction. dash et al protein model having > % of the residues in the core and allowed regions can be considered a high-quality model. the hypothetical model was further analyzed using errat and verify d. for a good model, structure should retain an errat score > . , against which the model in this study obtained an errat score of . . verify d graph indicates that . % of residues of this model had an averaged d- d score of . , which is good. along with the qmean analysis, the protein model in our interest resulted in a z-score of − . , and the total score was . . this value denotes a higher quality of the model, where the acceptable score ranges between and ( figure b ). on the basis of the results obtained from the aforementioned structural validation programs, the model ( figure c ) showed much reliability and was considered for further study. molecular docking simulation revealed that the proposed epitopes bound in the cleft of the hla-a* : ( figure s ), where the highest binding affinity was − . kcal/mol (table s , observed for the hkegaffly epitope). the chimera program was used to visualize the interactions of docked hla-a-epitope complexes, as shown in figures and s . then, binding energy calculation was carried out to understand the binding of hla with epitopes. here the binding free energies of mm-gbsa and mm-pbsa are approximate free energies of binding, so a more negative value denotes stronger binding. from mm-gbsa analysis, the highest binding free energy was observed for hla-a* : with epitope (hkegaffly) of - . kj/mol (table s ). on the contrary, the lowest binding free energy was obtained for atedpssgy epitope, i.e. - . kj/mol. in contrast of mm-gbsa, the hkegaffly epitope was also resulted the highest binding energy of - . kj/mol, while the lowest binding free energy was seen for tedpss-gyy epitope, - . kj/mol. since hkegaffly epitope obtained the highest docking affinity and binding free energy, its complex subjected for molecular dynamics simulation. table interaction, binding, and conservancy of identified t-cell epitopes validation of predicted t-cell epitope as described in the "materials and methods" section, the hypothetical structure of hla-a* : protein was generated using the homology technique. the structure was then analyzed through various web-based protein validation software. as shown in figure , the ramachandran plot generated by the procheck server showed that about . % of the residues of protein are located in the most favored region, as against % in the outlier region and . % in the generously allowed region. it should be noted that the and remained stable in the range from . Å to Å. in case of epitope, similar rmsd pattern was observed, where the order of magnitude was seen to fluctuate in some range. the average energy of the simulation was - . kj/mol; the average coulombic charge and van der waals interactions was - . kj/mol, . kj/mol, respectively. we also calculated the contribution of each residue for both hla and epitope in the simulation, in terms of rmsf and rmsd. as seen in figure a , highest rmsd was observed the ns md simulation of hla-epitope (hla-a* : -hkegaffly) complex was carried out using amber force field, following the energy minimization protocol. the stability of the hla-epitope complex by means of rmsd was calculated and rendered in figure a . from the results, it is revealed that the hla molecule was stabilized after ns simulation and tended to remain in plateau phase thereafter for rest of the period. the rmsd value of hla was observed to grow up quickly from . for arg residue at the position of in hla, while lowest rmsd observed for cys . however, this residue was also resulted highest rmsf value of . Å, while the rests of the residues were in lowest fluctuation. in case of epitope, the histidine residue at the first position and the tyrosine residue in th position were seen to be very much flexible, as these residues were resulted with highest rmsd and rmsf ( figure b ). in the meanwhile, we calculated the number of hydrogen bond formed between the epitope and hla molecule during the simulation. the results represented in figure b , showed that hydrogen bond at initial stage was , and the range decreased to . during the simulation, the number of hydrogen bond was at a range of - , in silico-based vaccine design against ebov gp potentiality to express the b-cell response. furthermore, the surface accessibility of the protein was also analyzed using the emini surface accessibility prediction methods, since a potent b-cell epitope should be accessible through the surface. as shown in figure s and table , higher accessibility was found in regions - and - amino acid residues. figure s represents the β-turns region identified by chou and fasman β-turn methods. according to the result, the region from to (in the region of - and - ) is regarded as β-turns as well as hydrophilic in nature. these are two properties required to be a potent b-cell epitope. experimentally, antigenicity is related to the protein flexibility. that is why we implemented the karplus and schulz flexibility prediction method, where it was evident that the regions of - and - were regarded as the most flexible ( figure s ). finally, based on the hidden markov model, the bepipred linear epitope prediction tool was utilized to predict linear b-cell epitopes. the predicted result is rendered and tabulated in figure s and table . hence, by comparing the foregoing results, the peptide sequences ranging from to are which indicates the strong binding of epitope-hla complex. hence, all analyses lead to the conclusion that hkegaffly is one of the most prominent t-cell epitopes for gp based designing of vaccine. for the identification of potential b-cell epitopes, amino acid scale base methods have been used in this study. consistent with this protocol, we used diverse investigation processes for the calculation of an incessant b-cell epitope. according to the analysis of kolaskar and tongaonkar's antigenicity prediction method, the average antigenicity was . , while . and . were the maximum and minimum, respectively. the kolaskar and tongaonkar antigenicity prediction uses a semiempirical method to predict antigenicity on the basis of physicochemical properties of the residues in a protein and their diversity in experimentally known epitopes, where values > . were considered to denote a potential antigen. as summarized in table and figure s [ ] [ ] [ ] [ ] [ ] however, the information representing the population coverage in the worldwide are still limited. in such case, computational based epitiope screening is very much efficient in context of hla class i molecules, and also much safe, high specificity and cost effective. therefore, this study incorporated various immunoinformatics and molecular modelling tools to identify potential epitopes present in ebov gps. initially, a set of glycoprotein sequences from the different strains of ebov has been subjected to perform multiple sequence alignment. previous gp sequences analysis of different strains of each ebov species revealed a high degree of sequence similarity, , and thereby, it is believed that targeting gp from old strain could provide strong and cross reactive immunity against the new strain and previous outbreaks in . interestingly, in our molecular analysis, we have found ~ - % conservation for the amino acid sequences of different strains within the species, which confers the degree of f lss ngvatdvpsatkrwgfrsgvppkvvnyeagewae kkpdgseclpaapdgirg vsgtgpca tf kdf plrepvnatedpssgyys tgfgtne ytsgkrsntt peidtti tgilqlpr tsfflwviilfqrtfsiplgvihnstlqvsdvdklvcrdk lrsvgln atdvpsa rsgvppkvvny gseclpaap fprcryvhkvsgtgpcagdfafh egafflydrlastviyr aegvvaflilpqa fsshplrep sgyysttiryq lfevdnltyvqlesr tpqfllqlnet iwkvnpe able to provoke the immune response as b-cell epitope for gp-based designing of vaccine. in recent trends, the primary focus of vaccine development is very much rely on gps, as they are involved in cell attachment, fusion and entry as well as assist in invasion; and thus plays the role of pathogenesis of disease. the central role in silico-based vaccine design against ebov gp similarity and support the previous analysis. from this set of gps, the most antigenic protein sequence was determined by vaxijen server. based on auto cross covariance (acc), the vaxijen server transform the protein sequence into uniform vectors of physicochemical properties of proteins. with % sensitive, % accuracy and specificity, the l -cv (leave one -out cross validation) was used to identify antigenicity of protein for viral species. the resultant antigenic protein (vaxijen score ≥ . ) was then subjected for various immunoinformatics analysis, followed by iedb web server. at the beginning five potent -mer epitopes have been predicted from netctl . server and selected for further study. using the threshold of . , the netctl . server predicts maximum number of epitopes without compromising the specificity or sensitivity levels, covering all mhc class i supertypes. the five most potent epitopes are represented in table , and the scores are the predicted mhc class i affinities in the form of -logic and ic value. for mhc-i binding prediction, peptides with ic values < nm are considered high affinity, < nm for intermediate affinity, and < nm for low affinity. therefore, we selected maximum alleles having binding affinity < nm. it is advocated that t-cell epitope binding to specific multiple hla supertypes are termed as promiscuous in vaccine design, since they effectively increase the coverage of higher proportions of human populations. , according to the results, both hkegaffly and lfevdnlty bind to the highest number of alleles. however, hkegaffly represents highest conservancy and was hence considered as epitope of choice. we also validated each epitope by molecular docking simulation and mm-gbsa/mm-pbsa studies with hla-a* : protein, as it was found common in the results from mhc-i binding interaction analysis. prior of docking simulation, the three dimensional structure of hla molecule was prepared by using the phyre , followed by intensive mood. as a result, residues ( %) of hla-a* : modelled at > % accuracy. in these study, bck_chain a, crystal structure of hla-a* showed the highest similarity of % ( figure s ). the selected model has been chosen from the twenty models generated by phyre , on the basis of similarity and confidence level. phyre is one of the best protein prediction servers that allows remote fold reorganization and homology detection. using hidden markov model (hmm), this server predicts the structure of given protein sequence by constructing backbone, loop modelling and adding side chains. however in intensive mode, additional ab initio approach is used for reconstruction of missing region, backbone and side chain. in docking simulation, among the other epitopes, hkegaffly obtained highest binding affinity (table s , supplementary material). in addition, mm-pbsa and mm-gbsa techniques are frequently to re-rank docking poses from molecular docking study, as they achieve a much better performance than docking scoring functions. nevertheless, the success rate of the absolute binding free energy prediction strongly depends on the systems. thence, we used both of these solvation models to predict binding energy more accurately, where the results from mm-pbsa examine the accuracy and reliability of the results from mm-gbsa. results of binding energy calculation are shown in table s . the relative magnitude of binding free energy obtained from gb methods is found to be consistent with those calculated using pb method, despite of the differences in the absolute value of salvation energy. as a corollary, these results also demonstrated the consistence with relative stabilities of hla-epitope complexes. in previous published reports regarding in silico epitope identification of ebov, [ ] [ ] [ ] [ ] the studies are limited to sequence-based scoring function techniques and some extend to docking simulation. these techniques have certain limitations, though these are very useful. in docking simulation of peptide and protein, it faces problem like peptide's flexibility. , whereas, energy based approach like molecular mechanics and interaction energy scoring can add valuable information to sequence based results. , therefore, we have performed md simulation study of ns long to enhance the predictive power of the peptide affinity calculations to mhc molecule. in molecular dynamics simulation, both epitope and hla protein were seen to achieve equilibration, while different fluctuations of rmsd were seen by the time evolution. higher rmsd values of epitope indicate the flexibility in binding with hla molecule, during the simulation. these results were further confirmed by the analysis of per residue contributions in dynamics simulation by means of rmsf and rmsd. low values of rmsf indicate the core region of the hla protein was stable, while high values of rmsd demonstrated the motion of the protein during the simulation. in like manner, the rmsd and rmsf profiles of eptiope confirm the synergic conformation changes to accommodate the binding pocket of hla. the hydrogen bond occupancy analysis between the hlaepitope further confirmed the stability of the complex during the simulation. overall, these results evidently demonstrate that both hla and epitope have remarkable conformation changes to facilitate the binding and formed stable complex in thermodynamic environment (figure ). submit your manuscript | www.dovepress.com dash et al it is one of important factors in vaccine design that the distribution of hla varies according to the diverse ethnic groups and geographic regions around the world. therefore, wide range of population coverage must be considered during the designing of an effective design. according to the results from population coverage analysis, the epitope hkegaffly showed wide range of population coverage in different regions of the world (table ) , where the highest coverage was observed central africa; one of the most ebov infected areas. this result indicates that it will specifically bind with the prevalent hla molecules in the target population, where the vaccine will be employed. in other aspects, the b-cell epitope stimulates minimal immune unity, which is very much strong enough to elicit a potent humoral immune response, causing no harmful side effects to human body. thereby, we are also calculated and found that the sequences ranging from - as a b-cell epitope, by taking consideration of amino acid property, hydrophilicity, accessibility, flexibility, turns, exposed surface, polarity and antigenic propensity. this study could provide a solid base for vaccine design. in recent years, most vaccines have been developed based on b-cell immunity; however, the current strategy relies mostly on t-cell epitope owing to long-lasting immunity. both b-cell and t-cell epitopes are offered in this study for stimulating immunity in several ways. the resulting peptides showed b-cell and t-cell selectivity, better conservancy, population coverage, and significant interaction with mhc- allele with good affinity. above all, the predicted epitopes are anticipated to offer long-term and high protective immunity against ebov. the authors report no conflicts of interest in this work. computational biophysics; chemoinformatics and drug design; in silico adme/tox prediction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit 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is assisted by bound water molecules key: cord- -ofp tupv authors: kühl, a.; pöhlmann, s. title: how ebola virus counters the interferon system date: - - journal: zoonoses public health doi: . /j. - . . .x sha: doc_id: cord_uid: ofp tupv zoonotic transmission of ebola virus (ebov) to humans causes a severe haemorrhagic fever in afflicted individuals with high case‐fatality rates. neither vaccines nor therapeutics are at present available to combat ebov infection, making the virus a potential threat to public health. to devise antiviral strategies, it is important to understand which components of the immune system could be effective against ebov infection. the interferon (ifn) system constitutes a key innate defence against viral infections and prevents development of lethal disease in mice infected with ebov strains not adapted to this host. recent research revealed that expression of the host cell ifn‐inducible transmembrane proteins – (ifitm – ) and tetherin is induced by ifn and restricts ebov infection, at least in cell culture model systems. ifitms, tetherin and other effector molecules of the ifn system could thus pose a potent barrier against ebov spread in humans. however, ebov interferes with signalling events required for human cells to express these proteins. here, we will review the strategies employed by ebov to fight the ifn system, and we will discuss how ifitm proteins and tetherin inhibit ebov infection. the ebola virus (ebov) is an enveloped, negativestranded rna virus of the filovirus family. infection of humans with ebov causes ebola haemorrhagic fever. at present, neither antivirals nor vaccines are available to combat this lethal disease, and ebov is classified as a category a priority pathogen (niaid, ) . four ebov species have been defined (kuhn, ) , zaire ebolavirus (zebov), sudan ebolavirus (sebov), côte d'ivoire ebolavirus (ciebov) and reston ebolavirus (rebov), and a fifth species has been proposed, bundibugyo ebolavirus (bebov) (towner et al., ) . african fruit bats are a natural reservoir of the second filoviral genus, marburg virus (marv), and have also been proposed as a natural reservoir of ebov (leroy et al., ) . african fruit bats may transmit the virus to humans either directly or via an intermediate host (groseth et al., ) . outbreaks of summary zoonotic transmission of ebola virus (ebov) to humans causes a severe haemorrhagic fever in afflicted individuals with high case-fatality rates. neither vaccines nor therapeutics are at present available to combat ebov infection, making the virus a potential threat to public health. to devise antiviral strategies, it is important to understand which components of the immune system could be effective against ebov infection. the interferon (ifn) system constitutes a key innate defence against viral infections and prevents development of lethal disease in mice infected with ebov strains not adapted to this host. recent research revealed that expression of the host cell ifn-inducible transmembrane proteins - (ifitm - ) and tetherin is induced by ifn and restricts ebov infection, at least in cell culture model systems. ifitms, tetherin and other effector molecules of the ifn system could thus pose a potent barrier against ebov spread in humans. however, ebov interferes with signalling events required for human cells to express these proteins. here, we will review the strategies employed by ebov to fight the ifn system, and we will discuss how ifitm proteins and tetherin inhibit ebov infection. zebov, sebov, ciebov and bebov have been recorded in africa and were associated with case-fatality rates of up to % in larger outbreaks. rebov has been detected in swine in the philippines and is believed to be apathogenic for humans with an intact immune system (barrette et al., ; hartman et al., ) . the determinants accounting for the differential pathogenicity of the different ebov species are poorly understood. the ebov genome encodes three non-structural (sgp, ssgp, d-peptide, as discussed later) and seven structural proteins (fig. ) . the n protein (np), the l protein, viral protein (vp ) and vp are associated with the viral rna, forming a ribonucleoprotein (rnp) complex. the l protein has a polymerase function and is essential for genome replication and transcription, and these processes are regulated by vp and vp (dolnik et al., ) . the minor matrix protein, vp , contributes to nucleocapsid formation, while the major matrix protein, vp , facilitates budding of progeny particles from infected cells (huang et al., ; hartlieb and weissenhorn, ; dolnik et al., ) (fig. ) . the structural glycoprotein, gp , , mediates binding and infectious entry into host cells (kawaoka, ) . innate defences of the host are crucial to successfully fight ebov and other acute infections. the interferon (ifn) system is an integral part of the innate immunity against viral infections. sensor molecules of the ifn system recognize viral components and induce signalling that triggers expression of ifns (baum and garcía-sastre, ) . subsequent binding of ifn to ifn receptors on uninfected cells activates signal transducer and activator of transcription (stat)-dependent signalling, which commandeers the cell to express ifn-stimulated genes (isgs), which can function as antiviral effectors molecules (schindler et al., ; sadler and williams, ) . ebola virus strains lethal in humans were found to be unable to produce fatal disease in adult mice. however, when essential components of the ifn system were inactivated in mice, fatal disease was observed (bray, ) . similarly, adaptation of ebov to efficient replication in adult mice (bray et al., ) resulted in the generation of viruses with mutations allowing efficient interference with components of the murine ifn system . thus, the ifn system is generally capable of restricting filovirus spread and pathogenesis. however, several ebov proteins are well adapted to block processes essential for the establishment of a vigorous ifn response in human cells, potentially explaining why the ifn system frequently fails to protect humans from lethal ebov infection, as discussed below. the molecular pathways leading to expression of ifninduced antiviral effector molecules, and thus to the transition of cells into an antiviral state, are well characterized (baum and garcía-sastre, ) . however, the antiviral effector molecules induced by ifn and the molecular mechanisms underlying their antiviral action are incompletely understood. recent, groundbreaking studies attempted to close this gap (schoggins et al., ) and identified novel isgs, among them the tetherin and ifninduced transmembrane proteins (ifitms) (neil et al., ; van damme et al., ; brass et al., ). tetherin exhibits an unusual topology and restricts release of several enveloped viruses and filovirus-like particles from infected cells, while ifitm proteins inhibit infection by filoviruses and other enveloped viruses at the stage of viral entry, as discussed below. in the present review, we will summarize current knowledge on ebov interference with the ifn system. in addition, we will discuss how tetherin and ifitms block filovirus infection. the interferon system constitutes a major innate defence against infections by viruses and other pathogens. the components and signalling pathways of the ifn system have been described in several recent reviews (garcía-sastre and biron, ; baum and garcía-sastre, ; liu et al., ) and are only briefly summarized here. three classes of ifns have been defined according to the receptors bound by these cytokines. type i ifn, including ifna and ifnb, are produced by many cell types as a direct result of viral infection. ifnc, the only type ii ifn, is generated by activated t cells and nk cells. type iii ifns, which include ifnk - , are incompletely characterized, but are believed to regulate the antiviral response. the ifn system integrates two major sensor and signalling networks. in cells exposed to viruses, receptors for pathogen-associated molecular patterns sense the presence of the invading pathogens. these receptors are located in the cytoplasm, like retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene (mda- ), or in the extracytoplasmic space, like toll-like receptor (tlr)- and tlr- / / , and, upon pathogen recognition, induce ifn regulatory factor (irf)- -and irf- dependent signalling cascades that lead to the expression of type i ifns (fig. ) . secreted type i ifns then bind to cells expressing the type i ifn receptor, which consists of two subunits, ifna receptor (ifnar ) and ifnar . ligand binding to ifnar and ifnar triggers receptor dimerization and signalling. stat and stat are integral components of the signalling pathway induced by ifnars (fig. ) . homodimers of stat bind to ifncactivated sites (gas), while stat /stat heterodimers recognize ifn-stimulated response elements, resulting in the transcription of isgs, many of which have antiviral activity, like the well-characterized myxovirus resistance guanosine triphosphatases (mx gtpases) (haller et al., ; sadler and williams, ) . however, the full spectrum of isgs has only been recently characterized (schoggins et al., ) , and novel isgs that target discrete steps in the viral life cycle, like ifitms and tetherin, have been identified, as discussed below. vp is the smallest of the seven ebov-encoded structural proteins and constitutes the minor matrix protein relative to the major matrix protein vp (han et al., ) . it is required for the assembly of fully functional nucleocapsids (huang et al., ; hoenen et al., ) and contributes to the budding of virus-like particles (vlps) (han et al., ; licata et al., ) . furthermore, vp can shut down the host's ifn-a/b and ifn-c recognition of viral pathogen-associated molecular patterns (pamps) by prrs (purple) in infected cells initiates a signalling cascade including adaptor molecules (blue) and kinases (green), which activate transcription factors (red) that induce the expression of type i ifns and isgs (left panel). binding of ifn-a/b and ifn-c to their receptors induces phosphorylation (by jak /tyk ), dimerization and nuclear translocation of stat transcription factors, which induce the expression of several isgs such as tetherin, ifitm, pkr and others (right panel). ifn, interferon; tlr, toll-like receptor; rig-i, retinoic acid-inducible gene i; mda- , melanoma differentiation-associated gene ; myd , myeloid differentiation primary response protein ; trif, tir domain-containing adaptor-inducing ifn-b; ips- , ifn-b promoter stimulator ; traf , tumour necrosis factor receptor-associated factor ; irak, interleukin- receptor-associated kinase; ikke, ijb kinase e; tbk , tank-binding kinase ; irf, ifn regulatory factor; nf-jb, nuclear factor j light chain enhancer of activated b-cells; isg, ifn-stimulated gene; ifnar, ifn-a receptor; ifngr, ifn-c receptor; jak , janus-activated kinase ; prr, pathogen recognition receptor; tyk , tyrosine kinase ; stat, signal transducer and activators of transcription. a. kü hl and s. pö hlmann response to viral infection (reid et al., ) . for this, vp inhibits the nuclear translocation of the transcription factor stat (reid et al., ) (fig. ) , a key component of the ifn-induced signalling pathway controlling the expression of isgs, as discussed below. upon activation, stat is tyrosine-phosphorylated (py-stat ) and either heterodimerizes with py-stat for type i ifn signalling or homodimerizes with py-stat for type ii ifn signalling. the dimerization leads to the exposition of a nuclear localization signal (nls) in stat , which is recognized by the nuclear import adaptor protein importin a, specifically importin a , a and a (karyopherin a , a , a ; np- subfamily of importins). importin a is then bound by the nuclear import factor importin b (sekimoto et al., ) , which facilitates import of stat dimers into the nucleus ( fig. ) . however, in the presence of vp , which like stat binds to importins a , a and a (reid et al., (reid et al., , , the recognition of the py-stat nls by these importins is disrupted, and py-stat is not imported into the nucleus. blockade of nuclear transport of py-stat is not because of a global inhibition of nuclear import, as vp does not bind to importin a (karyopherin a ; rchi subfamily), a or a (karyopherin a , a ; qip subfamily) (reid et al., ) and does not affect nuclear import mediated by these factors. mutational analyses revealed that vp binds to amino acids - in importin a , which constitute armadillo repeat (arm) and comprise part of the binding site for py-stat (reid et al., (reid et al., , . predictions on vp structure offer insights into the mechanisms potentially underlying vp inhibition of importin a. ). thus, it was suggested that vp , like importin a, importin b and exportins (nuclear export factors important for recycling of importin a to the cytoplasm), belongs to the arm family of proteins. furthermore, it was posited that binding of vp to importin a mimics that of exportin, which also targets arm repeat . as a consequence, vp may block release of the autoinhibitory nls of importin a, and this would inhibit binding of other proteins to the nls binding site of importin a, including py-stat . alternatively, targeting the arm repeat of importin a by vp may block binding of py-stat because of competitive inhibition . in vp , the amino acid residues and - were found to be crucial for the inhibition of ifn-b-induced signalling and py-stat accumulation in the nucleus (mateo et al., ) . the lack of inhibition of ifn-b-induced signalling by a vp mutant with amino acid exchanges at these positions correlated with absence of importin a binding, highlighting that binding of vp to importin a is essential for ifn antagonism (mateo et al., ) . the ifn system can protect immune-competent mice from lethal ebov infection (bray, ; mahanty et al., ) . adaptation of zebov to lethal infection of mice was associated with mutations in vp and np . however, both wild-type vp and vp of the mouse-adapted (ma) strain were able to bind to human and mouse np- importins and to disrupt the interaction with py-stat (reid et al., ) . similar findings were documented for vp of rebov, which is believed to be non-pathogenic for humans, and it was shown that zebov, rebov and ma vp can suppress ifn-b-induced gene expression (reid et al., ) . thus, alterations in vp interference with the ifn response might not account for the acquisition of virulence of ma zebov in mice and for the lack of virulence of rebov in humans, respectively. the ebov-encoded protein vp fulfils several functions important for viral amplification. as essential polymerase cofactor, vp is involved in the formation of the ebov rnp complex and therefore crucial for transcription and viral replication (mühlberger et al., ) . furthermore, it is required for nucleocapsid assembly (huang et al., ) . consequently, interference with expression of vp attenuates viral growth and virulence (enterlein et al., ; hartman et al., a; prins et al., b) . additionally, vp blocks multiple steps of the innate antiviral defence (fig. ) , such as the signalling pathways leading to the expression of type i ifns and type i ifn-induced genes, double-stranded (ds) rna-dependent protein kinase (pkr) translation inhibition and rna silencing cárdenas et al., ; feng et al., ; haasnoot et al., ) . the key role of vp in the defence against innate immunity is exemplified by studies demonstrating that vp mutated viruses are unable to block the ifn-dependent induction of antiviral factors (cárdenas et al., ; hartman et al., a,b) . moreover, vp impairs the maturation of dendritic cells and thus prevents the establishment of an adaptive immune response (jin et al., ) , an important feature of ebov haemorrhagic fever pathogenesis (hartman et al., a) . the ability of vp to antagonize the type i ifn response was identified by basler et al. ( ) who showed that vp can functionally replace the influenza a virus (fluav) non-structural protein (ns ) protein, a viral type i ifn antagonist. vp was found to inhibit the virus-induced activation of type i ifn promoters by targeting the transcription factor irf- . in contrast, activation of ifn promoters through exogenous ifn was not modulated by vp . upon activation by viral infection, irf- is phosphorylated by cellular kinases such as tank-binding kinase (tbk- ) and ijb kinase epsilon (ikke) (prins et al., ). subsequently, ifr- dimerizes and translocates into the nucleus where it activates transcription of isgs (fig. ) . vp blocks irf- phosphorylation and therefore subsequent nuclear accumulation of irf- cárdenas et al., ) . for this, vp binds to ikke and tbk- and inhibits substrate binding and kinase activity (prins et al., ) . mutational studies revealed that a n-terminal coiledcoil domain in vp mediates homooligomerization, which was found to be essential for ifn antagonism exhibited by the c-terminus of the protein (reid et al., ) . several amino acid residues (r , k , r , k , r ) in the central basic patch of the c-terminal ifn inhibitory domain (iid) of vp were shown to be required for inhibition of virus-induced activation of ifn-regulated promoters and production of ifn-b (cárdenas et al., ; prins et al., b) , with r being of particular importance (hartman et al., ; cárdenas et al., ) . mutations in the iid attenuate viral growth in cell lines and the c-terminal basic patch residues are highly conserved between filoviruses, highlighting their importance for the function of vp (hartman et al., ; prins et al., b) . notably, fig. . interference of ebola virus vp with the expression of ifn and isgs. transcription of the ebov genome results in the production of dsrna intermediates, which are recognized by the cytoplasmic pathogen recognition receptor (prr) rig-i. single-stranded rna is recognized by the endosomal prrs tlr- and tlr- . activation of these prrs induces signalling cascades leading to the expression of type i ifns. in addition, dsrna is recognized by the rna interference machinery that facilitates rna degradation and thereby suppresses the expression of viral genes. vp is able to block several of the above-described processes. because of its dsrna-binding domain, it is able to sequester dsrna from recognition by rig-i and to protect it from degradation through the rna-induced silencing complex. furthermore, it inhibits the phosphorylation of irf- and therefore irf- dimerization, translocation into the nucleus and the expression of ifn-induced genes. in addition, vp is able to induce su-moylation of irf- and irf- , which interferes with dimerization and nuclear translocation. ifn, interferon; tlr, toll-like receptor; trbp, hiv- trans-activation response rna-binding protein; pact, protein activator of pkr; rig-i, retinoic acid-inducible gene i; myd , myeloid differentiation primary response protein ; trif, tir domain-containing adaptor-inducing ifn-b; ips- , ifn-b promoter stimulator ; traf , tumour necrosis factor receptor-associated factor ; irak, interleukin- receptor-associated kinase; ikke, ijb kinase e; tbk , tank-binding kinase ; irf, ifn regulatory factor; isg, ifn-stimulated gene; pkr, dsrna-dependent protein kinase; ifitm, ifn-induced transmembrane protein. the disruption of the irf- inhibitory activity of vp does not influence the function of vp in viral replication and transcription , as amino acid residues essential for polymerase cofactor function of vp differ from those required for ifn antagonism (prins et al., a) . sequences in the c-terminus of vp resemble the dsrna-binding domain of fluav ns , which is important for ifn antagonism (donelan et al., ; hartman et al., ) . indeed, vp is able to bind dsrna, but not single-stranded (ss) rna or dsdna and mutations in the central basic patch of iid, which abrogate ifn antagonism were found to be incompatible with dsrna binding (cárdenas et al., ; leung et al., ; prins et al., b) . the structure of vp iid in complex with dsrna has been determined at the atomic level (kimberlin et al., ; leung et al., ) , and the protein was found to display a unique fold compared with known dsrna-binding proteins, including that of fluav ns (leung et al., ) . interestingly, vp displays a bimodal dsrna-binding strategy. upon dsrna recognition, vp builds asymmetric dimers; one monomer binds the dsrna phosphate backbone, whereas the other binds terminal nucleotides of the dsrna molecule (kimberlin et al., ; leung et al., ) . this dsrna-binding mode of vp dimers seems to mimic that of rig-i, a key cellular sensor of dsrna, and vp and rig-i might occupy overlapping binding sites on dsrna (kimberlin et al., ; leung et al., ) . these findings suggest that vp may sequester dsrna from recognition by rig-i and potentially other dsrna-binding molecules such as mda- or dicer (kimberlin et al., ; . in addition, differences in the structural organization and dsrna binding of zebov and rebov vp were identified, which might contribute to the differential pathogenicity of these viruses in humans (kimberlin et al., ; leung et al., ) , although it needs to be noted that both viruses are highly pathogenic in macaques. vp promotes sumoylation of ifr- via pias ifr- but not irf- is critical for the production of type i ifns (honda et al., ) , and a recent study shows that vp inhibits irf- by promoting its sumoylation (chang et al., ). thus, vp forms a complex with irf- and pias (the small ubiquitin-like modifier (sumo) e ligase protein inhibitor of activated stat) and promotes irf- sumoylation via pias (chang et al., ) (fig. ) . this study also revealed that vp displays an ifn inhibitory activity independent of its ability to recognize dsrna, and this activity was mapped to the n-terminus, which is essential for interactions with irf- and pias . the interferon-inducible dsrna-dependent protein kinase (pkr) recognizes dsrna produced in the context of infection by rna viruses and dna viruses encoding opposite open reading frames from which overlapping mrna sequences are generated (george et al., ). subsequently, it phosphorylates the eukaryotic translation initiation factor a (eif a), which results in the arrest of protein synthesis from cellular and viral mrnas (garcía et al., ) . in the context of ebov infection, it was noted that the expression of vp and eif- a phosphorylation inversely correlate and that autophosphorylation of pkr is disrupted in the presence of vp (feng et al., ) . thus, vp inhibits pkr activation (fig. ) . in fact, zebov was shown to not only block, but to reverse activation of pkr (schumann et al., ). feng et al. ( suggested that vp inhibition of pkr might not involve interactions of these proteins and might not depend on dsrna binding by vp . instead, a role of the n-terminal sequences in vp was suggested. subsequent work by schumann et al. ( ) indicates that mutations in the iid can be sufficient to relief the block to pkr activation imposed by vp and that prk antagonism by vp is therefore functionally separate from dsrna binding and irf- inhibition. vp inhibits rnai by targeting the components of the rna-induced silencing complex rna interference allows cells to specifically recognize and destroy viral rna and thus to combat viral infection. the observation that several viruses encode rnai silencing suppressors (rss), like the ns protein of fluav or the tat protein of the human immunodeficiency virus type (hiv- ), underlines the potency of this cellular defence mechanism (bivalkar-mehla et al., ). haasnoot et al. ( ) demonstrated that ebov vp is an rss, which inhibits shrna-mediated suppression of reporter gene expression and rescues production of tat-defective hiv- from suppression by rnai. the rss activity of vp was dependent on its dsrna-binding capability (haasnoot et al., ) , suggesting that vp may sequester and protect dsrna from recognition by dicer, a central molecule of the rnai pathway (fig. ) . however, a subsequent study demonstrated that vp interacts with components of the rna-induced silencing complex (risc) independent of the presence of sirna (fabozzi et al., ) . specifically, vp was found to interact with the two dsrna-binding proteins hiv- trans-activation response rna-binding protein (trbp) and protein activator of pkr (pact), which are part of the risc complex, but not directly with dicer (fabozzi et al., ) . in addition, evidence was presented that also vp and vp act as rss, and vp like vp was shown to bind to components of the risc complex, while no such interactions were seen for vp , which might employ a different strategy to inhibit rnai (fabozzi et al., ) . the tetherin protein (hm . , bst- , cd ) was identified as a type i interferon-inducible host cellular factor, which restricts release of progeny hiv- particles from infected cells (neil et al., ; van damme et al., ) . tetherin is counteracted by the hiv- accessory protein vpu (viral protein u), which allows efficient hiv- release form tetherin-expressing cells (neil et al., ; van damme et al., ) . the antiviral action of tetherin is not limited to hiv- ; several recent studies found that tetherin restricts release of vlps and progeny particles of several enveloped viruses, including members of the retroviridae, arenaviridae, paramyxoviridae, filoviridae, rhabdoviridae and herpesviridae families (jouvenet et al., fig. . countermeasures of ebola virus against ifn-induced antiviral proteins. ebola virus infection commences with viral uptake and transport of virions into endosomal compartments, where the ebov-gp is activated by cathepsins. subsequently, gp mediates fusion of the viral envelope with the endosomal membrane, allowing release of the viral genome into the cytosol. subsequently, the viral genome is transcribed into mrnas and replicated. viral proteins are translated and transported to the cellular membrane for assembly of new particles, which eventually bud from the host cell membrane. virus entry is inhibited by the ifn-induced ifitm proteins. the exact step that is blocked by ifitms remains to be defined. the protein kinase r, which senses dsrna intermediates of viral replication, phosphorylates the translation initiation factor eif a, which results in the arrest of translation of viral and cellular mrnas. the tetherin protein tethers budding ebov-like particles to the cell surface and is counteracted by ebov-gp. gp, glycoprotein; vp, viral protein; l, rna-dependent rna polymerase; np, nucleoprotein; ifitm, ifn-induced transmembrane protein; pkr, dsrna-dependent protein kinase; ebov-gp, ebola virus glycoprotein. ; mansouri et al., ; sakuma et al., ; pardieu et al., ; radoshitzky et al., ; weidner et al., ; watanabe et al., ; yondola et al., ) (fig. ) . the broad spectrum antiviral action of tetherin is intimately linked to its unusual structural organization. tetherin encodes a short cytoplasmic domain at its n-terminus, followed by a single transmembrane (tm) domain, an extracellular coiled-coil region and a c-terminal glycosylphosphatidylinositol (gpi) anchor (kupzig et al., ) . thus, the protein spans the membrane twice and is able to insert one end into the cellular membrane and the other end into the viral envelope. by this mechanism, tetherin retains budding virions at the surface of the infected cell and inhibits their transmission to new target cells (perez-caballero et al., ; hammonds et al., ). an artificially constructed tetherin, which displays the same topology as the original molecule, but is assembled from completely unrelated sequences, is able to inhibit virus spread as well, confirming that it is tetherin's unusual architecture that is responsible for tethering virions to cells (perez-caballero et al., ) . antagonism of tetherin is not limited to vpu. hiv- uses its envelope (env) protein to counteract tetherin (le tortorec and neil, ) , while the simian immunodeficiency virus (siv) uses either the accessory protein nef or vpu or env, depending on the origin of the virus (gupta et al., ; sauter et al., ) . the kaposi's sarcoma-associated herpes virus encodes the e ubiquitin ligase k as a tetherin antagonist (mansouri et al., ) . for the ebov, the gp , serves as tetherin antagonist (kaletsky et al., ). this ability of gp , is conserved between the gps of the different ebov species (zaire, sudan, côte d'ivoire and reston), the proposed species bebov and the second filoviral genus marv (kaletsky et al., ; lopez et al., ; radoshitzky et al., ; kühl et al., a,b) . the ebov glycoprotein and hiv- vpu employ different strategies to counteract tetherin the ebov glycoprotein (ebov-gp , ) is the only viral surface protein and mediates viral entry into the host cell that requires binding of gp , to the endosomal membrane protein niemann-pick c (npc ) (carette et al., ; cô té et al., ) . ebov-gp , is synthesized as a precursor protein, which is post-translationally cleaved by subtilisin-like proteases into its two subunits gp and gp (volchkov et al., ) . the large and heavily glycosylated, extracellular domain gp mediates attachment to the host cell; the smaller tm unit gp facilitates fusion of the viral envelope with the membrane of host cell endosomes (takada et al., ; wool-lewis and bates, ) . a recent study demonstrated that gp , also inactivates one of the cell's innate defences against infection, the tetherin protein (kaletsky et al., ) . vpu allows hiv- to evade tetherin by mediating cell surface downregulation and relocalization of tetherin into intracellular compartments (van damme et al., ) . in addition, vpu facilitates degradation of tetherin in lysosomal or proteasomal compartments goffinet et al., goffinet et al., , mangeat et al., ; mitchell et al., ; dubé et al., ) . in contrast, no evidence for downregulation, relocalization away from the cell surface or degradation of tetherin was observed in the presence of ebov-gp , or marv-gp , (kaletsky et al., ; lopez et al., ; radoshitzky et al., ; kühl et al., a) . furthermore, ebov-gp , is active against tetherin homologues from different monkeys, in accordance with the ability of ebov to infect several non-human primates (kühl et al., a) . in contrast, tetherin antagonism by vpu is limited to tetherin of human, chimpanzee and gorilla origin, and these species are infected by hiv- and vpu encoding siv, respectively (goffinet et al., ; jia et al., ; mcnatt et al., ). thus, vpu and ebov-gp , have a different specificity for tetherin and employ different strategies to counteract tetherin. tetherin interacts with the gp subunit of the ebov-gp , four different proteins are expressed from the ebov-gp gene: (i) the primary transcript from the gp gene is sgp, a soluble, secreted form of the gp (volchkov et al., ; sanchez et al., ) , which has an anti-inflammatory property (wahl-jensen et al., ) . (ii) a soluble d-peptide is released upon proteolytic processing of sgp by furin (volchkova et al., ) . (iii) the full-length, membrane-bound form gp , , which is incorporated into the virions, is produced by rna editing of the primary transcript (volchkov et al., ; sanchez et al., ) . (iv) furthermore, a small soluble gp (ssgp) has recently been identified, but the function of this protein is at present unknown (mehedi et al., ) . in addition, the tnfa converting enzyme induces shedding of gp , d from the surface of infected cells by cleaving gp , near the tm domain (dolnik et al., ) . all different forms of the gp might play a role in combating the host's immune system as, for example the shedded form of gp might be able to act as a decoy for neutralizing antibodies (dolnik et al., ) . however, neither sgp nor gp , d are able to counteract tetherin (kaletsky et al., ). in addition, a gp , mutant that is retained in the er failed to counteract tetherin, suggesting that the correct localization of the ebov-gp , at sites of viral budding is crucial for tetherin antagonism (kaletsky et al., ) . indeed, because of the expression of the gpi-anchor, tetherin is localized to lipid rafts, which are used by hiv- and ebov as platforms for budding (ono and freed, ; bavari et al., ) . vpu and tetherin interact via their tm domains, and the interaction is critical for tetherin antagonism . in contrast, the sequences of tetherin's cytoplasmic tail (ct) and tm domain do not determine counteraction by ebov-gp , . thus, tetherin chimeras in which the tm region and the n-terminus of tetherin were exchanged against similar domains of the transferrin receptor type (tfr) displayed antiviral activity and were counteracted by ebov-gp , (lopez et al., ) . nevertheless, zebov-gp , is able to efficiently coimmunoprecipitate human tetherin (kaletsky et al., ) , and the gp subunit of ebov-gp , was shown to be critical for the interaction (kühl et al., a) . which domain of tetherin interacts with zebov-gp remains to be determined, and, because of topological constraints, the tm unit and the ct are interesting candidates. however, the tm and ct have been shown to be irrelevant for tetherin counteraction by zebov-gp , (lopez et al., ) , and the importance of tetherin binding for tetherin inhibition by ebov-gp , remains to be determined. the ebov-gp , might relocalize tetherin within the plasma membrane or interfere with the structural integrity of tetherin tetherin and vp , which is essential for ebov budding, colocalize at the plasma membrane and release of vp based vlps is inhibited by tetherin (jouvenet et al., ) . it is thus possible that the ebov-gp , rescues the block to particle release by interfering with the integrity of tetherin at filoviral budding sites (kühl et al., a) . alternatively, the extracellular, heavily glycosylated ebov-gp subunit might interfere with the formation of the 'tetherin-clamp' between the cellular and the viral membrane, because of steric hindrance. finally, ebov-gp , might relocalize tetherin within the plasma membrane, thereby excluding it from membrane domains used by ebov for budding. indeed, it was observed that tetherin is excluded from plasma membrane sites positive for gp , in ebov-infected cells (radoshitzky et al., ) , lending support to the idea that ebov-gp , blocks tetherin's antiviral action by inducing its mislocalization within the plasma membrane. regardless of the mechanism underlying tetherin counteraction by ebov-gp , , it remains to be determined whether endogenous tetherin can reduce ebov release from infected cells, as modest effects were observed in one study (kühl et al., a) but not in another applying a substantially higher multiplicity of infection (moi) (radoshitzky et al., ) , and might thus restrict viral spread in the infected host. in addition, it will be interesting to determine whether african fruit bats, the potential natural reservoir of ebov, encode a tetherin-like protein and whether this tetherin homologue restricts ebov spread. the ifitm , and were recently discovered as inhibitors of host cell entry of several enveloped viruses, including ebov (fig. ) . ifitms , and are ubiquitously expressed in human cells and tissues upon exposure to type i (a) and type ii (c) ifn, and homologous proteins are present in many vertebrates (siegrist et al., ) . ifitm proteins were shown to play a role in early development, cell adhesion and control of cell growth (siegrist et al., ) . their antiviral activity was discovered in a sirna screen designed to identify host cell factors modulating fluav infection (brass et al., ). ifitm was identified as a potent inhibitor of host cell entry of fluav and members of the family flaviviridae, west nile virus and dengue virus serotype , but not hepatitis c virus (brass et al., ; jiang et al., ) , and the induction of ifitm expression was shown to be largely responsible for the blockade to fluav entry imposed by treatment of target cells with ifns (brass et al., ). ifitm and were also shown to restrict viral infection although to a lower extent, and the antiviral activity of ifitms was conserved between human proteins and murine orthologues (brass et al., ) . thus, ifitms are novel ifn-induced antiviral effector proteins, which could modulate viral spread in humans and animals. ifitms restrict viral entry into host cells while antiviral activity of ifitms was initially reported for influenza and flaviviruses (brass et al., ; jiang et al., ) , subsequent studies showed that ifitms inhibit entry of additional enveloped viruses such as vesicular stomatitis indiana virus (vsiv), severe acute respiratory syndrome coronavirus (sars-cov) and filoviruses huang et al., ) , which, very much like fluav and flaviviruses, depend on endo-/ lysosomal acidic ph for host cell entry. inhibition of filoviruses and sars-cov was demonstrated employing lentiviral vectors pseudotyped with the respective viral gps and with replication competent virus (huang et al., ) , but the inhibitory efficiency was modest. in contrast to fluav, ifitm showed the most prominent antiviral effects against replication competent ebov and marv, while inhibition by ifitm was less efficient (huang et al., ) . entry of marv and ebov was reduced by the expression of several ifitm orthologues of mouse and chicken, although with different efficacies compared to the human proteins (huang et al., ) . depletion of ifitm by shrna was sufficient to rescue entry of fluav pseudotypes, whereas a knockdown of both, ifitm and ifitm , was required to increase entry of ebov and marv pseudotypes (huang et al., ) . finally, a different study also detected inhibition of hiv- infection (lu et al., ) , which does not depend on low ph, and it is at present unclear which structure or process shared by the viruses listed above for host cell entry is targeted by ifitms. determinants of the antiviral activity of ifitm proteins domains and modifications important for antiviral activity of the ifitms have been identified, although most studies were not conducted in the context of filovirus infection. abrogation of s-palmitoylation of ifitm by mutation of crucial cysteine residues inhibited ifitm clustering in membrane compartments as well as the antiviral activity of ifitm against fluav (yount et al., ) . the sites of s-palmitoylation are highly conserved in members of the ifitm protein family, suggesting a general role in the antiviral activity. in addition, the sequence of the n-terminus is distinct for the different ifitms as ifitm is lacking an n-terminal amino acid stretch present in ifitm and . part of the n-terminus might be crucial for their different antiviral activity (siegrist et al., ) . indeed, the characterization of ifitm /ifitm chimeras revealed that sequences within the n-and c-terminus of ifitm are important for antiviral activity against vsiv . furthermore, lu et al. ( ) could show that only ifitm- and ifitm- inhibit the hiv- life cycle at the step of viral entry, and that deletion of the n-terminal region abrogated this ability. ifitm , in contrast, needs an intact intracellular domain to inhibit hiv- replication, whereas the n-and c-terminus are dispensable (lu et al., ) . what is known about the mechanism underlying ifitm dependent inhibition of host cell entry of ebov and other viruses? one possibility is that ifitms interfere with receptor expression. however, ifitm proteins do not interfere with the level of surface expression of sialic acids and ace , the receptors for fluav and sars-cov, respectively (brass et al., ; huang et al., ) . ifitm expression is compatible with fluav access to low ph compartments, and inhibition of sars-cov by ifitms could be rescued by forcing the virus to fuse with the plasma membrane instead of an internal membrane (huang et al., ) . thus, viruses might reach internal compartments in ifitm expressing cells in which fusion of viral and compartment membrane could normally occur, but membrane fusion might be blocked by ifitms. one possibility could be that ifitms interfere with the activity of cathepsins, ph-dependent endo-/lysosomal proteases essential for proteolytic activation of sars-cov and filoviruses in vitro (chandran et al., ; simmons et al., ) . no appreciable decrease of cathepsin activity was observed in ifitm expressing cells (huang et al., ) . the endosomal membrane protein npc has recently been reported as an essential host cell factor for ebov entry (carette et al., ; cô té et al., ) . according to the model of cô té et al., npc expression or activity is required for endosomal fusion after cathepsin-mediated processing of gp , . it is conceivable that ifitms interfere with npc expression or activity. furthermore, it is possible that a step in the filovirus life cycle different from membrane fusion could be affected by ifitms, as it was reported that ifitm and inhibit hiv- entry, whereas ifitm acts later in the viral life cycle by suppressing gag translation (lu et al., ) . thus, the mechanisms underlying inhibition of filoviruses and other viruses remain to be defined, including the possibility that ifitms require cellular cofactors to exert their antiviral effects. in sum, ifitms inhibit filovirus entry into host cells, and the level of constitutive ifitm expression might shape the choice of early target cells in filovirus infection. further research is needed to define the basal expression of ifitms in filovirus target cells and to elucidate the mechanism by which the different ifitm proteins restrict filovirus infection. the ifn system can potently restrict ebov spread and pathogenesis in infected mice, but is tuned down by viral proteins in infected humans. vp and vp play key roles in suppressing the ifn response by preventing nuclear translocation of stat and by targeting irf- and irf- , respectively. tetherin and ifitm proteins are novel isgs, which could suppress ebov infectious entry into target cells (ifitms) and release of ebov particles from infected cells (tetherin). efficient suppression of ebov release has so far only been demonstrated with vlps and remains to be shown with infectious ebov. for this, the sequences in gp , , which facilitate tetherin antagonism, need to be identified and altered, which should render ebov susceptible to inhibition by tetherin. the gp subunit is an excellent candidate for these endeavours (kühl et al., a) . the mechanism underlying ebov inhibition by ifitms is at present unknown, and its elucidation might require the identification of potential interaction partners of gp , and ifitms in host cell endosomes. in addition, it will be interesting to determine to which extent basal (in the absence of ifn) expression of ifitms and tetherin impact ebov spread. a recent study demonstrated that basal tetherin expression is broader than initially appreciated and raised doubts concerning the sole regulation of tetherin expression by ifns (erikson et al., ) . moreover, the role of the ifn system in the ebov infection of the potential reservoir host, fruit bats (leroy et al., ) , is of high interest. thus, ebov infection of these animals does not seem to induce disease (swanepoel et al., ; hayman et al., ) , and it is tempting to speculate that the bat ifn system efficiently controls viral replication. finally, the interesting insights into ebov interference with the ifn system open new avenues to the development of filovirus inhibitors. proof of concept comes from studies with hiv- , which demonstrated that stabilization of antiviral effectors molecules of the ifn system or inhibition of viral ifn antagonists by small molecules can suppress viral spread (cen et al., ; jiang et al., ) . the authors have no potential conflicts to declare. the ebola virus vp protein functions as a type i ifn antagonist the ebola virus vp protein inhibits activation of interferon regulatory factor 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infection ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ectodomain shedding of the glycoprotein gp of ebola virus filoviruses: interactions with the host cell a recombinant influenza a virus expressing an rna-bindingdefective ns protein induces high levels of beta interferon and is attenuated in mice vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {beta}trcp-dependent mechanism antagonism of tetherin restriction of hiv- release by vpu involves binding and sequestration of the restriction factor in a perinuclear compartment molecular determinants of ebola virus virulence in mice vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice in vivo expression profile of the antiviral restriction factor and tumor-targeting antigen cd /bst- /hm . /tetherin in humans ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway the vp protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr the dsrna protein kinase pkr: virus and cell control type interferons and the virus-host relationship: a lesson in detente tipping the balance: antagonism of pkr kinase and adar deaminase functions by virus gene products hiv- antagonism of cd is species specific and involves vpu-mediated proteasomal degradation of the restriction factor antagonism of cd restriction of human immunodeficiency virus type (hiv- ) particle release and depletion of cd are separable activities of hiv- vpu the ecology of ebola virus simian immunodeficiency virus envelope glycoprotein counteracts tetherin/bst- /cd by intracellular sequestration the ebola virus vp protein is a suppressor of rna silencing interferoninduced mx proteins in antiviral host defense immunoelectron microscopic evidence for tetherin/bst as the physical bridge between hiv- virions and the plasma membrane biochemical and functional characterization of the ebola virus vp protein: implications for a role in virus assembly and budding filovirus assembly and budding a c-terminal basic amino acid motif of zaire ebolavirus vp is essential for type i interferon antagonism and displays high identity with the rna-binding domain of another interferon antagonist, the ns protein of influenza a virus reverse genetic generation of recombinant zaire ebola viruses containing disrupted irf- inhibitory domains results in attenuated virus growth in vitro and higher levels of irf- activation without inhibiting viral transcription or replication inhibition of irf- activation by vp is critical for the high level of virulence of ebola virus whole-genome expression profiling reveals that inhibition of host innate immune response pathways by ebola virus can be reversed by a single amino acid change in the vp protein ebola and marburg hemorrhagic fever long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses infection of naive target cells with virus-like particles: implications for the function of ebola virus vp irf- is the master regulator of type-i interferon-dependent immune responses the assembly of ebola virus nucleocapsid requires virion-associated proteins and and posttranslational modification of nucleoprotein distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus. plos pathog species-specific activity of siv nef and hiv- vpu in overcoming restriction by tetherin/bst identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections the vp protein of ebola virus impairs dendritic cell maturation induced by virus and lipopolysaccharide broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein how ebola virus infects cells ebolavirus vp uses a bimodal strategy to bind dsrna for innate immune suppression the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin comparative analysis of ebola virus glycoprotein interactions with human and bat cells filoviruses. a compendium of years of epidemiological, clinical, and laboratory studies bst- /hm . is a raft-associated apical membrane protein with an unusual topology antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type envelope glycoprotein fold prediction of vp protein of ebola and marburg viruses using de novo fragment assembly fruit bats as reservoirs of ebola virus structure of the ebola vp interferon inhibitory domain structural basis for dsrna recognition and interferon antagonism by ebola vp contribution of ebola virus glycoprotein, nucleoprotein, and vp to budding of vp virus-like particles new developments in the induction and antiviral effectors of type i interferon ebola virus glycoprotein counteracts bst- /tetherin restriction in a sequence-independent manner that does not require tetherin surface removal the ifitm proteins inhibit hiv- infection protection from lethal infection is determined by innate immune responses in a mouse model of ebola virus infection hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation molecular mechanism of bst /tetherin downregulation by k /mir of kaposi's sarcoma-associated herpesvirus ebolavirus vp binding to karyopherins is required for inhibition of interferon signaling species-specific activity of hiv- vpu and positive selection of tetherin transmembrane domain variants a new ebola virus nonstructural glycoprotein expressed through rna editing vpu antagonizes bst- -mediated restriction of hiv- release via beta-trcp and endo-lysosomal trafficking comparison of the transcription and replication strategies of marburg virus and ebola virus by using artificial replication systems tetherin inhibits retrovirus release and is antagonized by hiv- vpu plasma membrane rafts play a critical role in hiv- assembly and release the ring-ch ligase k antagonizes restriction of kshv and hiv- particle release by mediating ubiquitindependent endosomal degradation of tetherin tetherin inhibits hiv- release by directly tethering virions to cells ebola virus protein vp impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk- basic residues within the ebolavirus vp protein are required for its viral polymerase cofactor function mutations abrogating vp interaction with double-stranded rna render ebola virus avirulent in guinea pigs infectious lassa virus, but not filoviruses, is restricted by bst- /tetherin homooligomerization facilitates the interferon-antagonist activity of the ebolavirus vp protein ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation ebola virus vp proteins inhibit the interaction of npi- subfamily karyopherin alpha proteins with activated stat interferon-inducible antiviral effectors inhibition of lassa and marburg virus production by tetherin the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv- strains jak-stat signaling: from interferons to cytokines a diverse range of gene products are effectors of the type i interferon antiviral response ebola virus vp antagonizes pkr activity through its c-terminal interferon inhibitory domain extracellular signal-dependent nuclear import of stat is mediated by nuclear pore-targeting complex formation with npi- , but not rch the small interferon-induced transmembrane genes and proteins ebola virus counters the interferon system a. kü hl and s. pö hlmann inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry experimental inoculation of plants and animals with ebola virus a system for functional analysis of ebola virus glycoprotein newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda. plos pathog. , e gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases processing of the ebola virus glycoprotein by the proprotein convertase furin delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sgp of ebola virus effects of ebola virus glycoproteins on endothelial cell activation and barrier function influenza virus is not restricted by tetherin whereas influenza vlp production is restricted by tetherin interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines budding capability of the influenza virus neuraminidase can be modulated by tetherin palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm we thank t.f. schulz for support. key: cord- - tknscm authors: sztuba-solinska, joanna; diaz, larissa; kumar, mia r.; kolb, gaëlle; wiley, michael r.; jozwick, lucas; kuhn, jens h.; palacios, gustavo; radoshitzky, sheli r.; j. le grice, stuart f.; johnson, reed f. title: a small stem-loop structure of the ebola virus trailer is essential for replication and interacts with heat-shock protein a date: - - journal: nucleic acids res doi: . /nar/gkw sha: doc_id: cord_uid: tknscm ebola virus (ebov) is a single-stranded negative-sense rna virus belonging to the filoviridae family. the leader and trailer non-coding regions of the ebov genome likely regulate its transcription, replication, and progeny genome packaging. we investigated the cis-acting rna signals involved in rna–rna and rna–protein interactions that regulate replication of egfp-encoding ebov minigenomic rna and identified heat shock cognate protein family a (hsc ) member (hspa ) as an ebov trailer-interacting host protein. mutational analysis of the trailer hspa binding motif revealed that this interaction is essential for ebov minigenome replication. selective ′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the ebov minigenomic rna indicates formation of a small stem-loop composed of the hspa motif, a ′ stem-loop (nucleotides – ) that is similar to a previously identified structure in the replicative intermediate (ri) rna and a panhandle domain involving a trailer-to-leader interaction. results of minigenome assays and an ebov reverse genetic system rescue support a role for both the panhandle domain and hspa motif in virus replication. ebola virus (ebov) can cause large and highly lethal human disease outbreaks. because of the scarcity of effective treatments against ebov, there is an urgent need to identify novel viral inhibitors that target specific viral processes. as of yet, relatively little is known about the molecular biology of ebov replication, and, in particular, the interplay between host factors and the viral genome. the ebov genome is a negative-sense, single-stranded rna organized into a leader non-coding region (ncr), followed by seven discrete transcriptional units encoding np (nucleocapsid protein), vp (polymerase cofactor and interferonresponse modulator), vp (matrix protein), gp , (glycoprotein), vp (transcriptional enhancer), vp (secondary matrix protein, ion channel and interferon-response modulator), l (rna-dependent rna polymerase) and a trailer ncr ( , ) . each gene is separated by intergenic regions of varying lengths that modulate transcript levels ( ) . the ncrs of rna virus genomes have highly conserved primary and secondary structures ( ) ( ) ( ) ( ) . however, the functions of ebov ncrs are not well characterized. replicon systems consisting of the complete trailer and leader sequences and short segments of the l and np protein genes that flank a reporter gene such as chloramphenicol acetyl transferase, green fluorescent protein, or luciferase have been developed ( ) ( ) ( ) ( ) . in these systems, transcription and replication from a replicon genome is supported by the ebov proteins np, l, vp , and vp , which are driven from co-transfected expression plasmids. such systems allow examination of virus specific processes involving the rna and these proteins. addition of vp and gp expres-sion plasmids can drive production of infectious vlps ( ) . using the minigenome replicon systems and/or computerassisted secondary structure predictions, some functions have been attributed to the ncrs. computer modeling of the ebov trailer and leader indicate that a panhandle structure may form between the ncrs starting with base paring of nucleotide (nt) and . almost identical hairpin structures for the leader of the ebov genome and ri rna are possible ( ) . computer modeling of ebov mini-ri rna and mutational analysis suggested that the terminal nts of the ebov leader are not essential for infection ( ) . chemical probing of the ebov ri rna revealed a stem-loop within the end that is involved in regulating transcription ( ) . additional probing analyses of mini-ri rna suggest that the trailer and leader do not interact, but that the terminal nts of the leader are important for replication ( ) . recently, a direct interaction between vp and the leader has been described, suggesting that vp clamps the rna template to prevent the polymerase complex (vp /l) from dissociation and allows productive transcription initiation in the presence of secondary structures in the template ( , ) . the optimal rna substrate for vp binding is a single-stranded rna that is linked to a stem-loop, as found in the region of the replication promoter element of the ebov genomic leader ( ) . other studies further emphasize the importance of non-terminal ebov rna secondary structures in transcription and replication. for instance, ebov gp mrna editing is dependent on such secondary structures ( ) ( ) ( ) . interrupting secondary structure formation inhibits synthesis of the mrna encoding gp , , which mediates virion entry into host cells. host proteins bind to rna secondary structures to modulate lifecycle processes for many positive-sense rna viruses, such as hepatitis c virus (hcv) ( ) , hepatitis a virus ( ) , poliovirus type ( ) , dengue viruses ( ) , bovine coronavirus ( ) , and murine hepatitis virus ( ) ( ) ( ) . however, few host protein-viral rna interactions have been characterized for negative-sense, single-stranded rna viruses. previously, the la autoantigen (sjögren syndrome antigen b) was shown to interact with the leader of rabies virus ( ) , vesicular stomatitis new jersey virus ( , ) , and rinderpest virus ( ) . la autoantigen is an rna polymerase iii transcription factor that shuttles between the nucleus and cytosol and may play a role in mrna stability for translation. interactions between the viral leader and la autoantigen are thought to play a role in replication. replication is increased when the concentration of la autoantigen is increased ( ) indicating a necessary functional role for host proteins to directly interact with the negative-sense viral genome. however, no specific viral rna secondary structures that interact with la or other host proteins have been identified. host dna topoisomerase (top ) facilitates ebov genome transcription and replication ( ) . in the case of retroviruses, such as human immunodeficiency virus- and rous sarcoma virus, top interacts with their viral genome through a genomic rna stem-loop, suggesting that the top -viral genome interaction directly regulates transcription and replication ( ) ( ) ( ) . whether a similar interaction occurs with ebov remains unclear, although, ebov np and l genes both contain a potential top target sequence (tcctt) ( , ) . considering the length ( nt) and structural complexity of ebov trailer, host and viral factors likely interact with trailer rna motifs modulating the virus lifecycle. the goal of this study was to determine the secondary structure of the ebov e- e-enhanced green fluorescent protein ( e- e-gfp) minigenome rna, identify host proteins that interact with the ebov trailer, define their rna binding motifs, and establish a functional role for the protein-rna interactions. e- e-gfp minigenome rna secondary structure and host protein interactions were examined using selective -hydroxyl acylation analyzed by primer extension (shape) ( , ) , antisense-interfered shape (aishape) ( ) , electrophoretic mobility shift assays (emsa), sirna, and mutational analysis, using both the e- e-gfp minigenome system and ebov reverse genetics. the human kidney embryonic cell line, t (atcc, manassas, va, crl ) used in e- e-gfp minigenome assays was maintained in dulbecco's modified eagle's medium (lonza) supplemented with % calf serum (sigma-aldrich, st. louis, mo, usa) with % v/v penicillin/streptomycin (lifetechnologies, grand island, ny, usa) at • c in a % co atmosphere. hela cells (atcc ccl- ) were similarly maintained and used for ebov infection assays as described below. grivet (chlorocebus aethiops) vero e cells (atcc #crl- ) were maintained similarly and used for cell lysate preparation for emsa as previously described ( ) . lysates were collected by scraping t- flasks of confluent cells into - ml of phosphate-buffered saline (pbs) followed by centrifugation at × g for min. pellets were resuspended in . ml of hypotonic buffer consisting of mm hepes (sigma-aldrich) . mm mgcl (sigma-aldrich), mm kcl (sigma-aldrich) and . mm pmsf (sigma-aldrich) and incubated on ice for min. non-ionic detergent (igepal , sigma-aldrich) was added to a final concentration of . % (v/v), and the suspension was vortexed twice for s followed by incubation on ice for min. the suspension was centrifuged at × g for min to obtain a clarified lysate. supernatant was removed and glycerol was added to a final concentration of % (v/v). supernatants were aliquoted, frozen immediately on dry ice and stored at − • c. a portion of the lysate was quantified using the pierce bca protein assay kit (thermo fisher scientific, waltham, ma, usa). primers listed in table were used for emsa template preparation, protein pull-down assays, and site-directed mutagenesis for minigenome assays as indicated. transcription templates for emsas were generated by polymerase chain reaction (pcr) amplification using the ebov e- e-gfp plasmid as a template. pcr products were resolved by nucleic acids research, , vol. , no. - . % low melting temperature agarose gel electrophoresis (seakem gtg, sigma-aldrich) and purified using the qi-aquick gel extraction kit (qiagen, valencia, ca, usa). products were dialyzed to remove excess salt and used as templates for transcription with the megascript t kit (thermo fisher scientific, waltham, ma, usa) following the manufacturer's protocol. radiolabeled probes were prepared by transcription in the presence of utp-␣ p. depending upon the length of the probe, pcr products were purified by either phenol:chloroform extraction and ethanol precipitation or megaclear transcription cleanup kit (thermo fisher scientific, waltham, ma, usa) and subsequently quantified. five micrograms of protein lysate was incubated with . pmol of radiolabeled probe in a final volume of l. reactions were incubated at • c for min in × reaction buffer consisting of mm kcl, mm tris ph . , mm mgcl , mm dtt, ng poly (i)-(c) (ther-mofisher waltham, ma, usa). reactions with unlabeled specific wild-type (wt) competitor and non-specific trna competitor at , , and × molar excess were carried out in parallel. the l reaction was loaded onto a × tris borate edta buffer, % non-denaturing polyacrylamide gel and electrophoresed for . h. gels were transferred to whatman chr paper (whatman, maidstone uk), dried at • c under vacuum for h, and exposed to a phosphorimager screen (ge healthcare life sciences, little chalfont, uk). at least three independent assays were performed per probe. typhoon software was used to analyze the gels and determine relative signal intensities of the shifted bands. background subtracted signal intensity for each sample was determined as a ratio to wt in the absence of competitor. relative average intensities were calculated and compared by paired t-tests using graphpad prism. the - probe ( pmol) was biotinylated using the pierce rna end biotinylation kit (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's protocol. this probe was purified by size-exclusion chromatography using probequant g- microcolumns (ge healthcare life sciences) and incubated with streptavidin magnetic dynabeads (thermo fisher scientific, waltham, ma, usa) in binding buffer for h at • c with constant rocking. lysate was prepared as described above and incubated with the prepared dynabeads. beads were harvested with a magnetic separator and washed in binding buffer three times. protein was eluted by boiling in × lithium dodecyl sulfate buffer for min. samples were loaded on a - % -(n-morpholino)propanesulfonic acid (mops) polyacrylamide gel and electrophoresed for h at v. the gel was silver-stained followed by mass-spectrometry of the whole lane for control and the - probe samples. peptide fragments were generated, and protein identities were determined by amino acid sequence homology. immunoprecipitation-reverse transcriptase-pcr (ip-rt-pcr) was performed as described previously ( ) . briefly, vero e lysate was incubated with - probe followed by immunoprecipitation with . g of anti-hspa (hsc ) antibody (santacruz biotechnology, dallas, tx, usa) and protein g dynabeads (thermo fisher scientific, waltham, ma, usa) and three rounds of washing with binding buffer. bound material was eluted from the beads in l of elution buffer ( mm tris-hcl, ph . , mm edta, % sodium dodecyl sulphate (sds)) at • c for min. the eluate ( l) was loaded on sds-mops - % gels and probed for hspa . the remainder was digested by g of proteinase k for min at • c, extracted by phenol:chloroform, and precipitated with ethanol. the pellet was resuspended in l of deionized rnase-dnase free water, and pmol of the resuspension were reverse transcribed using superscript iii (thermo fisher scientific, waltham, ma, usa) followed by pcr with hifi taq (thermo fisher scientific, waltham, ma, usa) and the - primer set (table ). negative control samples were processed in parallel by excluding the - rna. the ebov e- e-gfp minigenome system was kindly provided by dr. kawaoka, university of wisconsin-madison ( , ) . the nt ebov egfp minigenome rna comprises trailer (nt - ), a partial np gene (nt - ), egfp (nt - ), a partial l gene (nt - ) and leader (nt - ) sequences. hek t cells were seeded at . × cells and incubated overnight at • c and % co . cells were transfected using the calcium phosphate method by combining an equal volume of a solution containing . m cacl and minigenome plasmids to × hbss ( . m nacl, . m hepes, . m na hpo . h o) dropwise. the plasmids were in the following ratios per well: p e- e-gfp ( g), pcaggsnp ( ng), pcaggsvp ( ng), pcaggsvp ( ng), pcaggsl ( g) and pt ( g). after min incubation, the mixture was added to cells. at h posttransfection, cells were harvested, washed, and resuspended in % pluronic acid and % paraformaldehyde fixative for analysis by flow cytometry (becton dickenson, ca, usa). measurements were gated relative to vp -negative control samples. statistical analysis was performed by one-way anova using graphpad prism . (graphpad software). total rna from minigenome assay cells was extracted with trizol (thermo fisher scientific). five microgram of total rna was digested with rq dnase (promega) for h at • c. the rna samples were electrophoresed for h at v in a × mops and % formaldehyde agarose gel and transferred to hybond-n+ membrane (ge healthcare life sciences). probes specific for genomic and ri rnas (table ) were labeled with p atp using t polynucleotide kinase (promega). membranes were prehybridized for h in perfecthyb plus buffer (sigma-aldrich), and probe was added and hybridized overnight at • c for genomic and • c for ri, respectively. membranes were exposed to phosphor screen overnight, imaged on ge typhoon fla variable mode imager and quantified with imagequant software (ge healthcare). signal was normalized to vp (-) sample for the genomic probe and normalized to wt sample for the ri probe. various sirnas targeting hspa (table ) were used to transiently reverse-transfect hela cells ( cells per well, -well format) in triplicate at a nm final concentration, using hiperfect reagent (qiagen) as previously described ( ) . cells were washed the following day. twentyfour hours later, cells were infected with ebov-zsgreen at multiplicities of infection of , or for h. cells were fixed with % formalin (val tech diagnostics), and stained for high-content quantitative image-based analysis. the assay was repeated twice. in three wells on each plate, cells were transfected with a negative control sirna (nt, sicontrol non-targeting sirna # , dharmacon d- - ). cdna clones encoding ebov/h.sapiens-tc/cod/ /y ambuku-mayinga (ebov; genbank #af ) wt and variants thereof (a u, a u/a u or the terminal nt deletion variant) were constructed by standard cloning techniques. for recovering recombinant viruses, hek t cells in -well plates were transfected in duplicate with g of full-length ebov cdna clone-encoding plasmid and support plasmids ( g of pcaggs-np, . g of pcaggs-vp , . g of pcaggs-vp , g of pcaggs-l and g of pcaggs-t ) using lipofectamine (invitrogen carlsbad, ca, usa) according to the manufacturer's instructions. as a negative control, pcaggs-l was omitted from one of the samples. at day post-transfection, supernatants were collected, cell debris was removed by centrifugation, and an aliquot of the viruscontaining media (termed passage ) was used to infect a fresh monolayer of vero e cells. one week later, when cytopathic effects were observed in the wt-ebov samples, supernatants (termed passage ) were harvested, cleared by centrifugation, and stored at − • c. vero e cells that did not exhibit cytopathic effects (those transfected with the ebov mutants) were replenished with fresh growth media and incubated for an additional days. supernatants were harvested, cleared by centrifugation and stored at − • c. all ebov rescue experiments were conducted under biosafety laboratory (bsl- ) conditions. virus-containing supernatants (wt-ebov and mutants thereof) were used to infect vero e or hela cells in well plates ( cells/well). cells were inoculated with virus for h, washed with pbs, and replenished with fresh growth media. cells were fixed h later, blocked with % bovine serum albumin in pbs, and stained with murine monoclonal antibodies against ebov gp , ( d , : dilution) and with alexa fluor -conjugated antibodies ( : dilution, life technologies). infected cells were also stained with hoechst and hcs cellmask deepred (life technologies) for nuclei and cytoplasm detection, respectively. infection rates were determined by high-content quantitative image-based analysis on an opera quadruple excitation high sensitivity confocal reader (model and ; perkin-elmer, waltham, ma, usa) as described ( ) . all infections were conducted under bsl- conditions. rna was extracted using the zymo directzol kit (zymo research) following the manufacturer's instructions, including the optional on-column dnase-treatment. rna was prepared for sequencing and enriched for ebov-specific reads using the illumina truseq rna access kit with modifications to the manufacturer's recommended procedures as described previously ( ) . libraries were sequenced on an illumina miseq desktop sequencer using a version , cycle kit ( × ) and analyzed using in-house scripts. dna templates for in vitro transcription were generated by pcr amplification of plasmids encoding the ebov e- e-gfp minigenome and the corresponding replicon mutants, using primers listed in table . all pcr experiments were performed using platinum ® taq dna polymerase high fidelity (thermo fisher scientific, waltham, ma, usa). transcripts were synthesized with the t -megascript system (thermo fisher scientific, waltham, ma, usa) following the manufacturer's protocol. rnas were purified by denaturing m urea/ % polyacrylamide gel electrophoresis, followed by elution and ethanol precipitation. purified rnas were dissolved in sterile water and stored at − • c. six pmol of rna were heated at ºc for min and slowly cooled to • c. the volume was adjusted to l in a final buffer of mm tris-hcl (ph . ), mm nacl, mm mgcl . samples were incubated at • c for min. folded rna was divided into two equal portions ( l each) treated with l of mm -methyl- -nitroisatoic anhydride ( m ) ( ) ( ) . electropherograms were processed using the open-source shapefinder program ver. . following the software developer's protocol, including the required precalibration for matrixing and mobility shift for each set of primers as previously described ( ) . briefly, the area under each negative peak was subtracted from that of the corresponding positive peak. the resulting peak area difference at each nt position was divided by the average of the highest % of peak area differences, calculated after discounting any results greater than the third quartile plus . × the interquartile range. normalized intensities were introduced into open-source rnastructure version . ( ) . locked nucleic acid (lna)/dna chimeras were purchased from exiqon, woburn, ma, usa, and the sequences of which are provided in table . chimeras were added at × molar excess after folding the rna. samples were subsequently incubated at • c for min prior to m treatment (see above). to quantify alterations induced by antisense oligonucleotides, raw data were processed as described above. the secondary structure of the ebov e- e-gfp minigenome rna predicted by rnastructure software version . ( ) and chemical probing data from shape were used to generate three-dimensional ( d) models for the trailer-to-leader panhandle interaction in the wt-ebov genome and variants, a u and a u/a u, using open-source rnacomposer, version . (http://rnacomposer.cs.put.poznan.pl/). the quality of predicted models was evaluated using open-source molprobity and king tools ( , ) . identification of regions of the ebov trailer that interact with host proteins was performed by emsa ( figure a ) using the primers listed in table . initially, probes truncated at the end of the trailer were evaluated to identify host protein binding regions (supplementary table) . because each of the larger truncated probes was positive, further emsas were performed to define minimal rna regions of the trailer. overlapping probes were evaluated in triplicate (supplementary table) . background-corrected signal intensity was compared to wt probe and averaged for the replicates. ribonucleoprotien (rnp) complex formation of probe - was reduced in the presence of × molar excess of trna with a % reduction in binding (p = . ) indicating that the complex can be competed, but only at high concentrations. no reduction in rnp complex formation was observed when competed with wt, unlabeled probe - ( figure b) . the - probe was significantly competed with unlabeled wt probe at ×, ×, × and ×, and × trna ( figure c ). the - probe followed a similar pattern as the - probe and could be significantly competed with wt unlabeled probe and × and × trna ( figure d ). the data suggests that the rnp complex formation is specific because low to moderate concentrations of wt competitor and the highest concentrations of trna were required for significant competition. the - probe was chosen for use as bait in pull-down assays. host proteins were eluted from the beads, resolved and analyzed by mass-spectrometry ( figure a ). rattus norvegicus (peptide score . ), bos taurus (peptide score . ) and mus musculus (peptide score . ) hspa were identified as specifically binding to the - probe by peptide score and selectivity when compared to the control lane ( figure b ). other host proteins binding specifically to the - probe included atp a, mitofilin, aldehyde dehydrogenase and cops , but not as consistently or with lower peptide scores (data not shown). ip-rt-pcr confirmed the hspa : - interaction ( figure c and d). specific - probe pcr products were detected only when cell lysates and - probe were immunoprecipitated with anti-hspa antibody. - probe could not be detected by pcr in control samples excluding antibody, thus supporting a specific interaction between hspa and the - probe. following identification of hspa , a literature search was performed indicating that hspa interacts with a pentanucleotide motif, auuua , ( ) ( ) ( ) ( ) . closer examination of the ebov trailer sequence identified three of these motifs at nt positions - , - and - . here, these are referred to as hspa -binding motifs , and , respectively. based on the emsa data, hspa motif was chosen for further functional analysis. variants of motif were generated containing either a single point mutation or clustered multiple point mutations. the effect of these variants on ebov transcription/replication was examined in the context of the e- e-gfp minigenome assay using t cells. the e- e-gfp minigenome assay is a transfectionbased replicon system. expression plasmids encoding the ebov vp , vp , l and np proteins are co-transfected with a plasmid encoding the e- e-gfp minigenome that is driven by a t promoter and an expression plasmid encoding the t polymerase (t pol). the minigenome is described in detail in watanabe et al. ( ) . briefly the and ends of the ebov genome flank a egfp open reading frame (orf) as a reporter gene and is replication and transcription competent. single-nucleotide changes in motif of the minigenome indicated that an a u ( auuuu ) mutation resulted in a statistically significant (p < . ) decrease in both the total number of gfp-positive cells ( % decrease, figure a ) and in the mean fluorescence intensity of the gfp signal ( % decrease) ( figure b ). the a u/a u double mutant ( uuuuu ) motif resulted in a significant decrease in gfp-positive cells ( %, p < . , figure a and b). northern blots specifically targeting the genome and ri rnas were performed on the wt and mutant minigenome samples to determine which step of the virus lifecycle was impacted by mutagenesis of motif . based on previous identification of vp as essential for replication and transcription in an ebov minigenome assay, ( ), a control omitting expression of vp (vp (−)) was included to determine the level of t pol produced minigenome rna. this control is necessary to determine the level of minigenome produced by the viral proteins. if the mutant does not impact minigenome synthesis, then the northern blot signal intensity will be increased when compared to vp (−) and equal to the wt sample. if the mutant impacts minigenome rna synthesis, then the northern blot signal intensity will be lower than wild type or equal to the vp (−) signal or greater than the wt signal. the data indicate that four of five single point mutations, a u, u a, u a and a u, in motif reduced minigenome rna synthesis. the a u mutant demonstrated the greatest impact and was below the vp (−) signal ( figure c and supplementary figure s a ), however the u a mutant did not impact minigenome rna synthesis. the double mutants a u/u a, a u/u a, and a u/a u also reduced minigenome rna synthesis when compared to wt. these data indicate that motif is involved in minigenome rna synthesis. to assess the impact of motif in ri rna synthesis, a northern blot specifically targeting the ri rna was developed ( figure d and supplementary figure s b ). ri rna is not produced by t pol and will only be produced when the appropriate complement of viral proteins and a suitable rna template is present, therefore the northern blot signal intensity for wt ri rna was used as a basis of comparison. mutants u a, a u and a u/a u decreased ri rna synthesis when compared to wt sequence. mutants a u, u a, u a, a u/u a, a u/u a and a u/u a demonstrated variable but near wt levels of ri rna synthesis. these data indicate that motif also plays a role in ri rna synthesis. interestingly, the vp (−) control indicates that vp is also required for ri rna synthesis. transfection-based viral replicons reflect basic viral process, but do not reflect all aspects of the viral lifecycle. therefore, evaluation of the mutations with the greatest effect in the e- e-egfp minigenome assay, a u, a u/a u and an additional - mutant, was carried out using a full-length infectious clone ( table ). the a u/a u and - mutants could not be recovered in four of four replicates. however, the a u ebov variant was rescued in / replicates, although with slower kinetics. sequence analysis of one of three replicates indicated an 'a' insertion at position in % of viral rnas, which is in the gp orf. as expected, wt-ebov was recovered in all replicates. these data support the importance of motif in the virus lifecycle. based on e- e minigenome assay data, the a u mutant was evaluated for rnp formation by emsa, using the - probe. as shown in figure e and f significant competition was observed with cold wt competitor similar to the initial experiments described above. competition with cold trna did not reach significance at or × molar concentration, but was observed at and × molar concentration suggesting specificity of the interaction between hspa and the trailer in this probe. emsa of the - probe containing the a u point mutation indicated a % decrease in rnp formation (p < . , one-way anova std dev . ) ( figure e and f), compared to wt probe. rnp complex formation was competed with both unlabeled wt probe and trna supporting our hypothesis that a of motif is necessary for rnp complex formation (p < . one-way anova, graphpad prism). chemical modulation and sirna screening was performed to verify the role of hspa in the virus lifecycle. cells were pre-treated with oxymatrine, which is used as an hcv inhibitor that modulates hspa mrna stability ( ) , but failed clinical trial evaluation ( ) . oxymatrinetreated cells were infected at an moi of with ebov. oxymatrine treatment of cells minimally reduced viral titer, and semi-quantitative western blot did not support reduction of hspa expression (data not shown). thus, a previously established sirna screening assay ( ) was used to evaluate four commercially available sirnas against hspa . on-target sirna reduced relative ebov infection when compared to no sirna and negative control sirna at an moi , and ( figure g ). shape interrogates rna secondary structure by examining backbone flexibility (directly related to base pairing) at each nucleotide position via reactivity with a specific electrophilic reagent ( ) . we applied this technique to the ebov e- e-gfp minigenome rna, which contains all essential cis-acting elements for efficient gfp translation and ebov minigenome replication in vitro ( , , ) . reactivity to the shape reagent m for ebov minigenome rna is shown in figure a (supplementary figure s ) . the most reactive residues, and thus, the least structurally constrained residues have a reactivity > . . nucleotide positions with reactivities < . are indicative of fully base-paired residues. minimum free-energy modeling using shape data as pseudo free energy constraints indicated formation of a panhandle duplex structure between trailer and leader (figure a ). this long-range rna-rna interaction spans the first and last nt of trailer and leader, respectively. the trailer-to-leader panhandle is interrupted by an internal bulge on the leader side (nt - ) and a three-way - and - ) . the three-way junction embeds a short stem-loop (nt - ) containing hspa motif . the region upstream of the leader forms a stem-loop structure (nt - ) with an au-rich apical loop, similar to the ri hairpin previously identified as a putative vp binding site ( ) . residues a -u of this hairpin reacted more strongly with m than residues u -u . thus, the possibility exists that this hairpin (nt - ) forms an h-type pseudoknot structure with the upstream complementary region (nt - ). pseudoknots play a critical role in many biological activities, from regulation of viral gene expression to catalysis of mrna splicing and repeat-addition processivity of human telomerase ( , ) . subsequent experiments involving lna-directed displacement of the putative pseudoknot interaction (aishape) ( ) did not change reactivity of apical loop residues or flanking regions (data not shown). conceivably, weaker reactivities of the apical loop of this hairpin can be attributed to intraloop base-stacking interaction between a:u residues. hspa motifs (nt - ) and (nt - ) are located in an unstructured single-stranded region forming the internal loop (nt - , nt - ) of a hairpin preceding the gfp sequence ( figure a ). importantly, the gfp sequence base pairs independently (supplementary figure s ) . to validate the trailer-to-leader interaction, we applied aishape ( ) . experimentally, one strand of an rna duplex is displaced by hybridizing an antisense dna/lna oligonucleotide, and disrupted rna-rna interactions are characterized by enhanced m reactivity of the displaced nucleotides. two chimeric lna/dna oligonucleotides, -lna and -lna (table ) , were hybridized to the leader (nt - and nt - ) to disrupt base pairing interaction with the trailer (wt + lnas). in the control sample, these oligonucleotides were omitted (wt). aishape indicated changes in chemical reactivity within trailer residues ( figure b ). in particular, nts c -a and u -a of the experimental sample (wt + lnas) were more sensitive to m modification (median reactivity . ) than the corresponding residues in the control sample (wt, median reactivity of . ). moreover, nts a -u of wt + lnas were less reactive (median value . ) than their wt counterparts (median value . ). the rnastructure algorithm, when provided with pseudo energy constraints retrieved from aishape experiments, predicted that in the absence of its interacting partner, the trailer forms an independent stem-loop structure involving nts c -g ( figure b ). the stem of this hairpin is mainly composed of a:u base pairs interrupted by only two g:c pairs and one g:u wobble. the specificity of the aishape strategy was verified by formation of an extensive barrier to reverse transcription at the sites of lna/dna hybridization, as revealed during capillary electrophoresis separation of the reverse transcription products (data not shown). the m reactivity profile in the presence of both lnas indicated minor off-site changes, which could reflect perturbation of tertiary contacts within ebov minigenome rna ( supplementary figure s ). we performed structural analysis of ebov minigenome rna mutants - , a u and a u/a u to address how changes introduced within the trailer affect the conformation of the trailer-to-leader panhandle. although the lack of trailer sequence-induced structural rearrangements in deletion mutant - ( figure d ; supplementary figures s and ) , certain structural domains specific for wt ebov minigenome rna remained unchanged. these include a bifurcated stem-loop at the terminus (nts - ), a stem-loop occluding hspa motifs and (nts - ) and the hairpin structure previously proposed as vp binding site (nts - ) ( figure d ). in contrast, the a u mutation within motif affected only the conformation of the trailer-to-leader panhandle ( figure e and supplementary figure s ). this single nucleotide substitution eliminated formation of the nt - hairpin (containing hspa motif in wt rna), and introduced an a:c mispair (nt a , c ), and an asymmetric internal loop (nt - and ) ( figure e) . similarly, the a u/a u point mutations caused structural rearrangements of the panhandle duplex, again eliminating the nt - stem-loop and introducing a mismatch at position and an additional internal loop (g -u and c -u ) ( figure f and supplementary figure s ). a d structural model of the ebov trailer and leader was generated using rnacomposer ( ) (figure ). since the server does not accept sequences > nts, a -nt derivative sequence was created by deleting the sequences downstream of nt and upstream of nt and closing the remaining short helical region (g -a and u -c ) with a -g-a-g-a tetraloop. a dot-bracket notation generated by rnastructure software was manually adjusted to account for this deletion, and subsequently provided to rnacomposer. ten d rna models were generated and analyzed, taking into account their secondary figure . three-dimensional projection models of the trailer-to-leader interaction in ebov minigenome wt, a u and a ua u rnas. the nt of internally deleted ebov wt and mutant minigenome rna are depicted. specific cis-acting motifs and domains are color-coded as shown in the key. the models indicate that the previously defined vp -binding site stem-loop is near to hspa motif . a u and a ua u mutations affect the spatial arrangement between these stem-loop structures. structure topology, sequence homology, structure resolution, and free energy. in addition, the quality of predicted models was evaluated using molprobity ( . ) and king tools ( , ) . the models with the best topological score are presented in figure , indicating the position of trailer, leader, hspa motif and the nt - hairpin. the wt model reveals close spatial arrangement of hspa motif (green) with the hairpin (blue). the presence of trailer a u and a u/a u mutations changed the distance between these rna motifs. the model presented here provides useful insight into how rna substructures may interact during the course of ebov replication. accumulation of viral proteins during infections often leads to cellular stress and upregulation of heat shock protein expression ( ) . the role of hsps in the viral lifecycle is only just being unveiled ( ) ( ) ( ) . numerous studies also indicate that specific interactions of host proteins with viral secondary and tertiary rna motifs modulate the lifecycle ( , ) . in this manuscript, we provide novel insight into the structural conformation of ebov ncrs, identify a specific rna motif within the trailer that interacts with a host cell chaperone, hspa , and define the role of this protein/rna interaction in viral replication. emsa (figure ) , followed by protein pull-downs, mass spectrometry, and ip-rt-pcr ( figure ) allowed us to identify and confirm that hspa interacts with the first nts of the ebov trailer region. hspa , a member of the hsc family, is a host chaperone that assists mis-folded polypeptide chains to (re)fold into functional proteins and is crucial for cell survival during stress ( ) . hsc also interacts with hcv particles; hsc downregulation significantly reduced virus production either via modulation of viral assembly or release ( ) . in addition, hsc was shown to be part of a protein complex that includes hcv ns a and host proteins hsp and hsp ( ) and was demonstrated to assist the ns a/hsp complex essential for hcv ires-mediated translation. hsc is also recruited to reovirus viral factories ( ) and is present in influenza a virus ( ) , and vesicular stomatitis indiana virus viral particles ( ) . the pentanucleotide motif auuua is an hspa interacting motif ( ) ( ) ( ) ( ) . sequence analysis of the e- e-gfp minigenomic rna suggested three putative hspa motifs in the trailer. however, the exact structural conformation of these regions was undefined. thus, using chemical acylation techniques and site-directed mutagenesis, we determined the secondary structure of e- e-gfp ebov minigenome rna and demonstrated that its and ncrs form complex long-range rna interactions including a trailer-to-leader panhandle ( figure a and supplementary figure s ). closer analysis indicates that motif forms part of a small stem-loop (nts - ) that is in near proximity to the vp binding stem-loop (nts - ). to further investigate the role of the terminal nt in the - interaction and the importance of hspa motif , a deletion mutant was evaluated by chemical probing and in the context of an ebov infectious clone. shape data indicate that deleting trailer sequences caused structural changes within the e- e-gfp minigenome rna, mainly due to the release of the leader sequences for base pairing with complementary regions ( figure d ; supplementary figures s and ). these structural rearrangements did not affect the topology of other rna domains. experiments with the infectious clone system indicated that ebov mutant - was not viable (table ). since hspa motif is located within the terminal nts of the trailer, it is difficult to unambiguously determine if the panhandle structure or the hspa motif is necessary for virus growth, but such data do reinforce the importance of hspa motif . probing analysis of the a u and a u/a u minigenome rnas showed that hspa motif point mutations affect the panhandle conformation, eliminating the nt - stem-loop containing hspa motif ( figure e and f). no other rna domains outside of the - interaction were changed (supplementary figures s and ) , suggesting that the trailer-to-leader interaction forms an independent structural/regulatory element essential for efficient virus replication. hybridization of lna/dna chimeras to the leader did not induce extensive structural changes within upstream domains. these data suggest that the trailer-to-leader panhandle might indeed function as an autonomous element (supplementary figure s ) . the infectious clone system experiments also indicated that ebov mutant a u/a u was not viable. on the other hand, the a u mutant was rescued but with slower kinetics. sequence analysis indicated an a insertion into the the gp open reading frame in % of the viral rnas at position . the effect of this insertion, if any, on viral replication remains to be determined (table ) . automated d structure modeling of the trailer-to-leader interaction in the wt e- e-gfp minigenome rna implied a close association of hspa motif with the - hairpin, recently shown to bind vp ( ) (figure ). the first nts of the interacting - regions form an extended arm that is positioned orthogonally to the interacting hspa / - motifs. the spatial proximity of these elements suggests a potential molecular bridging between hspa /vp and their specific rna-binding motifs. a u and a u/a u mutations changed the spatial arrangement between these stem-loop structures possibly disrupting these complex long-range interactions (figure ). mutational analysis of hspa motif further confirmed its importance in the ebov lifecycle ( figure a and b). in particular, single-and double-point mutations indicated that residue a plays an essential role in viral replication and transcription. furthermore, northern blot data indicated a reduction in both minigenome and ri rna production of the a u mutant ( figure d and supplementary figure s ). the a u/a u double mutation also resulted in reduced ri rna and minigenome synthesis. since the ri rna serves a template for synthesis of the minigenome rna, reduction in minigenome rna most likely directly affects production of ri rna. in addition, emsa analysis of the a u mutant demonstrated reduced host protein/viral rna/rnp complex formation ( figure e ). it is interesting that sirna-directed ( figure g ) or chemical inhibition (data not shown) of hspa moderately reduced infectivity and viral titer, respectively, whereas ebov genome mutagenesis that targets the hspa binding motif led to nonviable virus. taken together, our structural probing, mutagenesis and reverse genetics data indicate that the conformation of the ebov trailer and its interactions with host cell hspa are essential regulators of the ebov lifecycle. our studies indicate that hspa plays a critical role in production of viral genomic and ri rnas. during transcription and replication, the viral genome must become a template for synthesis of progeny rna. this process involves uncoating or at least relaxation of the viral rnp. host factors likely interact with the viral rnp to maintain proximity of necessary factors and aid complex formation to initiate and complete these processes ( ) ( ) ( ) . it is likely that many of these interactions are weak and transient, acting as scaffolds for virus-driven processes. these transient interactions may assist proper rna folding for ri rna synthesis, transcription complex formation, or proper confirmation of packaging signals to drive discrimination by viral components to ensure effective packaging of full-length genomes and reduction of defective particles. ebola protein analyses for the determination of genetic organization sequence analysis of the ebola virus genome: organization, genetic elements, and comparison with the genome of marburg virus analysis of the highly diverse gene borders in ebola virus reveals a distinct mechanism of transcriptional regulation conserved rna secondary structures and long-range interactions in hepatitis c viruses the genetic code as expressed through relationships between mrna structure and protein function conserved rna secondary structures in flaviviridae genomes the structure and functions of coronavirus genomic and ends comparison of the transcription and replication strategies of marburg virus and ebola virus by using artificial replication systems termini of all mrna species of marburg virus: sequence and secondary structure production of novel ebola virus-like particles from cdnas: an alternative to ebola virus generation by reverse genetics high-throughput, luciferase-based reverse genetics systems for identifying inhibitors of marburg and ebola viruses characterization of the l gene and trailer region of ebola virus analysis of the role of predicted rna secondary structures in ebola virus replication ebola virus vp -mediated transcription is regulated by rna secondary structure formation the ebola virus genomic replication promoter is bipartite and follows the rule of six dynamic phosphorylation of vp is essential for ebola virus life cycle rna binding of ebola virus vp is essential for activating viral transcription rna binding specificity of ebola virus transcription factor vp ebola virus rna editing depends on the primary editing site sequence and an upstream secondary structure the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases hur displaces polypyrimidine tract binding protein to facilitate la binding to the untranslated region and enhances hepatitis c virus replication hepatitis a virus (hav) proteinase c inhibits hav ires-dependent translation and cleaves the polypyrimidine tract-binding protein differential utilization of poly(rc) binding protein in translation directed by picornavirus ires elements identification of proteins bound to dengue viral rna in vivo reveals new host proteins important for virus replication host protein interactions with the end of bovine coronavirus rna and the requirement of the poly(a) tail for coronavirus defective genome replication evaluation of the role of heterogeneous nuclear ribonucleoprotein a as a host factor in murine coronavirus discontinuous transcription and genome replication effect of mutations in the mouse hepatitis virus (+) protein binding element on rna replication mitochondrial aconitase binds to the untranslated region of the mouse hepatitis virus genome nucleotide sequence and host la protein interactions of rabies virus leader rna a host protein (la) binds to a unique species of minus-sense leader rna during replication of vesicular stomatitis virus leader rna of rinderpest virus binds specifically with cellular la protein: a possible role in virus replication dna topoisomerase facilitates the transcription and replication of the ebola virus genome cellular topoisomerase i activity associated with hiv- the role of topoisomerase i in hiv- replication topoisomerase i and atp activate cdna synthesis of human immunodeficiency virus type dna topoisomerases: structure, function, and mechanism rna structure analysis at single nucleotide resolution by selective -hydroxyl acylation and primer extension (shape) selective -hydroxyl acylation analyzed by primer extension (shape): quantitative rna structure analysis at single nucleotide resolution the rna transport element of the murine musd retrotransposon requires long-range intramolecular interactions for function ebola virus vp -vp interaction is sufficient for packaging e- e minigenome rna into virus-like particles shapefinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis ) sirna screen identifies trafficking host factors that modulate alphavirus infection infectious lassa virus, but not filoviruses, is restricted by bst- /tetherin molecular evidence of sexual transmission of ebola virus selective -hydroxyl acylation analyzed by protection from exoribonuclease (rnase-detected shape) for direct analysis of covalent adducts and of nucleotide flexibility in rna high-throughput single-nucleotide structural mapping by capillary automated footprinting analysis rnastructure: software for rna secondary structure prediction and analysis molprobity: all-atom structure validation for macromolecular crystallography modeling conserved structure patterns for functional noncoding rna mammalian hsp and hsp proteins bind to rna motifs involved in mrna stability cytokines direct the regulation of bim mrna stability by heat-shock cognate protein thermodynamics and kinetics of hsp association with a + u-rich mrna-destabilizing sequences analysis of sequence-specific binding of rna to hsp and its various homologs indicates the involvement of n-and c-terminal interactions three of the four nucleocapsid proteins of marburg virus, np, vp , and l, are sufficient to mediate replication and transcription of marburg virus-specific monocistronic minigenomes heat stress cognate host protein as a potential drug target against drug resistance in hepatitis b virus medicinal herbs for hepatitis c virus infection: a cochrane hepatobiliary systematic review of randomized trials pseudoknots: rna structures with diverse functions inhibition of hepatitis c virus in mice by a small interfering rna targeting a highly conserved sequence in viral ires pseudoknot automated d structure composition for large rnas identification of cellular proteome modifications in response to west nile virus infection broad action of hsp as a host chaperone required for viral replication virus-heat shock protein interaction and a novel axis for innate antiviral immunity heat shock protein modulates influenza a virus polymerase activity identification of proteins bound to dengue viral rna in vivo reveals new host proteins important for virus replication virus-host protein interactions in rna viruses structure and function of rna replication the cis-acting replication element of the hepatitis c virus genome recruits host factors that influence viral replication and translation rna virus replication complexes we are grateful to peter b. jahrling, jennifer sword, cindy allan, krisztina b. janosko, stacy l. agar, richard bennett, michael r. holbrook and the entire evps and irf-frederick team for their support and fruitful discussions about these experiments. we thank dr john l. casey and brittany l. griffin for their discussions about shape. we thank dr y. kawaoka, university of wisconsin for the ebov e- e egfp minigenome system. we thank laura bollinger and jiro wada for editing the manuscript and for preparing figures, respectively. the content of this publication does not necessarily reflect the views or policies of the us department of the army, the us department of defense, us department of health and human services (dhhs) or of the institutions and companies affiliated with the authors funding supplementary data are available at nar online.nucleic acids research, , vol. , no. key: cord- -a tv authors: yates, mary k.; raje, mithun r.; chatterjee, payel; spiropoulou, christina f.; bavari, sina; flint, mike; soloveva, veronica; seley-radtke, katherine l. title: flex-nucleoside analogues – novel therapeutics against filoviruses date: - - journal: bioorganic & medicinal chemistry letters doi: . /j.bmcl. . . sha: doc_id: cord_uid: a tv abstract fleximers, a novel type of flexible nucleoside that have garnered attention due to their unprecedented activity against human coronaviruses, have now exhibited highly promising levels of activity against filoviruses. the flex-nucleoside was the most potent against recombinant ebola virus in huh cells with an ec = μm, while the mcguigan prodrug was most active against sudan virus-infected hela cells with an ec of μm. Ó elsevier ltd. all rights reserved. since the first reported fatal outbreak in the mid s, members of the filoviridae virus family, including the ebola virus (ebov), the sudan virus (sudv), and the marburg virus (marv), have continued to devastate many areas across the globe, with mortality rates as high as %. , one of the worst outbreaks of ebov occurred in west africa from to , with over , documented infections and claiming more than , lives, including nearly health care workers. filoviruses are a group of enveloped, single-stranded, negative-sense rna viruses that cause fatigue, vomiting, and severe hemorrhagic fevers. , , members of the filoviridae family are zoonotic viruses, where the primary reservoir is speculated to be fruit bats, however, it is unclear if this is the only reservoir or how the transmission to humans occurs. the filoviruses are highly contagious and can easily spread through interaction with an infected individual by direct contact with bodily fluids including vomit, sweat, saliva, and respiratory secretions. , with the high potential for re-emergence of these lethal viruses, particularly due to ''super-spreaders", , it is imperative that a viable treatment option be identified in order to better fight these crippling pathogens before the next outbreak occurs. to date there are no available fda approved treatments for filovirus infections. while various therapeutic options have been pursued including vaccines, monoclonal antibodies, , and recom-binant proteins, , many of these have yet to reach clinical trials and may ultimately not translate well to effective treatments that can be made readily available during an outbreak, particularly in suboptimal conditions. one therapeutic option for the development of antiviral treatments is the use of nucleoside analogues. nucleoside analogues have long been the cornerstone of antiviral therapies due to their ability to inhibit viral replication because they mimic the structure of the natural nucleosides. , as such, they can be recognized by cellular or viral enzymes, including the viral dna or rna polymerases. moreover, because they contain various structural modifications, this leads to cessation of viral replication, typically due to chain termination. various nucleoside analogues against filoviruses such as ebov have already been proposed including s-adenosylhomocysteine hydrolase (sahase) inhibitors c ado and c nep ( fig. ) , , and the monophosphate derivative of bcx , an adenosine analogue that acts as a non-obligate chain terminator, however, none of these have progressed to the clinic. most recently gs- , a monophosphoramidate prodrug adenosine analogue which targets ebov rna-dependent rna polymerase (rdrp), exhibited very potent activity against both ebov and marv, , further demonstrating the potential for finding effective nucleoside inhibitors of filoviruses. over the past several years, research in our laboratory has focused on the development of flexible nucleoside analogues, termed ''fleximers". [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] one type of fleximer features a purine ring that is ''split" into its imidazole and pyrimidine moieties. the two pieces remain connected by a single cac bond, thus introducing free rotation between the two heterocyclic components without losing the necessary groups needed for recognition (fig. ) . , this strategic design retains the hydrogen bonding patterns needed for recognition, while allowing the flex-nucleoside to interact with alternative binding moieties, such as different amino acids in the binding pocket, that were previously unattainable by the parent nucleoside. , , studies within our lab have also shown that their inherent flexibility allows for an increase in bind-ing affinity compared to corresponding rigid inhibitors, as well as the ability to overcome point mutations in biologically relevant enzymatic binding sites, thus providing potential for overcoming the development of drug resistance. [ ] [ ] [ ] [ ] [ ] [ ] [ ] more importantly, recent work with some fleximer versions of the fda-approved nucleoside acyclovir, revealed significant activity against human coronaviruses middle east respiratory syndrome (mers-cov) and severe acute respiratory syndrome (sars-cov), representing the first nucleoside analogues to exhibit low micromolar levels of anti-cov activity. this was groundbreaking since nucleoside analogues had to that point failed to show viable levels of activity against these deadly viruses. as a result, this prompted further evaluation of the flex-analogues against other viruses, particularly given the dual anti-cov and anti-ebov activity recently noted by gs- . herein, we report the anti-filovirus activity for these analogues, as well as the corresponding phosphoramidate prodrug (fig. ) . the synthesis of the target compounds began with the substituted imidazole , utilizing the routes previously employed in our group (scheme ). treatment with sodium sulfite in a % ethanol/water solution resulted in simultaneous deacetylation and selective deiodination to provide key intermediate . acetylation of then generated , the protected intermediate needed for the prodrug synthesis. in parallel, the organometallic coupling reagent was synthesized starting from the commercially available -amino- -methoxypyrimidine. , stille coupling of to gave . alternatively, using the acetylated , stille coupling provided the desired double prodrug . synthesis of the mcguigan protide - started with commercially available l-alanine and utilized literature procedures to generate the phosphoramidate (scheme ). reaction of with fleximer in the presence of tert-butyl magnesium chloride then provided the desired mcguigan protide in % yield. after the successful synthesis of the three flex-analogues , , and , the compounds were screened against a panel of filoviruses including ebov, marv, and sudv, as well as other hemorrhagic fever viruses such as lassa and rift valley fever. the first series of assays utilized hela cells infected with live-virus isolates of ebov (makona), sudv (gulu), and marv (ci ). activity against all three viruses was observed for the mcguigan prodrug , with the best activity against sudv ( table ) . the second series of assays utilized huh cells infected with recombinant reporter ebov, lassa, and rift valley fever viruses. as observed in the first series of assays, compound was active against ebov at a similar concentration, however, compound (table ) . infectious diseases such as ebov continue to pose a serious health threat due to the high mortality rates associated with these deadly viruses. while ongoing studies have identified various therapeutics as potential ebov treatments, there are currently no fda approved vaccines or therapeutics, and as such, it is imperative that an effective treatment option is developed. within this study we found that both compounds and exhibited antiviral activity against a recombinant reporter ebov in huh cells, though surprisingly the mcguigan prodrug was -fold less potent (ec = . ± . lm and . ± . lm respectively). against wild-type viruses in hela cells, compound had no detectable activity, though compound inhibited both ebov and sudv (ec = ± and ± lm respectively). the difference in activity of in the huh cells compared to the hela cells is most likely due to a difference in specific metabolism of the compound in those cells lines, however, further studies are needed to confirm this hypothesis. efforts are currently underway to better understand the mechanism of action of these compounds and how they might interact with the viral rdrp or other viral replication enzymes. the results of those studies will be reported as they become available. disease 'superspreaders' accounted for nearly two-thirds of ebola cases, study finds this work was supported by the national institutes of health-t gm and r ai (ksr). we would also like to thank usamriid/mts colleagues dima gharaibeh, ylenia cau, tara kenny, cary retterer, rouzbeh zamani, glenn gomba and collin dube for assistance. work at usamriid was funded by the joint science and technology office for chemical and biological defense (jsto-cbd) of the defense threat reduction agency (dtra). opinions, interpretations, conclusions, and recommendations reported herein are those of the author and are not necessarily endorsed by the u.s. army. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.bmcl. . . . key: cord- - j zmuub authors: hunt, catherine l.; lennemann, nicholas j.; maury, wendy title: filovirus entry: a novelty in the viral fusion world date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: j zmuub ebolavirus (ebov) and marburgvirus (marv) that compose the filovirus family of negative strand rna viruses infect a broad range of mammalian cells. recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. details of this entry pathway are highlighted here. virus entry into cells is initiated by the interaction of the viral glycoprotein( ) subunit (gp( )) with both adherence factors and one or more receptors on the surface of host cells. on epithelial cells, we recently demonstrated that tim- serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. within the acidified endosome, the heavily glycosylated gp( ) is cleaved to a smaller form by the low ph-dependent cellular proteases cathepsin l and b, exposing residues in the receptor binding site (rbs). details of the molecular events following cathepsin-dependent trimming of gp( ) are currently incomplete; however, the processed gp( ) specifically interacts with endosomal/lysosomal membranes that contain the niemann pick c (npc ) protein and expression of npc is required for productive infection, suggesting that gp/npc interactions may be an important late step in the entry process. additional events such as further gp( ) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. once this has been achieved, sequences in the filovirus gp( ) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. this multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses. filoviruses (family mononegavirales, genera ebolavirus (ebov) and marburgvirus (marv)) are single-stranded, negative-sense rna viruses that exhibit a unique heterogeneous filamentous structure. both ebov and marv infect a wide variety of mammals and this wide tropism has complicated the identification of cellular proteins required for viral entry. a hemorrhagic fever is caused by these viruses in humans, non-human primates and perhaps other mammals and is associated with high morbidity and mortality during outbreaks. no therapeutic drugs or vaccines are currently available to treat or prevent filoviral infection. because of this and the high lethality associated with infection, filoviruses are considered category a priority pathogens by niaid and, in recent years, much research has focused on understanding how these viruses bind to and enter permissive cells. the ebov genome encodes seven genes, including the glycoprotein (gp) gene. the gp gene encodes for two known soluble gp forms (sgp and ssgp) [ , ] . the precise functions of the soluble forms of ebov gp are unknown, but it is speculated that they are involved in immune evasion, as most of the antibodies during a natural zaire ebov (zebov) infection are against sgp [ ] . full-length ebov gp is not directly encoded by the gp gene, but is produced by a transcriptional editing event that shifts the reading frame of about % of the gp transcripts. in contrast, while marv also encodes seven genes, its gp is directly encoded by the gp gene and soluble forms of marv gp are not thought to be synthesized from viral transcripts. precursor gp is cleaved by the host enzyme furin in the golgi apparatus, resulting in the formation of two gp subunits, gp and gp . gp contains the receptor binding domain (rbd) and is responsible for interacting with one or more cellular receptors. this interaction is believed to mediate virus entry into the endosomal compartment. gp contains a fusion loop, heptad repeat regions, the transmembrane domain and a short cytoplasmic tail. while furin processing of the filovirus gp routinely occurs within the golgi apparatus before the glycoprotein is expressed on the plasma membrane, proteolytic clipping is not required for virion infectivity [ ] . the cleaved subunits are linked by a disulfide bond to generate a gp , heterodimer that is located on the surface of virions and is approximately kda in size [ ] . as a class i viral fusion protein, gp , exists as a trimer of gp and gp heterodimeric subunits and is found in its full-length, heavily glycosylated, pre-fusion form on the surface of newly budded virions. this pre-fusion class i glycoprotein conformation is thought to be a metastable state, that upon the appropriate set of triggers, will convert to its post-fusion, low-energy form [ ] . the conformational change from a pre-fusion to post-fusion structure provides the energy to permit the viral and cellular membranes to fuse and thereby release the viral core into the cytoplasm. the events that are required for filovirus/cellular membrane fusion to occur have yet to be completely elucidated, but current studies are unraveling these steps and are highlighted here. several ebov gp crystal structures have been instrumental in understanding the conformational alterations that are required as the glycoprotein changes from the metastable, pre-fusion state to the low-energy, post-fusion form. lee et al. elucidated the trimeric structure of pre-fusion, mucin domain-deleted ebov gp , ectodomain [ ] , whereas, two groups independently solved the trimeric gp six helix bundle structure that is formed during fusion events [ , ] . mature gp is composed of three distinct domains: the rbd, the glycan cap and a heavily o-linked glycosylated mucin-like domain. the rbd in mature zebov gp is located from amino acid ~ to and composed of a base region and a region that interacts with one or more receptors on the surface of cells ( figure ) [ ] . while rbds of the different ebov strains are relatively conserved, only % identity between ebov and marv rbds exists. nonetheless, ebov and marv gp pseudovirions compete with each other for filoviral gp , -dependent entry into permissive cells, indicating that a common receptor or receptors are used by both viruses [ , ] . amino acids through and three additional short downstream regions interact with gp , serving as the base of the rbd. several linearly discontinuous regions from amino acids to sit above the base forming a series of beta sheets. residues both in beta sheets and adjacent loops have been implicated in cell binding, leading to the conclusion that the receptor binding site (rbs) is located in this region of the rbd [ , ] . gp residues to encode for a "glycan cap" that is extensively n-linked glycosylated and sits distal to the rbd from the surface of the virion. the glycan cap may protect the rbs from antibodies [ ] . this glycan cap also interacts with two regions of gp , including the internal fusion loop of gp that is critical for gp -mediated membrane fusion [ ] . the glycan cap/gp interaction restricts the availability of the fusion peptide, preventing pre-mature fusion events. finally, filovirus gp contains a mucin-like domain at the carboxy terminus from amino acids to . this region is heavily glycosylated with both n-and o-linked glycans [ ] . while the ebov mucin domain is not required for virus entry [ , ] , several roles for this domain have been suggested. like the glycan cap, it may shield gp rbs residues from immune recognition on free virus [ ] . in addition, the mucin domain causes cell rounding, masking of a number of cell surface markers and cytotoxicity that is not observed upon expression of mucin domain-deleted ebov gp [ , ] . the shielding effect of the bulky mucin domain of the rbd of gp is thought to also obstruct rbs interactions with adherence factors and receptors since removal of this domain enhances ebov titers. similar attempts to delete the marv mucin domain have proved unsuccessful [ ] . two of the three trimers are shown as space filling structures with gp in lighter grey and gp as dark grey/black. the third gp , heterodimer of the trimer is depicted as a ribbon structure with gp shown in teal and the gp subunit shown in tan. (c,d) ribbon diagrams of a single heterodimer of gp , . domains in gp are highlighted in c, whereas domains in gp are highlighted in d. in panel c, the base domain of gp that interacts with gp is shown in royal blue, the head domain is shown in teal with the beta-strands and adjacent loop region containing the rbs highlighted in red and the glycan cap is shown in gold. gp is shown in grey. in panel d, the internal fusion loop (ifl) that is flanked by beta-strands is shown in dark brown and heptad repeat region is shown in tan. the interaction of the ifl with gp residues from an adjacent subunit is evident in panel a. all ebov gp graphics (pdb accession number csy) were produced with pymol. filovirus gp is similar to other class i viral glycoproteins that mediate fusion events. gp is composed of a fusion loop found near the amino terminus followed by a n-terminal heptad repeat region, a c-terminal heptad repeat region, a transmembrane region and a short cytoplasmic tail [ , , ] . in the pre-fusion form on the surface of newly budded virions, the fusion loop and n-terminal heptad region are integral regions of the gp /gp trimeric structure, forming a platform or base upon which gp sits ( figure ). in contrast, the c-terminal heptad repeat region in the pre-fusion form may contain little structure and was deleted in the crystallization studies and it is not thought to contribute to the metastable, pre-fusion form of gp [ ] . not surprisingly, both the fusion loop and n-terminal heptad repeat are conserved (> % identity) between zebov and the musoke strain of marv, but the remaining portions of gp have limited identity. site directed mutagenesis studies of ebov or marv gp [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] suggest a similar chain of events to those previously reported for other class i viral glycoproteins leads to viral/cell membrane fusion [ , , ] . these events will be discussed in detail below. while progress is being made to identify cell surface proteins that enhance filovirus transduction/infection, the advancement of this area of research has been slow. in part this is due to the broad tropism of filoviruses for a variety of different cell types as well as the ability of these viruses to infect cells from a wide range of species [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one classical approach to identifying virus receptors that has been used is the introduction of a cdna library from a permissive cell into a cell that is not permissive for the virus [ ] [ ] [ ] . however, for reasons that are not entirely clear, this type of study has not been successful in identifying cell surface proteins that directly interact with ebov gp to mediate virus entry [ , ] . instead, another screening approach that correlated gene expression in a large panel of human cells with ebov gp-dependent transduction proved more productive and allowed us to identify a surface receptor for these viruses [ ] . the cell surface protein we identified, tim- , as well as other cell surface proteins that enhance filovirus infectivity are discussed in detail below. c-type lectin family members l-sign, dc-sign and hmgl have been shown to enhance filovirus entry [ , [ ] [ ] [ ] [ ] [ ] . studies have demonstrated that both the mucin domain and the glycan cap of gp interact with c-type lectins [ , ] . however, as both of these regions can be deleted from ebov gp without loss of viral transduction efficiency [ , [ ] [ ] [ ] , it is likely that c-type lectins increase filovirus attachment to cells rather than serving as cellular receptors that mediate internalization of the virus into endosomes [ ] . a similar adherence function for c-type lectins has been identified for other enveloped viruses such as hiv [ ] . early studies found that surface expression of some integrins was down regulated upon transfection of full-length ebov gp [ , , ] . in addition, ebov gp-mediated pseudovirion entry is reduced by either antibodies targeting β integrins or a soluble form of β integrins [ ] . these characteristics suggested that one or more integrin subunits might serve as receptors for filoviruses. however, no direct interaction between any portion of ebov gp and a member of the β integrin family has been identified. more recent studies implicate β integrin in stimulation of endosomal protease events that are required for productive ebov transduction, thus reducing the likelihood of β integrins serving as bona fide filoviral receptors [ ] . the tam family member axl was first implicated in filovirus entry through a cdna screen that introduced vero e cell cdna into poorly permissive jurkat t cells [ ] . axl is a tyrosine kinase receptor that is found on the plasma membrane in a variety of different cell types and enhances cell migration, division and viability upon activation [ ] . shimojima et al. demonstrated that anti-axl antibodies blocked ebov transduction of some cells, whereas these antibodies had no effect on transduction of other cells [ ] . mapping studies indicated that amino acid residues in both the ectodomain and the cytoplasmic tail of axl were required for filovirus entry enhancement [ ] . subsequently, a screen performed in our laboratory also identified axl as being important in ebov gp-dependent entry [ ] . through the use of multiple biochemical inhibitors, sirna and anti-axl antibodies, we defined a role for axl in ebov uptake [ ] , demonstrating that axl expression enhances macropinocytosis in some cells. as macropinocytosis is a principal uptake mechanism of filoviruses [ , ] , increased axl surface expression leads to greater virus internalization. however, axl does not appear to interact directly with ebov gp to promote viral internalization and therefore is unlikely to serve as a filoviral receptor [ ] . our lab performed a comparative genomic analysis (cga) screen [ ] to identify cellular genes whose expression highly correlated with ebov pseudovirion transduction [ ] . this screen showed a positive correlation between ebov transduction and expression of a series of cellular proteins that were previously appreciated to enhance ebov transduction (c-type lectins, integrins and axl). interestingly, expression of the t-cell immunoglobulin mucin domain- (tim- ) gene proved to be one of the strongest positive correlations. subsequent studies demonstrated that expression of tim- in poorly permissive cells enhanced ebov entry and loss of surface-expressed tim- in highly permissive cells abrogated filovirus infection/transduction. furthermore, tim- and the mucin domain-deleted ebov gp interacted, and removal of the glycan cap enhanced the specificity of gp interaction with tim- -expressing cells [ ] . in total, these findings have led us to propose tim- as a cell surface receptor for filoviruses. as epithelial cells are the only relevant cell type that expresses tim- , it is likely that other as of yet unidentified surface receptors will also prove to be important in mediating filovirus entry. the cellular uptake pathways mediating filovirus entry remain controversial despite numerous studies. the three most common and well studied endocytic pathways-caveolin-dependent endocytosis, clathrin-dependent endocytosis and macropinocytosis-have all been implicated in filovirus entry. early studies reported that the caveolae vesicular system and/or lipid raft domains were important for ebov gp-mediated entry [ ] [ ] [ ] . however, it was demonstrated that overexpression of caveolin in the poorly permissive lymphocytic cell line cem did not enhance levels of ebov gp-dependent transduction, suggesting that caveolae may not play a role in filovirus entry [ ] . since that time several groups have also implicated clathrin-dependent entry mechanisms in filovirus entry [ ] [ ] [ ] [ ] . in parallel, other groups using virus-like particles (vlps) and/or infectious filovirions identified macropinocytosis as a main and perhaps sole mechanism of filovirus uptake [ , ] . several groups have found that ebov gp-dependent entry can occur through a variety of different uptake mechanisms including caveolae, clathrin-coated pits and through actin-and microtubule-dependent pathways such as macropinocytosis [ , , ] within the same cell population. a number of these uptake studies that identified caveolin and/or clathrin as important in entry used ebov gp pseudotyped lentiviruses or vesicular stomatitis virus (vsv) as the cargo and an argument has been made that these smaller viral particles do not accurately represent the size constraints that may limit the uptake options of a filovirion that can be greater than µm in length [ ] . while a role for caveolin-and clathrin-dependent pathways in infectious filoviral entry remains to be demonstrated, it should be noted that several large, intracellular bacteria or parasites have been found to use either caveolin-or clathrin-dependent pathways, suggesting that the size of these vesicles can be expanded to meet the size of the cargo [ ] [ ] [ ] . in addition, it is likely that the mechanism of ebov uptake may be cell-dependent as both green african monkey cells (vero) [ , ] and human neuroblastoma cells (snb- ) [ ] both primarily use macropinocytosis for ebov uptake. however, the signaling pathways that are required for virus entry is cell-specific since vero cells require activation of the pi k/akt pathway, whereas snb- cells require plc activation [ ] . unlike some other class i viral glycoproteins, no evidence exists that ebov gp undergoes conformational changes upon binding to a cell surface receptor. presumably receptor/virus interactions lead directly to virus internalization into endosomes; however, this has not been demonstrated directly to date. characterization of the endosomal vesicles mediating ebov uptake is limited. saaed et al. have demonstrated that the early endosome antigen (eea ) colocalizes with ebov virus particles in endosomes early during infection of vero cells [ ] , whereas nanbo et al. have shown association of ebov particles lacking vp associated with the sorting nexin (snx ) within minutes of transduction [ ] . early and late endosomal proteins, rab and rab , respectively, have also been shown to be required for productive infection [ , ] . several gtpases previously implicated in endocytosis, rhob, rac and cdc , are also important for ebov gp-dependent transduction, providing additional insights into trafficking pathways used by these viruses [ , ] . as the vesicles traffic into the cell, they become acidified. for some viral fusion proteins, the combination of receptor engagement and endosomal acidity is sufficient for conformational changes that lead to viral/cellular membrane fusion [ ] [ ] [ ] . however, that is not the case for filoviruses. instead, multiple low ph-dependent endosomal and lysosomal proteolytic proteins are involved in ebov gp processing, priming a multi-step process that ultimately results in a small ebov gp , trimer that serves as the fusion-ready form of the glycoprotein. it is believed that this processed, fusion-ready form is then triggered by additional events to a conformationally stable state, resulting in fusion. as endosomal vesicles mature into late endosomes and the vesicular ph drops, activation of endosomal cysteine proteases cathepsin l and b occurs. these cathepsins sequentially process ebov gp into smaller forms [ , ] . cathepsin l proteolysis first removes a substantial portion of ebov gp , generating an approximate kda gp , species that lacks both the glycan cap and mucin domain of gp [ , ] . subsequently, gp is further trimmed by both cathepsin l and b to generate a much smaller form of gp . the exact size of this smaller gp remains controversial, but is between and kda [ , ] . irrespective of the exact size of the processed form, prevention of endosomal acidification or inhibition of cathepsin b activity abolishes ebov infectivity [ ] [ ] [ ] ] . interestingly, this processing may be insufficient for productive ebov infection as studies have demonstrated that the smaller - kda cathepsin l and b-cleaved form cannot infect cells entirely lacking cathepsins [ ] . thus, it has been proposed that additional cathepsin-dependent gp processing is required to generate the fusion-ready form of the glycoprotein [ ] . the size and composition of this smaller form is not known. in contrast to these studies, a recent study demonstrated that a thermolysintrimmed gp that is believed to generate a gp that is similar to the cathepsin l and b-cleaved form can be triggered to bind to liposomes at elevated temperatures under low ph and mildly reducing conditions [ ] . this new study suggests that at least under these conditions this gp conformation is a fusion-ready form. given the apparent importance of cathepsin cleavage for the generation of a fusion-ready form of the filovirus glycoprotein, it is surprising that studies have demonstrated that cathepsin l and b cleavage events can be sidestepped by the virus [ , ] . martinez et al. have shown that in monocyte-derived dendritic cells, cathepsin b is required for ebov infection, but not cathepsin l [ ] . these observations were confirmed in vero cells in a recent study by wong et al. [ ] . this group also extended the finding by selecting an infectious, recombinant vsv encoding ebov gp over several passages to become resistant to the cathepsin b inhibitor ca [ ] . six different mutants were identified that conferred cathepsin b independence. many of these mutations sit at the interface of gp and gp and the selected mutations were thought to alter the gp structure such that enhanced proteolysis by one or more currently undefined cysteine proteases was possible. thus, while cathepsin b and l independence can be achieved by filoviruses, processing by one or more additional cysteine proteases is still required for production of the fusion-ready form. most recently, two groups independently identified a novel host protein essential for ebov infection. cote et al. screened a library of small chemical molecules to find those that inhibited ebov gp pseudovirion entry into vero cells [ ] , whereas carette et al. utilized a genome-wide screen in mutagenized haploid human cells to look for those cells resistant to ebov gp-dependent entry [ ] . each group was able to deduce that disruption of one endosomal/lysosomal membrane protein, niemann-pick c (npc ), could significantly reduce ebov entry into a variety of cell populations. npc is primarily a membrane bound, late endosomal/lysosomal protein that is critical for cholesterol absorption and homeostasis. those individuals lacking a functional npc exhibit an abnormally high accumulation of cholesterol in the lysosomes of their cells, leading to altered protein and lipid trafficking with most cases resulting in fatality by months of age [ ] . cells where npc function has been biochemically disrupted or cells lacking npc showed a resistance to ebov infection [ , ] . clearance of cholesterol from npc null cells by cultivation in lipoprotein-depleted media did not rescue ebov infection, indicating that the npc protein itself, and not aberrant cholesterol transport, was important for ebov entry [ ] . cote et al. also showed that the expression of npc and not its cholesterol transport activity were critical for ebov entry [ ] . as the npc protein is primarily located on the endosomal and lysosomal membranes, npc has been proposed to serve as an entry factor downstream of ebov gp engagement of attachment factors/receptor(s) at the cell surface. consistent with a vesicular role for npc , cote et al. showed that a soluble form of thermolysin-cleaved ebov gp, but not uncleaved gp containing the glycan cap, bound to lysosomal membranes of npc -transfected cho cells [ ] . thus, at least in this over expression system, proteolytically-processed ebov gp appeared to interact with npc -containing membranes, suggesting that these interactions may be important for filovirus entry events that occur in late endosomes and/or in lysosomes. ebov gp-mediated attachment and entry into early endosomes was unaffected in npc -defective cells; however, electron micrographs of npc null cells infected with ebov gp pseudotyped virus show the accumulation of perinuclear vesicles laden with ebov gp pseudovirions that were positive for the lysosomal marker lamp [ ] . therefore, carette et al. have proposed that ncp is crucial for viral membrane fusion and escape from the lysosomal vesicle [ ] . at present, the precise role of npc during the ebov entry process remains to be fully elucidated. in addition, investigations into the precise location of filovirus fusion events within endosomal compartments will provide important insights into these events. interestingly, an inhibitor of npc , u a, has recently been shown to block entry of several pathogens, including the flavivirus dengue [ ] . this inhibitor also inhibited entry of hepatitis c virus [ ] as well as prions [ ] , suggesting that this cholesterol transporter may be critical for passage of a number of viruses, and perhaps other pathogens, through endosomes. most recently, a more definitive role for npc in hepatitis c virus entry has been determined through the use of additional biochemical agents [ ] . future studies exploring lipid accumulation and changes in lipid composition within the endosomal pathway could significantly enhance the understanding of the novel role of npc specifically in filoviral entry and more generally in endosomal trafficking of a number of enveloped viruses. as described above, the gp portion of ebov gp , allows delivery of the filovirion to an endosome where conditions become progressively more favorable for generating the fusion-ready form of gp , . trimming of gp , by host cathepsins (or artificially by thermolysin) enhances interaction of gp with tim- [ ] and permits npc binding [ ] . whether ebov gp interaction, with either of these molecules, directly mediates gp fusion events remains to be determined. filovirus gp contains an n-terminal internal fusion loop of residues defined by a disulfide linkage between cysteines and [ , ] . a core hydrophobic sequence of amino acids within this loop is thought to insert into host endosomal membranes, initiating membrane fusion events. within the intact gp heterodimer, the hydrophobic, internal fusion loop is flanked by antiparallel β-strands composed of the most n-terminal portion of the internal fusion loop and the n-terminal region of first heptad repeat region. the fusion loop is restrained by gp residues from a neighboring subunit, preventing premature fusion events [ , , ] . cathepsin-dependent processing alone is insufficient to trigger insertion of the fusion loop into liposomes [ , ] . when the ebov internal fusion loop interacts with liposomal membrane mimetics, lipid mixing is promoted with a parallel structural change in the loop [ ] . in a neutral ph, lipid environment, the antiparallel β-strands that flank the fusion loop lose their structure, generating a more alpha helical content with a flattened extended loop structure. under low ph conditions (<ph . ) in the presence of lipids, this flattened loop structure broadens out, forming a more hook-like structure [ ] . it has been previously shown that specific proline residues contained within the central portion of the fusion loop facilitate this lipid membrane interaction [ , , , ] . the insertion of gp into host membranes causes an extension of the gp trimers into an energetically unfavorable state. this also causes the two heptad repeat regions (hrs) within gp to be fully exposed to the physiological conditions within the acidified host endosome, which may aid in further triggering of gp to promote final collapse of the two hrs [ ] . the n-terminal gp heptad repeat region (hr ) is a highly ordered, alpha-helical structure that serves as a platform or base for gp and contains residues that are necessary for interactions with other gp hr s to form the trimeric structure of the pre-fusion gp [ , ] . in the pre-fusion form, hr does not interact with the carboxy terminal heptad repeat region (hr ). within the intact, pre-fusion trimer, hr appears to be disordered and was not included in the crystallized structure [ ] . following insertion of ebov gp fusion loop into the host membrane, the ebov gp trimeric heptad repeats collapse and form a six-helix bundle containing three hr and hr domains. the collapse into a coiled coil structure draws the two membranes into proximity allowing partial fusion (hemifusion) of the viral membrane and the host endosomal membrane [ , ] . hemifusion eventually leads to complete fusion of the viral and host endosomal membranes, and an opening through which the viral rna and its associated proteins can be released into the host cell cytoplasm, where the viral life cycle continues. this complex set of entry events summarized in figure provides numerous potential avenues for the development of antiviral therapies against filovirus infection. these possibilities include: ( ) interfering with adherence to permissive cells by blocking c-type lectin (or other adherence factor) interactions with the filoviral gp; ( ) blocking binding of gp to tim- and/or other cellular receptors that are identified in the future; ( ) preventing virus uptake by blocking macropinocytosis; ( ) interfering with cathepsin activity; ( ) inhibiting availability of npc within the lysosomal compartment and ( ) blocking hr /hr (coiled coil) interactions. it is likely that some of these steps will be more amendable than others to the development of antivirals that have minimal or no cytotoxicity. regardless, elucidation of these entry events provides clear targets for the development of drugs that may prevent both filovirus infection and disease. model for filoviral entry. trimers of filoviral gps on virions interact with both attachment factors (c-type lectins) and receptors (tim- ) on the surface of permissive cells. attachment factors are likely to concentrate virions on cells before receptor engagement and virion internalization by macropinocytosis. macropinocytosis is enhanced by tyrosine kinase receptors such as tam family members. following endosomal acidification, cathepsins l and b trim the ebov gp to a smaller form that needs at least one as yet undetermined factor to elicit gp fusion with host endosomal membranes. this smaller form of gp is able to interact with both tim- and the endosomal portion of the npc protein; however, whether gp and tim- interact within endosomes is not known. the energetically unfavorable insertion of the ebov gp fusion loop into host endosomal membranes (i) is followed by the energetically favorable collapse of ebov gp into a six-helix bundle (ii) allowing for lipid mixing and hemifusion of host and viral membrane lipids (ii). finally, the hemifused host and viral membranes resolve and a complete pore is formed (iii) through which the viral genomic complex passes into the cytoplasm, allowing the viral replication cycle to continue. structure-function analysis of the soluble glycoprotein, sgp, of ebola virus a new ebola virus nonstructural glycoprotein expressed through rna editing ebolavirus glycoprotein structure and mechanism of entry reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture biochemical analysis of the secreted and virion glycoproteins of ebola virus structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme structure of the ebola virus glycoprotein bound to an antibody from a human survivor core structure of the envelope glycoprotein gp from ebola virus at . -a resolution crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor characterization of marburg virus glycoprotein in viral entry ebola virus glycoprotein : identification of residues important for binding and postbinding events the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding covalent modifications of the ebola virus glycoprotein identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway effect of flanking residues on the conformational sampling of the internal fusion peptide from ebola virus functional importance of the coiled-coil of the ebola virus glycoprotein involvement of viral envelope gp in ebola virus entry into cells expressing the macrophage galactose-type c-type lectin permeabilization of the plasma membrane by ebola virus gp distribution of hydrophobic residues is crucial for the fusogenic properties of the ebola virus gp fusion peptide mutational analysis of the putative fusion domain of ebola virus glycoprotein phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of ebola virus cell entry of enveloped viruses the role of the transmembrane and of the intraviral domain of glycoproteins in membrane fusion of enveloped viruses ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis ebola virus: new insights into disease aetiopathology and possible therapeutic interventions pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines distinct cellular interactions of secreted and transmembrane ebola virus glycoproteins a search for ebola virus in animals in the democratic republic of the congo and cameroon: ecologic, virologic, and serologic surveys, - . ebola virus study teams distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses association of ebola-related reston virus particles and antigen with tissue lesions of monkeys imported to the united states folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells a putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor tyro family-mediated cell entry of ebola and marburg viruses folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus modulation of virion incorporation of ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity c-type lectins do not act as functional receptors for filovirus entry into cells the multiple facets of hiv attachment to dendritic cell lectins ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence downregulation of beta integrins by ebola virus glycoprotein: implication for virus entry alpha beta -integrin controls ebolavirus entry by regulating endosomal cathepsins tam receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer the mechanism of axl-mediated ebola virus infection tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein the tyro receptor kinase axl enhances macropinocytosis of zaire ebolavirus cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses association of the caveola vesicular system with cellular entry by filoviruses differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions ebola virus uses clathrin-mediated endocytosis as an entry pathway analysis of filovirus entry into vero e cells, using inhibitors of endocytosis, endosomal acidification, structural integrity, and cathepsin (b and l) activity ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis interactions with the host cell macropinocytosis: an endocytic pathway for internalising large gulps vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization role of caveolae in leishmania chagasi phagocytosis and intracellular survival in macrophages rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors receptor binding and low ph coactivate oncogenic retrovirus envelope-mediated fusion endocytosis and a low-ph step are required for productive entry of equine infectious anemia virus alpharetrovirus envelope-receptor interactions a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion relevant conformational change zaire ebola virus entry into human dendritic cells is insensitive to cathepsin l inhibition small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry requires the cholesterol transporter niemann-pick c niemann-pick type c disease: molecular mechanisms and potential therapeutic approaches u a, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication hepatitis c virus egress and release depend on endosomal trafficking of core protein prevention of prion propagation by dehydrocholesterol reductase inhibitors in cultured cells and a therapeutic trial in mice identification of the niemann-pick c -like cholesterol absorption receptor as a new hepatitis c virus entry factor structure and function of the complete internal fusion loop from ebolavirus glycoprotein ollmann saphire, e. ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion structure of the ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts roles of a conserved proline in the internal fusion peptide of ebola glycoprotein we would like to thank sven moller-tank, bethany rhein and andrew kondratowicz for critically reading the manuscript and their helpful suggestions and discussions. the authors declare no conflict of interest. key: cord- - ycgd h authors: markosyan, ruben m.; miao, chunhui; zheng, yi-min; melikyan, gregory b.; liu, shan-lu; cohen, fredric s. title: induction of cell-cell fusion by ebola virus glycoprotein: low ph is not a trigger date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ycgd h ebola virus (ebov) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. currently, how ebov fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. we successfully measure cell-cell fusion mediated by the ebov fusion protein, gp, assayed by the transfer of both cytoplasmic and membrane dyes. a small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in ebov gp known to reduce viral infection, all greatly reduce fusion. by monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that ebov gp-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. ebov gp must be cleaved by late endosome-resident cathepsins b or l in order to become fusion-competent. cleavage of cell surface-expressed gp appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal ph, or an inhibitor of anterograde trafficking. treating effector cells with a recombinant soluble cathepsin b or thermolysin, which cleaves gp into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed gp is not cleaved. whereas the rate of fusion is increased by a brief exposure to acidic ph, fusion does occur at neutral ph. importantly, the extent of fusion is independent of external ph in experiments in which cathepsin activity is blocked and ebov gp is cleaved by thermolysin. these results imply that low ph promotes fusion through the well-known ph-dependent activity of cathepsins; fusion induced by cleaved ebov gp is a process that is fundamentally independent of ph. the cell-cell fusion system has revealed some previously unappreciated features of ebov entry, which could not be readily elucidated in the context of endosomal entry. ebola virus (ebov) outbreaks continually occur and up to % of those infected die; currently there are no approved vaccines or antiviral therapeutics against the virus [ , ] . ebov initiates infection by fusion from within endosomes. experimentally, endosomal interiors are difficult to control, but systems that track the entry of several other viruses into cells have been developed and employed [ , , , ] . historically, these methods have relied on fusion of infectious virus or pseudovirus within cells; cell-cell fusion has not been among the systems in use for ebov. it is surprising that a cell-cell fusion system has not been developed, as the processing of the ebola fusion protein, gp, and other conditions necessary for fusion have been elaborated [ ] . (some years ago there was an isolated report of ebov gp-mediated cell-cell fusion, but this study has not been followed up by any other laboratory, including the original [ ] ). cellcell fusion has several important advantages over intracellular fusion assays, including complete control of the aqueous solution bathing the ectodomain of the fusion protein. in the present study we describe a direct and sensitive system to measure ebov gp-mediated cell-cell fusion with high time resolution, thereby providing fusion kinetics. the system exhibits the well-known central properties of ebov entry, providing strong support for the utility of the cell-cell fusion system to explore mechanisms of ebov entry that are not possible or practical with whole infectious virus. ebov gp is a prototypic class i viral fusion protein [ ] . it is synthesized as a homotrimer; each monomer is cleaved into gp -gp subunits by proteases within the golgi apparatus [ , ] . the gp subunit is responsible for binding to the intracellular receptor niemann pick type c , (npc ) and possibly to other molecules [ ] , and the gp subunit is responsible for membrane fusion [ , , , , , , ] . the two subunits of each monomer remain linked through a disulfide bond and a multitude of weak interactions [ , , , ] . after endocytosis of the virus, the gp subunit is cleaved by the endosomal proteases cathepsin b and/or l [ , , , , ] , while remaining attached to gp [ ] , and then binds to npc [ , ] . the low ph within endosomes is necessary for viral fusion. but it has not been known whether low ph directly triggers fusion by causing conformational changes in gp or whether it augments fusion by increasing the activities of the cathepsins [ , ] . after developing our system, we discovered that an ebov gp-induced fusion pore that connects two plasma membranes does not readily enlarge over time, in contrast to the pores of other viral fusion proteins. this anomalous lack of growth may be the reason cell-cell fusion has not been successfully observed in many prior attempts that used less sensitive assays to detect fusion. on the question of low ph, we found that activation of cathepsins by acidity is the sole cause for augmentation of fusion: if ebov gp on the cell surface is artificially cleaved by thermolysin in the presence of cathepsin inhibitors, the extent of fusion is independent of ph. ebov gp mediates cell-cell fusion mediated fusion is much longer than for some viral fusion proteins, such as iav ha [ ] , but comparable to others, such as hiv env in some studies [ ] . we thus tested, at various times, whether some of the cell pairs that had not yet fully fused had hemifused: the addition of . mm cpz to cell pairs ruptures hemifusion diaphragms that have formed between cell pairs, and this is a standard means to test for hemifusion [ , , ] . we used thermolysin-treated effector cells to maximize cleavage of ebov gp and found that adding cpz either , , or min after reneutralization did not induce any dye spread above that already observed, strongly indicating that a negligible percentage of cells were hemifused, but not fused, at any given time. npc is an intracellular receptor for ebov gp [ , ] . we compared extents of fusion for target parental hek t cells versus target hek t cells that stably overexpressed npc . effector cells that were not treated with thermolysin yielded fusion at ph . ( fig a, bar ) , and a greater extent of fusion after a -min acidic ph . pulse (fig a, bar ) . the extent of calcein spread was greater for target cells overexpressing npc (fig a, bars and ) than for parental t cells (fig a, bars and ) for matching conditions. fusion was still ph-dependent for target cells overexpressing npc : calcein spread was greater hr after a -min ph (merged) . the viral proteins expressed by transfecting effector cells are shown to the right of the images. cells expressing ebov gp were treated with μg/ml thermolysin for min; fusion was augmented with a -min ph . pulse. for cells expressing jsrv env, a -min ph . pulse was used to trigger fusion. effector cells expressing influenza virus (iav) ha were treated with trypsin and neuraminidase as described [ ] , bound to hek t cells, and fusion was triggered with a -min ph . pulse. for mock-transfected effector cells, a -min ph . pulse was employed. fused cells are marked by arrowheads. for this set of experiments, the extent of fusion hr after reneutralization was about % for ebov gp, % for jsrv env, and % for iav ha. thermolysin treatment results in greater extents of fusion between cells. (a) schematic of the experimental protocol is shown above the bar graph. e, effector cells; t, target cells, th, thermolysin. bar graph: fusion of thermolysin-treated effector cells expressing ebov gp (columns and , dark red) was greater than for untreated cells (columns and , dark yellow). for both thermolysin-treated and non-treated cells, a -min ph . pulse applied at room temperature augmented fusion, measured after an additional h incubation at neutral ph. for each condition, at least experiments were performed. typical images used to obtain the data of the bar graph are shown on the right: in top images, cells were treated with μg/ml thermolysin; in bottom images, cells were not treated. cells that have fused are marked by arrows. (b) the kinetics of fusion for thermolysin-treated (dark red squares) and untreated (dark yellow circles) effector cells. cleaving ebov gp by thermolysin speeds fusion kinetics, but extents of fusion are the same for treated and untreated cells after a ph . pulse at °c is followed by a h reneutralization. * p < . ; *** p < . . doi: . /journal.ppat. .g . pulse (fig a, bar ) than in the absence of the pulse (fig a, bar ) . we confirmed that fusion was dependent on the presence of npc by generating and purifying a recombinant soluble protein consisting of domain c of npc fused to gst (denoted snpc ). the purity and size of snpc was confirmed (fig b, inset) . we added snpc to the external solution and found that the extent of fusion increased monotonically with the amount of snpc added ( fig b) , in accord with the prior demonstration that by binding npc , ebov gp undergoes conformational changes favorable for fusion [ ] . the augmentation of fusion by snpc indicated that, although there was a sufficient amount of npc on cell surfaces to stimulate fusion, this amount was relatively small and fusion was consequently limited. npc is an endosomal protein [ ] , but a small fraction of npc may be present on the plasma membrane of a cell. we assessed this possibility by using flow cytometry to measure overexpression of npc (second set of two bars) led to greater fusion with effector cells than did mock-transfected target cells (first set of bars). for these experiments, the effector cells were not thermolysin-treated (i.e., these experiments relied on endogenous levels of gp cleavage). for each set of experiments, a -min ph . pulse (labeled "pulse ph +") led to more fusion than when ph was never lowered (-). for each condition, n = . binding with an antibody against npc (from lifespan biosciences) on parental cells; shrna that targeted npc was stably expressed in one set of these cells, and npc was overexpressed in another set (fig c and d ). the level of binding of the secondary fitclabeled antibody against endogenous npc (as measured by mean fluorescence intensity, mfi) was -fold greater than in the absence of the primary ab ( fig c, bar vs. bar , and fig d) . expression was reduced for cells in which npc was knocked down by shrna (bar ), and was greater for cells in which npc was overexpressed (bar ). these results demonstrate that copies of npc reside in the plasma membrane of the cells we used as targets in cellcell fusion experiments. ebov gp is certainly cleaved within endosomes as part of viral infection [ ] . because we observed cell-cell fusion at acidic ph without adding thermolysin, it is extremely likely that a fraction of gp on the cell surface was cleaved into a fusion-competent form. an antibody that only recognizes cleaved gp has not been reported, so we had to devise an alternate means to quantitatively measure the extent of cleavage. we were able to distinguish between the two forms of gp by using the property that npc binds to cleaved, but not uncleaved, ebov gp. we used a snpc to examine cleaved gp by flow cytometry; in parallel, we measured the total amount of gp on cell surfaces by using an anti-flag antibody that bound to the flag tag on our gp construct. we also created a gp construct that was intrinsically more likely to be cleaved on the cell surface: we inserted the furin recognition site rrkr at amino acids - of gp (referred to as gp furin ), the putative cleavage site for catl in gp [ , ] . we reasoned that because exogenous expression of furin facilitates cleavage at this inserted site, generating the fusion-active - kda subunit, the extent of cleavage of gp on the plasma membrane as well as the extent of cell-cell fusion would be greater for this construct than for wt. we experimentally confirmed our expectations: we determined the amount of cleaved gp on the cell surface by adding snpc (fused with gst) to cells expressing either ebov gp or gp furin , and measuring their binding to an anti-gst antibody. this antibody was detected by a fitc-conjugated secondary antibody (fig ) . the fraction of wt gp cleaved on parental cells ( fig a, bar ) was the same for cells that were transfected with both gp and furin (bar ). the specificities of snpc and antibody binding were confirmed by the - fold higher fluorescence than was seen for cells that did not express gp (bar ). it is notable that cotransfection of cells by gp furin and furin resulted in greater cleavage (bar vs bar ). we found that the expression of total wt gp as measured by the anti-flag antibody was not significantly altered by coexpression of furin ( fig b, columns and ), but cells that coexpressed gp furin and furin consistently showed a decreased total gp (compare bar and ), possibly due to non-specific degradation of gp furin . to determine the relative percentage of cleaved gp, we normalized cleaved gp by total gp. (these are relative and not absolute percentages because different antibodies were used to detect cleaved vs. total gp.) we found that a higher percentage of gp on the plasma membrane was cleaved for cells coexpressing gp furin and furin than for cells expressing wt gp or gp furin alone ( fig c) . western blot analyses, using an anti-flag or an anti-gp antibody (kind gift of james cunningham), showed that the addition of furin increased the amount of cleaved gp furin construct as compared to gp furin alone (fig d, lanes and in left and right panels). furin did not cleave any wt gp (lanes ). we used these constructs to verify that an increased cleavage of ebov gp led to a greater extent of fusion ( fig e) . cotransfecting cells with gp and furin (bar ) led to the same extent of fusion as did transfection of gp alone (bar ). in contrast, cotransfecting with gp furin and furin led to more fusion (bar ) than transfecting only gp furin (bar ). control experiments of transfecting only furin showed that furin, per se, did not promote fusion (bar ). these experiments, taken together, establish that ebov gp does appear on the cell surface, that some of it is cleaved, and that for the gp furin construct, cleavage is augmented by coexpression of furin. to further confirm that the observed fusion was indeed mediated by ebov gp, we utilized mutations that had previously been shown to greatly reduce viral infection [ ] . we used mlv pseudovirus expressing gp, and observed that, indeed, the level of infection caused by the point mutant w a (fig , bar ) , the double mutant g a/g a (bar ), and the point mutant i a (bar ) were all substantially less than for wt gp ( bar ) . we then measured the extents of cell-cell fusion mediated by each of the mutant proteins. the extent of fusion in absence of thermolysin treatment supported by all three of the mutants (fig b, bars , , and ) was much less than for wt gp (bar ). flow cytometry measurements, using the same cells as for fusion experiments, showed that each of the mutant gps was well expressed on the cell surface ( fig c) . these experiments provide support that reduced infectivity by ebov correlates with reduced gp-mediated fusion. we next tested . , a small molecule inhibitor against npc , which prevents ebov entry, as well as testing a neutralizing antibody (kz ) against ebov gp. we found that both significantly reduced ebov gp-mediated cell-cell fusion (fig a and b ). the inhibitor . greatly reduced ebov gp-mediated fusion but did not significantly alter cell-cell fusion induced by either semliki forest virus (sfv) e /e or iav ha (fig a, . at μm). similarly, the neutralizing antibody kz , which recognizes the interface between gp and gp [ ] , reduced ebov gp-mediated fusion, but not sfv-e /e or iav ha-induced fusion ( another central fingerprint of gp-mediated fusion is inhibition of ebov infectivity by bafilomycin a (bafa ). by neutralizing endosomes, bafa inhibits infection, at least in part, by reducing cathepsin activity which in turn results is reduced cleavage of gp . we found that addition of bafa ( or nm) reduced the amount of cleaved gp that appeared on the cell surface ( fig c, bar vs bar ). this occurred despite a consistently greater amount of total gp in the plasma membrane after the addition of bafa (fig d) . (this greater amount was unexpected. possibly, bafa prevented lysosomal degradation of gp.) normalizing the amount of cleaved gp by the total shows that cleavage of cell surface gp was significantly reduced by bafa ( fig e) . thus, all data support the conclusion that the aqueous dye spread we observe is due to fusion induced by ebov gp. many of the effects of ph on kinetics and extents of ebov gp-induced fusion we found were unexpected and quite different than those of ph-dependent fusion for other viral proteins. notably, the extents of fusion did not monotonically increase as ph was progressively lowered, and the apparent ph dependence qualitatively varied with the times of reneutralization (fig ) . after a ph . pulse, the extents of fusion were always greater than those achieved after more acidic pulses; following a ph . pulse (at short incubation times (i.e., min) after the shift to neutral ph), more fusion was observed than for a less acidic pulse ( fig a) . however, for ph pulses of . and above, as the reneutralization time was increased, the extents of fusion became effector cells were labeled with dio and target cells with dii. both dyes were excited by a nm laser; dio emission was detected at nm and dii emission was recorded for nm. for facs measurements, trypsin and edta were added to cells prior to assaying; this treatment separates bound cells back into individual cells, but does not separate fused cells. representative data for lipid dye mixing is shown for a -min ph . pulse (left panel), and a ph . pulse (right panel). (e) average percentage of fusion as determined by lipid dye mixing as a function of ph (bars , , and ). lipid dye spread was negligible when using effector cells that were mock-transfected (bar ). thresholds for dio and dii were the same for all experiments and indicated on the two panels. fusion was scored as the percentage of fluorescent particles above both thresholds (i.e., the third quadrant). error bars are sem (n = , for each bar) and extents of fusion were statistically compared to the extent for a ph . pulse. * p < . ; *** p < . . doi: . /journal.ppat. .g less dependent on ph; fusion was independent of ph for . and above after a h reneutralization ( fig a) . in contrast, effector cells expressing iav ha showed the typical and expected response of greater extents of fusion for lower ph values at all times after reneutralization; fusion events reached their full extents after a min reneutralization (fig b, using the same protocol as for ebov gp experiments). thus, iav ha induces fusion more rapidly than does ebov gp. in separate experiments, we compared extents of ebov gp-mediated fusion after a h and h reneutralization that followed min, room temperature, acidic ph pulses ( fig c) . after the h reneutralization, fusion was relatively independent of the acidity of the ph pulse, and a low ph pulse did not greatly augment fusion (compare filled bars to open bars, fig c) . equality in final extents of fusion at ph . and . could be a consequence of all cell pairs quickly fusing at low ph, thereby eliminating the possibility of further fusion, although we consider this unlikely. in addition to single cell measurements of aqueous dye transfer, we also monitored lipid dye continuity between effector cells (treated with thermolysin) and target cells. we labeled effector cells with the lipophilic fluorescent dye dio and labeled target cells with dii and determined extents of fusion by flow cytometry (facs). the double positive cells (i.e., the third quadrant) are clearly products of hemifusion or cell-cell fusion ( fig d) . for effector cells treated with thermolysin, the percentage of fusion for the representative experiment was . % at ph . , the optimal ph for fusion ( fig d, second panel) and only . % at ph . (first panel). averaging six separate experiments for each condition, after a -h reneutralization, lipid mixing was greatest for a -min ph . pulse, and less for a ph . pulse than for cells maintained at neutral ph ( fig e) . the approximately two-fold greater fusion determined by flow cytometry at ph . than at . is also in agreement with the data for spread of calcein (fig ) . for mocktransfected effector cells, virtually no lipid dye spread was observed between effector and target cells (fig e) , in agreement with the aqueous dye spread measurements. therefore it is clear that ebov gp mediates a considerable amount of cell-cell fusion, and does so at an optimal ph of . . once calcein movement from effector to target cell commenced, it continued for ebov gpmediated fusion, but at an extremely slow rate. in general, the fluorescence due to calcein never equalized between target and effector cells for ebov gp-induced fusion (fig ) . in contrast, for fusion pores created by other viral fusion proteins [ , ] , such as jsrv env ( fig a, upper images) , the fluorescence did equalize. it is possible that the ebov gp pores eventually closed, preventing calcein from attaining the same concentration in effector and target cells. we therefore quantified the rate of transfer of calcein by plotting calcein fluorescence of effector and target cells as a function of time ( fig b) . for ebov gp-induced pores (red curve), the transfer occurred over a time course of tens of minutes, and over this period the increasing fluorescence of a target cell never equalized the decreasing fluorescence of an effector cell ( fig b) . the fluorescence of the effector and target cells, on the other hand, equalized within a minute or so for jsrv env-mediated pores (fig b, blue curve) . the exceedingly slow transfer of calcein is a further indicator that ebov gp-mediated pores remained extremely small. the fact that calcein transferred, albeit slowly over long times, shows that the fusion pores did not irreversibly close (or if they did, new pores opened) within tens of minutes of formation. as a control, we added saponin to effector cells and measured release of calcein to be sure that the dye did not become compartmentalized and therefore failed to transfer for reasons unrelated to the size of the fusion pore. release was fast from the saponin-treated cells and was almost complete within s, demonstrating that the overwhelming majority of calcein was, in fact, free and mobile. we further studied the size and rate of growth of ebov gp-mediated fusion pores by assessing the size of dyes that can permeate these pores over time. we loaded effector cells with cmtmr in addition to calcein. cmtmr forms disulfide bonds with the tri-peptide glutathione, and these complexes are somewhat larger than calcein. the complexed glutathione can also form disulfide bonds with cytosolic proteins and hence cmtmr fluorescently labels proteins that are much larger than calcein. as a consequence, the size distribution of molecules labeled by cmtmr is expected to be quite diverse, some only somewhat larger than calcein and others very much larger. we found that cmtmr transferred for only - % of the cell pairs for which calcein exchange occurred (fig c) . the relative inability of cmtmr to spread indicates that fusion pores typically did not enlarge sufficiently to allow passage of a molecule of the size of the nucleocapsid of ebov. in actual viral infection, factors absent in our model the small difference in fluorescence of effector and target cells connected by the jsrv env pore indicates that a small percentage of calcein is not free to transfer, possibly because it is bound to cellular elements. (c) images of effector cells loaded with calcein am (green) and cmtmr (red) and target cells loaded with cmac (blue) taken after a hr reneutralization at °c that followed a -min ph . pulse. calcein has transferred (white arrows, left panel) from most of the effector cells in contact with target cells. cmtmr (middle panel) mixed with cmac for only one cell (brown arrow). an overlay of calcein and cmtmr is shown in the third panel, with calcein (white arrows) and cmtmr (brown arrow) transfer shown. doi: . /journal.ppat. .g system are probably promoting expansion of the fusion pore connecting an envelope and endosomal membrane. we used electrical capacitance measurements to directly and quantitatively assess fusion pore size. the slow time course for ebov gp-mediated fusion necessitated that the tight electrical seal between the patch pipette and plasma membrane be maintained for long times. this proved difficult in practice. we were able, however, to electrically observe pores between cell pairs in three cases, and in these cases the pores never enlarged within s of formation and generally fluctuated within small values of conductance (fig a) . the conductance of the fluctuating pores did not return to baseline, showing that the pores did not close, but instead remained restricted to a small size. by way of comparison, it can be readily seen from representative traces of electrically measured fusion pores created by other viral proteins (fig b) that fusion pores generally significantly enlarge over time. the absence of pore enlargement for ebov gp suggests that many of the prior attempts at monitoring cell-cell fusion mediated by this fusion protein did not succeed because the reporter molecules that needed to permeate the fusion pore for detection of fusion were too large to pass through the pore. although only three pores were electrophysiologically measured, the finding that each of them did not exhibit increased conductance over time implies that the slow passage of fluorescent dyes through them was not due to structures that prevent their access to the pores. slow pore enlargement could be due to a number of factors, including slow recruitment and incorporation of additional copies of cleaved gp into the wall of the pore, or slow accumulation of lipids into the wall. we added nh cl to external media to test whether acidic intracellular compartments were essential for ebov gp-mediated cell-cell fusion. the addition of mm nh cl greatly reduced fusion after a -min ph . pulse in the absence of thermolysin treatment, so as to avoid activating uncleaved ebov gp on the cell surface (fig a) . in contrast, the addition of mm nh cl did not affect fusion induced by an optimal ph pulse for either sfv e /e or iav ha (fig a) . similarly, μm chloroquine inhibited cell-cell fusion mediated by the fusion protein of ebov, but not by the proteins from either sfv or iav (fig a) . the elimination of fusion by the addition of mm nh cl (bar ; same conditions as in fig a) was most likely caused by reducing cathepsin activity through neutralization of intracellular compartments: it was largely reversed by adding a recombinant cathepsin b to the external solution ( fig b, bar ) . because the normally acidic intracellular compartments were neutralized by nh cl, the pool of ebov gp on the cell surface that was previously uncleaved must have been cleaved by the added membrane-impermeant recombinant protease. the dose-response curves for inhibition of fusion by chloroquine (fig c) or nh cl (fig d) verified that inhibition of fusion is increased with increasing concentration of the neutralizing agent. therefore, even if the external solution is acidified, ebov gp-mediated cell-cell fusion does not occur unless the acidity of intracellular organelles is maintained. we conclude that ebov gp present on the cell surface requires an intracellular compartment for cleavage, as is consistent with previous reports. it is possible, however, that there are copies of cathepsins in the plasma membrane, and acidification of the external solution activates them to cleave ebov gp. proteinase k (pk) has proved useful for assessing conformational changes that viral proteins undergo at different stages of fusion [ , ] . we found that ebov gp was pk-sensitive for all steps of the fusion process (s text and s a fig) , that fusion was restored over time after removing pk (s b fig), and that normal cellular trafficking of protein led to replacement of proteolytically digested gp with newly delivered intact gp (s fig). we also used brefeldin a (bfa, μm)-an inhibitor of trafficking from endoplasmic reticulum to golgi-to further characterize the consequences for fusion of altering intracellular trafficking of ebov gp. here we found that treatments expected to increase the amounts of cleaved ebov gp on the cell surface led to greater extents of fusion (s text and s fig). ph-dependent cathepsin activity is essential for gp-mediated fusion for virus internalized in endosomes, ebov gp is believed to be cleaved by cathepsins b and l, but not by cathepsins a or d. we prevented cathepsin-induced cleavage by treating bound effector and target cells with a cathepsin b inhibitor (ca- ) or a cathepsin l inhibitor (z-fy-cho). in the absence of thermolysin treatment, the inhibitors led to significantly reduced fusion at both neutral and low ph (fig a, compare "untreated" and "treated": as always, changes of solutions containing membrane-impermeant buffers were used to control ph). using inhibitors against cathepsin a (lactacystin) or cathepsin d (pepstatin a)-neither of which is thought to cleave ebov gp-did not lead to reduced fusion using the same protocol as for the cathepsin b and l inhibition experiments (fig a) . these results provide strong support that fusion observed in our experiments in the absence of thermolysin treatment is due to, at least in part, copies of ebov gp on the cell surface that have their gp subunits cleaved by cathepsins. these results also document that neither cathepsin a or d cleaves ebov into a fusion-competent form. from the results as a whole, it is clear that low ph does not induce fusion unless the gp subunit has been cleaved. it is known that cathepsin activity is increased by acidity. we suggest that low ph acts, at least in part, by augmenting cathepsin activity on the cell surface. the same pattern of ph-dependence of fusion was observed for effector cells treated with thermolysin while cathepsin activity was continually inhibited: fusion was dependent on ph and was significantly reduced by the cathepsin b inhibitor or the cathepsin l inhibitor (fig a, thermolysin-treated, bars and of each set of columns), but was relatively unaffected by cathepsin a or d inhibitors (bars and ). cell-cell fusion exhibits a maximum at ph . (column compared to column - ). several cathepsins exhibit maximal activity in the ph range of . to . [ ] , so the maximum extent of fusion at ph . would likely be due to the ph dependence of cathepsin activity on the cell surface. control experiments provide additional support for the conclusion that cathepsins aid ebov gp-mediated fusion between cells. blocking cathepsin b (by adding the cathepsin b inhibitor) immediately after application of an acidic ph pulse resulted in a substantial . for effector cells not thermolysin-treated, fusion experiments were performed in the absence of a low-ph pulse (first set of columns) and with a ph . pulse (second set of columns). for cells treated with thermolysin, cathepsin inhibitors were added prior to the thermolysin treatment and maintained throughout the experiments. for thermolysin-treated cells, fusion was measured for the case without (third set of bars) and with a low ph pulse (fourth set). only inhibitors of cathepsin l or b diminished fusion, and they did so for all conditions. each of the four cathepsin inhibitors was added (separately) at the time of mixing effector and target cells. the cathepsin l and cathepsin b inhibitors reduced fusion to the same extent at ph . and ph . . the cathepsin b and d inhibitors were without effect. fusion was measured hr after the low ph pulse. the concentration of all inhibitors was μm, a high concentration to ensure maximal inhibition. (b) in contrast to the experiments in (a), the cathepsin b inhibitor was added subsequent to the low ph . pulse (second bar). less fusion occurred than for control (filled bar). (c) a recombinant human cathepsin b (rh catb, μm) was added to effector cells that had not been treated with thermolysin. the addition of rh catb (hashed bar) led to substantially increased fusion (filled bar, control). a -min ph . pulse was applied to promote fusion. the extents of fusion were measured after a -hr reneutralization for all experiments of this figure. error bars are sem. (d) t cells were co-transfected with plasmids encoding ebov gp and cathepsin b. cleaved gp on the plasma membrane was measured by flow cytometry using snpc , and the cleaved form was normalized by the total gp (measured by anti-flag), as described in fig . alternatively, gpexpressing cells were treated with thermolysin, and cleaved gp was measured and normalized as described. **p< . ; ***p< . . reduction in the extent of fusion after a -h reneutralization (fig b, effector cells were thermolysin-treated). the reduction from the control was~ -fold; a -fold reduction also occurred when the cathepsin b inhibitor was constantly present (see fig b) . the nearly equal percentages of inhibition of fusion are expected, since in the presence of the cathepsin inhibitor, uncleaved copies of ebov gp would not be cleaved during the period of reneutralization. thus, low ph appears to promote cleavage of ebov gp by cathepsins on the cell surface. incubating effector cells that were not treated with thermolysin with a recombinant human cathepsin b (rhcat b) (fig c, bar ) increased fusion significantly over the control (bar ). the simplest explanation for this increase is that the recombinant protein led to a higher level of gp cleavage than that induced by endogenous cellular cathepsins. to explicitly test whether increasing the activity of cathepsin increased the likelihood that gp on the cell surface was cleaved, we cotransfected cells to express gp and cathepsin b and used snpc to measure the percentage of gp in the plasma membrane that was cleaved (as described for fig ) . this percentage was greater (fig , column ) than the control (column ) in the presence of cathepsin b transfection. using the same techniques, we also showed that adding thermolysin to solution did indeed increase cleavage of cell surface gp (fig d) . does low ph directly cause conformational changes in ebov gp to induce fusion, or does it work via increasing the activity of cathepsins, or both [ , ] ? we were able to approach these questions by using the ability of bfa to effectively block delivery of ebov gp to the cell surface and, independently, by using cathepsin inhibitors to prevent gp cleavage. we incubated effector cells with bfa for min to prevent further delivery of ebov gp to the plasma membrane prior to a thermolysin-treatment, and maintained the presence of the drug during all solution changes. the extent of fusion was independent of ph, and considerably less than when the trafficking inhibitor was not employed (fig a) . the clear conclusion is that, with bfa present, all fusion was caused by copies of ebov gp that had been cleaved by thermolysin and that remained on the cell surface. the finding that ph pulses did not affect the extent of fusion at all shows that acidity did not promote the conformational changes in cleaved ebov gp that would lead to fusion. we inhibited cathepsin activity to further test the conclusion that once cleaved, ebov gp no longer requires low ph to induce fusion. we performed experiments in which ca- , a cathepsin b inhibitor, was continually present (fig b) . the inhibitor was added to isolated effector cells and maintained for min to ensure that all ebov gp delivered to the plasma membrane was not cleaved. effector cells were then thermolysin-treated, always maintaining the inhibitor. these cells were bound to target cells, and the external solution was acidified to ph . ; after reneutralization, cells were maintained for h at °c, with all manipulations performed in the presence of the inhibitor (fig b, experimental protocol illustrated on top) . the extent of fusion was greater when the inhibitor was not added (control): this indicates that delivery to the cell surface of ebov gp cleaved by endosomal cathepsin (subsequent to thermolysin treatment) significantly contributes to fusion. more importantly-and central to the mechanism of ebov gp-mediated fusion-the extent of fusion was independent of ph. this finding strongly implies that acidic conditions have no direct effect on ebov gp-mediated fusion. the ph dependence of fusion is solely due to the ability of cathepsin to cleave ebov gp; once cleaved, acidic conditions directly induce conformational changes in cleaved ebov gp that lead to fusion. using both cytoplasmic and membrane dye transfer assays, we established that known properties of ebov fusion occurring within endosomes are replicated by our cell-cell fusion system and that specific inhibitors of ebov infection-the small molecule inhibitor . and a neutralizing antibody kz -block fusion. the inhibition of ebov gp-mediated cell-cell fusion (but not iav ha or sfv e /e fusion) by the lysosomotropic agents nh cl and chloroquine is expected: ebov gp cleavage is eliminated because cathepsin activity is greatly reduced by neutralization of endosomes; inhibiting cathepsin activity reduces cleavage of ebov gp [ , ] . we also showed that copies of npc reside in the target membrane and some gp resides in the plasma membrane, and that a fraction of the gp is properly cleaved. it is virtually certain that the cell-cell fusion process investigated in the present study is mediated by ebov gp. it is likely that past lack of success in observing cell-cell fusion is attributable to the fact that the fusion pore mediated by ebov gp on the cell surface remains small. over the time scales of electrical measurements, the pore does not enlarge at all. based on fluorescence dye spread measurements, it enlarges more slowly and to a lesser extent than any other pore mediated by a viral fusion protein of which we are aware, and it may even tend to close. the ebov gp fusion pore is large enough to allow the passage of calcein, but just barely. there is little doubt that the fluorescent dye cmtmr does not permeate the pore because virtually all of it complexes with proteins; the complex becomes permeable only after a pore enlarges. electrical measurements directly demonstrate that the ebov gp-induced pore remains small. over the course of time in our cell-cell fusion experiments, ebov gp-mediated fusion pores do not significantly enlarge. the question now becomes: how readily does a fusion pore enlarge when connecting an ebov envelope with an endosomal membrane? this fusion pore must expand to sizes that permit passage of the large viral nucleocapsid. four major possibilities present themselves: (i) the necessary enlargement is extremely slow for the endosome-viral pores; (ii) elements engaging plasma but not endosomal membranes, such as cytoskeleton, retard the growth of fusion pores; (iii) a protein (such as the two-pore calcium channel, present in the endosomal compartments that support ebov fusion [ ] ) is required for pore enlargement; (iv) control of calcium concentrations (e.g., through the two-pore channels) regulates fusion pore formation or enlargement in endosomes. methods to monitor the formation and growth of fusion pores of ebov gp-bearing viral particles within endosomes will be needed to answer these questions [ ] . we have unambiguously shown that a fraction of ebov gp on the cell surface is cleaved. by using the gp furin construct we also demonstrated that increased cleavage correlates with greater fusion. in addition, we functionally evaluated the cleavage status of ebov gp on the cell surface by adding a water-soluble recombinant cathepsin b or thermolysin to solution and found that these proteases promoted fusion. late endosomes and lysosomes are generally thought to be the cellular site of cleavage of ebov gp by cathepsins [ ] ; it is likely that a fraction of ebov gp is cleaved within endosomes and then recycled to the plasma membrane where it mediates cell-cell fusion, independent of ph (fig ) . we suggest that uncleaved ebov gp that reaches the surface is cleaved upon acidification of the external solution by cathepsins within the plasma membrane. thermolysin cleaves uncleaved copies of ebov gp that are delivered to the cell surface, accounting for the enhancement of fusion by the addition of the protease. regardless of the site of gp cleavage, an appreciable fraction of the gp subunit is indeed cleaved into its fusion-competent form after the addition of thermolysin. npc serves as receptor for ebov gp in endosomes, and is essential for the virus to infect a cell. we have now shown that npc is not confined only to intracellular membranes, but rather that some copies reside in plasma membranes. our finding that snpc promotes ebov gp-mediated cell-cell fusion suggests that domain c of npc alone is sufficient to induce the needed conformational changes in the fusion protein. it is well established that the extents of cell-cell fusion correlate with the levels of viral fusion protein expression on cell surfaces [ , ] . thus, it is not surprising that the extents of cell-cell fusion induced by ebov gp are affected by its delivery to, and loss from, plasma membranes. for some viral fusion proteins, such as the paramyxovirus hendra and nipah virus f proteins, and sars coronavirus s protein, cell-cell fusion is sensitive to protein cycling [ , , ] . these proteins require acidic intracellular compartments for cleavage: endosomes for nipah virus [ ] , and endosomes and the trans-golgi network for hendra virus [ ] . but the dependence of cell-cell fusion on protein trafficking is unusual for typical ph-dependent viral fusion proteins; for these proteins acidity does not promote cleavage, but instead directly induces conformational changes [ ] . once activated, these typical low-ph-dependent fusion proteins quickly inactivate if they do not promote fusion [ ] . hence, protein delivered to the cell surface subsequent to an acidic pulse will not be able to promote fusion. in contrast, proteins that induce fusion at neutral ph will promote fusion once they are delivered to the cell surface. our results show that ebov gp cleaved by endosomal cathepsins are no longer sensitive to ph and therefore can induce fusion once they arrive at the plasma membrane. infectivity is subject to processes other than fusion, and so infectivity need not always correlate with extents of cell-cell fusion. for example, it has recently been shown that the activity of two-pore calcium channels in endosomes is required for ebov infection [ ] , and that ebov infects by fusing to endosomal membranes that contain both npc and the two-pore channel [ ] . tetrandrine blocks these channels and inhibits ebov infection. we found that tetrandrine ( nm) did not affect ebov gp-mediated cell-cell fusion. it is notable that the extent of fusion that occurs after h at neutral ph was roughly equal to the extent that follows a ph . pulse. because fusion induced by cleaved gp is ph-independent, we interpret the continual increase in fusion at neutral ph over time to be a consequence of intracellular trafficking: new copies of ebov gp continually replace or supplement old copies and these new/supplemented, cleaved copies can cause fusion between cells that had not previously fused. thus, acidification likely promotes more fusion at early times through activation of surface cathepsins that cleave ebov gp. however, it is not presently clear why fusion kinetics is faster after a ph . pulse than after a ph . (or more acidic) pulse. the ph dependence of cathepsin activity is complicated [ ] . while activity generally increases with acidification, some cathepsins exhibit the same activity in the range of ph as at lower values of ph [ ] . for others, activities are maximal at an intermediate ph, such as . [ ] . also, the ph dependence of cathepsin activity varies with environment and conditions, such as redox potentials on each side of the membrane in which a cathepsin resides [ , ] . any relevant cathepsins (e.g., b or l) on the cell surface can, at their optimal ph, cleave ebov gp. a direct test of whether ebov gp on the cell surface is maximally cleaved by cathepsins at ph . will require methods to measure the percentage of ebov gp that is cleaved as well as cathepsin activity at the cell surface. the role of acidic ph in ebov fusion has been debated in the field [ , , ] . our data unambiguously show that cell-cell fusion is regulated by extracellular ph. the acidity of the extracellular solution can, in principle, augment both the activity of cathepsins that reside in the plasma membrane and directly promote conformational changes of cleaved ebov gp on the cell surface. (although cathepsins are regarded as endosomal membrane proteins, some copies also likely reside in plasma membranes from which many endosomes derive [ ] ). the great reductions in fusion caused by inhibition of cathepsin activity and recovery of fusion by addition of a recombinant cathepsin establish that cathepsins' activity at the cell surface is consequential. independent experiments in which cathepsin activity was inhibited or delivery of protein to cell surface was blocked show that the ph-dependence of fusion is eliminated once ebov gp is cleaved. this demonstrates that fusion mediated by the cleaved form is intrinsically ph-independent. that is, cleaved ebov gp is essentially a neutral ph fusion-inducing protein; all the experimentally observed and biological relevant ph-dependence is a consequence of cathepsin activity. the faster kinetics of cell-cell fusion after a ph . pulse than for pulses at higher values can be accounted for by greater cleavage of ebov at the cells surface at ph . . a previous study used model peptides to mimic the six-helix bundle of ebov gp and found that low ph increased bundle stability [ ] . the stage of fusion in which bundle formation occurs has not been identified for ebov gp. it may be, for example, that the bundles form subsequent to pore formation, as occurs for hiv env [ ] , and that increased bundle stability aids pore enlargement, but not fusion itself [ ] . alternatively, the model peptide may not mimic bundle stability within a full length, structurally intact gp. as a general rule, fusion kinetics for viral proteins that induce fusion at neutral ph are slower than for proteins that utilize low ph as a trigger. this could explain the slow fusion kinetics of ebov gp mediated fusion, despite classification as a low ph-requiring process. alternatively, a need to continually deliver ebov gp could be the reason ebov gp-mediated cell-cell fusion is slow. the development of an experimentally convenient system of ebov gpmediated fusion should make it possible to determine molecular mechanisms by which ebov releases its genome into infected cells. our results and conclusions are diagrammatically summarized in fig . npc is an intracellular receptor for ebov gp within endosomes. but, as we have shown, npc can also reside in the plasma membrane. endosomal cathepsins cleave ebov gp, and any cathepsins that reside in plasma membranes will also cleave surface gp upon acidification of the external solution. both cleaved and uncleaved copies of ebov gp are continually delivered to and retrieved from the surface, and hence intracellular trafficking contributes to extents and kinetics of fusion. but binding of ebov gp to the target membrane should inhibit endocytotic retrieval. consequently, ebov gp (both cleaved and uncleaved) should accumulate at potential fusion sites, leading to more fusion over time. preventing acidification of endosomes to block cleavage of ebov gp, or inhibiting delivery of the protein to the cell surface, greatly reduces fusion. acidification of the external solution to ph . increases the activity of cathepsins that reside in the cell surface, and this results in additional cleavage of ebov gp. the addition of thermolysin converts all surface gp to the cleaved form, thereby resulting in the maximal extent of fusion. that cell-cell fusion induced by cleaved ebov gp does not depend on ph, provides critical insight into the mechanism of ebov entry and infection. . for this work, we mainly used an n-terminal flag-tagged, mucin-deleted ebov gp construct by replacing the signal peptide of gp with that of preprotrypsin followed by a flag sequence. all gp mutants, including gp furin , were made by overlapping pcr using the flag-tagged gp construct as the template. the plasmid to express jsrv env has been described [ ] . to express iav ha for dye spread experiments, we used the x strain (plasmid provided by judith white, university of virginia, charlottesville, va). a standard calcium phosphate method was used to express sfv e /e via transfection of the pcb -wt vector, plasmid provided by margaret kielian, albert einstein college of medicine, bronx, ny [ ] . a small molecular inhibitor, . , was a gift of james cunningham (harvard medical school, boston, ma). hek t and cos cells employed have been previously described [ ] . the hek t cells stably expressing were generated by transducing cells with pbabe retroviral vector expressing npc (gift of kartik chandran) followed by puromycin selection (sigma, μg/ml). all cells were grown in dulbecco's modified eagle's (dmem) medium, supplemented with . % penicillin/streptomycin plus % fetal bovine serum (fbs). schematic diagram illustrating control of fusion by cleavage of ebov gp and its transport to the cell surface. ebov gp is synthesized in er, transported to golgi complexes where it is processed into gp and gp subunits, and ultimately targeted to the plasma membrane. ebov gp can undergo endocytosis from the plasma membrane and eventually reach late endosomes and lysosomes. it is then further cleaved by cellular cathepsins, bound by npc , and recycled back to plasma membrane. alternatively, ebov gp is directly cleaved by cathepsins on the plasma membrane or by thermolysin treatment. ebov gp proteins do not permanently remain on the surface, but rather undergo continual delivery and removal. cleaved gp accumulates at potential fusion sites, leading to the observed increased fusion over time. solid arrows denote pathways definitively established in the present study. dashed arrows denote pathways that are likely to occur based on data of the present study. light dashed arrows denote pathways suggested by data of the present study. for all experiments using ebov gp, cos cells were maintained in eagle's medium with glucose, l-glutamine, and sodium pyruvate, supplemented with % cosmic calf serum (hyclone, logan, utah), pen strep (gibco), and . mg/ml g sulfate (cell gro, manassas, va), and transfected to express ebov gp by a standard calcium phosphate procedure [ ] . about~ x cells were loaded with . μm calcein-am as previously described [ ] and sometimes coloaded with μm -(and- )-((( -chloromethyl)benzoyl)amino)tetramethylrhodamine) (cmtmr) [ ] . if these effector cells were thermolysin-treated to cleave ebov gp, μg/ml thermolysin was incubated with the cells for min at room temperature. exchanging the solution with dmem removed thermolysin; residual thermolysin was further removed by spinning down the cells three times and replacing the aqueous solution. hek t cells were maintained in the same media and in the same way as cos cells and were used as targets.~ x cells were loaded with μm cmac. effector cells were mixed, including a gentle vortex-in a tube containing either pbs ++ (sometimes supplemented with mg/ml bsa) or dmem-with the labeled target hek t cells. the cells were added into polylysine-coated ( mg/ml) wells of an -well slide (thermo fisher) [ ] and allowed to settle and adhere to the bottom for min at room temperature. the ph was lowered (or not) for min at room temperature to the indicated value (ph . unless stated otherwise, using an exchange solution consisting of mm nacl, . mm kcl, . mm mgcl , . mm cacl , mm mes), the solution was then reneutralized to ph . by an exchange of solutions, and the temperature raised to °c. after this reneutralization for the indicated time, generally h, fusion was scored as a function of time by the transfer of calcein into target cells, as described [ ] . for fusion experiments utilizing iva ha as control, the expressed ha in effector cells were was cleaved into ha -ha subunits with trypsin, as previously described [ ] . to label effector cells,~ x cells/ml were incubated with μm dio for min at °c. the day before an experiment, target cells were split and plated on glass cover slips placed in culture dishes so as to allow convenient transfer. these target cells were labeled by μm dii for min at °c. labeled effector cells were thermolysin treated ( μg/ml) and added above labeled target cells. binding was allowed to occur for min at room temperature before washing out unbound effector cells. the solutions bathing the cover slips (one cover slip per culture dish) was acidified to the indicated ph for min at room temperature, and the culture dish placed in a °c incubator for h. cells were detached from cover slips by adding μg/ml trypsin to a divalent-free solution containing . mm edta for min at room temperature, followed by vigorous, repeated pipetting to dissociate bound (i.e., neither hemifused or fused) cells. colocalization of the two lipid dyes was monitored by flow cytometry (guava easy cite, guava technology, millipore), using two channels emission, one for each dye ( nm for dio and nm for dii; both excited by a nm laser). the same protocols were followed for mock-transfected cos cells; this data was subtracted from data obtain for cos cells expressing ebov gp to obtain percentages of fused cells. the domain c of npc was cloned into a pgex- t vector that had a gst tag on the n-terminus (ge healthcare life sciences, pittsburgh, pa). the expression of fusion protein was induced in e. coli. by iptg ( . mm) and purified by glutathione sepharose b (ge healthcare life sciences). protein was quantified by a bradford assay and used for cell-cell fusion and for measurements of cleaved gp. the expression of npc on t cell surfaces was determined by using anti-npc (against n-terminus - aa; lifespan biosciences). determination of ebov gp cleavage. hek t cells were transfected with ebov gp or gp furin in the presence or absence of a plasmid that encodes furin (kind gift of paul bates, university of pennsylvania). transfected cells were detached by pbs containing mm edta. one portion ( million cells) was used to measure the cleaved gp by incubating cells with μg snpc for h on ice, followed by adding a mouse monoclonal anti-gst antibody; the fluorescence signal was quantified by adding a fitc-conjugated secondary anti-mouse antibody using flow cytometry. another portion of the transfected cells (also million) was used to measure the total gp expression on the cell surface, using an anti-flag antibody. the total gp, as determined by mean fluorescence intensity (mfi), was used to determine the percentage of gp on the plasma membrane that was cleaved: the amount of cleaved normalized by total gp. cleavage of gp furin in cell lysates was determined by western blotting using anti-flag or an anti-gp antibody. production of mlv retroviral pseudotypes bearing ebov gp and viral infection were as described previously [ ] . briefly, gp/lapsn packaging cells stably expressing mlv gag-pol and alkaline phosphatase (ap) were transfected with plasmids encoding the ebov gp or mutants, and the viruses produced were used to infect htx cells (a subclone of ht ). viral infectivity was determined by counting ap + foci h after infection. we proteolytically determined the stages of fusion at which ebov gp was proteinase k-sensitive by treating cells with . mg/ml proteinase k for min, and maintaining or removing it as indicated, at various points in our protocol (s fig). we cleaved ebov gp with thermolysin and used our standard protocol (a -min ph . pulse), incubated the cells for h at ph . , and then measured fusion. all experiments were performed in parallel on the same days; as controls, proteinase k was not employed and extents of fusion were measured. brefeldin a ( μm) was used to inhibit anterograde protein trafficking as described in the experiments of s fig. we determined the latency between lowering ph and fusion by using cells mixed within a tube, placed over poly-lysine coated cover slips within a culture dish maintained at °c and allowed to settle for min. the ph was lowered to the indicated value for min at room temperature through an exchange of solutions. the cover slips were then transferred into dishes at °c, neutral ph, for indicated times. the time of transfer is defined as time = . for thermolysin-treated cells, the effector cells were treated prior to binding to target cells. the extents of fusion were quantified at varied times by spread of calcein. we used the rate of accumulation of calcein into the target cell and its depletion in effector cells to access the size of the fusion pore as a function of time [ ] . the fluorescence of both effector and target cells was proportional to calcein concentration, as verified by the procedure detailed in [ ] . neutralizing intracellular compartments nh cl ( mm unless otherwise noted) was added to the bathing solution and maintained throughout the course of an experiment, including during any low ph pulses, to obtain neutralization. the same procedure was used with chloroquine ( μm unless stated otherwise) or bafa ( or nm) to cause endosome neutralization. the ionic and buffering contents of the nh cl-containing low ph solutions were pre-adjusted to the required ph so as to maintain osmotic strength at mosm during low ph pulses. to monitor ebov gp expression and its recovery after proteinase k treatment, cells were treated with . mg/ml of the protease for min at room temperature, and the staining protocol now described was used immediately or after cells were maintained for h in dmem at °c, as indicated. ebov gp-expressing cos cells were transferred to ml tubes and incubated for h at room temperature with a primary antibody (human anti-ebov gp-kz stock at . mg/ml, ibt bioservices, gaithersburg, md) that was diluted : in pbs ++ that was supplemented with % fetal bovine serum. after three washes with the % fbs-pbs ++ solutions, a secondary fitc-conjugated goat anti-human antibody (fisher scientific) was added at a final concentration of μg/ml and maintained for min at room temperature in the dark. after cells were washed twice, they were added to -well slides that had been treated with poly-l-lysine (m.w. , - , , sigma aldrich, st. louis, mo). the cells were then fixed for min at room temperature with % paraformaldehyde, and washed twice with dmem. pore size over time was also quantified by using patch clamp time-resolved admittance measurements [ ] , often referred to as capacitance measurements. fusion was promoted by a -min ph . pulse at room temperature. because the fusion pore took considerable time to form, and a seal between the patch pipette and cell could only be maintained once solutions and temperature were established, temperature was raised to °c for~ min before attempting electrical measurements. (this procedure reduced the time between establishing the seal and fusion pore formation). the cover slip was then placed in a temperature-controlled chamber on a microscope stage, and the seal established. the external solution consisted of mm n-methyl-glucamine aspartate- mm mgcl - mm hepes (ph . ); the solution within the patch pipette was mm cesium glutamate- mm mgcl - mm bapta [ , -bis(o-aminophenoxy)ethane-n,n,n ,n -tetraacetate]- mm hepes (ph . ). to electrically characterize fusion pores created by iav ha, aslv env, and hiv env, we used cell lines that stably express the fusion protein and target cells that stably express the cognate receptor: hab cells that express iav ha as effectors and t cells as targets [ ] ; nih t enva cells that express aslv env and t tva cells as targets [ , ] ; and tf cells that express hiv env and hela t cells as targets [ ] . pair-wise student t-tests were used to compare the outcome of a manipulation on fusion as compared to the control. in figures, unless otherwise indicated, a single asterisk ( à ) denotes p < . , two asterisks ( Ãà ) denotes p < . , and three asterisks ( ÃÃà ) denotes p < . . the periods in which proteinase k (pk, μg/ml) was present are marked in the schematic protocol, and numbers correspond to bar numbers below. regardless of whether pk was present prior (bar ) or subsequent (bar ) to treating effector cells with thermolysin, the presence of pk virtually eliminated fusion. similarly, adding and then maintaining pk to effector cells as they were bound with target cells led to greatly reduced fusion (bar ). but adding pk immediately after the low ph pulse (bar ) hardly affected fusion. (b) ebov gp-mediated fusion recovered over time after proteinase k treatment: left-hand bars of each pair denote that a ph pulse was not applied; a ph . pulse was applied for the right hand bars. adding proteinase k and washing out immediately prior to thermolysin treatment virtually abolished fusion (first set of two bars). allowing hr between proteinase k removal and thermolysin treatment restored most of the fusion (second set of bars). waiting h completely restored fusion (third set of bars). the effect of pk treatment on ebov gp expression was assessed using volocity imaging software (perkin elmer). integral fluorescence per field ( image fields per datum point) was calculated after subtracting the fluorescence background determined from the mock-transfected images. this quantification shows that expression of ebov gp was greatly reduced by the proteinase k treatment and significantly recovered after the protease was absent for h. this demonstrates that the ebov gp expression levels were steady over time. (tif) s fig. inhibitors of trafficking show that ebov gp is dynamically exchanged between plasma and intracellular membranes. the presence of brefeldin a (bfa, μm) at all points of the fusion protocol that utilizes thermolysin-treated effector cells and a ph . pulse reduced fusion greatly (bar ) compared to the control (bar , bfa was not included). washing out bfa and immediately treating effector cells with thermolysin led to greater fusion (bar ). waiting min after the washout before thermolysin treatment led to fusion (bar ) comparable to control. adding and maintaining bfa after binding effector and target cells, but before applying a low ph pulse led to substantially reduced fusion (bar ). applying bfa after the low ph pulse led to less fusion than the control (bar ), but to greater fusion than when the drug was added prior to the low ph pulse (bar , extent of fusion higher than for bar ). thermolysin was used to cleave ebov gp just prior to measuring fusion for all conditions of, allowing meaningful comparisons. (tif) ebola haemorrhagic fever progress of vaccine and drug development for ebola preparedness similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized madin-darby canine kidney cells visualizing infection of individual influenza viruses hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change detection of cell-cell fusion mediated by ebola virus glycoproteins the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding a system for functional analysis of ebola virus glycoprotein endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions ebola virus glycoprotein : identification of residues important for binding and postbinding events ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity comprehensive analysis of ebola virus gp in viral entry ebola virus entry requires the host-programmed recognition of an intracellular receptor characterization of the receptor-binding domain of ebola glycoprotein in viral entry ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion cell entry of enveloped viruses biochemical analysis of the secreted and virion glycoproteins of ebola virus zaire ebola virus entry into human dendritic cells is insensitive to cathepsin l inhibition proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection structure and function of the complete internal fusion loop from ebolavirus glycoprotein evolution of intermediates of influenza virus hemagglutinin-mediated fusion revealed by kinetic measurements of pore formation evidence that the transition of hiv- gp into a six-helix bundle, not the bundle configuration, induces membrane fusion the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion a point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion single residues in the surface subunits of oncogenic sheep retrovirus envelopes distinguish receptor-mediated triggering for fusion at low ph and infection the niemann-pick c protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for npc expression studies of the "chain reversal regions" of the avian sarcoma/leukosis virus (aslv) and ebolavirus fusion proteins: analogous residues are important, and a his residue unique to enva affects the ph dependence of aslv entry structure of the ebola virus glycoprotein bound to an antibody from a human survivor fusogenicity of jaagsiekte sheep retrovirus envelope protein is dependent on low ph and is enhanced by cytoplasmic tail truncations intermediates in influenza virus pr/ haemagglutinininduced membrane fusion structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors ebola virus (ebov) infection: therapeutic strategies characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment the ebola virus glycoprotein directs fusion through npc + endolysosomes ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density the initial fusion pore induced by baculovirus gp is large and forms quickly a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l cathepsin l is involved in proteolytic processing of the hendra virus fusion protein inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry the nipah virus fusion protein is cleaved within the endosomal compartment subcellular localization and calcium and ph requirements for proteolytic processing of the hendra virus fusion protein mutations in the fusion protein cleavage site of avian paramyxovirus serotype increase cleavability and syncytium formation but do not increase viral virulence in chickens acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology the lysosomal cysteine proteases exocytosis of active cathepsin b enzyme activity at ph . , inhibition and molecular mass cathepsin b responsiveness to glutathione and lipoic acid redox cathepsin b stability, but not activity, is affected in cysteine:cystine redox buffers a new player in the puzzle of filovirus entry properties of a plasma membraneassociated cathepsin b-like cysteine proteinase in metastatic b melanoma variants designed protein mimics of the ebola virus glycoprotein gp alpha-helical bundle: stability and ph effects hiv- envelope proteins complete their folding into sixhelix bundles immediately after fusion pore formation structural basis for differential neutralization of ebolaviruses the transmembrane domain and acidic lipid flip-flop regulates voltage-dependent fusion mediated by class ii and iii viral proteins effects of membrane potential and sphingolipid structures on fusion of semliki forest virus enzootic nasal tumor virus envelope requires a very acidic ph for fusion activation and infection two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity we thank kartik chandran, james cunningham, david sanders, and gary kobinger for provision of reagents that made this work possible. key: cord- -vvbjulm authors: brinkmann, constantin; hoffmann, markus; lübke, anastasia; nehlmeier, inga; krämer-kühl, annika; winkler, michael; pöhlmann, stefan title: the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vvbjulm vesicular stomatitis virus (vsv) release from infected cells is inhibited by the interferon (ifn)-inducible antiviral host cell factor tetherin (bst- , cd ). however, several viruses encode tetherin antagonists and it is at present unknown whether residual vsv spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. here, we show that the viral glycoprotein (vsv-g) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the hiv- vpu protein. tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a gxxxg motif in the transmembrane domain of vsv-g. however, mutation of the gxxxg motif did not modulate tetherin sensitivity of infectious vsv. these results identify vsv-g as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread. vesicular stomatitis virus (vsv) is a negative-stranded rna virus within the rhabdoviridae family, and vsv new jersey and indiana are major vsv serotypes. vsv is transmitted from insects to ungulates (mainly cattle, horses and pigs), in which it can cause mucosal lesions [ ] [ ] [ ] . in addition, the virus can be transmitted to humans and such infections usually induce influenza-like symptoms [ ] . vsv replicates fast, is highly immunogenic and is frequently used to model infection by negative-stranded rna viruses. moreover, vsv is used as a tool for diverse scientific endeavors [ ] . for instance, vsv has oncolytic properties [ ] and is developed for cancer therapy [ ] . moreover, vsv variants in which the open reading frame for the viral glycoprotein (vsv-g) has been replaced by that of the ebola virus (ebov) glycoprotein (gp) are currently tested as vaccines against ebov infection [ ] [ ] [ ] . the interferon (ifn) system is an integral component of innate immunity and constitutes the first line of defense against viral infection. sensors of the ifn system, including toll-like receptors and retinoic acid inducible gene i-like receptors, can detect pathogen-associated plos one | https://doi.org/ . /journal.pone. december , / a a a a a molecular patterns (pamps), which triggers signals that commandeer the cells to express ifn [ , ] . binding of ifn to uninfected cells in turn triggers further signaling events that induce the expression of ifn-stimulated genes (isg), many of which exert antiviral activity [ , ] . vsv spread can be blocked by ifn in cell culture, although the viral matrix protein vsv-m interferes with ifn signaling [ ] [ ] [ ] . the isg-encoded proteins that are responsible for ifninduced blockade of vsv infection are not fully known, although ifitm and tetherin were shown to block vsv infection in transfected cells [ , ] . the ifn-induced antiviral host cell protein tetherin (cd , bst- ) blocks release of diverse enveloped viruses from infected cells [ , ] . the particular membrane topology of tetherin is key to its antiviral activity: tetherin harbors an n-terminal transmembrane domain and a c-terminal gpi-anchor which allows the protein to simultaneously insert into viral and cellular membranes, thereby forming a physical tether between virus and host cell [ ] . several viruses encode tetherin antagonists which allow viral spread in tetherin-positive cells [ ] . the prototypic tetherin antagonist, the hiv- protein vpu, and most other viral tetherin antagonists block tetherin by reducing its expression at the plasma membrane [ ] [ ] [ ] , which is used by these viruses as platform for budding of progeny particles. in contrast, the ebov-gp, another tetherin antagonist, interferes with tetherin's antiviral activity without modulating tetherin expression or cellular localization [ ] [ ] [ ] [ ] and the mechanism underlying tetherin antagonism by ebov-gp is largely unclear. two studies reported that vsv is inhibited by tetherin. weidner and colleagues showed that directed expression of tetherin resulted in a profound decrease in vsv release from infected cells [ ] . liberatore and coworkers dissected cell-cell spread of vsv from viral dissemination to distal cells via free particles and found that only the latter process was markedly inhibited by tetherin [ ] . however, it is at present unknown whether vsv encodes a tetherin antagonist, which is responsible for residual viral spread in tetherin-positive cells. here, we show that vsv-g counteracts tetherin in transfected cells. however, no evidence for a contribution of tetherin-antagonism to spread of authentic vsv in tetherin-positive cells was obtained. human embryonal kidney- t, vero (african green monkey, kidney) and hela (human, cervix carcinoma) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs, biochrome, berlin) and penicillin/streptomycin (pan-biotech, aidenach; final concentration penicillin units/ml, streptomycin . µg/ml). bhk- cells (baby hamster kidney) were cultivated in dmem supplemented with % fbs (biochrome) and penicillin/streptomycin. cells were cultured at ˚c in humidified atmosphere containing % co . for seeding and subcultivation, cells were washed with phosphatebuffered saline (pbs) and detached by incubation in a trypsin/edta solution (pan-biotech, aidenach; hela, vero and bhk- cells) or by directly resuspending them in dmem ( t). cell numbers were determined under a light microscope using a neubauer chamber. transfection of vero, bhk- and hela cells was performed using lipofectamine (thermofisher scientific, dreieich) according to manufacturer's protocol, while t cells were transfected using the calcium phosphate method. selection and maintenance of vero cells stably transfected with the retroviral pqcxip vector was achieved using puromycin (cayman chemical, ann arbor). t cells were obtained from dsmz (acc- , leibniz institute dsmz-german collection of microorganisms and cell cultures). vero and bhk- cells were obtained from collaborators. hela cells were obtained through the nih aids reagent program, division of aids, niaid, nih and their identity was confirmed by str dna typing employing a published protocol [ , ] . plasmids encoding vpu, ebov-gp and hiv- p -gag were previously described [ , ] . expression plasmids for the coding sequences of vsv nucleoprotein (vsv-n), phosphoprotein (vsv-p), matrix protein (vsv-m), glycoprotein (vsv-g) and rna-dependent rnapolymerase (vsv-l) were generated by amplifying the respective open reading frame (orf) from a plasmid-encoded vsv anti-genome (indiana strain, kindly provided by g. zimmer) and inserting them via standard cloning procedures into plasmids pcaggs (vsv-n, vsv-p, vsv-m (mutant ncp, harboring four amino acid substitutions associated with reduced cytotoxicity [ ] ) and vsv-l) or pcg (vsv-g). this was achieved by overlap-extension pcr technique using primers that introduce the desired nucleotide exchanges. generation of vsv-g mutants a r and lxxxl was achieved by the same strategy using the expression plasmid for wt vsv-g as a template. the expression plasmid for human tetherin was generated by amplifying the orf from a previously described plasmid [ ] and inserting it into plasmid pcaggs using kpni and xhoi restriction sites. similarly, porcine tetherin was pcr amplified and cloned via ecori and nhei restriction sites. for detection of human tetherin expression, the sequence for a c-myc antigenic tag was added to the ' end of human tetherin using pcr. in order to generate transgenic cells stably expressing human tetherin, the genetic information for human tetherin was cloned into the retroviral vector pqcxip-mcs, a modified form of pqcxip (clontech, palo alto), in which the multiple cloning site was modified to contain sites for noti-bamhi-agei-hpai-mlui-xhoi-nrui-ecori. to generate recombinant vsv that expresses egfp (enhanced green fluorescent protein), we constructed a plasmidencoded vsv anti-genome (genbank: j . ) with an additional transcription unit for egfp between vsv-g and vsv-l orfs. first, unique mlui and nhei restriction sites were introduced upstream and downstream of the orf for vsv-g, respectively. next, a cassette consisting of the coding sequences of vsv-g and egfp, separated by a minimal intergenic region [ ] was cloned and inserted into the parental vsv genome making use of mlui and nhei restriction sites, thereby replacing the genetic information of vsv-g and thus generating vsv à (the asterisk stands for egfp). for convenient exchange of vsv-g or egfp orfs by other transcription units, unique asci and noti restriction sites were added upstream and downstream of the vsv-g and egfp orfs, respectively. in order to generate vsv à mutant vsv-g (lxxxl), the respective orf was amplified from the corresponding vsv-g (lxxxl) expression plasmids with primers adding ' mlui and ' asci restriction sites and inserted into vsv à , thereby replacing the parental orf coding for wt vsv-g. the integrity of all pcramplified sequences was confirmed by automated sequencing. release of virus-like particles (vlps) and its inhibition by tetherin has been examined as described [ , ] . in brief, t cells were seeded in -well plates and cotransfected with plasmids encoding hiv- p -gag, tetherin and a potential tetherin antagonist or empty plasmid, using the calcium phosphate method. at h post transfection, the transfection medium was replaced by fresh culture medium. for blockade of vsv-g-dependent tetherin antagonism, anti-vsv-g hybridoma supernatant (i , concentrated mouse hybridoma supernatant from crl- , atcc) was added to the culture medium at a final dilution of : , . at h post transfection the supernatants were collected and cells were lysed in μl of x sds-containing lysis buffer ( mm tris [ph . ], % glycerol, % sds, % β-mercaptoethanol, . % bromophenol blue, mm edta). the lysates were incubated at ˚c for min. the supernatants were cleared by centrifugation and vlps were pelleted from cleared supernatants by centrifugation through a % sucrose cushion. the concentrated vlps were lysed in μl x sds loading buffer and incubated at ˚c for min. subsequently, cell lysates and supernatants were analyzed for the presence of gag employing western blot. for immunoblotting, the proteins were separated via sds-polyacrylamid (paa) gel electrophoresis using a . % paa gel and transferred onto a nitrocellulose membrane (ge healthcare life sciences, solingen, . μm). the membranes were blocked in % milk powder in pbs supplemented with . % tween and gag-protein was detected using : diluted supernatants of hybridoma cells secreting a mouse anti-gag antibody ( -h - c). expression of vsv-g wt and mutants was detected using the aforementioned anti-vsv-g hybridoma supernatant at a dilution of : while vsv-m was detected using mouse monoclonal antibody h (kerafast, boston). tetherin expression was detected employing anti c-myc-hybridoma supernatants (c-mycl- e (ecacc )). expression of vsv proteins, vpu and ebov-gp was detected using previously described rabbit sera [ ] [ ] [ ] . expression of ß-actin was detected using mouse anti-ß-actin antibody (sigma-aldrich, munich) at a dilution of : , . bound antibodies were detected using hrp-coupled anti-mouse and anti-rabbit secondary antibodies (dianova, hamburg) at a final concentration of . μg/ml. binding of secondary antibodies was detected using a self-made ecl solution ( . m tris-hcl ph . , μg/ml luminol (sigma-aldrich, münchen), mg/ml para-hydroxycomaric acid (sigma-aldrich, münchen); . % h o ) and signals were visualized using the chemocam imaging system and the chemostar-professional software (intas). for quantification of the signal intensity, the program imagej (fiji distribution) [ ] was used. gag signals detected in the supernatants were normalized against the respective signals obtained in cell lysates. for the rescue of vsv à harboring wt or mutant vsv-g, we employed a strategy developed by others [ ] with slight modifications. first, bhk- cells were grown in -well dishes until they reached~ % confluency. at this point, the cells were infected with recombinant modified vaccinia virus ankara expressing t polymerase ( [ ] , vmva-t , kindly provided by g. sutter) at a multiplicity of infection (moi) of . at h post infection, the inoculum was removed and the cells were transfected with expression plasmids for vsv-n, -p and -l (see above) as well as the respective, plasmid-encoded vsv à anti-genome (the genome is preceded by a t promotor sequence and followed by a hepatitis delta virus ribozyme and a t terminator sequence for cytoplasmic production of negative-sensed viral genomes that are template for transcription and genome replication) using lipofectamine (thermofisher scientific, dreiech) as transfection reagent. the following dna concentrations were used (per well): . μg vsv-n, . μg vsv-p, . μg vsv-l and . μg plasmid-encoded vsv à or vsv à (lxxxl) genome. transfection was carried out in optimem medium (thermofisher scientific, dreiech) for h, before the transfection medium was replaced by standard culture medium. at h post transfection, the culture medium was replaced by medium containing μg/ml rifampicin and μg/ml cytosine β-d-arabinofuranoside (both from sigma-aldrich, münchen) to limit rmva-t replication. after an additional h, the culture medium was collected, clarified from cellular debris by centrifugation ( , rpm, min, ˚c) and twice filtered through a membrane filter with a pore size of . μm to eliminate rmva-t from the preparation. next, fresh bhk- cells grown in t- flasks were inoculated with a : dilution of the passage (p ) to amplify vsv à for virus stock production (p ). quantification of titers from vsv à stocks and cell culture supernatant was carried out on confluently grown bhk- cells seeded in -well plates. after removal of the cell culture supernatant, cells were inoculated with -fold serial dilutions of virus (diluted in serum-free medium). at h post infection, the inoculum was removed and cells were overlaid with culture medium containing % methylcellulose (sigma-aldrich, münchen). after an incubation of - h, egfp-positive cells were counted under the fluorescence microscope to determine viral titers (displayed as focus forming units per ml, ffu/ml). in addition, vsv à titers were also determined for hela cells as this cell line is less susceptible to vsv infection (~ x) and therefore titers determined on bhk- cells are not useful to calculate the optimal infectious dose for hela cells. to verify the authenticity of wt and mutant vsv-g in vsv à , viral rna was isolated from p stocks and reverse-transcribed into cdna using the superscript iii first-strand synthesis system (thermofisher scientific, dreiech) according to the manufacturer's protocol (for random hexamers). next, a~ , bp fragment was amplified with primers binding in the intergenic region upstream of vsv-g (forward) and the ' end of the egfp orf (reverse) using phusion polymerase (thermofisher scientific, dreiech), separated by agarose gel electrophoresis and extracted from the gel by commercial kits (macherey & nagel, düren), before being subjected to automated sequence analysis (seqlab, göttingen). to assess the effect of directed tetherin expression on the spread of vsv à , t cells were transfected with increasing amounts of expression plasmid encoding for human tetherin. to avoid unspecific effects due to differences in the total dna amounts of the tetherin vector, empty expression plasmid was used for equilibration. cells transfected only with empty expression vector served as controls. at h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for h before they were infected. additionally, vero cells stably expressing human tetherin (vero-tetherin) or stably containing empty vector were used. in order to analyze the impact of sirna-mediated knockdown of endogenous tetherin expression on vsv spread, hela cells were transfected with sirna specific to human tetherin or scrambled sirna (control) (both santa cruz, dallas; pm final concentration) using lipofectamine and optimem medium (both from ther-mofisher scientific, dreieich). at h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for h before they were infected. for infection, vsv à stocks were diluted with serum-free medium to obtain the desired moi and inoculated onto target cells for h. afterwards, the inoculum was removed and cells were washed with pbs before receiving fresh culture medium. next, cells were further incubated and supernatants were collected for quantification of vsv à titers at different time points. to analyze vsv-g-driven host cell entry, vsv vectors were pseudotyped with either vsv-g wt or mutants a r and lxxxl. for this, t cells were transfected with the respective expression plasmids or empty plasmid as negative control. at h post transfection, cells were inoculated with vsv-g trans-complemented vsv à Δg that lacks the genetic information for vsv-g but codes for egfp and firefly luciferase from two independent transcription units [ ] (kindly provided by g. zimmer) at an moi of . at h post infection, the inoculum was removed and the cells were washed with pbs. next, medium containing a neutralizing antibody directed against vsv-g (i , produced from crl- hybridoma cells, atcc) was added to the cells and left for h in order to neutralize residual input virus that had not entered the cells so far. subsequently, the cells were again washed and further incubated with fresh culture medium for - h. then, the supernatant was collected, clarified from cellular debris by centrifugation ( , rpm, min, ˚c) and used for transduction experiments. for this, t cells were inoculated with identical volumes of the respective vsv pseudotypes and firefly luciferase activity in cell lysates was quantified at h post inoculation using a plate luminometer (hidex) and commercial substrates (pjk and promega) as described elsewhere [ ] . for analysis of the surface expression of tetherin, t cells were cotransfected with tetherin plasmid and plasmid encoding viral antagonist or empty plasmid as negative control. at h post transfection, the cells were washed and harvested in pbs. expression of tetherin at the cell surface was detected by employing a tetherin-specific mouse monoclonal antibody (biolegend) at a dilution of : and an alexa -conjugated anti-mouse secondary antibody at a dilution of : (thermofisher scientific, dreieich). subsequently, cells were fixed with % paraformaldehyde and staining was analyzed employing a lsr ii flow cytometer (bd biosciences, heidelberg) and the facs diva software (bd biosiences, heidelberg). the data were further analyzed using the fcs express flow research software (de novo software). to ensure that directed tetherin expression did not lead to unwanted cytotoxic side-effects at the concentrations used, we analyzed cell viability employing the celltitre-glo kit (promega, mannheim). for this, t cells grown in -well plates were transfected with increasing amounts of tetherin expression plasmid or empty vector, which was also used to equilibrate total dna amounts. at h post transfection, the transfection medium was replaced by fresh culture medium and the cells were further incubated for h. next, the cell culture supernatants were removed and μl of the celltitre-glo reagent were added. after an incubation period of min at room temperature, μl of the lysates were transferred into a white, opaque-walled -well plate, before luminescence was recorded using a plate luminometer (plate chameleon v, hidex, turku). all samples were analyzed in triplicates. for normalization, viability of cells transfected only with empty expression vector was set as % and relative viability of tetherin-transfected cells was calculated. if not explicitly stated in the figure legends, unpaired and paired student t-tests were performed to analyze data pairs originating from individual experiments or mean data of multiple experiments, respectively. one-way anova with bonferroni post-test analysis was carried out for combined comparison of multiple groups ( à , p . ; Ãà , p . ; ÃÃà , p . ). in order to analyze tetherin antagonism by vsv proteins, we employed a previously reported virus-like particle (vlp) release assay, which measures release of hiv-gag-based vlps from transfected t cells and its blockade by tetherin [ , ] . release of vlps was markedly diminished upon coexpression of tetherin and tetherin's antiviral activity was blocked by the well-established tetherin antagonists vpu and ebov-gp (fig a and b) , as expected. in addition, vsv-g but not vsv-l, m (mutant m(ncp), [ ] ) and p interfered with tetherin's antiviral activity, although less efficiently than vpu and ebov-gp (fig a and b) . finally, none of the viral proteins tested modulated vlp release from tetherin-negative control cells, indicating that the effects observed in tetherin-positive cells were specific. thus, vsv-g can antagonize tetherin in transfected cells, although with reduced efficiency as compared to ebov-gp and vpu. the studies described above were conducted with human tetherin. since vsv can infect livestock, we next analyzed if vsv-g also antagonizes pig-derived tetherin. we found that vpu failed to antagonize porcine tetherin (fig c) , in keeping with the well-established notion that the anti-tetherin activity of vpu is highly species specific [ ] [ ] [ ] . in contrast, both ebov-gp and vsv-g rescued vlp release from blockade by porcine tetherin (fig c) , indicating that vsv-g might be able to promote viral spread in infected pigs by antagonizing tetherin. evidence that tetherin antagonism by vsv-g can be blocked by a gprotein specific antibody and is not due to vsv-g overexpression we next sought to investigate whether tetherin counteraction by vsv-g can be inhibited by antibodies. this question was triggered by our previous observation that antibodies directed against ebov-gp can interfere with gp-mediated tetherin counteraction [ ] . to this end, we added supernatant of hybridoma crl- (which secretes the vsv neutralizing antibody i ) to cells expressing vsv-g and releasing vlps in the presence and absence of tetherin. the hybridoma supernatant did not modulate vlp release from tetherin-negative control cells coexpressing vsv-g but abrogated vlp-release from cells coexpressing tetherin and vsv-g (fig d and e) . we cannot exclude that the antibody triggered vsv-g internalization and a control antibody remains to be tested. however, the results available at present suggest that antibodies directed against vsv-g might not only block viral entry into target cells but may also interfere with tetherin antagonism. moreover, our findings indicate that the ectodomain of vsv-g plays an important role in tetherin counteraction. we next investigated whether the vsv-g expression levels attained in transfected cells exceeded those found in infected cells, which would suggest that tetherin counteraction by vsv-g could have been due to overexpression. for this, vsv-g expression levels in cells transfected to express vsv-g and cells infected with a recombinant vsv encoding gfp (vsv à ) were compared. cells were harvested at the same time points at which vlp release and (figs a- e and e and f) and vsv release (fig a- c and fig a- d ) from tetherin-positive cells were examined within functional assays. the immunoblot revealed that less g-protein was expressed in transfected as compared to infected cells (fig f) , indicating that tetherin antagonism was not due to overexpression. in this context, it should be noted that the lack of correlation between infectious dose and g-protein expression levels resulted from the fast replication kinetics of vsv, which led to % infected cells at the time of analysis. moreover, the reduced arrow heads indicate bands exhibiting the molecular weight expected for unglycosylated (white), partially (grey) and fully (black) n-glycosylated tetherin. (c) quantification of four experiments performed as described for panel (b). the intensities measured for all tetherin signals with a molecular weight between and kda were added to yield total tetherin expression. tetherin expression in the absence of antagonist (mock) was set to %. error bars indicate sem. one-way anova with bonferroni post-test analyses were performed for panels a and c to test for statistically significant differences between samples with and without (mock) antagonist (*, p . ). signals for actin in vsv-versus mock-infected cells can be attributed to the well-established cytotoxic effects associated with vsv infection [ , , ] . collectively, the results discussed tetherin antagonism by viral proteins is usually due to removal of tetherin from the site of viral budding, the plasma membrane [ , ] . in order to investigate whether vsv-g also interferes with tetherin expression at the plasma membrane, we performed flow cytometry with cells transfected to coexpress tetherin and viral antagonists. coexpression of vpu but not ebov-gp reduced tetherin surface levels (fig a) , as expected from previous reports [ , ] . in contrast, coexpression of vsv-g did not modulate surface levels of tetherin (fig a) . in agreement with these findings, neither vsv-g nor ebov-gp coexpression reduced tetherin levels in cell lysates while a marked decrease in tetherin levels was observed upon coexpression of vpu (fig b and c) . thus, vsv-g, like ebov-gp, employs a mechanism different from that of vpu for tetherin counteraction. upon coexpression of vsv-g and ebov-gp, a band with the size expected for unglycosylated tetherin accumulated (fig b) , which is known to exert reduced antiviral activity as compared to the fully glycosylated form [ ] . therefore, it is conceivable that vsv-g and ebov-gp inhibit tetherin by interfering with its n-glycosylation, similarly as reported for sars-coronavirus orf a protein [ ] . weidner and colleagues reported that vsv spread can be efficiently blocked by directed expression of tetherin [ ] , and a subsequent study demonstrated that tetherin mainly inhibits cell-free but not cell-associated vsv spread [ ] . we sought to confirm these findings and to establish a cellular system which allows investigating the potential contribution of vsv-gmediated tetherin counteraction to viral spread. for this, we transfected t cells with rising amounts of tetherin plasmid, infected the cells with vsv and quantified the number of infectious units present in culture supernatants at h post infection. we observed a modest but dose-dependent reduction of viral titers upon transfection of increasing amounts of tetherinplasmid (fig a) in the absence of appreciable cytotoxic effects (s fig). moreover, sirnamediated knock-down of endogenous tetherin expression in hela cells reduced the amount of cell surface associated tetherin (fig b) and increased viral titers roughly -fold as compared to cells transfected with scrambled sirna or mock transfected cells (fig c) . collectively, these findings confirm that vsv is inhibited by tetherin and indicate that knock-down of tetherin expression in hela cells affords the opportunity to examine whether vsv-g-mediated tetherin antagonism is operative in the context of infected cells. program is presented. expression of vsv-g wt was set to %. (c) incorporation of vsv-g wt and mutant lxxxl into vsv pseudotypes was investigated by western blot analysis using an anti-vsv-g antibody (concentrated supernatants from hybridoma cl- ). to ensure that similar amounts of pseudotypes were analyzed, levels of particle-associated m proteins were determined using an anti-vsv-m antibody. pseudotypes harboring no glycoprotein (mock) served as negative control. the results of a single immunoblot are shown from which irrelevant lanes were cut out. similar results were obtained in two separate experiments. (d) t cells were transduced with equal volumes of the vsv pseudotypes described in panel (c). at h post transduction, luciferase activity in cell lysates was measured. transduction driven by vsv-g wt was set as %. the average of three independent experiments is shown. error bars indicate sem. (e) t cells were cotransfected with plasmids encoding gag, the indicated glycoproteins and tetherin. expression of gag in supernatants and cell lysates was determined by western blot. detection of β-actin expression served as loading control. (f) the average of three independent experiments conducted as described for panel (e) and quantified via the imagej program is presented. error bars indicate standard error of the mean (sem). release of gag from cells coexpressing the highest amount of vsv-g and tetherin was set to %. for all graphs (b, d, f), paired two-tailed students' t-tests were performed to assess whether differences between vsv-g wt and lxxxl mutant were of statistical significance. https://doi.org/ . /journal.pone. .g a gxxxg motif in the transmembrane domain of vsv-g contributes to tetherin antagonism in transfected cells but is dispensable for vsv spread in tetherin-positive cells having established that vsv-g can antagonize tetherin upon directed expression and that vsv spread is reduced but not abrogated by tetherin, we finally sought to determine whether vsv-g-mediated tetherin antagonism contributes to viral spread. for this endeavor, we needed to identify a mutation in vsv-g that selectively interferes with tetherin antagonism. to this end, we tested a vsv-g mutant in which a gxxxg motif in the transmembrane domain was changed to lxxxl (fig ) . this mutation was chosen, since our unpublished results indicate that a gxxxa motif in the transmembrane domain of ebov-gp contributes to tetherin antagonism. in addition, we analyzed vsv-g mutant a r. this mutation is known to interfere with viral entry by rendering vsv-g defective for membrane fusion [ ] . both mutation a r and mutation of the gxxxg motif (mutant lxxxl) were compatible with robust g-protein expression (fig a and b ) and particle incorporation (fig c) . moreover, mutation of the gxxxg motif (mutant lxxxl) did not interfere with host cell entry of vsv-g pseudotypes (fig d) . in contrast, mutation a r markedly reduced entry efficiency (fig d) , as expected [ ] . finally, mutant a r was able to counteract tetherin while mutant lxxxl failed to efficiently antagonize tetherin (fig e and f ). thus, mutation of the gxxxg motif afforded an opportunity to investigate whether g-protein-mediated tetherin antagonism contributes to viral spread. for this, the lxxxl mutation was introduced into the vsv genome, employing a reverse genetics system, and infectious vsv was rescued. infection of sirna-transfected hela cells with vsv wt and mutant lxxxl at equal moi revealed that knock-down of tetherin expression increased vsv wt release, as expected. however, a comparable rescue of viral release was observed for the lxxxl mutant (fig a and b) . moreover, spread of vsv wt and mutant lxxxl was comparably reduced by directed expression of tetherin in vero cells (fig c and d) , suggesting that tetherin antagonism by vsv-g might not appreciably contribute to viral spread in tetherin-positive cells. tetherin and other effector proteins of the ifn system are responsible for the establishment of an antiviral state in ifn exposed cells [ , ] . many viruses evolved countermeasures against these antiviral effectors by either multi-functionalizing their structural proteins or by acquiring non-structural proteins with antagonistic activity. whether vsv, which is sensitive to blockade by tetherin [ , ] , encodes an antagonist that allows residual viral spread in tetherin-positive cells is unknown. here, we show that the surface protein of vsv, vsv-g, can antagonize tetherin, at least upon directed expression. tetherin counteraction by vsv-g did not involve modulation of tetherin expression and was partially dependent on a gxxxg motif in the vsv-g transmembrane domain. however, mutation of the gxxxg motif in the context of infectious vsv did not affect viral spread in tetherin-positive cells. thus, vsv-g is a novel tetherin antagonist but the potential contribution of its antagonistic activity to viral spread in tetherin-positive cells requires further investigation. previous studies reported that directed expression of tetherin in nih t cells and cells markedly diminished viral release [ , ] . however, tetherin expression failed to reduce viral release to background levels in both studies, although tetherin was expressed at high levels, which leaves the possibility that vsv encodes a modestly active tetherin antagonist. in keeping with such a scenario, we found that vsv-g antagonizes human and pig tetherin but does so less efficiently than vpu and ebov-gp. tetherin antagonism could only be demonstrated in the context of directed vsv-g expression in the present study. nevertheless, tetherin counteraction was not due to g-protein overexpression, indicating that vsv-g could block tetherin in infected cells, as discussed below. several viral tetherin antagonists inhibit tetherin by interfering with its expression or appropriate cellular localization. for instance, vpu can interfere with the anterograde transport of tetherin [ , ] and directs tetherin towards lysosomal degradation [ ] [ ] [ ] , which ultimately results in reduced tetherin levels at the cell surface. in contrast, vsv-g did not modulate total tetherin expression or tetherin levels at the cell surface. these findings are similar to those previously reported for ebov-gp [ , ] , which antagonizes tetherin by a so far unknown mechanism. moreover, vsv-g, like ebov-gp, was active against diverse tetherins, as demonstrated by the counteraction of porcine tetherin, which shares only % sequence identity with human tetherin. finally, our finding that antibody i directed against the vsv-g ectodomain interferes with tetherin antagonism recapitulates findings previously made for ebov-gp [ ] and suggests that both vsv-g and ebov-gp depend on their ectodomains for tetherin counteraction. how vsv-g (and ebov-gp) counteracts tetherin remains unknown and at present a role of direct interactions (potentially disrupted by anti-ectodomain antibodies) cannot be discounted. our finding that coexpression of vsv-g resulted in increased generation of unglycosylated tetherin, which is known to display little antiviral activity [ ] , suggests that vsv-g might block tetherin, at least in part, by interfering with its posttranslational modification. a similar inhibitory strategy has previously been reported for the sars-coronavirus orf a protein [ ] but the underlying mechanism is incompletely understood. in order to get initial insights into the contribution of vsv-g-mediated tetherin antagonism to vsv release from tetherin-positive cells, we examined previously characterized mutations in the ectodomain (a r) and the transmembrane domain (g l and g l, mutant lxxxl) of vsv-g for their effect on tetherin antagonism. a r markedly reduced vsv-gdriven entry, as expected [ ] , but did not affect tetherin antagonism. in contrast, mutation of the gxxxg motif in the transmembrane domain had no impact on entry but reduced tetherin antagonism by roughly %, as discussed below. the finding that the gxxxg motif was dispensable for vsv-g-driven virus-cell fusion was unexpected, since the same motif has been reported to be required for cell-cell fusion induced by exposure of vsv-g expressing cells to low ph [ ] . in this context, it is important to state that the integrity of the lxxxl mutations after amplification of the rescued viruses in bhk- cells (passage stocks) has been confirmed by sequencing. therefore, the gxxxg motif seems to be important for vsv-g-driven cell-cell but not virus-cell fusion and the underlying reasons remain to be determined. the finding that the gxxxg motif is required for tetherin antagonism is in keeping with our unpublished observation that a gxxxa motif in the ebov-gp transmembrane domain, which has been reported to be important for ebov-gp-mediated cellular detachment [ ] , is required for full tetherin antagonism by ebov-gp. although mutation of the gxxxg motif in vsv-g reduced tetherin antagonism in transfected cells, it had no impact on viral spread in hela cells, which express endogenous tetherin [ ] , and vero cells, which were engineered to express tetherin. these observations could indicate that vsv-g-mediated tetherin antagonism is not operative in vsv infected cells. in fact, one could speculate that in infected cells the interaction between vsv-g and other viral proteins, which is required for assembly of progeny particles [ ] , compromises the ability of vsv-g to counteract tetherin. alternatively, it is conceivable that the modest reduction of tetherin antagonism observed in transfected cells upon mutation of the gxxxg motif precluded detection of anti-tetherin effects in infected cells. the identification of a mutation in vsv-g that selectively and efficiently reduces tetherin antagonism is required to address this question. collectively, our results and published data indicate that vsv-g, ebov-gp [ ] and possibly other viral glycoproteins can interfere with tetherin's antiviral activity via a mechanism that is relatively independent of the tetherin sequence. glycoprotein-mediated interference with tetherin's n-glycosylation status might contribute to this effect. moreover, our results indicate that findings made for tetherin-antagonism in transfected cells might not always adequately reflect the situation in infected cells. finally, our findings suggest the possibility that vsv might acquire robust tetherin antagonism upon infection of humans. supporting information s fig. directed tetherin expression is not associated with major cytotoxic effects. t cells were transfected with increasing amounts of expression vector for human tetherin. empty vector was used for equilibration of total dna amounts. at h post transfection, intracellular atp levels were quantified. the average of three independent experiments performed with triplicate samples is shown, error bars indicate standard error of the mean (sem). a paired two-tailed student's t-test was used to examine whether differences in cell viability between cells transfected with empty vector ( μg tetherin plasmid) and tetherin-expressing cells were of statistical significance (ns, not significant; à , p . ). (pdf) vesicular stomatitis transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice emergence and re-emergence of vesicular stomatitis in the united states vesicular stomatitis virus: re-inventing the bullet oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p , ras, or myc function and involves the induction of apoptosis oncolytic vesicular stomatitis virus as a viro-immunotherapy: defeating cancer with a "hammer" and "anvil assessing the safety and immunogenicity of recombinant vesicular stomatitis virus ebola vaccine in healthy adults: a randomized clinical trial six-month safety data of recombinant vesicular stomatitis virus-zaire ebola virus envelope glycoprotein vaccine in a phase double-blind, placebo-controlled randomized study in healthy adults safety and immunogenicity of the rvsvg-zebov-gp ebola virus vaccine candidate in healthy adults: a phase b randomised, multicentre, double-blind, placebo-controlled, dose-response study prrs are watching you: localization of innate sensing and signaling regulators pattern recognition receptors and the innate immune response to viral infection interferon-stimulated genes: a complex web of host defenses a diverse range of gene products are effectors of the type i interferon antiviral response the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter factors affecting the sensitivity of different viruses to interferon rhabdovirus evasion of the interferon system tetherin inhibits cell-free virus dissemination and retards murine leukemia virus pathogenesis interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms tetherin inhibits retrovirus release and is antagonized by hiv- vpu the antiviral activities of tetherin tetherin inhibits hiv- release by directly tethering virions to cells counteraction of the multifunctional restriction factor tetherin differential effects of human immunodeficiency virus type vpu on the stability of bst- /tetherin vpu directs the degradation of the human immunodeficiency virus restriction factor bst- /tetherin via a {beta}trcp-dependent mechanism vpu antagonizes bst- -mediated restriction of hiv- release via beta-trcp and endo-lysosomal trafficking tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein the ebola virus glycoprotein and hiv- vpu employ different strategies to counteract the antiviral factor tetherin ebola virus glycoprotein counteracts bst- /tetherin restriction in a sequence-independent manner that does not require tetherin surface removal anti-tetherin activities of hiv- vpu and ebola virus glycoprotein do not involve removal of tetherin from lipid rafts recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues short tandem repeat typing technologies used in human identity testing the tetherin antagonism of the ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by gp -specific antibodies fusionactive glycoprotein g mediates the cytotoxicity of vesicular stomatitis virus m mutants lacking host shutoff activity tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv- strains the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes human immunodeficiency virus type vpu protein is an oligomeric type i integral membrane protein modulation of virion incorporation of ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization fiji: an opensource platform for biological-image analysis recombinant vesicular stomatitis viruses from dna non-replicating vaccinia vector efficiently expresses bacteriophage t rna polymerase a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon the glycoproteins of all filovirus species use the same host factors for entry into bat and human cells but entry efficiency is species dependent hiv- antagonism of cd is species specific and involves vpu-mediated proteasomal degradation of the restriction factor species-specific activity of siv nef and hiv- vpu in overcoming restriction by tetherin/bst species-specific activity of hiv- vpu and positive selection of tetherin transmembrane domain variants the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup severe acute respiratory syndrome coronavirus orf a inhibits bone marrow stromal antigen virion tethering through a novel mechanism of glycosylation interference molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein g, a class iii viral fusion protein hiv- vpu antagonizes bst- by interfering mainly with the trafficking of newly synthesized bst- to the cell surface hiv- vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor cd /tetherin to overcome the virion release restriction the transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus g protein inhibition of ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein the following reagents were obtained through the nih aids reagent program, division of aids, niaid, nih: anti-hiv- nl - vpu polyclonal antiserum from dr. klaus strebel, hela cells from dr. richard axel. polyclonal antiserum against vsv was kindly provided by dr. georg herrler. key: cord- -d h bp authors: carrion, ricardo; ro, youngtae; hoosien, kareema; ticer, anysha; brasky, kathy; de la garza, melissa; mansfield, keith; patterson, jean l. title: a small nonhuman primate model for filovirus-induced disease date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: d h bp ebolavirus and marburgvirus are members of the filovirus family and induce a fatal hemorrhagic disease in humans and nonhuman primates with % case fatality. to develop a small nonhuman primate model for filovirus disease, common marmosets (callithrix jacchus) were intramuscularly inoculated with wild type marburgvirus musoke or ebolavirus zaire. the infection resulted in a systemic fatal disease with clinical and morphological features closely resembling human infection. animals experienced weight loss, fever, high virus titers in tissue, thrombocytopenia, neutrophilia, high liver transaminases and phosphatases and disseminated intravascular coagulation. evidence of a severe disseminated viral infection characterized principally by multifocal to coalescing hepatic necrosis was seen in ebov animals. marv-infected animals displayed only moderate fibrin deposition in the spleen. lymphoid necrosis and lymphocytic depletion observed in spleen. these findings provide support for the use of the common marmoset as a small nonhuman primate model for filovirus induced hemorrhagic fever. ebolavirus (eboz) and marburgvirus (marv), members of the filoviridae family, are causative agents of hemorrhagic fever. filovirusinduced disease begins with flu-like symptoms and progresses rapidly to the final stages of viral hemorrhagic fever infection, which are characterized by fever, hemorrhage, and hypotensive shock (groseth et al., ; zampieri et al., ) . infection is fatal in up to % as in the case of zaire ebolavirus or marburg angola. the increased frequency of filovirus outbreaks in central and western africa and the potential use of such agents as biological weapons underscore the need to understand pathogenesis of these viruses and to develop effective intervention strategies (groseth et al., ; peterson et al., a,b) . nonhuman primates (nhps) are the preferred animal model for human filovirus infection because infection with eboz and marv isolated from humans results in fatal hemorrhagic disease. numerous species of nhps have been used, including baboons, african green monkey, rhesus and cynomolgus macaques, to study filovirus pathogenesis because the pathology of infected nhps is similar to that seen in humans (bente et al., ; geisbert et al., b) . the sporadic nature of disease outbreaks and the ethical issues associated with conducting a human vaccine trial make such a study difficult to execute. early-stage development of vaccines has historically occurred in mouse and guinea pig models of filovirus disease, but these models use adapted viruses obtained through sequential passage in the rodent species because the wild-type virus does not cause uniform lethality (bray et al., ; connolly et al., ) . the disease observed in rodents does not reproduce the extensive disseminated intravascular coagulation seen in human filovirus disease. besides the requirement for an adapted virus, there are reports that the rodent models are not necessarily predictive of efficacy in nhps (feldmann et al., ; geisbert et al., ; jahrling et al., ) . nhps are relevant models to study infectious disease as their immune system is similar to humans and they are good predictors of efficacy in vaccine development and other intervention strategies. licensure of a filovirus vaccine will require testing in a nhp species. the macaque model is the most frequently used model in filovirus efficacy studies involving vaccines or therapies. given the drawbacks of rodent models of filovirus disease, there is a critical need for development of a small animal model that develops disease consistent with human infection using wild-type virus. the common marmoset (callithrix jacchus) is an anthropoid primate weighing between and g that is used extensively in biomedical research and provides an attractive alternative to other nhps (mansfield, ) . this small-bodied primate is especially valuable in maximum containment research, where space is generally at a premium. additionally, reagents are available to characterize immunological response to vaccines. marmosets have been used to study a number of viral diseases including hemorrhagic fever viruses such as lassa and junin viruses, as well as eastern equine encephalitis virus, sars-cov, and hepatitis gb virus b, as well as other human syndromes (adams et al., ; avila et al., ; bright et al., ; carrion et al., a; greenough et al., ; jacob et al., ; lukashevich et al., ; mansfield, ; weatherford et al., ; weissenbacher et al., ) . marmoset models of lassa hemorrhagic fever and argentine hemorrhagic fever have been used to validate countermeasures against the hfvs (avila et al., ; carrion et al., b; samoilovich et al., ; weissenbacher et al., a,b) . here we report that a single intramuscular injection of a common marmoset with as little as plaque forming units (pfu) of either marv or ebov resulted in fatal hemorrhagic disease. the experimental filovirus infections induced a systemic disease with histological features similar to human infection, most notably hepatocellular necrosis and extensive fibrin deposition. the availability of a marmoset model for filovirus induced disease is especially attractive since it provides a small animal model for preclinical trials that is susceptible to viruses isolated from humans without adaptation. all animals appeared normal until days post-infection (dpi) when weight loss and anorexia was observed. animals infected with pfu ebov were also febrile (n °f) at this time point. animals infected with pfu of ebov developed a fever dpi. similarly, marv infected animals developed a fever at days and for the high dose and low dose, respectively. after the onset of fever, the behavior of animals changed: they became depressed, had reduced stool production, and weight loss ( - % from baseline). just before death, febrile response resolved and some animals became hypothermic. marmosets infected with ebola virus succumbed to disease at to days dpi while marv infected animals succumbed to disease at six to eight dpi. complete blood counts and biochemical analyses (table ) were performed on blood collected from marmosets. beginning at dpi, blood contained elevated levels of serum alanine aminotransferases (alt), a marker of hepatocellular necrosis, which continued to increase until the animal was euthanized. ggt (gamma-glutamyltransferase) and alp (alkaline phosphatase), measured to evaluate excretory liver functions, were elevated n -fold above baseline. further evidence of hepatic involvement was noted in marv animals as a significant increase in total bilirubin (tbil) observed just prior to death (n mg/dl). hematological data showed a reduction in numbers of platelet in all animals over the course of infection with development of leukocytosis ( fig. ) . all animals experienced an increase in neutrophils and concomitant decrease in lymphocytes beginning at dpi (fig. ). the virulence of filoviruses in humans is related to viremia, suggesting that rate of virus replication is an important factor of pathogenesis (towner et al., ) . at necropsy, tissue was collected from marmosets for determination of viral load by qrt-pcr. virus was detected by dpi, resulting in viremia greater than to log genome equivalents (ge) per milliliter on the day of necropsy. all tissues contained amounts of viral rna comparable with levels of viremia (table ) . at necropsy, differences in peak viral rna level in the tissues of animals infected with low ( pfu) and high ( pfu) doses of ebov was observed. viral load in blood from marv infected animals ranged from . log at day to . log at day . lesions seen in both ebov marmosets and marv marmosets were similar. hepatomegaly was seen in all animals and was often accompanied by multifocal pigmentation and overall discoloration of the liver. splenomegaly was also a common finding at necropsy. most animals had lung abnormalities in most lobes, primarily hemorrhage and congestion. hemorrhage was frequently noted at the site of venipuncture. other findings included hemorrhage in bladder and adrenals as well as light pigmentation present in lymph nodes. microscopic examination of tissue sections stained with hematoxylin and eosin revealed a number of pathologic lesions (tables and ) . renal lesions were observed in all ebov-infected animals ( fig. a) , and consisted of fibrin microthrombi lodged within capillary beds of the glomerular tufts. thrombi were apparent diffusely and often associated with expansion of bowman's space and accumulation of proteinaceous fluid. polymorphonuclear cells were infrequently observed in glomerular tufts and glomerular cells were necrotic. in conjunction, hypoxic nephrosis was evident in the proximal convoluted tubules of most animals and was likely secondary to microthrombosis of the glomerular tufts. marv-infected animals were dissimilar: renal morphology was within normal limits (fig. a ). lungs from all animals revealed fibrin thrombi present within large-to medium-sized pulmonary arteries (fig. b ). thrombi often contained increased numbers of neutrophils and pyknotic debris. perivascular lymphatics adjacent to these vessels were often dilated and contained protein. lungs were diffusely congested and multiple foci of fibrin microthrombi were evident in many small arterioles, extending into capillary beds and associated with congestion and hemorrhage. occasionally extravasated erythrocytes were mixed with evidence of proteinaceous exudates in alveolar spaces. in contrast, pulmonary changes were minimal to mild in marvinfected marmosets. one animal ( ) receiving pfu and one receiving pfu developed mild edema surrounding small pulmonary arteries. in addition, three animals had evidence of a mild focal interstitial pneumonitis characterized by neutrophilic infiltration, septal necrosis and variable alveolar edema. it is unlikely that the pulmonary findings were of clinical significance. hepatic lesions in ebov-infected marmosets were observed in all animals but differed dependent on the dose and day of death (fig. c ). in animals inoculated with pfu of ebov, hepatic lesions were mild and consisted of multifocal infiltrates of polymorphonuclear cells within hepatic sinusoids associated with evidence of early necrosis of sinusoidal cells. viral inclusions were not observed. in animals inoculated with the pfu and surviving to dpi, hepatic disease was more severe and characterized by multifocal to coalescing hepatocellular coagulative necrosis infiltrated by small to moderate numbers of neutrophils. hepatocytes within and adjacent to these regions of necrosis often contained large amphophilic intracytoplasmic inclusions (fig. d ). inclusions had an asymmetric perinuclear distribution. these more severe changes were accompanied by intracellular biliary stasis. the findings suggest initial targeting of resident sinusoidal cells (kupffer and monocytes) with subsequent extension to adjacent hepatocytes. marv-infected animals all displayed multifocal to coalescing hepatocellular necrosis. necrotic foci varied from individual hepatocytes to small aggregates of cells up to μm in diameter. necrotic hepatocytes had rounded and deeply eosinophilic homogeneous to granular appearing cytoplasm with pyknotic or karyorrhectic nuclei (fig. b ). occasionally, hepatocytes in the early phases of degeneration had well defined eosinophilic bodies present within the cytoplasm (eosinophilic degeneration) but the amphophilic inclusions were infrequently observed (fig. c) . dispersed within and adjacent to these regions of necrosis were increased numbers of neutrophils found within sinusoids and mixed with the necrotic debris. in four animals there was evidence of mild to moderate hepatocellular dissociation. the degree of hepatocellular necrosis was similar regardless of inoculum. spleens of all ebov-infected animals were depleted of lymphocytes and contained deposits of fibrin (fig. e) . extensive fibrin deposits were observed within the red pulp. these changes were accompanied by necrosis of cells within the cords of billroth in severely affected animals. lymphocyte depletion was severe and appears to be focused on periarteriolar sheaths (t cell regions) and to a lesser extent follicular (b cell) regions. in most residual areas of white pulp, pyknotic and karyorrhectic nuclear debris was evident and these regions were infiltrated by polymorphonuclear cells at multiple foci. in marmosets given pfu of virus, involvement of b cell regions was significantly less. in contrast, all animals receiving pfu marv inoculum had evidence of marked lymphocyte depletion and lymphoid necrosis in the spleen (figs. d and e). periarteriolar sheaths and follicular b cell areas were reduced in size and contained pyknotic and karyorrhectic debris. the changes were present to a lesser degree in the pfu group, suggesting that viral dose may have been responsible for the observed differences. fibrin deposition was observed in the medullary cords and correlated with the degree of lymphoid necrosis and lymphocytic depletion. in two ebov animals, mild to moderate lymphocytic hyperplasia was observed in cortical parafollicular regions, accompanied by increased numbers of tingible body macrophages. in these nodes, scattered areas of hemorrhage and necrosis were occasionally apparent. in contrast, in the two remaining animals, there was evidence of extensive lymphocytic necrosis that was focused in medullary and parafollicular regions, while follicles were spared. these changes were accompanied by hemorrhage, neutrophilic infiltration and extensive fibrin deposition (fig. f) . changes in lymph node morphology were apparent in only of marv-infected animals, and consisted of mild to moderate lymphoid necrosis and lymphocytic depletion. in the remaining animals, there was an absence of secondary follicles with germinal centers but lymphocyte numbers appeared normal (fig. f) . fibrin microthrombi were observed in the adrenal glands of some ebov-infected animals and were most frequently observed in the subcapsular region in the zona glomerulosa. thrombi were accompanied by congestion and hemorrhage. multifocal dissociation and necrosis of cortical adrenocytes was evident in a subset of animals and was occasionally accompanied by infiltration by a small number of polymorphonuclear cells. no viral inclusions were evident. fibrin was not observed in marv animals. within adrenal glands there were multiple scattered areas of necrosis of individual and small aggregates of adrenocytes accompanied by the presence of erythrocytes, lymphocytes and mononuclear cells. lesions were observed inconsistently in other organs and were often related to the effects of disseminated intravascular coagulation and terminal hypoxia. these included evidence of myocardial hypoxic necrosis, and microhemorrhages in the brain, heart, urinary bladder and pancreatic islets. two animals infected with pfu of marv had evidence of a mild focal fibrosis, which dissected along muscle fascicles and was associated with scant histiocytic and lymphocytic infiltrates. findings in other organs were unremarkable. we report for the first time that the common marmoset is susceptible to experimental infection with viruses from the family filoviridae. the intramuscular inoculation of as little as pfu of either ebov or marv induced pathological features similar to those observed in human disease. most notably, animals experienced thrombocytopenia, neutrophilia and disseminated intravascular coagulation. furthermore, the small nonhuman primate experiences a disease syndrome comparable to what has been reported in other nonhuman primate models currently used to study filovirus disease. cynomolgus macaques are frequently used in preclinical testing of filovirus vaccine and therapeutic strategies and are considered the "gold standard" animal model (bente et al., ) . experimental ebov infection of macaques by intramuscular injection of pfu of virus results in a rapid fatal disease. beginning at dpi, macaques experienced anorexia coinciding with the onset of fever. shortly after these initial findings, petechial rash was seen with varying degrees of recumbency, culminating in prostration and death at days post-challenge (geisbert et al., d) . we observed a similar, although shortened (death - dpi), course of disease in marmosets infected with ebov. previous work has shown that intramuscular inoculation of macaques with marv results in an analogous course of disease; however, overall disease progression was delayed with death occurring between days and (bente et al., ; geisbert et al., ; jaax et al., ) . consistent with the macaque model, experimental inoculation of marmosets with marv also results in delayed onset of disease, with death occurring to days later than that seen with marmosets infected with ebov. however, the course of marburg disease in marmosets was more rapid than that seen in macaques, with death occurring at to dpi. although not statistically significant, it is interesting to note that marv infected marmosets had an average time to death day shorter than those receiving a high dose of virus. human filovirus infection is marked by neutrophilia, lymphopenia and thrombocytopenia. these findings are observed in the macaque model of filovirus disease and are used as surrogate markers of disease progression for in vivo studies (geisbert et al., b; simpson, ; simpson et al., ) . marmosets infected with either of the filoviruses also display these hematological abnormalities. beginning at dpi, overall platelet counts decreased while neutrophil numbers increased, with a concomitant decrease in lymphocyte numbers (fig. ) . autopsy findings from fatal human cases reveal hepatomegaly with necrotic foci observed throughout liver. likewise, experimentally infected macaques developed numerous hepatic lesions (geisbert et al., b (geisbert et al., ,c,d, jaax et al., ) . the marmoset showed biochemical signs of liver involvement early in infection: markers of liver function (alt, alp, ggt) reached levels in excess of -fold above baseline at necropsy. consistent with high-levels of liver enzymes, gross examination of the liver revealed hepatomegaly with pale foci throughout all lobes. microscopic examination of sections from the liver revealed necrosis with mild to moderate inflammation. hepatic changes did not appear to be virus dose-dependent, although further animal or morphometric studies would be needed to confirm this. evidence of mild to moderate hepatocellular fatty change was present. similar findings have been documented in the macaque model of filovirus infection and most importantly, the findings reported here are comparable to those reported in fatal human cases (geisbert et al., b (geisbert et al., ,c, jaax et al., ) . one difference between the macaque model of filovirus infection and marmosets is that marmosets do not develop a petechial rash. in this respect, they appear to be more similar to the african green monkey model of filovirus infection (bente et al., ; davis et al., ) . we speculate that the rapid disease progression preempts its development. nonetheless, clear signs of coagulation abnormalities are pronounced, as animals experience thrombocytopenia, hemorrhage and bleeding at sites of venipuncture. fatal human cases are characterized by hemorrhage and bleeding at site of venipuncture and other coagulation abnormalities. the coagulopathy observed in humans at times exists in the absence of rash: only % of patients infected with ebov develop a maculopapular rash (bwaka et al., ) . further evidence that the marmoset mimics human disease is that microscopic examination of tissue from ebov-infected animals reveals widespread fibrin deposition that is a hallmark of coagulation abnormalities (geisbert et al., c,d; jaax et al., ) . ebov infection of the marmoset caused a severe disseminated viral infection characterized principally by microthrombosis in multiple organs (disseminated intravasculature coagulation). to a lesser degree, marvinfected animals displayed moderate fibrin deposition in the spleen. a similar observation has been reported for rhesus macaques infected with marburgvirus angola where deposition of polymerized fibrin was less pronounced than in ebov infected macaques (geisbert et al., ) . these findings are similar to that seen in human infection and the macaque (geisbert et al., a,b,c; jaax et al., ) . signs of coagulopathy characteristic of primate infections are observed variably in rodent models (bente et al., ; bray et al., ; connolly et al., ) . high levels of viremia in human filovirus infections are associated with high mortality. a -log difference in titers is sufficient to predict clinical outcomes (towner et al., ) . inoculation of macaques with either ebov or marv results in high viral loads in both blood and tissue (geisbert et al., d ). in the current study, all marmosets had high virus loads (n ) in blood and tissue regardless of dose or agent. consistent with human cases, virus was widely disseminated, with the spleen and liver frequently having levels -log higher than other tissue. in animals given pfu ebov, viral load was -logs less than those in animals receiving pfu of virus. this difference could be a result of a rapid onset of morbidity affecting viral load. virus load in animals infected with marv was similar and likely the result of relatively longer time to death compared to ebov animals. it is worthwhile noting that recently it has been shown that filovirus replication results in a surplus of genomes with varying degrees of packaging efficiency and infectious particles. in the in vitro studies, the ratio of rna to focus forming units was -fold more in ebov versus marv (weidmann et al., ) . taking this into account, the difference in virus load between marv and ebov infected marmosets may be correspondingly different. differences in histopathology were observed between animals inoculated with marv and ebov. ebov infection resulted in widespread intravascular coagulation apparent in multiple organs including the spleen, adrenal gland, kidney and lung ( fig. and table ). there was evidence of extensive lymphocytic necrosis focused in medullary and parafollicular regions, while follicles were spared. these changes were accompanied by hemorrhage, neutrophilic infiltration and extensive fibrin deposition (fig. f) . findings suggest initial activation within t lymphocyte areas followed by subsequent and rapid lymphocytic depletion, most likely through apoptotic mechanisms. comparing changes to changes observed in the spleen suggests alterations in the lymph node lag behind those observed in the spleen. marv animals inoculated with pfu had moderate fibrin deposition in the spleen, widespread intravascular coagulation was not observed, suggesting a significant difference in pathogenesis between the two agents ( fig. and table ). the observation that hepatic lesions are less severe in animals receiving high dose of virus versus a lower dose is difficult to explain with a small sample size. the virus preparation used to infect the monkeys was prepared at a low moi in order to minimize the effect of di particles. it is known in filovirus infection, hepatic lesions early in infection are less severe than those at terminal endpoints (geisbert et al., a, b,c,d) . perhaps the relatively quick time to death in those animals infected with high dose of virus preempted the development of severe liver pathology. our study has shown that intramuscular inoculation of marmosets with ebov or marv causes hemorrhagic fever reminiscent of human infection. since nonhuman primates are more predictive of filovirus therapeutic efficacy than rodents, the small-sized marmoset is a viable alternative small animal model. the marmoset size is comparable to that of a guinea pig, making it attractive for testing therapeutic reagents that are only available in small quantities. in addition, the neotropical monkey can serve to alleviate the shortage of macaques available for research (patterson & carrion, ; satkoski et al., ) . that the common marmoset is susceptible to wild-type, non-adapted virus is especially attractive to those wishing to evaluate countermeasures in a small animal model with viruses derived from fatal human cases. biosafety eboz and marv are risk group , category a biothreat agents. all experiments with this virus were performed within a biosafety level (bsl ) facility by personnel in a biosafety suit at the texas biomedical research institute (san antonio, tx). both the institutional animal care and use committee and the institutional biohazards committee at texas biomed approved experimental protocols. virus strains ebov (kikwit) and marv musoke were provided by dr. peter jahrling (usamriid, fort detrick, maryland) . stocks were used to infect tissue culture flasks containing vero e cells at % confluency. the flasks were infected at an moi of . then incubated until cytopathic effect greater than % was observed then harvested and stored in aliquots in an ultralow temperature freezer. infectivity of virus stocks was determined by plaque titration (moe et al., ) . ten adult marmosets ranging in weight from g to g were obtained from the southwest national primate research center in san antonio, texas. one week before the start of the study, animals were transferred to the bsl facility at texas biomed and housed individually in a climate controlled caging. at day , animals were injected intramuscularly ( . ml) with either ebov ( pfu, n = ; pfu, n = ) or marv ( pfu, n = ; pfu, n = ) diluted in pbs. animals were sedated and blood was collected at days , , and for analysis. animals were euthanized when moribund using approved methods. all procedures were approved by the texas biomed institutional animal care and use committee and were performed in accordance with applicable federal guidelines. biochemical analysis of plasma samples was performed using mammalian liver enzyme profile rotor on vet scan analyzer (abaxis, inc., union city, ca). complete blood counts were performed using a vet hmt machine (abaxis, inc., union city, ca). tissue was collected using sterilized scalpels and forceps. to minimize contamination of tissue with blood, tissue samples were harvested after blood collection. aseptically collected tissues were fixed in phosphatebuffered % paraformaldehyde (ph . ) and embedded in paraffin for histological examination. paraffin-embedded tissues were cut in -μm sections, deparaffinized and stained with hematoxylin and eosin. total rna isolation and qrt-pcr viral load in tissues were measured by quantitative rt-pcr (qrt-pcr). tissue was homogenized with trizol reagent (invitrogen) using the tissuelyser (qiagen inc., valencia, ca) homogenization system and rna extracted per the manufacturer's recommendations. real-time qrt-pcr was performed using an abi prism sequence detection system (applied biosystems, foster city, ca) and rna ultrasense onestep real-time qrt-pcr system (invitrogen). the ebov qrt-pcr assays were performed as previously described (sun et al., ) . marv qrt-pcr was performed as described above using a primer/probe set directed at a region within the marv glycoprotein (magp-f: ′ggccttcaggg-caggtgta ′, magp-r : ′ cctgtgcatgagggttttga ′, mmgp-probe: ′ fam-ccttgctgttagatcctcctaccaa-tamra ′). common marmosets (callithrix jacchus) as a nonhuman primate model to 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ebola virus we thank jerritt nunneley, michele reynolds, robert geiger, hilary staples, and juan zapata for technical assistance and helpful discussions. we thank bernadette guerra and robert lanford of the texas biomed molecular core laboratory for assistance with qrt-pcr assays. we thank sandra rios, maria messenger and april hopstetter for assistance in preparing this manuscript and anthony griffiths for critical review of this manuscript.financial support: this work was supported by the national institutes of health (nih) for regional centers of excellence for biodefense and emerging infectious diseases (grants u ai and u ai ), an nih laboratory construction grant (c rr ), and a grant from the southwest foundation forum. we acknowledge the new england primate center (grant p rr - ) for histopathology support and the southwest national primate research center base grant (p rr ). key: cord- -v y xc authors: horigan, verity; gale, paul; kosmider, rowena d.; minnis, christopher; snary, emma l.; breed, andrew c.; simons, robin r.l. title: application of a quantitative entry assessment model to compare the relative risk of incursion of zoonotic bat-borne viruses into european union member states date: - - journal: microb risk anal doi: . /j.mran. . . sha: doc_id: cord_uid: v y xc this paper presents a quantitative assessment model for the risk of entry of zoonotic bat-borne viruses into the european union (eu). the model considers four routes of introduction: human travel, legal trade of products, live animal imports and illegal import of bushmeat and was applied to five virus outbreak scenarios. two scenarios were considered for zaire ebolavirus (webov, cebov) and other scenarios for hendra virus, marburg virus (marv) and middle east respiratory syndrome coronavirus (mers-cov). the use of the same framework and generic data sources for all eu member states (ms) allows for a relative comparison of the probability of virus introduction and of the importance of the routes of introduction among mss. according to the model webov posed the highest risk of an introduction event within the eu, followed by marv and mers-cov. however, the main route of introduction differed, with webov and mers-cov most likely through human travel and marv through legal trade of foodstuffs. the relative risks to eu mss as entry points also varied between outbreak scenarios, highlighting the heterogeneity in global trade and travel to the eu mss. the model has the capability to allow for a continual updating of the risk estimate using new data as, and when, it becomes available. the model provides an horizon scanning tool for use when available data are limited and, therefore, the absolute risk estimates often have high uncertainty. sensitivity analysis suggested virus prevalence in bats has a large influence on the results; a % reduction in prevalence reduced the risk of introduction considerably and resulted in the relative ranking of marv falling below that for mers-cov, due to this parameter disproportionately affecting the risk of introduction from the trade route over human travel. bats are natural reservoir hosts for many viruses which are recognised as serious potential threats to human and/or animal health (calisher et al., ) . the bat-borne viruses emerging in the african, asian and australian continents have come to the fore more recently with regards to their threat to human health and pandemic potential. since there have been a number of large-scale human outbreaks of bat-borne diseases e.g. zaire ebolavirus (ebov) and severe acute respiratory syndrome (sars) in western africa and asia respectively, whilst a significant number of human cases of nipah virus (niv) are reported in bangladesh every year (iedcr, ) . pteropid bats are known to be the natural host of hendra virus (hev) (halpin et al., ) , a member of the same genus (henipavirus) as nipah virus. since hev has been responsible for seven human cases in australia, four of which were fatal (smith et al., ) . bats have also been linked with marburg virus (marv) , and, more tenuously, with the emerging middle east respiratory syndrome coronavirus (mers-cov) (memish et al., ) . within the european union (eu), zoonotic incidents of bat-borne viruses have been restricted to european bat lyssavirus types and which have been responsible for less than human cases since (fooks et al., ) . to date, there have only been a few isolated reports of introduction of bat-borne viruses (e.g. marv, sars, mers-cov) from outside the eu mainly through entry of infected humans (puzelli et al., ; desenclos et al., ; who, ; reuss et al., ) . however, these incidents illustrate that introduction can occur and support the need for some level of surveillance activity to assess the probability of when and where further incursions may take place. for most bat-borne zoonotic diseases, primary transmission routes for human infection include direct or indirect contact with bat bodily fluids (leroy et al., ; luby et al., ) , or via intermediate animal hosts (parashar et al., ; hanna et al., ) . onward transmission of disease is then possible via human-to-human contact or contact with contaminated fomites or the environment, with nosocomial infections being particularly important in some instances (baron et al., ; chowell et al., ) . disease introduction into the eu could therefore potentially occur from a number of routes, including human travel, illegal and legal importation of food products and transport of live animals. these routes have previously been associated with incursion of other viruses into the eu. for example, human travel and immigration are thought to be the primary reasons why individual member states (mss) have a high prevalence of the same human immunodeficiency virus (hiv) subtypes as their historical african colonies (faria et al., ) . classical rabies has been detected in imported domestic pets (suárez-rodríguez et al., ; mcquiston et al., ) and avian influenza (h n type a) has been detected in illegal imports of crested hawk eagles from thailand into belgium (van borm et al., ) . in the case of trade, illegal importation of food products has been a suggested route of origin for the foot and mouth disease epidemic in the uk in (defra, ) , whilst legal trade in fresh produce, such as fruit and vegetables, has been associated with norovirus (hjertqvist et al., ) and hepatitis a outbreaks (dentinger et al., ) . virus specific transmission characteristics may influence the relative importance of these potential routes of disease introduction. in terms of government financial resource allocation, it is important to develop methods to assist in efficient targeting of surveillance activities e.g. to inform which pathogen(s) are most likely to enter the eu, where they are most likely to enter and what scenarios would have the most impact with regards to human or animal health and welfare or trade implications. to address these issues, a number of relative risk ranking tools have previously been developed, such as the eu wide discontools ( ) and the uk specific d r (gibbens et al., ) . however, these tools are qualitative and are generally based on chosen criteria rather than a defined quantitative assessment. there is, therefore, benefit in a quantitative model that can utilises freely available numerical data from datasets on trade and human travel such as eurostat ( ) and faostat ( ) . to address this need, a generic quantitative risk assessment framework for the entry of bat-borne zoonotic viruses to the eu was developed (simons et al., ) , considering the pathways: human travel, live animal movement, legal trade of food products and illegal trade of bushmeat. using current knowledge of virus characteristics such as environmental survival and host incubation periods, the framework was parameterised for niv, to provide an assessment of the relative risks of transmission through the known pathways of introduction into the eu. in this paper the model framework is parameterised for a number of other virus outbreak scenarios (marv, ebov, hev and mers-cov) and the relative probabilities of introduction to eu mss are compared and discussed. the impact of uncertainty in the parameter estimates is also investigated though scenario analyses. the entry assessment model parameterised for niv (simons et al., ) , was re-parameterised for five outbreak scenarios (marv, ebov, hev and mers-cov), to compare and assess the relative risk of introduction to the eu mss for these viruses of concern. for ebov two different outbreak scenarios were considered: ) disease geographically distributed in western africa, where the human cases are on a similar scale to that observed in the west africa outbreak (webov) (i.e. epidemic situation), ) disease geographically distributed in central africa, where human outbreaks have previously been relatively limited (cebov) (i.e. non-epidemic situation). it is acknowledged that the link between bats and mers-cov is more tenuous than initially thought when it first emerged (memish et al., ) , but the virus was included here to provide an example of a respiratory coronavirus circulating within the middle east area. recent evidence of replication and shedding of mers-cov in experimentally infected jamaican fruit bats (artibeus jamaicensis) (munster et al., ) and discovery of closely related mers-like cov (anthony et al., ) further support the hypothesis that bats are ancestral reservoirs for mers-cov. as neither live ebov nor mers-cov virus has been isolated from bats a very low prevalence of infection in bats was assumed. different prevalence values for all the viruses were considered in the scenario analysis. the assessment was conducted following the world organisation for animal health (oie) code for import risk analysis (oie, ) . under traditional oie guidelines, there are three components of risk assessment: entry, exposure and consequence. this model only considered the entry assessment (i.e. it ceases at the point at which virus is released into the eu) and did not consider subsequent potential exposure of the virus to humans, livestock or wildlife on entry to the eu. the model has been discussed in detail by simons et al. ( ) . briefly, the main outputs of the model were a relative estimate of the annual probability of at least one introduction event into each eu ms, j, p v (j), and an overall estimate of the probability of at least one introduction event for the eu as a whole. this estimate took into account factors such as the probability an individual unit is infected (or contaminated) in the exporting country, the survival of the virus over the duration of the journey, whether or not the animal/human displays clinical signs and the annual volume of products being imported. the model equations for the various routes are described by simons et al. ( ) . the model was coded in the r software package and is deterministic; as such no stochastic variability of specific parameters was considered in the baseline model. the relative risk estimate was derived by combining the probability of at least one introduction event from each of the routes included in the model (fig. ) from all the potential exporting countries to produce an overall probability for each ms: where r is the total number of routes considered for the virus (human travel (r = ), live animal movement (r = ), legal trade of 'at risk' products (r = ), illegal trade of bushmeat (r = )) and p r (j) is the probability of at least one introduction event via route r to ms j per year. the average number of years to an introduction event was calculated, y v (j) = /p v (j). in addition the eu mss were ranked according to their probability of disease introduction, z v (j) = { : } by comparing the average number of years, y v (j), to an introduction event for each route and ms and ranking the mss from to accordingly. this provided an indication of where in the eu an introduction event is more likely. note that the average number of years to an introduction event is based on the input data provided and so does not account for subsequent changes in future years of model factors such as trade patterns or disease prevalence; e.g. for webov it would be assumed that the same number of cases will occur in western africa every year as in . the model considered the four primary routes of introduction based on extensive literature reviews (simons et al., ) . while other potential routes exist such as direct exposure from bats through natural migration or accidental exposure via aeroplane strikes, they were not considered here for the viruses of concern. however, as the model framework is adaptable, and eq. ( ) is multiplicative with respect to the routes, the choice of routes can be amended and these pathways can be considered in the future, as and when appropriate data become available. the model was parameterised for hev, marv, mers-cov, webov and cebov. the genus ebolavirus includes five species, each with a single member virus (kuhn et al., ) . due to the potential differences in parameters for the different viruses only ebola virus (from species zaire ebolavirus) was parameterised here. the model considered the probability of introduction to eu mss from 'exporting countries', that is, those in which virus was strongly expected to be circulating in humans, livestock or wildlife (fig. ) . this was determined from peer-reviewed publications of where the viruses had been reported. for livestock and wildlife, including bats, only positive test results for isolation of live virus or detection of viral rna were considered (active bat infection). countries that had reported positive seroprevalence or those which had reported a human case known to have arisen from recent travel to another country were not considered as an 'exporting country'. the full details of the generic model parameters are presented in simons et al. ( ) . in this section an overview of the data sources used to parameterise each route is presented. human travel: passenger travel data from exporting countries to eu mss were obtained from the eurostat dataset aviapaexcc (eurostat, ) . for ebov, marv and hev for which outbreaks are sporadic, n hinf (k) was estimated using the average number of cases per outbreak over a year period. this value was assumed by the authors to encompass all relevant historical data. however, as human cases of mers-cov have been reported regularly since march , n hinf (k) was calculated by dividing the number of reported cases by the number of reporting years assuming a constant rate per year. to account for differences in prevalence between passenger types e.g. business, visiting family, and tourist etc., the baseline prevalence of infection in the exporting country was weighted by the average passenger duration of stay (days) in the exporting country. the ratio of passenger types was assumed to be the same for each exporting country. the sub-clinically infected population was estimated by multiplying the prevalence of infection in passenger type i, θ(i,k) by the incubation period of the virus. passenger detail such as healthcare employees potentially exposed to infected patients or eco-tourists with the intention of visiting bat caves was not accounted for here although it is acknowledged that these factors could influence the risk outcome as has been documented (who, ) . legal trade: to determine whether a product was considered contaminated, the concentration of virus on the product on arrival to an eu ms was estimated where c min is a threshold viral load upon arrival at the eu ms, below which the product was considered not to be contaminated. note, that this value was set to log tcid for all viruses as a worst case scenario. the model considered the prevalence of contamination in at risk raw products (see table for definition), the initial concentration of virus on a raw product in the exporting country and any reduction in viral load between initial contamination of the raw product and arrival in the eu ms, including the effect of processing. the default estimate for the prevalence of contamination in raw products, pgraw(k), was based on the estimated prevalence of active virus v. horigan et al. microbial risk analysis ( ) - shedding in bats, pbinf(k), the contact rate of the bat with the product, pbcontact(k) and seasonality of virus shedding, i.e. the proportion of the year that bats can shed the virus. data on volume of trade from exporting countries to eu mss were obtained from faostat ( ). bushmeat: in this model bushmeat was assumed to enter an eu ms via aircraft passenger luggage. the volume of contaminated bushmeat entering the eu was estimated by combining the probability of a passenger of type i bringing in bushmeat from exporting country k, p bm (i,j,k), and the probability that a consignment of bushmeat was contaminated. the actual number of bushmeat consignments entering the eu from country k was estimated based on the number of bushmeat consignments seized in the eu ms, nseized(i,j,k), and an under-reporting factor accounting for the proportion of passengers luggage that were searched. the under-reporting factor was estimated to be . % based on literature (falk et al., ) and assuming targeted testing of passengers occurs (simons et al., ) .the model did not account for any virus reduction that may occur from processing bushmeat such as smoking or salting and assumed that the possibility of luggage being searched for bushmeat was the same for each exporting country. live animals: this route considered the number of animals of species s arriving from an exporting country k in one year and the prevalence of live animal infection of species s in the exporting country to give the probability that at least one infected animal entered a ms. numbers of live animal exports from exporting countries to eu mss were obtained from the trans-european trade control and expert system (traces) database which provides data on the number of animals that are brought into the eu and issued with a common veterinary entry document (traces, ) . virus specific parameter estimates used in the model are given in table . estimates for niv are also provided for comparison (simons et al., ) . further information (including references) on these estimates is provided in appendix a. the model developed here is deterministic for ease of use in an outbreak situation where rapid parameterisation and data availability for all eu mss are key requirements. uncertainty and variability in the model were previously considered for niv by implementing a series of analyses using alternative parameter values (simons et al., ) . it was found that while some scenarios had an impact on the absolute values of probability of introduction of niv, the relative rankings, of both routes and mss were more robust. however, the estimate for the prevalence of niv in bats had considerable impact on the average number of years to an eu introduction of niv relative to the baseline model and much lower estimates for this prevalence were the only scenarios to have an impact on the relative ranking between the routes. given this, and the complexity involved in assessing multiple uncertainties between multiple scenarios, the scenarios considered here were a % and % reduction in the virus prevalence in bats as these reductions both had considerable impact in the previous model for niv; smaller reductions in virus prevalence had little impact. at the eu level the probability of viral introduction was ranked highest for webov with an overall average prediction of at least one introduction event occurring in one year (table ) , primarily via human travel and associated illegal importation of bushmeat. in relative terms, and given the uncertainties in the absolute value estimates, marv and mers-cov were of a comparable risk whilst the overall probability of introduction was lowest for hev. a % or % reduction in virus prevalence in the exporting country bat population only affected the risk estimates for the legal trade and bushmeat routes. consequently, for ebov and mers-cov, which had a relatively high probability of introduction from human travel, the decrease in risk from trade and bushmeat was not sufficient to affect the overall probability of disease introduction. for hev and niv, however, where the legal trade and bushmeat routes posed the highest risk in the baseline model the decrease in overall risk was substantial. human travel replaced legal trade and bushmeat as the route with the highest associated probability for hev, marv and niv introduction when the virus prevalence in bats was reduced by % ( % for marv). the number of imports of live animals was low for all exporting countries resulting in a relatively low probability of introduction via this route for all viruses (table ) . only dromedary camels (camelus dromedaries) have been shown to be a risk factor for mers-cov transmission (azhar et al., ) , but as there is no legal trade of live camelids to the eu from countries reporting cases of mers-cov, the risk from live animals was considered to be negligible. within the eu, individual mss demonstrated different relative probabilities for the various pathogens when the probabilities for all the routes of introduction were combined for each ms (fig. ) . the probability of introduction for mers-cov was quite high across most of the eu mss, but for other viruses it was mainly focussed in a few mss, usually in western europe with the probability of introduction for mss from eastern europe and scandinavia generally being much lower (fig. ) . overall, the probability of introduction was highest for individual viruses in those mss with strong historical links to relevant exporting countries, e.g. the united kingdom (uk) for niv and france for cebov. such links usually correspond to a relatively large volume of human travel or legal trade movements between the countries. it should be noted that this analysis does not consider movement within the eu after the initial entry. the countries are ranked according to the probability of introduction for each virus in table . overall there was a relatively wide variation in the relative ranking of many of the mss between the different viruses. different distributions of risk scores were observed between routes but considering the relative ranking of the mss ( - ), the uk, france, germany and the netherlands generally have the highest probability of introduction for all viruses considered here. the entry assessment described here shows the potential for application of a quantitative model framework for any pathogens, using zoonotic bat-borne viruses as an example. although a scarcity of data for virus specific parameters resulted in a high degree of uncertainty in the absolute risk values presented, the main strengths of this model lie in the estimates of relative risks between routes of entry and those mss which are at greater risk of virus introduction. the model has the capability to allow for a continual updating of the risk estimate using new research data as, and when, it becomes available. any increase in the model risk estimate output would allow the stakeholder to consider employing suitable risk reduction strategies or heightened surveillance providing a rapid and cost-effective response. of particular value was the model's ability to illustrate the relative importance of the different routes of entry between viruses; legal trade of foodstuffs was more important for hev, niv and marv while human travel was more important for mers-cov and both ebov scenarios. these differences could be partly attributed to the virus specific parameters. for example rapid decay of mers-cov influenced the relative risk of the transmission pathways; the half-life of mers-cov is very short compared to the other viruses ( min at pre-harvest temperatures (van doremalen et al., ) so it is unlikely to persist in high numbers on any produce imported via legal trade or in contaminated bushmeat. the probability of introduction to the eu via the pathways under consideration here varies across the eu at ms level; the uk, france, germany and the netherlands often had the highest probability of table the expected number of years to eu entry for different viruses, by individual route and all routes combined for the baseline model. results for % and % reduction in virus prevalence in bats are shown in brackets respectively for legal trade, bushmeat and all routes (the model assumes no effect on human travel and live animal routes). human travel introduction for all viruses considered. in general those countries which ranked the highest, with regard to probability of introduction (table ) corresponded to those with the highest population and the highest 'disposable income' (calculated as gross domestic product (gdp) derived from purchasing power parity) (see appendix b). other contributory factors could include immigration population densities and trade partner characteristics, both of which frequently have a historical basis. the netherlands was an exception to this in that it ranked highly in the probability of virus introduction yet only th for population density and th for gdp (see appendix b). one explanation for this could be that the netherlands is serving as a hub for travellers and trade entering the eu and that a reasonable proportion entering the netherlands are going onto other european countries. it is also possible that more dutch people, compared to other eu mss, travel to countries with these viruses. data from uganda suggest that in the netherlands was the th most popular country of origin for tourist arrivals, the only european countries with more arrivals were the united kingdom and germany, but dutch tourists represented a higher proportion of their population (republic of uganda, ). freely available statistical data on trade and human travel for western europe were, in general, more complete than for eastern european mss. data for countries such as estonia, latvia and lithuania were lacking for some routes resulting in a low ranking for these countries which may not be a true reflection of the actual risk. it is difficult to determine whether this is a true data gap or if the route genuinely has a low probability of introduction for these countries. this was a particular problem for cebov where twelve countries were lacking data for specific routes and, therefore, equally ranked as . . the generic parameters for which eu wide datasets exist have a relatively high degree of completeness although there is a concern that potentially high risk low volume trade products e.g. camel milk may be under-recorded therefore underestimating the risk via these trade products. virus specific parameters depend more upon focussed research studies and peer reviewed literature and rely upon detection of pathogens in reporting countries. uncertainty in these virus parameters, in particular, the prevalence of infection in bats in the exporting country, and viral persistence during processing and storage may limit the application of the model by introducing considerable uncertainty. the sensitivity analysis of virus prevalence in bats demonstrated that the results for the relative importance of the routes for ebov and mers were quite robust with human travel remaining the route with the highest probability of introduction. with regards to hev, niv and marv, however the sensitivity to the variation in prevalence indicates that further data for this particular parameter would strengthen the model results; this is particularly true of marv where a % reduction in virus prevalence changed the risk ranking order of the routes of introduction. note that this analysis is to consider the uncertainty about the true prevalence in bats, as such it has no impact on the parameterisation of the human prevalence, which is based on human outbreak data. it should be noted that the parameterisation of this model uses the best available data at the current time. some parameters are subject to high uncertainty and the probability of introduction of different viruses will be dynamic, changing over time if a virus spreads amongst different animal species populations or if new human outbreaks occur. simons et al. previously demonstrated that changes in the exporting country (e.g. if china were to get niv in the future) or 'at risk' product types can have a large effect on the model outputs (simons et al., ) . we have demonstrated here that changes in the virus prevalence in bats in the exporting country can have an impact on the average number of years to eu entry for the different viruses and on the relative ranking of the individual routes of entry. we have also highlighted the differences in probabilities for two ebola scenarios; a relatively small non-epidemic human outbreak and a large epidemic scale outbreak. it is acknowledged that the viruses considered here could have differing sensitivity to stochastic variability of specific parameters given the complex dynamics between the routes and viruses. alternative scenarios could, therefore, be considered in the future. all risk pathways were given equal weight within the model as the model predicts probability of introduction not risk of human/animal exposure and consequence as stated in the oie risk assessment (oie, ) . for example, the model results suggest that the legal trade (fruit) route has a high probability of introduction for hev, although human infection from consumption of contaminated fruit is not a proven transmission route for this virus. this route was considered in the model based on the knowledge that fruit bats are known to consume raw fruit in orchards (eby and lunney, ) and date palm sap is a known route of transmission for niv (another henipavirus) (luby et al., ; khan et al., ; nahar et al., ) . similarly, there is currently no evidence of human-to-human transmission of hev but as this has occurred for the bangladesh strain of niv (gurley et al., ) it is plausible that this could occur with or without mutation and adaptation of currently identified strains. real-time application of the model would allow for removal or addition of pathways if future scientific work provides suitable input data or if trade patterns between third countries and the eu alter. thus, all pathways were assessed for completeness according to the dogma 'absence of evidence is not evidence of absence'. whilst eu wide trade controls are implicitly accounted for within the model parameters, risk mitigation procedures put in place by individual mss such as targeted sampling are not taken into account. it is possible that there have already been introduction events of the diseases under consideration here within the eu, but these have remained undetected due to lack of subsequent human/animal infection and/or onward transmission within the individual ms. for example, although the importation of mers-cov cases to the eu remains possible, an ecdc risk assessment determined that the risk of sustained human-tohuman transmission is low (ecdc, a). however, the outbreak of mers-cov in south korea demonstrated that the potential exists for a serious risk of onward human spread, with > cases arising from the importation of human index case (su et al., ) . validation of such a model presented here is difficult as there are few independent resources for which to compare the results. however, it is of relevance that the five mss suggested to have the highest probability of introduction of mers-cov by the model (germany, uk, italy, france and the netherlands) have already had imported human cases of this pathogen (ecdc, ) . the model results are also consistent with other reports which predict more imported cases of mers-cov to arrive into the eu (ecdc, ; who, a; bialek et al., ; poletto et al., ) . all cases reported outside of the middle east have had a recent travel history to the middle east or contact with a case that had travelled from this region (su et al., ) . this is in line with the highest probability of introduction for mers-cov predicted by this model to be via human travel (table ) . overall, the approach developed here provides a high-level horizon scanning tool for the probability of introduction of bat-borne zoonotic viruses into the eu. the virus scenario with the highest probability was the webov scenario with an overall average prediction of just under one introduction event per year, primarily via human travel. due to the wide scope of the model, which necessitated using global datasets sometimes with incomplete data, there was a high degree of uncertainty in the absolute risk values presented. a general lack of data on virus specific parameters also contributed to this uncertainty. thus, the main strengths of this model lie in the comparison of the relative risks between viruses and routes of entry. whilst there have been several risk assessments carried out for the introduction of individual pathogens into the eu (rolin et al., ; durand et al., ; mur et al., ; snary et al., ) this model was able to assess a range of viruses and could be adapted for other pathogens, as it has the advantage of easy access to a number of relevant databases. the model also allows for a continual updating of the risk estimate enabling the stakeholder to respond in a rapid and risk appropriate manner, for example, by implementing risk-based surveillance and control strategies. (table a ). the beginning of saw the largest recorded outbreak of ebola emerge in western africa with sierra leone, liberia and guinea being the countries most affected. due to the potential differences in parameters for the different viruses only ebola virus (from species zaire ebolavirus), ebov, is parameterised here. the number of cases per outbreak estimated over a year period was used for n hinf (k).this is estimated at , for west africa, for the drc, for gabon and for roc. time to clinical signs, t ip (k). the recognised incubation period for ebov disease is - days (del rio et al., ) . a review of epidemiological parameters from ebola outbreaks including incubation period has recently been published (van kerkhove et al., ) . using a sum of all the estimated means divided by the number of studies the time to clinical signs used is . for ebov only (table a ) . bat infection prevalence in exporting country, p binf (k). evidence of ebov infection in bats (epomops franqueti, hypsignathus monstrosus, and myonycteris torquata) is currently based on seroprevalence and presence of viral rna. table a shows the relevant papers that have attempted isolation of ebov. combined, bats were tested with testing positive for viral shedding of ebola. as such the prevalence of ebov in bats for this model was assumed to be . % . product types (l): the potential for ebov to act as a foodborne pathogen has been addressed (bausch, ) . parallels have been drawn between the emergence of the reston strain of ebov in domestic pigs in the philippines in and the - outbreak of niv in malaysia and singapore. both imported fruit products and live pigs/pig products were therefore considered as potential risk factors for the introduction of ebov into the eu. proportion of the year bats may shed active ebov virus, p season (k). seasonal climate has been found to be associated with human ebov outbreaks (ng et al., ) . increased great ape mortality has frequently been reported during the dry seasons of july and december (pourrut et al., ) and the biannual birthing periods of the bat species identified as potential natural reservoirs also occur during the dry seasons when fruit is scarce (langevin and barclay, ) . in the absence of more definitive data the proportion of the year in which bats are presumed to be infectious is estimated as . . initial viral load on raw product, c (x). the initial concentration of ebov on the raw product is assumed to be equivalent to that shed by bats. as there have been no successful virus isolation attempts from bats, values are extrapolated from experimental evidence. nasal washes and oral and rectal swabs from pigs challenged via mucosal exposure with × pfu of the zaire strain of ebov had infectious titres ranging from × to × tcid /ml (kobinger et al., ) . infectivity of mucosal wash fluids obtained from monkeys experimentally infected with the reston strain of ebov ranged from < . log pfu/ml at initial infection to a maximum of . log pfu/ml in terminal animals (jahrling et al., ) . based on these data the initial viral load used here follows a log normal distribution with mean log tcid /ml (variance = log tcid /ml in the absence of any other data). virus decay in the environment and during transport, c hlenv (j,k), c hltrans (j,k). in a study on the survival of filoviruses in liquids and on solid substrates the half-life of ebov was calculated to range from . to . days at + °c in tissue culture media and sera respectively and days at room temperature (piercy et al., ) . no virus could be recovered from any solid substrate stored at room temperature but at + °c the virus had a half-life of ∼ . days on glass and ∼ days on plastic substrates. from the data available, a mean half-life of days ( h) is used as an estimate for virus reduction during transport at + °c and virus reduction pre-harvesting is estimated at days ( h) using liquid media data. minimum viral load to consider product contaminated in eu ms, c min . experimental infection of bats with the zaire strain of ebov was achieved with an inoculation dose of . ffu (fluorescent focus forming units) (swanepoel et al., ) . non-human primates (nhp) have been shown to be uniformly susceptible to intramuscular inoculation of pfu of the zaire strain of ebov (geisbert et al., ; smith and wang, ) . however, doses as low as pfu were sufficient to cause infection in rhesus macaques (kortepeter et al., ) and pfu of a guinea pig adapted strain of the virus was used to experimentally infect baboons (ignatiev et al., ) . johnson and colleagues (johnson et al., ) reported using a dose of pfus to infect rhesus monkeys with ebov by inhalation. in another experiment, three out of four orally inoculated rhesus monkeys were infected when using a dose of . log of ebov (jaax et al., ) . rhesus macaques were aerosol challenged with calculated doses between and , pfu of ebov delivered as a small-particle aerosol (twenhafel et al., ) whilst a lethal dose of ld in african green monkeys and - ld in baboons has been demonstrated (ryabchikova et al., ) . as a worst case scenario it is assumed that c min = log tcid . species of animal, s. detection of ebov by serology and pcr in animals has been recently summarised by pigott et al. ( ) . dogs and pigs are, so far, the only domestic animals identified as species that can be infected with ebov (weingartl et al., ; allela et al., ) . although dogs can be asymptomatically infected, they may excrete infectious viral particles in urine, faeces, and saliva for a short period before virus clearance. pigs have been shown to be susceptible to both the reston and zaire strains of ebola. conversely, the zaire strain is also capable of transmission from pigs to cynomolgus macaques without direct contact (weingartl et al., ) . pigs, challenged with ebov via mucosal exposure, replicated the virus to high titres mainly in the respiratory tract with shedding observed from oronasal mucosa up to days post-exposure. transmission to cohabiting naïve pigs was also observed (kobinger et al., ) . animal species in which evidence of natural/experimental ebov infection has been demonstrated and considered as potentially capable of introducing ebov into the eu include: pig, domestic dog, lord derby's scaly-tailed squirrel, duiker, non-human primates, small rodents and the shrew (morvan et al., (falk et al., , chaber et al., . a study on the species of bushmeat items confiscated at us ports of entry between and , suggested that bats accounted for around . % of all bushmeat (bair-brake et al., ) . thus, in the absence of other information, we assume that . % of bushmeat is bats. the same study suggested that around % of bushmeat is derived from nhp and % from rodents. rodents and blue duikers made up % of the total number of bushmeat carcasses detected at paris roissy-charles de gaulle airport from sub-saharan africa (chaber et al., ) . despite being herbivorous, duikers have been known to eat the flesh of decomposing carcasses and could become infected with ebov via this transmission route (rouquet et al., ) . number of human infection in exporting country, n hinf (k). human cases of hev have been restricted to the state of queensland in australia (table a ) . hendra virus was first described in since which time human infections have occurred in separate outbreaks with a % case fatality rate. no human cases have been reported for the last years (since ) which is attributed to horse keepers/vets greater awareness of the disease and an equine vaccination programme. the number of human infections in exporting country k in one year is assumed to be . time to clinical signs, t ip (k). estimates of average time to clinical signs in humans can be seen in table a . taking an average of all the estimates available, a value of . days was used for the average time to clinical symptoms of hev. bat infection prevalence in exporting country, p binf (k). in hev rna was detected in up to two-thirds of pooled-urine samples from bats near hev cases in horses (plowright et al., ) . variable virus excretion has been reported in urine, with prevalence in pooled urine samples collected under roosting flying-foxes ranging from - % in the one-in-four sampling events that yielded positive results. subsequent studies have detected excretion spikes as high as % on rare occasions (field et al., ) . based on documented evidence of virus isolation (table a ) the prevalence of hev in bats was assumed to be . %. product types, l. whilst there is currently no evidence of human hev infection as a result of contaminated fruit consumption it is plausible that this could occur with or without mutation and adaptation of currently identified strains. this framework models only the possibility that fruit products contaminated with hev could enter the eu. all products in the faostat database recorded under section -fruits and derived products, are therefore included. proportion of the year bats may shed active hev virus, p season (k). flying foxes appear to excrete hev at any time of year and spillover in horses can occur in any month but the majority of equine cases ( confirmed or possible cases as of december (smith et al., ) ) have occurred from june to september suggesting that there is a greater risk of infection at this time (field et al., ) . there was an initial coincidence of hev outbreaks with birthing seasons of australian fruit bat species and the isolation of hev from the uterine fluid and aborted foetus of a p. poliocephalus bat indicated that this may be a significant route of infection for horses (fogarty et al., ) . however, there now appears to be a temporal clustering of spillovers during the winter period with / spillovers to june occurring in june, july and august (goldspink et al., ) . the proportion of the year that bats are assumed to shed active virus is therefore estimated to be ∼ months or . of year. initial viral load on raw product, c (x): the initial concentration of hev on the raw product is assumed to be equivalent to that shed by bats. virus has been detected in the urine, faeces, saliva and birthing fluids of experimentally infected flying-foxes (williamson et al., (williamson et al., , halpin et al., ) , and in the urine, uterine fluid and foetal tissue of naturally infected free-living flying-foxes (halpin et al., ; field et al., ) . whilst these studies report on prevalence of virus there are no data on quantification of virus in bats available. however, in experimental inoculations of pigs virus titres of . log tcid /ml were found in nasal swab samples (li et al., a) . based on these data the initial viral load on the raw product follows a log normal distribution with mean . log tcid /ml (variance = log tcid /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): using an exponential decay model the half-life of hendra virus under laboratory conditions was calculated to be . min, . and h. for , and °c respectively (scanlan et al., ) . more recent modelling predictions using the same data calculated half-lives of . s, . h and h using a weibull distribution (martin et al., ) . when incubated in p. alecto urine (ph ∼ ) hev had a half-life of h. at °c and h at °c (fogarty et al., ) . the half-life in mango flesh ranged from . h. at ph to h. at ph at °c (fogarty et al., ) . the calculation of . h at °c was assumed to be the most accurate for this scenario and this value was therefore used for virus decay in the environment; a value of h was used for decay during transport consistent with the modelling prediction using weibull distribution. minimum viral load to consider product contaminated in eu ms, c min uniform disease occurred with an inoculation value of . × pfu of hev for guinea pigs and . × pfu for landrace pigs whereas an inoculate dose of × pfu in minipigs did not cause uniform fatality (li et al., b) . horses in a vaccine efficacy study were challenged oronasally with × tcid in experimental infection with hendra (marsh et al., , middleton et al., while cats orally challenged with × tcid succumbed to disease after a day incubation period (hooper et al., ) . as a worst case scenario it is assumed that c min = log tcid . species of animal, s. horses are moved internationally for competition, breeding, slaughter and as companion animals. all horses being permanently exported to europe from australia must complete a day pre-export isolation at an approved quarantine stable in australia. in horses, the incubation period is estimated to be - days although the incubation period in one horse may have been days (doh, ) . evidence suggests that horses have the potential to excrete virus in nasal secretions up to days before showing signs of infection (kung et al., ) and should be considered as potentially infectious from h prior to onset of clinical signs of disease. virus is recoverable from infected horse's urine and saliva for at least days. transmission of hev or niv via semen has not been investigated, although the likelihood of a stallion being infected, clinically healthy and having semen collected for export is considered remote (maf, ) . experimental inoculation of pigs has indicated that they could be a potential host for hev (li et al., b) . in a serological survey of swine herds in queensland, australia (black et al., ) no hev antibodies were found in the tested serum samples. two dogs have tested positive for hev antibodies on properties where horses developed hev infection in july and july . although the source of exposure for the dogs cannot be definitively ascertained, horse-to-dog transmission is the most plausible scenario. experimental hendra virus infections have been performed in horses, cats, ferrets, hamsters, african green monkeys and guinea pigs all of which developed fatal diseases. cats from australia are prohibited from entering the uk unless they are accompanied by a certificate from the australian veterinary authorities confirming that they had not been on a holding where hev has been confirmed during the days prior to export. pigs, dogs, cats and horses are all considered in the model. species of bushmeat, p bmsp (s). bushmeat is part of the traditional diet of indigenous australian people whilst australian game meat plays a part in (goldspink et al., ) a samples are the same reported in different articles. (kissling et al., (kissling et al., ) south africa unknown -possibly from zimbabwe visited sinoia caves - days prior to onset of symptoms yes (gear et al., ) kenya kitum cave (< miles from lake kyoga where monkeys originated) yes (smith et al., ) kenya kitum cave (< miles from lake kyoga where monkeys from) no (johnson et al., ) modern australian cuisine. some of the animals that were traditionally hunted for meat are now endangered and protected including the flying foxes (in new south wales and queensland) although aboriginal people are excluded from protection laws and have the legal right to hunt native animals for their own consumption. a study reported on the species of bushmeat items confiscated at us ports of entry between and , suggested that bats accounted for around . % of all bushmeat (bair-brake et al., ) . thus, in the absence of other information, we assume that . % of bushmeat is bats. a. . . human travelnumber of human infections in exporting country, n hinf (k). since marv outbreaks have originated in kenya, the drc, angola and uganda (table a ) . time to clinical signs, t ip (k). data from secondary cases of marv in kenya place the median incubation period at days. however the incubation period in index cases following exposure to a reservoir source is calculated as having a mean of . days (timen et al., ; amman et al., ) with a range of - days. based on documented cases of exposure and subsequent infection marv was calculated to have an incubation period ranging from to days (typically - days) the range being modulated by factors such as infectious dose and possibly by route of infection (brauburger et al., ) . using data available from historical marburg cases with precise exposure dates (n = ), the median incubation rate for marburg was calculated by pavlin to be days with no significant difference between primary and secondary cases (pavlin, ) . the value of days is used in the model. bat infection prevalence in exporting country, p binf (k). the estimate for the prevalence of marv in bats is based on published information from peer reviewed publications on the isolation of active marv (table a ). the number of bats actively shedding virus is taken as a percentage of the entire pool tested as it is assumed that if no rna is detectable then virus isolation would be highly unlikely as a direct correlation between rna levels and the ability to isolate virus has been demonstrated (towner et al., ) . due to the uncertainty surrounding this parameter, the prevalence of active virus shedding in bats was assumed to be p binfw (k) = . % as a worst case scenario. product types, l. marv has been isolated from rousettus aegypticus, a fruit bat which is known to discard hard particles in their food at foraging sites (herzig-straschil and robinson, ) . they are known to consume various fruit crops produced for human consumption such as date, fig, apricot and peach. a recent paper succeeded in isolating marv from both oral and rectal secretions of r. aegyptiacus experimentally infected with virus demonstrating potential avenues for viral shedding (amman et al., ) . thus all products in the faostat database recorded under section -fruits and derived products, are included. proportion of the year bats may shed active marv virus, p season (k). retrospective analysis of historical human infections found there was a temporal clustering of infections coinciding with the seasonal pulses in virus circulation in r. aegyptiacus covering months of the year in total (amman et al., ) . the observation of these distinct pulses of virus infection in older juvenile bats appears to coincide with the peak biannual birthing seasons. thus the proportion of the year that bats are presumed to be infectious with marv is . . initial viral load on raw product, c (x): the initial concentration of marv on the raw product is assumed to be equivalent to that shed by bats. successful isolation of marv roughly correlated with tissue samples that had rt-pcr ct values of or less (> tcid /ml) (amman et al., ) . the highest rna level, measured using ct values, corresponded to an approximate infectious titre of × pfu/ml (towner et al., ) . measurements of concentrations of marv in experimentally microbial risk analysis ( ) - infected guinea pig saliva, urine and faeces showed a virus concentration of . - . log ld (median lethal dose) (chupurnova et al., ) . the ld was calculated to be × − tcid of virus for wild type mice (qiu et al., ) . however, it should be noted that this will be a rodent adapted strain of the virus. the virus has also been found to be excreted at high levels of up to guinea pig infectious doses in urine of experimentally challenged monkeys (simpson, ) . marburg virus was isolated from oral secretions of r. aegyptiacus experimentally infected with virus from a naturally infected bat of the same species (amman et al., ) . viral loads were measured by qrt-pcr analysis of viral rna and reported as mean tcid equivalents. marburg virus positive oral swabs were obtained on day - post infection with highest viral loads detected on day ( . × tcid /ml equivalents) and cleared from oral secretions by day . based on these data the initial viral load on the raw product follows a log normal distribution with mean . log tcid /ml (variance = log tcid /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): in a study on the survival of filoviruses in liquids and on solid substrates the half-life of marburg virus in liquid media was calculated to be between . and . days at + °c and ∼ days at room temperature (piercy et al., ) . no virus could be recovered from any solid substrate stored at room temperature but at + °c the virus had a half-life of ∼ . - . days on glass and ∼ . - days on plastic substrates depending on the media in question. from the data available, a mean half-life of days ( h) is used as an estimate for virus reduction during transport at + °c and virus reduction pre-harvesting is estimated at days ( h) using liquid media data. minimum viral load to consider product contaminated in eu ms, c min : evidence of experimental infection in rodents is not considered here as these models generally use adapted viruses obtained through sequential passage in the rodent species as the wild-type virus does not cause uniform lethality (bray, ) . a single intramuscular injection of a common marmoset with as little as pfu of virus has resulted in fatal haemorrhagic disease (carrion et al., ) whilst pfu has proven to be a uniformly lethal dose of virus (thi et al., ; geisbert et al., ; hensley et al., ; smith et al., ) . however doses as low as - pfu and - pfu have been reported as causing disease by viral inhalation. the doses were equally fatal but symptoms were delayed by one day in the lower dose group (alves et al., ) . when bats were experimentally infected by subcutaneous inoculation with a dose of tcid marv there was evidence of infection in all bats although no clinical signs were observed. as a worst case scenario it is assumed that c min = log tcid . species of animals (s): all species of nhps were considered as susceptible to marv due to previous research (simpson, (chaber et al., ; falk et al., ) . a study reported on the species of bushmeat items confiscated at us ports of entry between and , suggested that bats accounted for around . % of all bushmeat (bair-brake et al., ) . thus, in the absence of other information, we assume that . % of bushmeat is bats. the same study suggested that around % of bushmeat is derived from nhp which is of relevance here as marv has been previously transmitted from nhp to humans. a. . . human travel number of human infections in exporting country, n hinf (k). sporadic cases have occurred in europe and the rest of the world but the index case of these outbreaks has always recently travelled to the middle east. a large number of cases reported in saudi arabia have been nosocomial; the recent outbreak of mers-cov in south korea illustrates the role the hospital environment can play in the spread of pathogens (park et al., ) . the number of human infections (table a ) were taken as the total number (as at november th ) divided by the period of time over which the infections have been recorded. time to clinical signs, t ip (k). the incubation period of mers-cov has been estimated using data from exposure of secondary cases to the index case in a hospital outbreak (assiri et al., ) and using traveller-related clusters (cauchemez et al., ) . the median incubation period for confirmed cases in the hospital outbreak was . days ( % ci . - . ), and . days ( % ci . - . ) for travel related clusters in the uk, france, italy and tunisia (fisher-hoch, ) . a figure of . days is used in this model. bat infection prevalence in exporting country, p binf (k). growing serological and molecular evidence suggests that the dromedary camel (camelus dromedarius) is an intermediate species for mers-cov (ecdc, b) but the virus is hypothesised to have originated from bats. a mers-cov sequence identical to that of the virus isolated from an index-case patient was detected in a faecal pellet from a taphozous perforatus bat sample (table a ) collected near the home of an index case in an area which is an important date palm production area in saudi arabia (memish et al., ) . as yet no evidence has been found of actual infection in chiropteran populations. product types, l. some human cases of mers-cov infection have had gastrointestinal symptoms including diarrhoea and vomiting. coronaviruses have been demonstrated to survive on fresh produce over a period of days (yepiz-gomez et al., ; mullis et al., ) . thus, it is possible that fruit and vegetables contaminated with coronavirus may be potential vehicles for transmission to humans. infected bats (or other animals) contaminating raw fruit via saliva or urine whilst foraging for food is considered a risk and thus all products in the faostat database recorded under section -fruits and derived products, are included. other legal imports considered as potential risk commodities are camel meat and camel milk. mers-cov rna and antibodies have been detected in milk collected from actively shedding dromedary camels in qatar providing evidence of the potential for virus transmission (reusken et al., a) . camel milk imports from uae to the uk began in and are transported by plane as a chilled product. the operation is stated to have obtained iso certification for both the farm and dairy processing facilities to fulfil the eu import requirements. there have been consignments in comprising of: proportion of the year bats may shed active mers-cov virus, p season (k). although there is no evidence of viral shedding in bats to observe any seasonal fluctuations, analysis of human infections since the first reporting of mers-cov found there was a temporal clustering of infections with a sharp rise in the number of cases in april and may and a smaller peak in august-september. the birthing period for taphozous perforates is in april and may. in line with the peak in human infections in saudi arabia the proportion of the year that the source is presumed to be infectious with mers-cov is months or . . initial viral load on raw product, c (x): experimental infection of common marmosets with . × tcid mers-cov exhibited viral loads of ∼ log tcid eq/g in nasal mucosa days after challenge (falzarano et al., ) . respiratory samples gave higher values than blood or other tissues. camels inoculated with doses of tcid gave viral titre of maximum of log tcid eq/ml of nasal swab samples (adney et al., ) . based on these data the initial viral load on the raw product follows a log normal distribution with mean log tcid eq/ml (variance = log tcid /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): the stability of mers-cov (isolate hcov-emc/ ) was evaluated under three different environmental conditions (van doremalen et al., ) . the mean half-life was calculated as: . (h) at °c % humidity and . h at °c % humidity indicating that temperature influences the environmental decay of the virus. in liquid matrices mers-cov was found to have a half-life of ∼ h in milk at + °c and h at + °c (van doremalen et al., ) . the mean half-life for mers-cov in the environment was taken to be . h, as it was assumed to most accurately mimic a real-life situation whereby fruit contaminated by either infected bat saliva or urine would then be exposed to temperatures of ∼ °c prior to harvesting (assuming a peak shedding prevalence in april/may and aug/sep time). however, after harvest when the fruit is assumed to be kept at + °c during transport the average half-life time of h, as calculated from milk, is used as virus survival time is assumed to be more similar between solid and liquid microbial risk analysis ( ) - matrices at this temperature than at °c. minimum viral load to consider product contaminated in eu ms, c min . the only animal models found to be naturally permissive to infection are nonhuman primates. rhesus macaques challenged with × tcid (de wit et al., ) and . × tcid (yao et al., ) showed mers-cov related pathology and had detectable levels of viral rna. challenge with a total viral dose of . × tcid caused a severe partially lethal disease in the common marmoset (callithrix jacchus) (falzarano et al., ) . camels inoculated with doses of tcid gave viral titre of maximum of log tcid eq/ml of nasal swab samples (adney et al., ) . as a worst case scenario it is assumed that c min = log tcid . species of animal, (s). there has been documented evidence of mers-cov viral rna (chu et al., ) , virus specific antibodies (alagaili et al., ; briese et al., ; corman et al., ; meyer et al., ; reusken et al., b) and virus isolation (raj et al., ; hemida et al., ) in dromedary camels, both from countries in the arabian peninsula and the african continent. according to the ec (commission regulation (ec) no / ) no camel imports are allowed from the arabian peninsula or countries in africa where antibodies to the mers-cov has been detected in camel sera. however, there is no evidence of camels from approved rd countries being tested for the mers-cov and, as this virus is suspected of being circulating in camels for some time, it is not impossible that they could be harbouring the coronavirus too. only dromedary camels were considered as 'at risk' for live animal imports to the eu. species of bushmeat, pbmsp(s): bats, or camel meat, illegally imported as bushmeat from the arabian peninsula and africa could be of potential risk in the transmission of the mers-cov. as has previously been stated, in the absence of other information, we assume that . % of bushmeat is bats. it is possible that camel meat could be present in generic 'red meat' illegally imported as bushmeat from the arabian peninsula. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome 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(pteropus poliocephalus) an animal model of mers produced by infection of rhesus macaques with mers coronavirus survival of respiratory viruses on fresh produce serological evidence of ebolavirus infection in bats key: cord- -kldu q x authors: oany, arafat rahman; sharmin, tahmina; chowdhury, afrin sultana; jyoti, tahmina pervin; hasan, md. anayet title: highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach date: - - journal: in silico pharmacol doi: . /s - - - sha: doc_id: cord_uid: kldu q x purpose: ebola virus (ebov) is such kind of virus which is responsible for , cases and deaths worldwide only in and with an average diseases fatality rate between % and %. although, medical technology has tried to handle the problems, there is no food and drug administration (fda)-approved therapeutics or vaccines available for the prevention, post exposure, or treatment of ebola virus disease (evd). methods: in the present study, we used the immunoinformatics approach to design a potential epitope-based vaccine against the rna-dependent rna polymerase-l of ebov. bioedit v . . sequence alignment editor, jalview v and clc sequence viewer v . . were used for the initial sequence analysis for securing the conservancy from the sequences. later the immune epitope database and analysis resource (iedb-ar) was used for the identification of t-cell and b-cellepitopes associated with type i and ii major histocompatibility complex molecules analysis. finally, the population coverage analysis was employed. results: the core epitope “fryeftapf” was found to be the most potential one, with % conservancy among all the strains of ebov. it also interacted with both type i and ii major histocompatibility complex molecules and is considered as nonallergenic in nature. finally, with impressive cumulative population coverage of . % for the both mhc-i and mhc-ii class throughout the world population was found for the proposed epitope. conclusion: to end, the projected peptide gave us a solid stand to propose for vaccine consideration and that might be experimented for its potency in eliciting immunity through humoral and cell mediated immune responses in vitro and in vivo. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. background evd, previously designated as ebola haemorrhagic fever, is a fatal disease in humans and other mammals (monkeys, chimpanzees and gorillas) (choi and croyle , leroy et al. , sullivan et al. . the fatality rate of edv is varied from to % with an average of about % (peters and peters ) (hoenen et al. ). the fifth one, reston virus (reston ebolavirus), is harmful to nonhuman primates, but not to humans (elisha and adegboro , geisbert et al. ). among the recognized species of ebolavirus, the notoriously deadly zaire ebolavirus is responsible for epidemics which have been taken place mainly in african countries including democratic republic of congo, uganda, sudan, the ivory coast, and gabon (baize et al. , chowell et al. , feldmann et al. , frieden et al. , hewlett and hewlett , kuhn et al. , li and chen , rouquet et al. . this virus is passed on people from wild animals and through human-to-human contact transmits in the human population. those are infected with this virus bear fearsome symptoms, including high fever, hemoptysis, impaired kidney and liver function and severe internal bleeding (gatherer , goeijenbier et al. , keiser et al. , peters and peters . in the fall of the ebola virus gained widespread attention when in west africa the largest outbreak has been reported in history. the ebov genome is a single-stranded, negative-sense, non-segmented rna approximately kb long. it codes for seven tandemly arranged viral genes which order is ′ leader-np (nucleoprotein) -vp (virion protein )-vp -gp (glycoprotein)-vp -vp -l (rna-dependent rna polymerase)-trailer − ′. transcription and translation of this viral genome result in the synthesis of seven structural proteins and a single non-structural, secreted glycoprotein (feldmann et al. ) . three of the structural proteins are membrane-associated proteins; gp is a type i transmembrane protein, while vp and vp are placed on the inner surface of the membrane. the remaining four, np, vp (transcription factor), vp (polymerase cofactor), and l (rna-dependent rna polymerase), are essential to viral genomic rna to form the ribonucleoprotein complex. these proteins have been shown to be necessary and sufficient for ebov transcription and replication (crary et al. , feldmann et al. , mühlberger et al. , takada et al. ). to date, information regarding the processing, structure and functions of ebola virus (ebov) protein l (ebol) demonstrates that it is an rna-dependent rna polymerase, with the assistance of vp . it also shows mrna (guanine-n ( )-)-methyltransferase, mrna guanylyltransferase and poly (a) synthetase activities which are essential for the replication and transcription of ebov (poch et al. ) . the viral mrna guanylyltransferase serves either as transcriptase or as replicase. the transcriptase synthesizes subgenomic rnas, assures their capping and polyadenylation. the transcriptase stutters on a specific sequence, leads to a co-transcriptional editing of the glycoprotein (gp) mrna. in replicase mode, the polymerase replicates the viral genome without recognizing the transcriptional signals. these reports suggest that ebol is an important cellular component for the transcription and replication of the ebov genome and, as such, plays a key role in the ebov life cycle. due to the emergence of ebola virus outbreak, there is an immediate need to determine novel therapeutic targets against this pathogen. the identification of specific epitopes derived from infectious pathogens has significantly advanced the development of epitope-based vaccines (evs). bettered understanding of the molecular basis of antigen recognition and hla binding motifs has resulted in the advancement of rationally designed vaccines depend on algorithms predicting the peptide's binding to human hla. in comparison to the conventional vaccines, peptide or epitope based vaccines are easy to develop, chemical stable, more specific, and free of any infectious or oncogenic potential hazard (holland and domingo , sette et al. ) . though evs have varied advantages, the wet lab based discovery of candidate epitopes is expensive and time consuming. furthermore, for the final selection of epitopes various immunological requirements are needed to be considered. as a result computational methods, an alternative in silico approaches (germain ) have recently been attracting growing interest of the researchers for predicting epitopes with reduced cost and time. the application of bioinformatics in immunology is termed as immunoinformatics. currently, numerous immunoinformatics tools are available for identifying b and t cell epitopes and human leukocyte antigen (hla) ligands (petrovsky and brusic , poland et al. , sette and fikes with high sensitivity and specificity. the 'immunoinformatics' approach has already proven its potency in the case of human immunodeficiency virus (wilson et al. ) , multiple sclerosis (bourdette et al. ) , tuberculosis (robinson and amara ) and malaria (lópez et al. ) with desired results. in the present study, we have followed immunoinformatics approaches for designing potential conserved epitope candidate for the utility of vaccine development against the deadly ebola virus, with an expectation of further wet lab validation. the protein sequences of the rna-dependent rna polymerase-l ) of the ebov were retrieved from the uniprotkb (apweiler et al. ) database in the fasta format. bioedit v . . sequence alignment editor (apweiler et al. ) was used for the identification of the conserved region among the sequences through multiple-sequence alignment (msa) with clustalw (hall ) . finally, jalview v tool (thompson et al. ) was used to retrieve the alignment and the clc sequence viewer v . . (http:// www.clcbio.com) was used for analysis of the divergence among the different strains of the ebov. vaxijen v . , a web-based server (waterhouse et al. , doytchinova and flower ) was used for the determination of the antigenicity of the conserved sequences. herein, we used the default parameters for the prediction, with a threshold value of . . for this study, two online servers were used. firstly, the netctl v . server (larsen et al. ) was used for predicting potential cytotoxic t lymphocyte (ctl) epitopes from the conserved peptides. here for predicting the epitopes, we used a combined algorithm including major histocompatibility complex class i (mhc-i) binding, transporter of antigenic peptide (tap) transport efficiency, and proteasomal c terminal cleavage prediction. depending on the score, the best candidates were picked for further investigation. the epitope prediction was confined to mhc-i supertypes. mhc-i binding and proteasomal cleavage were carried out through artificial neural networks and the weight matrix was used to estimate the tap transport efficiency. the threshold value for epitope identification was set at . for maintaining sensitivity and specificity of . and . , respectively during the analysis. this would support to assess the findings more decisively by developing more epitopes. finally, for confirming the prediction with default parameters, ctlpred (bhasin and raghava ) was employed additionally. furthermore, from the immune epitope database and analysis resource (iedb-ar), t cell epitope prediction tools was implied for the identification of mhc-i (hoof et al. , nielsen et al. ) and mhc-ii (wang et al. ; binding of the peptide. in order to calculate the half-maximal inhibitory concentration (ic ) values required for peptide binding to mhc-i molecules, stabilized matrix method (peters and sette ) was applied with a preset . -mer epitope. in case of mhc-ii binding analysis, the iedb-recommended method was used for the specific hla-dq, hla-dp, and hla-dr loci. herein, specific peptides were used to predict the mhc-ii interaction on the basis of mhc-i analysis and antigenic conservancy. population coverage for epitope was assessed by the iedb population coverage calculation tool (bui et al. ). here we used the allelic frequency of the interacting hla alleles for the prediction of the population coverage for the corresponding epitope. linear b cell epitopes are of different lengths of peptides from to in comparison to that of t cell epitopes. b-cell epitope produces immune response when it interacts with b lymphocytes. it then initiates the differentiation of b lymphocytes into plasma and memory cells (nair et al. ) . there are a number of web-based tools are available for the prediction of b-cell epitope which are hosted by iedb-ar. for the b-cell epitope prediction with high accuracy, multiple tools, including the emini surface accessibility prediction (emini et al. ) , kolaskar and tongaonkar antigenicity scale (kolaskar and tongaonkar ) , parker hydrophilicity prediction, (parker et al. ) and finally the chou and fasman beta turn prediction tool (chou and fasman ) were employed, because the antigenic parts of a protein belong to the beta turn regions (rini et al. ) . the structure of the conserved region was constructed by homology modelling using the modeller v (Šali et al. ) . modeller is a program that implements an automated approach to comparative protein structure modelling by satisfying spatial restraints (fiser et al. , sali and blundell ) . finally, the evaluation of the predicted model was verified by using two software tools, procheck (arnold et al. , laskowski et al. and qmean (benkert et al. ) . for predicting the disorder among the amino acid sequences, disopred v (ward et al. ) server was used. in order to calculate the protein variability index the protein variability server was implied where wu-kabat variability coefficient (garcia-boronat et al. ) has been used. the web-based allerhunter server (muh et al. ) was used to predict the allergenicity of our proposed epitope for vaccine development. this server predicts allergenicity through a combinational prediction, by using both integration of the food and agriculture organization (fao)/world health organization (who) allergenicity evaluation scheme and support vector machines (svm)-pairwise sequence similarity. allerhunter predicts allergens as well as nonallergens with high specificity. this makes allerhunter is a very useful program for allergen cross-reactivity prediction (liao and noble ) . epitope conservancy of the candidate epitopes was examined using a web-based epitope conservancy tool available in iedb analysis resource (bui et al. ). the conservancy level of each potential epitope was calculated by looking for identities in all rna-dependent rna polymerase-l protein sequences of different strains retrieved from database. a total of rna-dependent rna polymerase-l protein molecules from different variants of the ebov were retrieved from the uniprot database. the msa of the rnadependent polymerase-l proteins was retrieved from bioedit tool through clustalw with bootstrap replicates (additional file : figure s ). clc sequence viewer was used to construct phylograms from the msa obtained from bioedit, in order to analyze the divergence among the retrieved sequences. phylogram of rna-dependent rna polymerase-l is depicted in fig. . finally, the highly conserved region from the msa was retrieved for the further analysis. the selected conserved region is depicted in the fig. , from the msa number to . then the vaxijen v . server calculate the antigenicity of the conserved sequences with a score . . t-cell epitopes were selected firstly by using the netctl v . server where the epitope prediction was confined to mhc-i supertypes. based on the combined score, the top five epitopes (table ) were listed for further analysis. t-cell epitopes were again predicted by the ctlpred server (table ) . here a combined approach of artificial neural networks and support vector machines was applied. depending on the two analyses, the most common epitope-containing peptides, identified by both servers, was selected. the selected epitope was then used for the mhc-binding analysis. mhc-i-binding prediction, which was run through the stabilized matrix method, predicted a wide range of mhc-i allele interactions for the proposed t-cell epitopes. the mhc-i alleles for which the epitope showed higher affinity (ic < nm) are listed in table . the output of the mhc-ii interaction analysis is also shown in table . iedb population coverage tool analyzed the population coverage of the proposed epitope. the combined mhc-i and mhc-ii class were assessed against the whole world population with the selected mhc-i and mhc-ii interacted alleles (fig. ) . here, for predicting potential b-cell epitopes, we used amino acid-based methods. according to this procedure different analysis methods were applied for the identification of a continuous b cell epitope. the kolaskar and tongaonkar antigenicity scale was used for assessing the antigenic property of the peptides. the average antigenic propensity of the protein was . , with a maximum of . and a minimum of . . for the protein the antigenic determination threshold value was . , where all values equal or greater than . were potential antigenic determinants. the antigenic plot is depicted in the fig. . to be a potent b cell epitope, it must be surface accessible. hence, emini surface accessibility prediction was employed, with a maximum propensity score of . at threshold . (fig. ) . to strengthen our support for the prediction of the epitope to elicit b cell response the parker hydrophilicity and the chou and fasman beta turn prediction were employed. those are described in the figs. and . homology model of the conserved region was obtained by the modeller software, which is shown in fig. a and b. procheck server validated the stereochemical quality of the model through ramachandran plot (fig. c) , andqmean server also assessed the tertiary structure, with a qmean score of . . disopred v server predicted the disorder of the conserved peptide in order to get insight about the disorder among the conserved sequences, which is depicted in fig. . protein variability server predicted the variability of the conserved region of the rna-dependent rna polymerase-l ( fig. ) to ensure that the proposed epitope is within the invariable region. conservation analyses of the proposed epitopes were analyzed by the iedb conservancy analysis tool that is shown in table . allerhunter server predicted the our world is the habitation of more than seven billion people now. with the upgrade of medical science, new viruses along with their causing diseases are also emerging. ebola virus is such kinds of virus with a deadly outrage of their endemic nature especially in africa in recent time (evans and popova ) . till now there is no potential treatment for this virus to combat its deadly effects. recent time, the immunoinformatics approach give us some sort of hope for the design of an effective therapeutics, like vaccine, in association with the advancement of sequence based technology. similar approaches have been used successfully for identifying vaccine candidates in several pathogens viz. human corona virus (oany et al. ) , saint louis encephalitis virus (hasan et al. ) , crimean-congo hemorrhagic fever virus fig. parker hydrophilicity prediction of the epitope, with a minimum propensity score of − . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow fig. chou and fasman beta turn prediction of the epitope,with a minimum propensity score of . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow (oany et al. ) , chikungunya virus (hasan et al. ) and some others. the in vitro validation of this type of work has also been proven in recent time (khan et al. ) . though epitope-based vaccine designing has become a familiar approach, in the case of ebov no significant work yet has been done. ebov is an rna virus which has genetic blueprints made of rna instead of dna. creating vaccines is particularly difficult for rna viruses as they can quickly mutate their different exposed proteins (twiddy et al. ) . therefore the most potential way to create stable antiviral therapies against rna viruses including ebov is to target the transcription or replication machinery. scientists revealed that rnadependent rna polymerase-l (ebol) is an important cellular component for the transcription and replication of the ebov genome. when an ebov infects a cell, its rna genetic blueprint enters the cell along with rna-dependent rna polymerase-l. this polymerase normally "read" the rna genetic blueprint in order to synthesize mrna, which then leads to the formation of viral proteins as well as viral replication and more viral particles are produced. for these two vital involvements at the gateway, this protein was targeted to design most potential epitopes using in silico computational approaches. in the current study, firstly all the available sequences of rna-dependent rna polymerase-lwere retrieved from database. then antigenicity of the conserved peptides, generated by multiple sequence alignment was predicted by vaxijen, which suggested their ability to elicit potential immune response. sequence based bioinformatics approaches were applied to predict both b cell and t cell epitopes for conferring immunity in different ways. though at present, most of the vaccines are based on b cell immunity; vaccines based on t cell epitope have been encouraged recently. it is because, with time humoral response from memory b cells can be overcome easily by antigenic drift, while cell mediated immunity often provides long lasting immunity (bacchetta et al. , igietseme et al. ). cytotoxic cd + t lymphocytes (ctl) inhibit the spread of infectious agents by recognizing and killing infected cells or secreting specific antiviral cytokines (garcia et al. , shrestha and diamond ) . thus, vaccination based on t cell epitope is a unique approach to obtain strong immune response against infectious agents, such as, viruses (klein et al. ) . both netctl and ctlpred server were used to find epitopes for the activation of t-cell immunity with potential antigenicity. by examining the output it was predicted that fryeftapf would be the best epitope candidate and was further subjected for binding proficiency analysis. length is an important factor to consider for peptide antigen binding with mhc or tcr or both. t cell epitopes presented by mhc class i molecules are generally peptides between and amino acids in length. we therefore set peptide lengths at before making software based mhc class i t cell epitope identification using fig. protein variability index of the conserved peptides of all the sequences. the prediction suggests that our proposed epitope "fryeftapf" falls in the invariable region (blue line). notes: the conservancy threshold was . in this analysis. the x-axis indicates the amino acid positions in the sequences and the y-axis indicates the shannon variability score fig. disorder prediction of the conserved antigenic amino acid sequences. here, our proposed epitope lies outside ( - ) of the disordered region to secure its potentiality as an effective epitope. notes: amino acids in the input sequence are considered disordered when the blue line is above the gray dashed line, that is, when the confidence score is . . the orange line shows the confidence score of the disordered protein-binding residue predictions immune epitope database (iedb). analysis revealed that the core epitope "fryeftapf" would interact with ten different mhc class i alleles. on the other hand, the complete peptide "nlafryeftapfiey" interacts with the highest numbers of mhc class ii alleles (as many as alleles). along with the t-cell epitope, in our study, attention was also given to the b-cell epitope, which can induce both primary and secondary humoral immunity (trainor et al. ). multiple prediction methods were applied to determine the b-cell epitope considering several criteria of antigenicity, hydrophilicity, surface accessibility, and beta-turn. our proposed epitope has met all the criteria of the above b-cell prediction methods. the three-dimensional model of the conserved protein ensured the exact location of the epitope outside of the protein ( fig. a and b ) surface and the model validity was assessed by ramachandran plot (fig. c) , whereby . % amino acid residues were found within the favored region. the epitope was also treated as suitable candidate for vaccine through tenabled its position in the conserved sequence, by the discopred and protein variability server (figs. and ). conservancy is the most important criterion of an epitope to consider it for vaccine development. conservancy analysis of our proposed epitope showed % conservancy among all the available sequences. another important feature of the peptide vaccine is its allergenicity (mckeever et al. ) . in silico analysis revealed that the proposed epitope is nonallergenic in nature. wide range population coverage must be needed for a potential vaccine aspirant. at this point, our proposed epitope covers a remarkable population of . % for both types of mhc allele throughout the world population. that makes the epitope as a supreme candidate for vaccine consideration. finally, from the above in silico analysis, we are really optimistic that our proposed epitope would trigger an immune response in vitro and in vivo. a number of approaches exist for new vaccine development, such as recombinant vaccines, sub-unit protein and dna vaccines, auxotrophic organisms to deliver genes and so on. current study is an attempt to identify potential epitope targets against ebov using different computational tools. it is quite obvious that in order to minimize the deadly effects of ebov, highly potential drugs are immediately required and these in silico approaches will reduce the wet lab efforts with higher probability of success. therefore, it is concluded that the identified epitope may be exploited further for developing epitope-based vaccine against ebov. nevertheless, the initial hints we obtained will help to prioritize potential therapeutics for ebov. additional file : figure s . msa of the rna-dependent polymerase-l proteins of the different ebov. 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system for functional analysis of ebola virus glycoprotein clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mutation analysis of the fusion domain region of st. louis encephalitis virus envelope protein inferring the rate and time-scale of dengue virus evolution characterization of the l gene and ′trailer region of ebola virus a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach peptide binding predictions for hla dr, dp and dq molecules the disopred server for the prediction of protein disorder jalview version -a multiple sequence alignment editor and analysis workbench development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv- the authors declare that they have no competing interests.authors' contributions aro has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. ts and asc carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. tpj worked for computational analysis. mah conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com key: cord- -mejd blb authors: lewnard, joseph a; reingold, arthur l title: emerging challenges and opportunities in infectious disease epidemiology date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: mejd blb much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. initially submitted september , ; accepted for publication november , . much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. infectious diseases; methods; modeling; surveillance abbreviation: ebov, ebola virus. the priority afforded to infectious diseases within epidemiologic research has been fluid over the past years or longer. despite the lasting prominence of early investigations into measles, cholera, plague, typhoid fever, malaria, and yellow fever ( ) ( ) ( ) ( ) ( ) ( ) , the intellectual tradition of modern epidemiology stems largely from studies of chronic diseases dating to the post-world war ii era, when such conditions came to surpass infectious diseases in morbidity and mortality in highincome countries amid improvements in living conditions and the introduction of numerous antibiotics and vaccines. this epidemiologic transition co-occurred with a shift in focus for epidemiologic research (and training programs) toward the multifactorial etiology of chronic conditions ( ) . in parallel, early th-century work on the "dependent happenings" of communicable diseases ( ) ( ) ( ) ( ) yielded to the development of today's core biostatistical methods for chronic diseases, premised on the independence of outcomes among subjects ( ) ( ) ( ) . since the late th century, the emergence of human immunodeficiency virus and acquired immunodeficiency syndrome and other infections has renewed interest in infectious diseases and their health, economic, and security implications ( ) . outbreaks of severe acute respiratory syndrome, pandemic influenza a h n , ebola virus (ebov), and zika virus have prompted international responses, whereas influenza a h n and h n , lassa fever virus, nipah virus, and middle east respiratory syndrome coronavirus, among other agents, have been a source of regional concern. the incidence and endemic range of "neglected" infections, including dengue and cholera ( , ) , have expanded, and antimicrobial resistance has threatened to derail the control of tuberculosis, typhoid, malaria, gonorrhea, yaws, and invasive bacterial infections ( ) ( ) ( ) ( ) ( ) ( ) . although highly effective vaccines are available, measles and yellow fever have resurged on multiple continents, due to gaps in vaccine coverage ( , ) , while short-lived vaccine-induced protection has facilitated unexpected resurgences in diseases once on the path to elimination, such as pertussis and mumps ( , ) . after decades of relative obscurity in the mid- th century, infectious disease epidemiology has experienced an intellectual rebirth in response to disease emergence. repopulation of this field by scientists trained not only in clinical medicine but ecology, demography, and quantitative sciences has led to the adoption of methods scarcely addressed in traditional public health training programs. cluster-randomized trial designs, for example, have become commonplace for evaluating infectious disease interventions, quantifying indirect effects resulting from contagion ( ) . classical models of ecological dynamics have been adapted to address the transmission and control of infectious agents, while our expanding ability to integrate such models with epidemiologic data through bayesian statistics has enhanced their relevance to policymaking ( ) ( ) ( ) . most recently, sequencing and phylogenetic analysis have afforded an unprecedented view into the population structure and dynamics of pathogens ( , ) . here, we review developments in infectious disease epidemiology together with their implications for research and practice, and for the training of future epidemiologists. we first consider the role of epidemiologic surveillance in the context of pathogen emergence and the integration of surveillance data into quantitative studies of transmission. next, we discuss challenges in causal inference, including the evaluation of public health interventions against emerging pathogens and the difficulties of attributing clinical syndromes to microbial agents. surveillance justifiably has been seen as a core public health function, with its important role articulated by langmuir in ( ) . in the united states and elsewhere, surveillance for diverse infectious (and noninfectious) diseases has typically relied on an essentially passive system of reporting by healthcare providers or laboratories, often mandated by public health laws. although such passive systems of disease reporting have proven invaluable, their limitations, such as incomplete, often inconsistent detection of cases and delayed detection of outbreaks, are well documented ( ) . as a result, active surveillance systems that do not depend on providers and laboratories to report have been developed and promoted. the centers for disease control and prevention-funded emerging infections program and its components (e.g., foodnet, abcs), initiated in , is notable example of such a system, as are international versions promoted through the centers for disease control and prevention's global health security initiative ( ) . such systems, while expensive to develop and maintain, are of great value, especially when they collect biological specimens (e.g., isolates of bacterial and viral pathogens) for typing and support analytic epidemiologic studies (e.g., casecontrol studies of vaccine effectiveness ( ) ). nevertheless, these systems, too, may have limitations, particularly in their ability to detect in a timely fashion outbreaks caused by novel microbial agents, prompting interest in alternative methods. in this article, we consider proposed alternatives, highlighting their benefits and challenges. beyond efforts to quantify the incidence of infectious diseases of known etiology, disease surveillance has been advocated as a means of mitigating the threat posed by novel pathogens. large-scale efforts to identify pathogens with the potential to spill over from animals to humans have received notable investment, for instance from the us agency for international development emerging pandemic threats program ( ) . more recently, metagenomic sequencing has enabled the number of known viruses to be multiplied in such studies ( ) . the aim of those working on the global virome project, launched in , to characterize within years all . million viruses thought to exist ( ) . however, the pathway for translating data sets produced by such activities into actionable threat-reduction programs remains unclear. that only approximately viruses are known to infect humans (and an even smaller handful to cause major epidemics) may constrain the value of large-scale virus discovery for identifying high-risk pathogens, as well as viral determinants of pathogenic potential ( ) . spillover events causing recent epidemics-including h n in mexico, middle east respiratory syndrome in saudi arabia, and ebov in west africa-have been poorly predicted by factors long believed to drive disease emergence ( ) . accordingly, interest in expanding our catalogue of potential pathogens should be weighed against our persisting need to enhance the detection and control of outbreaks of known pathogens ( ) . for instance, ebov epidemics in the democratic republic of the congo in took weeks to be identified, with dozens of suspected cases having already accumulated ( , ) . serological studies have received increasing enthusiasm for monitoring emerging pathogens of significance to humans ( , ) . the prevalence of antibodies indicating previous exposure may provide valuable information about the frequency of animal-human spillover events and the potential for person-toperson spread, overcoming reporting biases that favor detection of large outbreaks under traditional surveillance. moreover, the low cost of multiplex assays makes integrated surveillance of multiple pathogens plausible. although serosurveys have bolstered recent efforts to understand the geographic range and clinical spectrum of ebov and zika virus infections ( , ) , the enhancement of dengue hemorrhagic fever risk by prior exposure ( ) , and the role of immunologic history in influenza susceptibility and vaccine response ( ) , there remain few examples of public health programs undertaking serological studies for routine surveillance, at least in civilian populations ( ) . outside of laboratory-based surveillance, the increasing availability of passively collected "big data" on the health and behaviors of individuals has prompted enthusiasm about enhancing disease surveillance through alternative data streams. initiatives such as the promed-mail network and healthmap ( , ) compile and disseminate news about outbreaks from media and other sources, aiming to trigger investigation by public health organizations. data such as emergency department visits, medication sales, online search queries, and social media postings have also been suggested as real-time indicators of outbreak activity, although their integration into public health responses remains a subject of debate ( ) ( ) ( ) . the need to overcome reporting biases is a central challenge, because observations may be too nonspecific to distinguish between meaningful and spurious signals in settings with high technological capacity while also being insensitive to even high-risk events in resource-poor settings ( , ) . although nontraditional data sources have, in some applications, supported inferences about epidemic dynamics ( ) , limited information about cases from such sources remains a barrier. for instance, models fitted from news reports of recent measles and mumps outbreaks have yielded considerable underestimates of vaccine coverage ( ) ( ) ( ) , underscoring the importance of field investigations. forecasting the incidence of diseases has been a more successful application of these emerging data streams and dataanalytic approaches. although the acknowledged failure of google flu trends-a prediction approach based on internet search behavior-yielded important lessons about nonmechanistic forecasts, approaches based on machine learning and crowdsourced human judgment have provided the most accurate within-season predictions of us influenza activity in recent comparisons ( ) ( ) ( ) . given expanding interest in forecasting among researchers, funding agencies, and other stakeholders, there is a clear and compelling need to evaluate whether such forecasts can enhance the success and efficiency of public health response efforts. mathematical modeling as a means to understanding infectious disease spread dates to studies by sir ronald ross ( ) . although the use of models to connect data such as age of infection to transmission dynamics of endemic infections has longstanding precedent ( , ) , assimilation of outbreak data for near-term assessments of control priorities is a comparatively recent phenomenon. integration of modeling with the public health response to epidemics of bovine spongiform encephalopathy and foot-and-mouth disease in the united kingdom and the severe acute respiratory syndrome epidemic ( - ) has led to expectations for near real-time modeling studies during major outbreaks. in recent experience, models of the spread of pandemic influenza a h n ( ), cholera ( ), middle east respiratory syndrome ( ), ebov ( , ) , chikungunya virus ( ), zika virus ( , ) , yellow fever ( ) , and plague ( ) have all been published within weeks of the respective outbreak notifications. although the circumstances of particular epidemics dictate what data may be available and pertinent, methods for fitting models to data have generally focused on exponential growth rates in cases ( ) or the distribution of the serial interval ( ) . methods based on the latter class of data offer the advantage of illustrating real-time changes in reproductive numbers ( ) ; however, the requisite information from patient line lists is seldom available. reliance instead on ecological data exposes models to numerous vulnerabilities, notably the inability to discern individual risk factors (and thus the population meaningfully at risk). these shortcomings may prevent models from predicting reductions in transmission before depletion of the susceptible population. a challenge thus lies ahead in determining the role of models in outbreak response and the best practices for communicating modeling results. although the ability of models to evaluate prophylactic strategies may be considered a benefit, recommendations to act against remote future risks have sometimes triggered resistance among stakeholders ( ) . during the west african ebov epidemic, for example, attention to worst-case model-based projections prompted some to question the reliability of the models ( ), reflecting an important discrepancy between public understanding of modeling as a forecasting tool and the intended uses of models for scenario-based comparisons ( , ) . this use of modeling has been better understood in attempts to communicate the impact of interventions after the fact ( ). the ease of sequencing pathogen genomes has afforded a new view into transmission during outbreaks. use of sequence data to identify transmission clusters in the presence of unsure epidemiologic links dates to the early years of the human immunodeficiency virus and acquired immunodeficiency syndrome epidemic ( ) . in recent years, sequencing has aided efforts to track the sources of unexplained epidemics of cholera in haiti ( ) and ebov in west africa ( ) , and has shown increasing utility for reconstructing the geographic spread of pathogens ( , ) . a particular advantage of phylogenetic analysis is the possibility of estimating unobserved epidemiologic quantities, such as the reporting fraction ( ) and reproductive numbers for subcritical transmission ( ) , which remain difficult to assess from traditional case-notification data. beyond reconstructing the demographic history of pathogen lineages, recent years have seen progress toward joint analysis of epidemiologic and sequencing data ( ) . such "phylodynamic" approaches have shown particular relevance for emerging infections, including distinguishing the role of repeated introductions and subsequent local transmission ( ) ( ) ( ) ( ) . whereas most applications have been tailored to specific data sets and assumptions, the development of generalized methods for joint inference of epidemiologic and phylogenetic parameters remains a priority ( ) to support real-time analysis. the ability to rapidly develop and deploy countermeasures to mitigate the threat posed by emerging infections has received increasing recognition as a component of public health preparedness. however, outbreaks are difficult environments in which to evaluate interventions. during the west african ebov epidemic, the feasibility of a new paradigm for development and evaluation of interventions in emergencies was demonstrated by accelerated vaccine safety, immunogenicity, and efficacy studies ( ) . the coalition for epidemic preparedness innovations was established in , with an initial focus on vaccines against nipah virus, middle east respiratory syndrome coronavirus, and lassa fever virus, in addition to adaptable vaccine platforms for novel threats ( ) . lessons learned in ebov vaccine trials will have an influential bearing on evaluations during future emergencies. despite efforts to accelerate evaluation of candidate vaccines, incidence had reached low levels by the time phase iii efficacy trials were ready to begin, posing a threat to their statistical power: a planned trial in liberia was canceled due to declining transmission ( ) , and no cases of disease occurred in a second trial in sierra leone ( ), preventing efficacy assessments. in a stepped-wedge trial in guinea, clusters of primary and secondary contacts of ebov disease cases were randomly assigned to immediate or delayed vaccination; no cases were reported among vaccine recipients during the trial ( ) or in subsequent field deployments of the vaccine, supporting a conclusion of near % vaccine efficacy. debates surrounding design of these trials highlight methodological questions requiring additional attention. in the guinean trial, a "ring" vaccination scheme helped maximize power by enrolling contacts of known cases ( ) . however, the choice of individual-or cluster-level randomization within rings was debated. because members of a vaccinated cluster are exposed to direct protection through vaccination and indirect protection due to reduced transmission within their clusters ( ), cluster-randomized trials have weaker statistical power than individually randomized trials of the same size ( ) . moreover, the direct effect measured in individually randomized studies may be a preferred, transportable efficacy measure ( ) . uses of simulation helped in planning vaccine trials tailored to the real-world circumstances of the ebov outbreak ( ) and enabled trialists to compare alternative designs in terms of ethical mandates ( ) . simulation-guided design further presents the opportunity for applying adaptive trial methods ( ) in the context of infectious disease outbreaks, where dynamic trends in incidence may highlight the benefits of such approaches. in addition to efficacy trials for new interventions, observational studies are needed to assess licensed interventions against evolving and re-emerging pathogens. most commonly applied in evaluations of influenza vaccines, test-negative designs have become popular in routine ( ) and exploratory ( ) studies of vaccine effectiveness. by measuring vaccine effectiveness from the exposure odds ratio of vaccination among individuals seeking care who test positive or negative for a pathogen of interest, this design seeks to overcome associations of health-care seeking with vaccination status ( ) . however, it is uncertain whether health-care seeking and other sources of confounding are appropriately controlled for, and whether measures accurately capture vaccine direct effects ( , ) . uncertainty about the validity of estimates that routinely inform vaccine policy-making demonstrates the need for formal evaluations of such studies and strategies to reduce bias. time series analyses of public health surveillance data provide another approach to measuring the real-world impact of vaccination on disease incidence, with the advantage of identifying the overall effect of a vaccination program resulting from direct and indirect protection ( ) . although the ecological nature of such designs permits the introduction of biases from changes in diagnostic practices or health-care seeking, such studies nonetheless have offered important insights where other approaches failed. limited reductions in influenza-related deaths among elderly persons amid increases in influenza vaccine coverage during the s provided an important indication that the "healthy vaccinee" effect accounted for astonishing and implausible protection against all-cause mortality among elderly influenza vaccine recipients in cohort and case-control studies ( ) ( ) ( ) . newer methods continue to improve public health inferences obtained from time-series data. in a recent evaluation of invasive pneumococcal disease incidence, trends expected under continued use of -valent pneumococcal conjugate vaccine provided a counterfactual condition for measuring the impact of the switch to a -valent vaccine targeting emerging serotypes ( ) . bayesian averaging of models encoding differing pre-and postvaccination trends and change points provides a generalized strategy for defining such counterfactual comparisons ( ) . other signals of transmission intensity in surveillance data, such as age of infection and sub-or multiannual periodicity, may provide additional insights while reducing sensitivity to fluctuations in reporting effort ( , ) . the re-emergence of pathogens against which vaccines are widely deployed, such as varicella, pertussis, and mumps in the united states, poses additional challenges for conducting vaccine effectiveness studies. situational factors may undermine researchers' ability to establish the extent to which cases owe to primary or secondary vaccine failure in all or certain vaccine recipients, and whether emerging pathogen lineages are escaping vaccine-driven immune pressure. for instance, high compliance with vaccine schedules may limit variation in individuals' vaccination status and exposures, necessitating large samples to detect factors influencing vaccine performance ( ) . because re-emergence most likely reflects the expansion of or several pathogen clades, limited pathogen diversity may hinder the application of conventional approaches to identifying microbial determinants of vaccine escape ( , ) . novel methods to distinguish null from vaccine-driven mutations in antigencoding regions ( , ) may streamline efforts to identify vaccine escape, while mathematical modeling provides a basis for comparing candidate hypotheses with observations ( , ) . because vaccine studies are typically powered for primary clinical endpoints, long-term observational studies are needed to monitor for rare vaccine-attributable adverse events. such studies have been crucial to identifying safety concerns such as intussusception after rotavirus vaccination ( ) and to refuting spurious links, such as autism onset after measles-mumpsrubella vaccination ( ) . however, unique challenges arise in vaccine safety studies; at the individual level, vaccination and adverse-event detection may be confounded due to health-careseeking behavior, whereas at the population level, age-related confounding may occur when vaccine recommendations are based on the individual's age. ecological designs taking advantage of natural experiments have proven useful in numerous studies of vaccine safety ( , ) but inconclusive for certain classes of rare events, including those that also result from the vaccine-targeted infection ( , ) . recent years have seen growing interest in case-only methods offering the ability to reduce or rule out individual-level sources of confounding. case-crossover methods are common among such approaches ( , ) and resemble matched case-control studies by sampling "control" periods from the person-time contributed by case individuals before an adverse event. self-controlled case-series methods similarly benefit from the use of cases as their own controls, following a cohort logic in estimating the relative incidence of adverse events after vaccination within specified risk periods ( ) ; with adequate sample size, researchers may be able to use such analyses to eliminate or greatly reduce potential time-or age-related confounding. in response to the growing threat posed by antimicrobial resistance, the world health organization and national governments have prioritized bringing novel antimicrobial drugs to market ( ) . these plans will necessitate phase iii trials in which the efficacy of new therapeutic agents is addressed and possibly phase iv studies, in which the optimal use of new and existing drugs, either singly or in combination, can be determined. whereas patients traditionally have been enrolled in antimicrobial treatment studies on the basis of target bacterial species infections or clinical syndromes, it is uncertain within what strata such trials may yield transportable inferences. rather than merely the infecting pathogen's baseline resistance or susceptibility phenotype, strata may be defined by factors such as pathogen lineage, mutational barriers to resistance development ( ) , and presence of horizontally transferable resistance elements in cocolonizing agents or environmental sources ( ) . stratification based on interpatient and even intrapatient tumor heterogeneity is an emerging feature of cancer therapy trials and may provide a template for such designs ( ) . in addition to clinical endpoints, carriage of susceptible and resistant bacteria, including commensal agents not purposefully targeted by treatment, can inform the impact of treatment on resistance selection in targeted and bystander species ( ) . whereas between-group differences in the absolute prevalence of colonization with resistant organisms ( ) are tested for routinely, stratified measurements (see the reports of shrag et al. ( ) and feikin et al. ( ) , for example) of the effect of treatment on acquisition and clearance of susceptible and resistant pathogens are more informative of the underlying biology ( ) and may detect signals of selection masked by simpler betweengroup comparisons ( , ) . studies are also needed to address optimal deployment of new and existing antimicrobial drugs in clinical practice. the tradeoff between maximizing a drug's impact and minimizing resistance selection has led policymakers to ration certain new drugs as last-resort treatments. however, in recent experience, such decisions have ignited ethical debates ( ) . coupling mathematical modeling with field-based studies has proven useful for understanding the effectiveness of antimicrobial use policies, as highlighted in recent evaluations of the risk of resistance under population-wide access of the tuberculosis drug bedaquiline ( , ) and antimicrobial cycling to limit resistance selection in hospital settings ( , ) . whereas certain efforts we have discussed have been made to identify novel microorganisms able to cause human infection ( , ) , most epidemics caused by emerging pathogens have been recognized first by clusters of anomalous syndromes-such as cardiopulmonary syndrome caused by new world hantaviruses, severe acute respiratory syndrome caused by a coronavirus, and congenital abnormalities caused by zika virus -before the role or even existence of the etiologic microorganism had been characterized. the problem of ascribing a clinical syndrome to an etiologic agent is among the oldest in epidemiology, dating at least to the th century, when robert koch laid out criteria for such inference (i.e., koch's, or more correctly, henle-koch's, postulates ( ) ). however, these postulates have long been recognized as inadequate, particularly for illnesses caused by viruses, and thus of largely historical interest ( ) ; for instance, the notion that a pathogen should be absent from healthy individuals is incompatible with the prominence of carriage and asymptomatic infection in the natural history of numerous pathogens. a growing appreciation of the complexity of the human microbiome, and the likelihood that intricate mixtures of microorganisms at diverse body sites may be either the cause or consequence of both detrimental and beneficial physiological states, has further highlighted the difficulty of linking a given health outcome to infection by a single microorganism. the now-recognized critically important role of persistent infection and the inflammation it can produce in diverse cancers and possibly other chronic diseases has further diminished the relevance of koch's postulates and the age-old distinction between infectious and chronic diseases, as illustrated in the well-known example of human papillomavirus causing cervical, anal, and oral cancers ( ) . the best causal understanding of such relationships has come from randomized trials demonstrating that infection-preventing interventions are efficacious against downstream chronic illness, such as peptic ulcers due to helicobacter pylori and chronic wheeze due to respiratory syncytial virus ( , ) . natural experiments following the same intuition have provided additional evidence of such relationships, such as measles-induced immunosuppression ( ) , malnutrition and stunting due to enteric infection ( ) , and complex or chronic otitis media due to tissue damage from acute early-life disease ( ) . such relationships have proven difficult to probe in the absence of a randomized or natural experiment, because of the likelihood that confounding factors influence individuals' risk for initial infection as well as chronic sequelae. new paradigms for ascribing an etiologic role to microorganisms and resulting host responses are clearly needed and may prove important in efforts to quantify the health impacts of infectious disease interventions. the recognition in the s and s that infectious diseases were not, in fact, disappearing 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years of age in resourcepoor environments prevention of early episodes of otitis media by pneumococcal vaccines might reduce progression to complex disease the authors received no specific funding for this article. conflict of interest: none declared. key: cord- - ihyiwgb authors: eickmann, markus; gravemann, ute; handke, wiebke; tolksdorf, frank; reichenberg, stefan; müller, thomas h.; seltsam, axel title: inactivation of ebola virus and middle east respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet c light and methylene blue plus visible light, respectively date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: ihyiwgb background: ebola virus (ebov) and middle east respiratory syndrome coronavirus (mers‐cov) have been identified as potential threats to blood safety. this study investigated the efficacy of the theraflex uv‐platelets and theraflex mb‐plasma pathogen inactivation systems to inactivate ebov and mers‐cov in platelet concentrates (pcs) and plasma, respectively. study design and methods: pcs and plasma were spiked with high titers of cell culture–derived ebov and mers‐cov, treated with various light doses of ultraviolet c (uvc; theraflex uv‐platelets) or methylene blue (mb) plus visible light (mb/light; theraflex mb‐plasma), and assessed for residual viral infectivity. results: uvc reduced ebov (≥ . log) and mers‐cov (≥ . log) infectivity in pcs to the limit of detection, and mb/light decreased ebov (≥ . log) and mers‐cov (≥ . log) titers in plasma to nondetectable levels. conclusions: both theraflex uv‐platelets (uvc) and theraflex mb‐plasma (mb/light) effectively reduce ebov and mers‐cov infectivity in platelets and plasma, respectively. has been transmitted through the transfusion of blood products. however, the fact that the mortality rate is high (> %) and the transmission mechanisms are still not completely understood highlight the need for increased awareness of the potential threat to blood safety by mers-cov. mers-cov is an enveloped, positive-sense single-stranded rna coronavirus. interestingly, viral rna was detected in the sera of asymptomatic patients during an outbreak of severe acute respiratory syndrome, which is caused by another coronavirus, severe acute respiratory syndrome-cov. consequently, there might be an asymptomatic viremic phase that could theoretically lead to transmission via blood transfusion. however, blood transfusions have not been implicated in the transmission of mers-cov to date. ebov, a member of the filoviridae family, is an enveloped pathogen containing negative-sense single-stranded rna as its genetic material. ebov disease is extremely fatal and is known to cause death rates of up to %. recent outbreaks of ebov disease in west africa have devastated guinea, liberia, and sierra leone, killing more than , out of nearly , persons affected. ebov is transmitted to humans from animals and spreads through human-to-human transmission, that is, via direct contact with infected blood and/or secretions. different pathogen inactivation (pi) techniques can effectively inactivate or incapacitate such viral agents, rendering them unable to replicate in blood components or products. theraflex uv-platelets (macopharma), a novel ultraviolet c (uvc)-based pi system for platelet concentrates (pcs), is currently undergoing clinical efficacy and safety testing. [ ] [ ] [ ] in contrast to other pi technologies, it works without exogenously added photoactive substances. shortwave uvc light ( nm) interacts with nucleic acids directly, inducing the formation of cyclobutane pyrimidine and pyrimidine pyrimidone dimers that prevent the elongation of nucleic acid transcripts. the theraflex mb-plasma (macopharma) is a photodynamic pi procedure for single units of fresh-frozen plasma. this system, based on the administration of methylene blue (mb) in combination with visible light (mb/light), was developed to increase the viral safety of plasma transfusions. , more than million units have been treated by this method and safely transfused to patients for more than years. both systems have been shown to effectively inactivate a broad range of different dna and rna viruses, including arboviruses such as west nile virus, chikungunya virus, and zika virus; recently, large outbreaks of the latter viruses have occurred outside of their previously known geographic ranges, seriously affecting thousands of people. , [ ] [ ] [ ] [ ] [ ] [ ] this study aimed to investigate the efficacy of theraflex uv-platelets and theraflex mb-plasma to inactivate mers-cov and ebov in pcs and plasma, respectively. all components were prepared from whole blood ( ml) collected from screened, unpaid volunteers and stored in ml of cpd according to german guidelines. blood was separated into red blood cells (rbcs), plasma, and buffy coat by standard blood banking procedures. plasma units were leukoreduced by filtration (plasmaflex, macopharma), pooled, split again, and stored at - c until further use. plasma-reduced pcs were prepared from pools of five buffy coats as described by eriksson and colleagues. the pools were mixed with ml of pas ssp (macopharma), which is equivalent to pas-e. the suspension was centrifuged for . minutes at g. pcs were isolated, leukoreduced with a high-efficiency filter system (autostop bc, haemonetics), and stored under agitation at c. the platelet (plt) concentration was approximately /ml, the plasma content approximately %, and the residual white blood cell (wbc) content was not more than per unit. air was routinely removed from all plasma units and pcs. the anti-mers-cov enzyme-linked immunosorbent assay (igg; euroimmun) was used to screen pcs and plasma units for the presence of antibodies against mers-cov. only products tested for antibodies to mers-cov and confirmed to be negative were used in the inactivation experiments. plts were treated using theraflex uv-platelets, a pi system consisting of a uvc illumination device (maco-tronic, macopharma) and a disposable set (ref xuv xu, macopharma). briefly, the pcs were irradiated with uvc light at a wavelength of nm to a total dose of . j/cm , with vigorous agitation to ensure the uniform treatment of all compartments. the amount of uvc energy delivered was expressed in j/cm . plasma units were mb/light-treated using theraflex mb-plasma (macopharma), a pi system consisting of a theraflex mb-plasma kit and the macotronic b ledbased illumination device. its closed-bag system contains a mb pill that delivers mg of mb per unit of plasma or approximately mmol/l per plasma unit. the standard treatment cycle includes initial filtration for leukoreduction (plasmaflex filter) followed by a second postillumination filtration step for the removal of mb and its photoproducts (blueflex filter). , plasma processing for pi efficacy testing deviated from the standard procedure in that the removal of mb and photoproducts (blueflex filtration) was not performed so as to exclusively determine the virus inactivation effects of illumination. the amount of led-based light energy delivered was expressed in j/cm . the total illumination dose was set at j/cm , as is routinely recommended for this system. endpoint titration assays for microtiter plates were employed to measure virus titers in eight serial -in- dilutions per parallel sample. vero e cells were used as the indicator cells. sample titration was performed at the initial dilution at which vero cells exhibited no cytotoxicity. the plates were incubated at c in a humid atmosphere with % co . after to days of incubation, the cell layers were assessed for virus-induced changes in morphology (cytopathology). the % tissue culture infectious dose (tcid ) was calculated according to the spearman-k€ arber method and was expressed as log tcid . , the effectiveness of each individual process step for each virus was calculated as the log reduction factor (rf) using the formula rf loga -loga n , where r is the reduction factor, a is the total virus load after spiking, and a n is the total virus load in the treated sample. the overall reduction factor was expressed as the sum of rfs for all steps. the limit of detection of the assay was defined as the lowest tcid achievable at noncytotoxic sample concentrations. vero e cells (atcc ccl- ) together with the zaire ebolavirus strain (mayinga- ) were grown to approximately % confluence in dulbecco's modified eagle's medium (dmem) with % fetal bovine serum (fbs), mmol/l l-glutamine, mg/ml streptomycin, and iu/ml penicillin. on day , the viral supernatant was collected, centrifuged, aliquoted, and frozen at - c until further use (spiking experiments). vero e cells (atcc ccl- ) together with mers-cov (emc/ ), obtained from ron a. fouchier, were grown to approximately % confluence in dmem with % fbs, mmol/l l-glutamine, mg/ml streptomycin, and iu/ml penicillin. on days to , the viral supernatant was collected, centrifuged, aliquoted, and frozen at - c until further use (spiking experiments). for each virus, two bags of pcs (volume, ml) and plasma (volume, ml) were spiked with % viral supernatant and treated as described with uvc or mb/light, respectively. each treatment was delivered incrementally up to the full light dose ( . j/cm for uvc and j/cm for mb/light). at different process steps, samples were collected and virus titrations were performed. reference samples were taken from each bag before treatment, stored at room temperature, and tested at the end of the experiments to account for any intrinsic virus inactivation by the blood product. virus titers after spiking were below the expected dilution factor in six of the eight blood units used. in one pc and in one plasma unit used for ebov inactivation and in all four blood units used for mers-cov inactivation, the virus titers before pi treatment were up to log lower than expected from a in- dilution. however, no additional reduction in virus titers was observed during the course of the experiment. the results of the infectivity assay demonstrated that uvc irradiation dose-dependently inactivated ebov and mers-cov in plasma-reduced pcs (table ). at . j/cm ( % of the full uvc dose), ebov and mers-cov infectivity levels were below the detection limit, resulting in virus reduction factors of greater than . for ebov and of greater than . for mers-cov. table , light doses as low as j/cm , or % of the standard full light dose of j/cm for led-based illumination, inactivated ebov and mers-cov in plasma to levels below the detection limit. these results correspond to at least . and at least . log reductions of ebov and mers-cov, respectively. the theraflex uv-platelets and theraflex mb-plasma systems are designed to inactivate or remove pathogens in plt and plasma products, as has been demonstrated in a wide range of pathogenic agents. , , , , [ ] [ ] [ ] the mechanism of theraflex uv-platelets is that shortwave uvc light penetrates blood fluids and cell membranes, causing direct covalent damage to the nucleic acids of pathogens and wbcs. this mechanism of action is broadly effective against extracellular and intracellular transfusion-transmitted dna/rna-containing pathogens, such as viruses, bacteria, and protozoa. theraflex mb-plasma combines mb-a phenothiazine compound that intercalates into viral nucleic acid-with visible light. the illumination of mb-treated plasma results in the generation of singlet oxygen, which leads to the destruction of viral nucleic acids and thus prevents viral replication. because mb only partially penetrates cell membranes, mb/light treatment is less effective against intracellular viruses. this is why two filters are integrated in the process chain. although the second filter mainly removes mb and its photoactivated products after illumination, both filters have multiple layers of small-meshed membranes capable of removing residual wbcs, rbcs, and plts that may carry intracellular pathogens. it was recently shown that the two filters efficiently remove high levels (up to . log colonyforming units per milliliter) of viable bacteria and bacterial spores. it can be assumed that parasites of similar sizes can also be removed by the theraflex mb-plasma procedure. a previous investigation showed that the filtration step before the addition of mb and visible light illumination was critical to achieving the complete and reproducible elimination of trypanosoma cruzi, the causative agent of chagas disease. it is estimated that there are more than known human pathogens, % of which are considered emerging or reemerging. the ability to provide proactive protection against emerging infectious agents complementary to the existing blood screening programs is a major additional benefit of pi systems for blood safety. thus, it is imperative that manufacturers continuously challenge their pi systems with new infectious agents. to our knowledge, this is the first study investigating the efficacy of uvc and mb/light against mers-cov and ebov or any other member of the coronaviridae and filoviridae families. our results showed that these two pi systems reduced mers-cov and ebov titers to below the limit of detection in the two blood units tested. the maximum viral titer reduction detectable in a cell culture infectivity assay is based on the starting viral concentration of the blood product and the detection limit of the assay which, in turn, is determined by the susceptibility of the cell culture to plasma and plt suspensions. as in a previous study, the observed reduction of ebov titers and mers-cov titers in the present study was beyond the expected dilution factor in pcs and plasma. this phenomenon may occur due to the presence of nonspecific immune mediators that neutralize viruses in plasma. , the mean log reduction factors of at least . (mers-cov) and at least . (ebov) in pcs treated with uvc and of at least . (mers-cov) and at least . (ebov) in plasma treated with mb/light suggest that both pi technologies used in this study are considerably effective against these two virus types. this study had a number of limitations. for example, the number of replicates was limited by safety constraints, as experiments with mers-cov and ebov are subject to the highest biosafety level. in addition, large-volume plating could not be performed, although this would have improved the detection limit and enabled more exact determination of log reduction factors. although plasma viral rna loads in mers-cov patients may be up to log genome copies/ml, and median viral rna loads of up to . log genome copies/ml have been detected in the blood of ebola patients, , virus titers in the plasma of asymptomatic infected or convalescent individuals recruited to donate blood may be significantly lower. as it is known for other infectious diseases, ebov viral titers expressed in genome copies per milliliter do not necessarily reflect the infectivity titer. while viral titers measured by quantitative polymerase chain reaction are based on the detection of a small fragment of the viral genome, infectivity titers describe the number of intact, functional viral units required for replication and disease transmission. although ebov is highly infectious and low doses of less than plaque-forming units of the virus are sufficient to cause disease, the ability of the uvc-and mb/ light-based systems to reduce mers-cov and ebov infectivity in blood products by several log steps may be sufficient to eliminate or significantly reduce the risk of transmission via the transfusion of pcs or plasma. in conclusion, this study further expands the list of pathogens that theraflex uv-platelets and theraflex mb-plasma can effectively inactivate in pcs and plasma, respectively. emerging infectious disease agents and their potential threat to transfusion safety current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections middle east respiratory syndrome coronavirus: five years later update on the epidemiology of middle east respiratory syndrome coronavirus cov) infection, and guidance for the public, clinicians, and public health authorities identification of a novel coronavirus in patients with severe acute respiratory syndrome ebola situation report update on the use of pathogenreduced human plasma and platelet concentrates a novel approach to pathogen reduction in platelet concentrates using shortwave ultraviolet light uvc irradiation for pathogen reduction of platelet concentrates and plasma mitochondrial dna multiplex real-time polymerase chain reaction inhibition assay for quality control of pathogen inactivation by ultraviolet c light in platelet concentrates inter-strand photoproducts are produced in high yield within a-dna exposed to uvc radiation main properties of the theraflex mb-plasma system for pathogen reduction methylene bluetreated fresh-frozen plasma: what is its contribution to blood safety? photodynamic virus inactivation of blood components updates on pathogen inactivation of plasma using theraflex methylene blue system virus inactivation in blood components by photoactive phenothiazine dyes inactivation of dengue, chikungunya, and ross river viruses in platelet concentrates after treatment with ultraviolet c light reduction of zika virus infectivity in platelet concentrates after treatment with ultraviolet c light and in plasma after treatment with methylene blue and visible light month transfusion transfusion richtlinien zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten (h€ amotherapie) k€ oln: deutscher € arzte-verlag platelet concentrates in an additive solution prepared from pooled buffy coats. in vivo studies storage of platelets in additive solutions: effects of phosphate two pathogen reduction technologies-methylene blue plus light and shortwave ultraviolet light-effectively inactivate hepatitis c virus in blood products the effectiveness of uvc pathogen inactivation system on reducing the trypansosoma cruzi and leishmania infantum burden in platelets the efficacy of the ultraviolet c pathogen inactivation system in the reduction of babesia divergens in pooled buffy coat platelets challenge study of the pathogen reduction capacity of the theraflex mb-plasma technology the efficacy of photochemical treatment with methylene blue and light for the reduction of trypanosoma cruzi in infected plasma emerging pathogens: the epidemiology and evolution of species jumps treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates ebola virus in vitro mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complementmediated virus neutralization viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection age and ebola viral load correlate with mortality and survival time in ebola virus disease patients clinical recognition and management of patients exposed to biological warfare agents key: cord- -tc qo k authors: gehring, gerrit; rohrmann, katrin; atenchong, nkacheh; mittler, eva; becker, stephan; dahlmann, franziska; pöhlmann, stefan; vondran, florian w. r.; david, sascha; manns, michael p.; ciesek, sandra; von hahn, thomas title: the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry date: - - journal: j antimicrob chemother doi: . /jac/dku sha: doc_id: cord_uid: tc qo k objectives: filoviruses such as ebola virus and marburg virus cause a severe haemorrhagic fever syndrome in humans for which there is no specific treatment. since filoviruses use a complex route of cell entry that depends on numerous cellular factors, we hypothesized that there may be drugs already approved for human use for other indications that interfere with signal transduction or other cellular processes required for their entry and hence have anti-filoviral properties. methods: we used authentic filoviruses and lentiviral particles pseudotyped with filoviral glycoproteins to identify and characterize such compounds. results: we discovered that amiodarone, a multi-ion channel inhibitor and adrenoceptor antagonist, is a potent inhibitor of filovirus cell entry at concentrations that are routinely reached in human serum during anti-arrhythmic therapy. a similar effect was observed with the amiodarone-related agent dronedarone and the l-type calcium channel blocker verapamil. inhibition by amiodarone was concentration dependent and similarly affected pseudoviruses as well as authentic filoviruses. inhibition of filovirus entry was observed with most but not all cell types tested and was accentuated by the pre-treatment of cells, indicating a host cell-directed mechanism of action. the new world arenavirus guanarito was also inhibited by amiodarone while the old world arenavirus lassa and members of the rhabdoviridae (vesicular stomatitis virus) and bunyaviridae (hantaan) families were largely resistant. conclusions: the ion channel blockers amiodarone, dronedarone and verapamil inhibit filoviral cell entry. members of the filoviridae family, which includes marburg virus (marv) and several species of ebola virus, are enveloped filamentshaped viruses with a non-segmented negative-sense rna genome. infection of humans causes a severe haemorrhagic fever syndrome. most cases occur in resource-poor settings in sub-saharan africa, with case fatality rates in the range of % - %. monocytes, macrophages and dendritic cells are thought to be sites of viral replication during the early and later stages of infection , yet viral amplification also occurs in endothelial cells and many other tissues. - consistent with rapid spread to a broad range of cell types and tissues in humans, filoviruses have been found to readily enter into a broad range of cultured cell types, with the notable exception of lymphocytes. , it has emerged over recent years that the filoviral route of cell entry is complex. interaction between the target cell and the incoming virion is mediated on the viral side by heterotrimers of the filoviral glycoprotein (gp) subunits (gp ) and (gp ) that are generated by the furin-mediated cleavage of a single precursor gp (gp ) and remain linked by a disulfide bond. , numerous factors on the surface of host cells have been reported to interact with gp /gp . these include the folate receptor, members of the tyro family of receptor tyrosine kinases (axl, dtk, mer) and t cell immunoglobulin and mucin domain , although the relevance of the folate receptor for filovirus entry was later disputed. , in addition, numerous c-type lectins, calcium-dependent carbohydratebinding molecules, including the asialoglycoprotein receptor, dc-sign and dc-signr, , human macrophage galactose-type c-type lectin and liver and lymph node sinusoidal endothelial cell c-type lectin have been reported to interact with gp and promote filovirus entry. following host cell binding, filoviruses are taken up into the cell most likely through macropinocytosis. , the low ph-dependent cathepsin b/l-mediated cleavage of filovirus gp is then required for fusion with a late endosomal membrane. in addition, the endosomal cholesterol transporter niemann -pick c (npc ) acts as an essential intracellular receptor. , this intricate multistep route of viral entry is likely to require some form of coordination, which may take advantage of cellular signalling, as has been shown for coxsackie group b viruses and hepatitis c virus. indeed, the phosphoinositide- kinase/akt pathway as well as protein kinase c and small cellular gtpases have been implicated in filovirus entry. although (as discussed below) several experimental antifiloviral approaches have been reported, no vaccine or specific antiviral treatment has yet been established. given low case numbers and geographical restriction to resource-limited regions, industry-sustained drug development programmes are unlikely. nonetheless, there is a clear unmet clinical need from naturally occurring cases as well as an additional potential need given that filoviruses are considered to be cdc category a bioterrorism agents (www.bt.cdc.gov/bioterrorism). given the complex filoviral route of entry, we hypothesized that drugs which perturb cellular signalling networks and are approved for other indications might also interfere with filovirus entry. we performed a small-scale screen and found that amiodarone, a multi-ion channel inhibitor used clinically as an anti-arrhythmic agent, acts as a potent and specific inhibitor of filoviral cell entry. the concentrations of amiodarone that are required for filovirus inhibition are well within the range that is achieved in serum during anti-arrhythmic therapy in humans, i.e. . - . mg/ml. pharmacological agents were purchased from sanofi-aventis (frankfurt, germany) (amiodarone), sigma-aldrich (taufkirchen, germany) [amiodarone, dronedarone, epigallocatechin- -gallate (egcg), ethosuximide, lidocaine, metoprolol], merck millipore (darmstadt, germany) [z-phe-tyr[t-bu]-diazomethylketone (fydmk)], toronto research chemicals (toronto, canada) (artesunate, cyclosporine a, erlotinib, fingolimod, imatinib, rapamycin, sorafenib, sotrastaurin, sunitinib), abbott (wiesbaden, germany) (verapamil), roche pharma (grenzach-wyhlen, germany) (bevacizumab), janssen-cilag (neuss, germany) (haloperidol) and astellas pharma (munich, germany) (theophylline). all immortalized cell lines were cultured in dulbecco's modified eagle medium (dmem) with % fetal calf serum (fcs), non-essential amino acids, l-glutamine and penicillin/streptomycin at c and % co . primary human umbilical vein endothelial cells were isolated from umbilical cords as previously described and cultivated in egm- medium (lonza, basel, switzerland) containing % fcs and penicillin/streptomycin. primary human hepatocytes were isolated using a modified two-step collagenase (roche p, mannheim, germany) perfusion technique as previously reported and cultured in williams' medium e (all biochrom ag, berlin, germany) supplemented with insulin ( mm), dexamethasone ( mm), penicillin/streptomycin, sodium pyruvate, hepes buffer, l-glutamine and % fcs. the generation of macrophages from primary human monocytes has recently been described. briefly, ficoll gradientpurified human monocytes from peripheral blood mononuclear cells were cultured in rpmi medium with . % human fibrin-depleted plasma and % penicillin/streptomycin for h. cells were then resuspended in x-vivo medium supplemented with % human fibrin-depleted plasma and % penicillin/streptomycin, seeded and allowed to differentiate for at least days. the medium was changed every - days. the plasmids csflucw encoding firefly luciferase in the context of a gutted lentiviral genome, hiv gag-pol, pcdna . and vesicular stomatitis virus (vsv) g-protein (vsv-g) that are the basis for the production of lentiviral pseudoparticles have previously been described, , as has the plasmid pcaggs-marvgp. pseudoparticles were generated by co-transfection of plasmids encoding: (i) a gutted lentiviral genome containing the firefly luciferase gene (csflucw ); (ii) hiv gag-pol; and (iii) the desired envelope gp. the day before transfection, × t cells were seeded in a mm well. the next day, a total of mg of dna was transfected per well using polyethylenimine. the medium was replaced after h. supernatants were harvested at h and h after transfection, pooled and filtered ( . mm pore size), and then used immediately or stored at c (, week) or c (. week). pseudoparticle transduction assays were performed in a -well format using × eahy cells per well. seeding numbers were adjusted for other cell lines. transductions were carried out with polybrene ( mg/ml) and with or without drugs. after h, the medium was exchanged. after h, cells were lysed and luciferase was measured as previously described. to separate the virus from the drug, we used an amicon ultra- centrifugal device with a kda cut-off (millipore). in this device, pre-treated pseudovirus was washed with ml of drug-free media three times by centrifugation at rpm. all work with the infectious mayinga strain of ebov (genbank accession number nc ) was performed under the highest safety precautions in the biosafety level (bsl- ) facility at the institute of virology, philipps-university marburg, germany. vero e cells were infected with ebov at a multiplicity of infection (moi) of tcid /cell. ebov particles were purified from the supernatant of infected cells at h post-infection by centrifugation over a sucrose cushion for min at rpm, c in a beckman ultracentrifuge using a sw rotor (beckman coulter, palo alto, ca, usa). pellets were resuspended in pbs. amiodarone was mixed with concentrated ebov gehring et al. particles with an moi of . tcid /cell and transferred to eahy cells. after h of incubation, the inoculum was removed and replaced by fresh dmem supplemented with % fcs. at h post-infection, cells were fixed in % paraformaldehyde for h, removed from the bsl- laboratory and subjected to quantitative immunofluorescence analysis. immunostaining of infected cells was performed as described below. immunofluorescence analyses were carried out as previously described. for the detection of nucleocapsid (np)-induced inclusion bodies in ebov-infected eahy cells (after fixation of the cells with % paraformaldehyde in dmem for h), we used a monoclonal anti-ebov np antibody and a secondary fitc-coupled goat anti-mouse antibody. numerical comparisons between groups were made by unpaired twosided student's t-test. differences with p values below . are referred to as significant. p, . and p, . are indicated by single and double asterisks throughout. graphpad prism software (la jolla, ca, usa) was used to fit concentration -response curves and determine the % inhibitory concentration (ic ). based on a literature review, we selected drugs for the initial screen. drugs were chosen that met several of the following selection criteria: (i) a known safety profile in humans; (ii) known to perturb cell signalling processes; (iii) recognized antiviral properties against viruses other than members of the filoviridae; - and (iv) coverage of a broad range of indications and drug classes. of these drugs, are approved by the ema and fda for various indications; one drug, the protein kinase c inhibitor sotrastaurin, is in advanced clinical development as an immunosuppressive agent for use in organ transplant recipients. human endotheliumderived eahy cells were transduced with pseudoparticles bearing either ebov gp or marv gp in the presence or absence of drugs. drug concentrations tested were based on serum levels achievable in humans. four concentrations were tested per drug and two rounds of screening were performed. the cathepsin b and l inhibitor fydmk, known to potently inhibit ebov gp-mediated cell entry, was included as a positive control ( figure ). while most drugs had no major effect on filovirus gp-mediated cell entry, amiodarone was found to reduce the detectable signal by over %, and thus more strongly than fydmk, which achieved % - % inhibition. amiodarone-mediated inhibition of ebov and marv gp-bearing pseudoparticles showed an ic of . mg/ml ( . mm) and . mg/ml ( . mm), respectively (figure a) . we then exposed eahy cells stably expressing firefly luciferase to different concentrations of amiodarone to test for cytotoxic and anti-proliferative effects. a % signal reduction (cc ) was seen at . mg/ml ( . mm) (figure b) . this corresponds to a calculated therapeutic index of and for ebov and marv, respectively. in a 'worst case' scenario (assuming that the true ic is ×sd above and cc ×sd below our determination), the index is still . for both ebov and marv. to assess the effect of amiodarone on authentic filovirus entry, eahy cells were exposed to purified ebov in the presence of different concentrations of amiodarone for h. the medium was exchanged, and h later cells were fixed with paraformaldehyde and stained for ebov np protein. dapi was used as a counterstain. there was a clear concentration-dependent reduction in the percentage of np-positive cells while the overall cell number was unchanged ( figure s , available as supplementary data at jac online). significant inhibition was observed at amiodarone concentrations of . mg/ml or higher ( figure ). on averaging all the experiments performed, the ic was . mg/ml, corresponding to a calculated therapeutic index of above . of note, the apparently flatter slope of the concentration response curve is most likely due to the fact that experiments performed on separate occasions were averaged. thus, authentic filoviruses may be more sensitive to amiodarone inhibition than pseudoparticles bearing filoviral gp. next we aimed to assess whether amiodarone inhibits entry driven by the gps of all filovirus species and whether entry mediated by the gps from other viral families may be affected. eahy cells were transduced with pseudoparticles bearing ebov or marv gp in the presence of the drugs indicated. out of four concentrations tested, the highest concentration without apparent signs of toxicity as judged by cell morphology is shown. the first and second numbers in the brackets indicate the drug concentration used in mg/ml and in mm, respectively. the cathepsin inhibitor fydmk, known to inhibit filovirus gp-mediated cell entry, was included as a positive control. columns represent the means of n ¼ independent measurements. #, since bevacizumab is an antibody, its concentration is given only in mg/ml. pseudoparticles bearing gp of the ebola virus species tai forest, reston, sudan and zaire, as well as marv, were similarly inhibited by amiodarone (figure a ). thus, amiodarone is a blocker of cell entry mediated by viral gps from across the filoviridae family. among a broader range of human pathogenic enveloped viruses that enter target cells through endocytosis and in a ph-dependent manner, we observed a significant inhibition of pseudoviruses bearing the gp of the new world arenavirus guanarito (figure b ). vsv-g-(rhabdoviridae family), lassa virus gp-(old world arenavirus) and hantaan (bunyaviridae family) virus gp-pseudotyped lentiviruses were affected to a lesser extent. since all pseudoviruses have the same lentiviral np and reporter genome, these differences strongly support the fact that amiodarone exerts its inhibitory effect on viral cell entry, i.e. a replication cycle step up to the point of fusion of the viral envelope with a cellular membrane. we next tested the effect of target cell pre-treatment. amiodarone was present either h before the cells were washed three times and then ebov or marv pseudoparticles were added to the cells, or during the h when the cells were exposed to the pseudoparticles, or both (figure a) . we observed the most marked inhibition when the drug was present throughout the experiment, i.e. from h prior to addition of pseudoviruses until h after, followed by the set-up where amiodarone was present only during the cell entry phase. in the case of marv, there was also significant inhibition when the drug was present only before the pseudoparticles were added to the cells. this is suggestive of a cell-directed mechanism of action. however, the effect on the target cells appears to be reversible upon removal of the drug. gehring et al. moreover, when ebov pseudovirus was incubated with a high concentration ( . mg/ml) of amiodarone and then washed in kda filter devices in order to remove drug, we observed a subsignificant % reduction of infectivity relative to vsv-g pseudoviruses ( figure s , available as supplementary data at jac online). next we assessed whether eahy cells could be adapted to amiodarone by growing them in the presence of . mg/ml amiodarone for weeks prior to experimentation. cell growth was largely unaltered and there were no obvious morphological alterations. we observed a comparable degree of inhibition of ebov and marv pseudoparticles, suggesting that prolonged exposure to amiodarone does not lead to compensatory changes in the host cell (figure b ). we next evaluated the amiodarone inhibition of cell entry mediated by filoviral gp in a range of different target cells. there was significant inhibition in all cell lines tested, although there appeared to be a trend towards a more pronounced effect on cell lines of endothelial origin compared with epithelial cell lines, with cell lines of fibroblast origin appearing least sensitive ( figure ). filoviral gp-mediated entry into primary human umbilical vein endothelial cells and in macrophages derived from primary human monocytes was significantly inhibited at amiodarone concentrations below mg/ml for both marv and ebov gp while amiodarone had no effect on the transduction of primary hepatocytes ( figure s , available as supplementary data at jac online). these cell type-specific differences are also in keeping with a host cell-targeted mechanism of action of amiodarone. the multi-channel inhibitor dronedarone as well as some calcium channel inhibitors share the anti-filoviral effect of amiodarone amiodarone inhibits or otherwise modulates a number of cellular targets that include potassium channels, sodium channels, calcium channels and a-and b-adrenergic receptors. we used more specific pharmacological agents to test whether the observed anti-filoviral effect was associated with any of the known pharmacodynamic properties of amiodarone. dronedarone, a newer drug with a pharmacodynamic profile similar to amiodarone, showed a marked concentration-dependent inhibition of marv and ebov gp-bearing pseudoparticles similar to amiodarone (figure a ). the l-type calcium channel blocker verapamil similarly inhibited filovirus gp-mediated entry, although higher absolute and molar concentrations were required (figure b) . conversely, the selective t-type calcium channel blocker ethosuximide had no effect. the sodium channel blocker lidocaine, the b -selective adrenoreceptor antagonist metoprolol, and the nonselective b-blocker and potassium channel blocker sotalol were also without detectable effects (figure c ). drug concentrations were chosen based on plasma levels reported to be achievable in humans. relevant toxicity or anti-proliferative effects at the concentrations used were excluded using an eahy cell line stably expressing a luciferase reporter ( figure s , available as supplementary data at jac online). thus, several agents that are either non-specific ion channel inhibitors (amiodarone, dronedarone) or inhibitors of l-type calcium channels (verapamil) inhibit cell entry mediated by filoviral gps, while other agents possessing some of the other multiple pharmacodynamic effects of amiodarone do not. in this paper we describe the identification of amiodarone and other drugs that act as inhibitors of ion channels (dronedarone, verapamil) as inhibitors of filovirus cell entry. we showed that amiodarone at concentrations that are routinely reached in human serum when amiodarone is employed clinically acts as a host cell-targeting agent that blocks viral entry. in the absence of approved treatments, the management of filoviral haemorrhagic fever is largely limited to supportive care and containment. mortality is very high in resource-poor settings, and even when modern intensive care is available, treatment is amiodarone inhibits filovirus entry jac challenging. several potential approaches have been suggested based on in vitro or animal data. these include monoclonal antibodies isolated from survivors, lipid-stabilized small-interfering rnas directed against viral genes and a recombinant vsv-based vaccine. however, it will be difficult to transfer these highly experimental approaches into routine clinical care. very recently, the oestrogen receptor modulator clomiphene, which is licensed for ovarian stimulation in humans, has been reported to inhibit filovirus infection, possibly by perturbing the role of npc in filoviral entry. , amiodarone is available both as an oral and an intravenous formulation and has been used for decades for both the long-term and short-term treatment of cardiac arrhythmias. as a generic drug, its use would be feasible in both resource-rich and resource-poor settings. it is known to have a narrow therapeutic window, but serum levels to treat cardiac arrhythmias are in the range of . - . mg/ml, which is -to -fold above the in vitro ic we observed for authentic filoviruses. the known side effects of amiodarone (which include ocular and pulmonary toxicity, thyroid dysfunction and pro-arrhythmic effects) are generally manageable, especially in the short term. importantly, they would clearly be acceptable if the drug at the same time increased the odds of survival in the setting of acute filoviral haemorrhagic fever. given that filoviral haemorrhagic fever involves the spread of infection to a broad range of cells and tissues, it is plausible that a potent entry inhibitor would limit systemic spread and hence might reduce mortality. however, clinical studies would be needed to determine this. at this point, we do not know the exact mechanism by which amiodarone and the other anti-arrhythmic agents studied inhibit filoviral cell entry. several observations suggest that amiodarone acts on the host cell rather than the incoming virion: (i) preincubation of target cells with the drug also had an inhibitory effect on marv; (ii) the most pronounced effect was seen when the drug was present before and during infection; and (iii) the inhibitory effect was cell type dependent in the sense that it was detectable to a variable extent on most cell types tested, with the notable exception of primary human hepatocytes. however, there was also a trend to reduced infectivity when pseudoviruses were pre-treated with amiodarone and then washed before being added to the target cell. this might suggest that either we were unable to completely separate the drug from the virus or that amiodarone inhibition requires some form of ternary interaction between drug, filovirus and host cell. there are at least two hypotheses for how amiodarone may render the host cell less susceptible to filoviral entry: on the one hand, both amiodarone and dronedarone have been reported to perturb a late step in endosomal processing, resulting in lipid accumulation and gross alterations in endosomal structure, a phenotype thought reminiscent of niemann -pick type c disease. , besides filoviruses, new world arenavirus guanarito was also strongly inhibited by amiodarone while other viruses tested were not. since both are thought to enter target cells via the late endosome, this would be consistent with an amiodaronemediated perturbation of this compartment. , , indeed shoemaker et al. have reported that clomiphene and other related inhibitors of filoviral entry they have identified have in common that they are cationic amphiphiles causing a niemann -pick-like accumulation of cholesterol in the endosomes. this could be due to inhibition of acid sphingomyelinase by a broad range of cationic amphiphiles. amiodarone, dronedarone and verapamil, with pka values of . , . and . , respectively, , all fit into that category. moreover, entry through late endosomes has also been suggested for hantaviruses, one of which was modestly inhibited by amiodarone in our research. in a different study, an inhibitory effect of very high concentrations of amiodarone on severe acute respiratory syndrome coronavirus was reported. that paper suggests that the inhibition of infection occurs after fusion of the viral envelope with the late endosomal membrane and delivery of the viral genome to the cytosol. since a block at a post-fusion step would be expected to affect all pseudoviruses equally, this does not explain our observation that some pseudoviruses are inhibited by amiodarone while others are not. the second hypothesis is that the inhibition of filoviral entry by anti-arrhythmic agents is linked to their primary pharmacodynamic properties. amiodarone is a pleiotropic drug known to target potassium channels, sodium channels, calcium channels, and a-and b-adrenergic receptors. when testing other agents with a narrower range of molecular target, only the l-type calcium channel blocker verapamil exhibited an anti-filoviral effect. these findings may suggest a role for calcium currents in filoviral entry. while their role is more obvious in excitable tissues such as muscle and neuronal cells, l-type calcium channels are also present in many non-excitable cells including endothelial and epithelial cells. , calcium fluxes can cause rapid shifts in intracellular calcium levels and are an important part of cellular signalling processes, most notably maybe the phospholipase c/ , , -inositol for each drug, a lower and a higher concentration was used as follows: ethosuximide, and mg/ml; verapamil, and mg/ml. individual data points represent the mean+sd of n¼ independent replicates from a representative of three independent experiments. (c) transduction in the presence of the sodium channel blocker lidocaine ( and mg/ml), the b-receptor blocker metoprolol ( . and . mg/ml) and the potassium channel blocker/b-receptor blocker sotalol ( and mg/ml). individual data points represent the mean+sd of n ¼ independent replicates from a representative of three independent experiments. amiodarone inhibits filovirus entry jac trisphosphate/diacylglycerol pathway. thus, interference with the calcium-mediated cell signalling events required for the orchestration of filoviral entry is another plausible mechanism. interestingly, it has very recently been reported that gabapentin, verapamil and other inhibitors of voltage-gated calcium channels, as well as the sirna-mediated knockdown of calcium channel subunits, inhibit infection by the new world arenavirus junin. ongoing work aims to test these hypotheses and determine the exact mechanism of filovirus inhibition by amiodarone and other anti-arrhythmic agents. the work presented here shows that a limited-scale screening of drugs with a known safety profile in humans can reveal novel antiviral properties that may be useful in addressing rare yet severe infections for which drug discovery programmes will likely not be initiated. the chances of identifying potentially useful agents may be best for viruses that employ complex cell entry routes such as filoviruses. the discovery of such unexpected antiviral properties in known drugs with cellular targets may at the same time offer novel insights into the molecular mechanisms of the virus -host interaction and new therapeutic approaches. ebola haemorrhagic fever pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells ultrastructural pathology of experimental ebola haemorrhagic fever virus infection marburg hemorrhagic fever: report of a case studied by immunohistochemistry and electron microscopy ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies filovirus tropism: cellular molecules for viral entry processing of the ebola virus glycoprotein by the proprotein convertase furin covalent modifications of the ebola virus glycoprotein folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses tyro family-mediated cell entry of ebola and marburg viruses t-cell immunoglobulin and mucin domain (tim- ) is a receptor for zaire ebolavirus and lake victoria marburgvirus folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha the asialoglycoprotein receptor is a potential liver-specific receptor for marburg virus c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus the ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions hepatitis c virus host cell entry phosphoinositide- kinase-akt pathway controls cellular entry of ebola virus practical guidelines for clinicians who treat patients with amiodarone. practice guidelines subcommittee, north american society of pacing and electrophysiology engulfment of apoptotic cells by microvascular endothelial cells induces proinflammatory responses isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies diverse cd proteins support hepatitis c virus infection role of the transmembrane domain of marburg virus surface protein gp in assembly of the viral envelope impact of occludin intra-and inter-species variation on its co-receptor function for authentic hepatitis c virus particles virus isolation and quantitation cyclosporine a inhibits hepatitis c virus nonstructural protein through cyclophilin a the green tea polyphenol, epigallocatechin- -gallate, inhibits hepatitis c virus entry egfr and epha are host factors for hepatitis c virus entry and possible targets for antiviral therapy viral drug sensitivity testing using quantitative pcr: effect of tyrosine kinase inhibitors on polyomavirus bk replication ion transport blockers inhibit human rhinovirus release infection of b lymphocytes by a human herpesvirus, epstein-barr virus, is blocked by calmodulin antagonists cellular electropharmacology of amiodarone acute liver failure, multiorgan failure, cerebral oedema, and activation of proangiogenic and antiangiogenic factors in a case of marburg haemorrhagic fever ebola virus can be effectively neutralized by antibody produced in natural human infection postexposure protection of non-human primates against a lethal ebola virus challenge with rna interference: a proof-of-concept study recombinant vesicular stomatitis virus vector mediates postexposure protection against sudan ebola hemorrhagic fever in nonhuman primates multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection amiodarone impairs trafficking through late endosomes inducing a niemann-pick c-like phenotype amiodarone alters late endosomes and inhibits sars coronavirus infection at a post-endosomal level different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses cell entry by human pathogenic arenaviruses identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model pka determination of verapamil by liquid-liquid partition hantaan virus enters cells by clathrindependent receptor-mediated endocytosis prolactin stimulates the l-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats update on vascular endothelial ca( +) signalling: a tale of ion channels, pumps and transporters calcium signaling sirna screen for genes that affect junin virus entry uncovers voltage-gated calcium channels as a therapeutic target we thank hyeryun choe, francois-loic cosset, jay hooper, stefan kunz, gary nabel, charles rice and anthony sanchez for providing reagents and sandra westhaus for help with data analysis. the authors declare that they have no conflicts of interest relevant to this work. figures s to s are available as supplementary data at jac online (http:// jac.oxfordjournals.org/). key: cord- -f vqn o authors: schmidt, rebecca; beltzig, lea c.; sawatsky, bevan; dolnik, olga; dietzel, erik; krähling, verena; volz, asisa; sutter, gerd; becker, stephan; von messling, veronika title: generation of therapeutic antisera for emerging viral infections date: - - journal: npj vaccines doi: . /s - - - sha: doc_id: cord_uid: f vqn o the recent ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. while human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. here, we compared adjuvanted virus-like particles (vlp) with recombinant modified vaccinia virus ankara and vesicular stomatitis virus (vsv), both expressing the ebola virus antigens. the neutralizing antibody titers of rabbits immunized with adjuvanted vlps were similar to those immunized with the replication-competent vsv, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. this approach also yielded high-titer antisera against nipah virus glycoproteins, illustrating that it is transferable to other virus families. multiple-step immunoglobulin g purification using a two-step – % ammonium sulfate precipitation followed by protein a affinity chromatography resulted in % recovery of functionality and sustained in vivo stability. adjuvanted vlp-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections. outbreaks of emerging viruses occur with worrying regularity, and while some are quickly controlled locally, others become public health events of global concern. among the pathogens involved, viruses that can be transmitted directly and cause severe disease with high mortality such as filovirus and henipavirus are considered world health organization (who) priority pathogens. until the recent west african ebola virus (ebov) epidemic, which resulted in , confirmed cases and , deaths, , ebov outbreaks were generally small and rapidly contained. , the same holds true for nipah (niv) and hendra (hev) viruses, which so far have mainly caused local clusters of infection, , or small outbreaks as the one currently ongoing in india. however, the first niv outbreak in malaysia and singapore in - resulted in documented infections with a case fatality rate of approximately %, , illustrating the challenges associated with predicting and preparing for such events. since licensed therapies or vaccines are often not immediately available, patient isolation, supportive care, and contact tracing are the main approaches used to control these outbreaks. , there is thus a renewed interest in therapeutic approaches such as convalescent sera or hyperimmune sera of animal origin, which can be rapidly available. passive antibody treatment with whole blood or plasma from convalescent patients has been used as experimental therapy during filovirus outbreaks since , , with variable results. whole blood from convalescent donors was given as a postexposure treatment during the kikwit outbreak with seven of eight patients surviving the infection. however, post-exposure treatment of nonhuman primates with whole blood from convalescent donors did not replicate the successes seen in human patients. more recently, patients treated with convalescent plasma failed to show significant improvement in survival during the west african outbreak, but definitive interpretation of these data is difficult because the antibody titers and virus-neutralizing activity of the convalescent plasma was not reported. post-exposure prophylaxis studies in ebov-infected nonhuman primates using high-titer purified immunoglobulin g (igg) from convalescent macaques is fully protective against subsequent challenge, indicating that functional antibody titers may be the determining factor. neutralizing antibodies are also important mediators of protection against niv. post-exposure passive transfer of sera from hamsters immunized with either vaccinia or vesicular stomatitis viruses (vsvs) expressing the niv-g protein provides protection from lethal virus challenge in a hamster model, , as do murine monoclonal antibodies that bind either niv-f or niv-g, and a human monoclonal antibody with neutralizing activity against the related hendra virus g protein in ferrets. in general, passive immunization has proven to be beneficial for numerous acute infections and is still commonly used as emergency treatment against life-threatening diseases. human igg preparations are the gold standard for rabies and tetanus post-exposure prophylaxis, and are available as emergency treatment for anthrax. pooled human igg for intravenous use (ivig) is used as a prophylactic and post-exposure treatment against hepatitis b, and is given to patients with complications during measles infection or to those that are at risk for severe disease. while human convalescent sera may not be readily available in outbreak situations, antisera of animal origin can be produced in a short time frame. animal-origin purified igg and igg fragment preparations are still routinely used as antivenoms, and even though immunization of horses with inactivated ebov failed to yield effective therapeutic antisera, equine hyperimmune sera produced using ebov virus-like particles (vlps) conferred protection against lethal challenge in rodents. here we compared different immunization platforms for the rapid induction of high functional antibody titers against ebov and niv, and optimized the subsequent igg purification process. squalene-containing adjuvants enhance ebov vlp-induced total and functional antibody titers vlps are an effective platform for the display of antigens in their native conformation. , to assess their ability for induction of high functional antibody titers, we produced ebov vlps by cotransfecting human embyonic kidney- (hek- ) cells with plasmids encoding for the ebov zaire strain mayinga matrix protein (vp ) and the full-length membrane-anchored glycoprotein (gp). after h, the supernatant was harvested, purified, and concentrated via ultracentrifugation. the resulting ebov vlp preparation contained two major proteins migrating in coomassie-stained gels at kda and around kda (fig. a) . western blot analysis identified the distinct band at kda as ebov-gp , the large cleavage fragment of ebov-gp, , and two bands between and kda as two isoforms of ebov-vp due to a second upstream in-frame start codon (fig. b) . to evaluate the effect of adjuvants on total and functional antibody responses, groups of mice were immunized intramuscularly with μg ebov vlp alone, or in combination with the squalene-containing water-in-oil sigma adjuvant, the oil-in-water titermax adjuvant, or % alhydrogel, all of which are routinely used for animal immunizations, and boosted and weeks later. serum was collected weeks after each immunization and kinetics of total and neutralizing antibody responses were followed. first antibodies against ebov-gp were detected after weeks, with approximately -fold higher titers in the sigma and titermax adjuvant groups. the titers in all groups gradually increased over the course of the immunizations, but while the final titers induced by non-adjuvanted ebov vlps were similar to those in the alhydrogel-adjuvanted and titermax-adjuvanted groups, sigma adjuvant resulted in -fold higher final titers (fig. c) . neutralizing antibody titers were determined by plaque reduction neutralization test (prnt) with the recombinant vsv fig. ebola vlp production and antibody response kinetics. a, b vlp preparations analyzed after transfection of hek- cells, harvest of the vlp-containing supernatant, purification, and concentration by ultracentrifugation through a sucrose cushion. samples were separated on sds-page gels and proteins were a stained with coomassie blue or b transferred to pvdf membranes. blots were stained with a polyclonal goat antiserum against ebov. lane : µg vlp sample containing ebola vp and gp; lane : µg vlp sample containing only ebola vp ; and lane : µl negative control (not transfected). all blots were derived from the same experiment and were processed in parallel. c, d mice were immunized i.m. with µg of vlps alone or in combination with either sigma adjuvant system, titermax gold, or alhydrogel, and boosted and weeks after the first immunization. final serum samples were collected weeks after the second boost. the total antibody response against recombinant ebov-gp c and neutralizing antibody response against vsvΔg/ebov-gp d are shown. antibodies against ebov-gp are reported as reciprocal serum endpoint titers using ipma and neutralizing antibodies were assessed by the % serum neutralization capacity (prnt ). symbols represent the mean of each group (n = ), and error bars indicate the standard error of the mean. the y-axis begins at the detection limit of the respective assays. statistical significance is indicated by *p < . and **p < . that expresses the gp of the closely related ebov zaire strain kikwit (vsvΔg/ebov-gp). first neutralizing antibodies were detected after the second immunization, but a robust increase was only observed in the adjuvanted groups, with a -fold increase in the titermax-adjuvanted and alhydrogel-adjuvanted groups and a -fold increase in the sigma adjuvant group (fig. d ), leading us to choose sigma adjuvant for all further experiments. induction of robust neutralizing antibody titers requires several immunizations vlps lack the ability for de novo protein production. to determine the importance of this aspect for the induction of functional antibodies, we compared adjuvanted ebov vlps with vector vaccines based on the replication-deficient modified vaccinia ankara virus (mva) that expresses either the gp protein of the zaire ebov guéckédou strain alone or in combination with vp (unpublished data), and the replication-competent vsvΔg/ebov-gp. due to the around % amino acid sequence identity and strong structural and functional conservation among zaire ebov-gp proteins, the induced immune responses are cross-reactive within the species. , as before, mice were immunized three times in -week intervals. total antibody titers against ebov-gp increased rapidly after initial immunization in all groups, but in contrast to the adjuvanted ebov vlps and the vsvΔg/ebov-gp, there was no further increase after the third immunization with the mva-based viral vectors (fig. a) . neutralizing antibodies were detected in mva/ebov-gp-immunized groups after the first and mva/ebov-vp /gp-immunized groups after the second immunization and slightly increased to yield final titers around (fig. b ). the first neutralizing antibodies in vsvΔg/ebov-gp animals were also detected after the first immunization with final titers reaching (fig. b) . even though the neutralizing antibodies were again only detected after the first boost in the group immunized with adjuvanted ebov vlps, titers ultimately reached levels around (fig. b) , illustrating that de novo protein expression is not required for efficient induction of functional antibodies. immunization with adjuvanted ebov vlps induces high neutralizing antibody titers in rabbits to generate larger volume of antisera for optimization of the purification process and to assess the scalability of antisera production, we repeated the immunization protocol in rabbits. animals were immunized with the adjuvanted ebov vlps, mva/ ebov-gp, or vsvΔg/ebov-gp, and boosted and weeks later. serum was collected week after the second boost. total anti-ebov-gp antibody titers (table , bottom row) and neutralizing antibody levels against vsvΔg/ebov-gp (table , bottom row) were slightly higher than the titers seen in mice, validating the immunization scheme in a different species. to put the titers obtained after immunization of rabbits in a clinically relevant context, total anti-ebov antibodies were quantified in a validated whole-virion ebov antibody capture enzyme-linked immunosorbent assay (elisa), used to assess the antibody responses in survivors of ebov virus disease during the west african outbreak, as well as a virus neutralization assay (vnt) with ebov zaire strain mayinga used to assess antibody responses in vsvΔg/ebov-gp vaccinated volunteers using the same assay. the optical density (od) values of rabbit sera tested in the validated whole-virion ebov elisa ranged from . to . and were thus comparable to high-titer convalescent sera after patients had cleared the virus. endpoint titrations revealed comparable total antibody titers against ebov and ebov-gp ( table ). the neutralizing antibody titers assessed in vnt with ebov were for mva/ebov-gp rabbit sera more than -fold and for vsvΔg/ebov-gp and vlp rabbit sera more than -fold higher than the average geometric mean neutralizing endpoint titers of fig. comparison of total and neutralizing antibody responses against ebov-gp induced in mice using different immunization approaches. animals were immunized i.m. with × pfu vsvΔg/ebov-gp, . × ffu of either mva/ebov-vp /gp or mva/ebov-gp, or µg of ebov vlps in combination with sigma adjuvant, and boosted and weeks later before final serum was collected weeks after the second boost. the total antibody response against recombinant ebov-gp a and neutralizing antibody response against vsvΔg/ebov-gp b are shown. antibodies against ebov-gp are reported as reciprocal serum endpoint titers using ipma and neutralizing antibodies were assessed by the % serum neutralization capacity (prnt ). symbols represent the mean of each group (n = ), and error bars indicate the standard error of the mean. the y-axis begins at the detection limit of the respective assays. statistical significance is indicated by *p < . table . comparison of total antibody responses against ebov and ebov-gp induced in rabbits using different antigen expression approaches generation of therapeutic antisera for emerging viraly r schmidt et al. ( ) or ( . ) in vaccines at day or ( table ), indicating that titers of the rabbit hyperimmune sera are within the therapeutic range. while more systematic bridging studies would be required to directly correlate our in-house with the official diagnostic assays, the similar relative proportions of the titers induced by the different immunization platforms indicates their suitability for initial analyses. vlp-based antisera production is applicable to other emerging viral infections immunization with recombinant viruses or vlps containing the niv fusion (f) and attachment (g) surface gps is sufficient to elicit a protective immune response, , , and there is considerable cross-reactivity with different niv and even hendra virus strains. to evaluate the applicability of the vlp-based immunization approach for the production of high-titer antisera against other emerging viruses, niv vlps were generated by co-transfecting hek- t cells with plasmids encoding the niv matrix protein (m) in combination with either the f or g protein of the niv malaysia strain. after h, supernatant was harvested, purified and concentrated via ultracentrifugation. the resulting niv-m/g and niv-m/f vlp preparations showed no distinct bands migrating in coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) gels ( fig. a ), but bands corresponding to niv-m ( kda), niv-g ( kda), and two bands representing uncleaved niv-f and cleaved niv-f (approximately and kda, respectively) could be readily detected by western blot analysis (fig. b) . to assess the total and functional antibody responses induced by immunization with the respective niv gp, two rabbits were immunized intramuscularly with μg niv-m/f or niv-m/g vlps in combination with the sigma adjuvant. animals were boosted and weeks later and serum was collected before each immunization and weeks after the second boost to follow the kinetics of the total and neutralizing antibody responses. antibodies against niv-f in rabbits immunized with niv-m/f vlps were detected after the first immunization, while induction of antibodies against niv-g required two immunizations with niv-m/g vlps (fig. c) . subsequent immunizations increased total antibody titers, and titers of niv-f antibodies ultimately reached levels approximately two times higher than niv-g antibodies (fig. c) . consistent with these findings, neutralizing antibody titers against the malaysia strain were found in the niv-m/f vlp group after the first immunization, while neutralizing antibodies against niv-m/g were first detected after the second immunization, and these titers increased in both groups of animals after the third and fourth immunizations (fig. d) . the neutralizing titers against the niv-f and niv-g glycoproteins (approx. ) were well above the level of neutralizing activity (> neutralizing units/ml) that protects against lethal niv challenge in passive transfer studies, , which suggests that our rabbit anti-niv gp sera would also provide a similar degree of protection. multi-step purification process yields igg and f(ab′) samples with high recovery of neutralizing activity acute and delayed adverse reactions to antisera, such as anaphylactic shock and serum sickness, are caused by the immunogenicity of xenogenic serum proteins or complement activation by the fc portion of whole igg preparations, respectively. to minimize the risk of unwanted side effects, we established a multi-step purification and concentration procedure using sera from ebov vlp-immunized rabbits. first, serum proteins were precipitated in a two-step precipitation using . and . m ammonium sulfate followed by igg affinity chromatography using protein a. next, igg was modified by a pepsin digestion to remove the fc portion to generate f(ab′) preparations (fig. a) . to accurately compare antibody titers over the course of the purification process, titers are expressed relative to the same volume for all samples. the purification and concentration is illustrated on a coomassie-stained non-reducing sds-page gel loaded with the same protein concentration of initial serum, purification intermediates after precipitation, and the igg and f(ab′) preparations (fig. b) . after ammonium sulfate precipitation, more than %, and even after subsequent igg affinity purification more than % of neutralization activity were recovered (fig. c) . pepsin digest led to around % loss of neutralization activity in f(ab′) preparations compared to unmodified serum, indicating that the multi-step purification process results in high yield purified igg preparations with only little loss of functional antibody titers, and f(ab′) fragmentation with at least % recovery of functional activity. comparable in vivo stability of homologous and xenogenic antibodies in mice antibody half-life is an important efficacy determinant of passive immune therapy. to assess the antibody stability in a different species, mice were treated intraperitoneally (i.p.) with purified homologous mouse igg, or heterologous rabbit igg preparations. doses were calculated based on total anti-ebov-gp antibody titers (fig. d) . the antibody levels in recipient mice remained stable over days after injection regardless of the origin of the igg, and treatment with a higher igg dose resulted in increased detectable titers in the recipients (fig. d) . since f(ab′) fragments reduce the incidence of adverse events due to the lack interaction with fc receptors, they are increasingly used to treat snake envenomation. however, they are associated with a shorter half-life, which may have a greater impact in the context of an amplifying virus than a single dose of venom. to determine the stability of the heterologous rabbit f(ab′) preparation, we injected mice intraperitoneally with different doses. in contrast to igg, f(ab′) fragments were only detected on the first day after transfer, even if the higher dose was used (fig. d) , indicating that the longer half-life of igg combined with its potential side effects may have to be weighed against the lower stability and reduced activity of f(ab′) fragments. the unprecedented magnitude of the western african ebov outbreak resulted in the deployment of novel vaccines and therapies that were already in advanced stages of clinical development. however, for many other emerging viruses, development is not that far advanced, so there is a need for treatment options that can be rapidly produced in clinically relevant quality and quantities. here we explored the potential of generating animal-origin hyperimmune sera in a -month time table . comparison of neutralizing antibody responses against ebov and vsvΔg/ebov-gp induced in rabbits using different antigen expression approaches vsvΔg/ebov-gp (n = ) mva/ebov-gp (n = ) µg vlps + adjuvant (n = ) α-ebov endpoint titer (log ) (vnt) α-vsvΔg/ebov-gp titer (log )(prnt ) generation of therapeutic antisera for emerging viraly r schmidt et al. frame using ebov and niv as model antigens. we found that adjuvanted vlps were more effective than mva-based or vsvbased expression vectors in inducing high-titer functional antibodies after repeated immunizations, reaching levels above the putative protective threshold for the respective pathogen. an optimized purification protocol combining ammonium sulfate precipitation followed by protein a affinity purification yielded concentrated polyclonal igg with maintained functional activity and in vivo stability, indicating that this approach may be feasible in emergency situations. monoclonal antibodies that neutralize ebov, such as kz , are protective in some small animal models, but often fail to provide protection in more relevant nhp models. the success of the zmapp monoclonal antibody cocktail demonstrated that targeting more than one antigenic site, including some non-neutralizing epitopes, improves efficacy. polyclonal antisera have the advantage of inherently targeting multiple neutralizing and nonneutralizing epitopes of a given antigen, thereby reducing the risk of escape mutant emergence, another known limitation of monoclonal antibody therapies. , this broader specificity is also a unique asset at early stages of an outbreak when the pathogen is not yet fully characterized, since cross-reactivity of already available antisera against related strains can be quickly ascertained and may provide an immediate treatment option. even though immunization of horses with inactivated ebov failed to produce effective therapeutic antisera, more recent approaches using adjuvanted recombinant ebov-gp ectodomain for immunization of sheep led to antisera that were effective as post-exposure treatment of rodents. , the production of fully human polyclonal antibodies by immunization of transchromosomal cattle with ebov-gp nanoparticles constitutes a further important step, but may be limited by availability of these purification and characterization of igg and f(ab′) preparations. antiserum from rabbits immunized three times with vlp-adjuvant combination was collected week after the final boost. a overview of the purification process showing two-step precipitation with and % ammonium sulfate saturation, buffer exchange, and desalting, followed by igg isolation using protein a affinity chromatography, and f (ab′) preparation by pepsin digest. intermediates and final preparations were resuspended at an initial volume or appropriately concentrated. b protein analysis of purification intermediates. ten micrograms of total protein from each preparation was separated by non-reducing sds-page and stained with coomassie blue. lane : initial antiserum; lane : resuspended proteins following - % precipitation; lane : elution of protein a affinity chromatography; and lane : igg after pepsin digest. total protein concentrations assessed by bca assay are shown below sds-page. all blots were derived from the same experiment and were processed in parallel. c neutralizing antibody response against vsvΔg/ ebov-gp of initial antiserum (n = ), intermediate after precipitation (n = ), affinity-purified igg (n = ), and f(ab′) preparation of affinitypurified igg (n = ) was assessed by the % serum neutralization capacity (prnt ). bars represent the mean of each group, and error bars indicate the standard error of the mean. d mice received single doses of either purified homologous mouse igg or heterologous rabbit igg or f(ab′) preparation by i.p. injection. detection of α-ebov-gp antibodies is reported as the reciprocal serum endpoint titers with the use of vsvΔg/ebov-gp in an ipma assay. symbols represent single animals and the corresponding lines for each group indicate the geometric mean (n = ). the y-axis begins at the detection limit of the respective assay generation of therapeutic antisera for emerging viraly r schmidt et al. expression but repeated exposure to the antigen in its native conformation is required for the induction of high functional antibody titers. the strong neutralizing antibody responses seen after immunization with adjuvanted vlps not only against ebov but also niv demonstrates the potential of this approach to be extended to other emerging pathogens. the determinants of vlp formation in other virus families such as flaviviruses, coronaviruses, and orthomyxoviruses are known, and medium-scale to large-scale transient transfection processes are increasingly used for customized antigen or protein production. this could conceivably form the basis of a "plug-and-play" system where antigens from emerging viruses are either synthesized or cloned, then expressed and purified using such production processes, and used for immunization of serum-producing animals. the regulatory requirements regarding the quality and safety of antigens and adjuvants used for the production of antisera differ considerably from vaccines, , as they are not applied directly to patients. since the manufacturing of therapeutic antisera is antigenindependent, existing processes and facilities could be used with little or no further adaptation to produce such pathogen-specific antisera in an emergency situation. furthermore, for pathogens with the potential of high public health impact, licensing under the food drug administration "animal rule" or the ema "orphan medicines" designation and subsequent stockpiling could be envisaged. , vlps have long been licensed as hepatitis b or human papillomavirus vaccines, and they are among the candidate platforms for emerging infections. for the generation of therapeutic antisera, vlps combine several advantages: they are non-infectious and can thus be handled without biosafety concerns, they can be adjuvanted, they are boostable, and they can be produced in a variety of cell lines. , for ebov, the combination of a non-neutralizing antibody targeting the glycan cap makes the monoclonal antibody cocktail zmapp more potent, indicating that the native glycosylation profile could be one crucial factor in the induction of effective polyclonal antibodies. in general, mammalian expression systems are able to produce post-translational modifications (ptms), but only human cell lines have the advantage of producing complex proteins with fully human ptms that are similar to those naturally synthetized in humans. a recent report demonstrates the efficacy of polyclonal antisera generated by immunization with vlps produced in an insect cell expression system. however, differences in nglycosylation and o-glycosylation profile between human and insect cell derived filovirus gps were reported, and may affect the quality of the antisera, and limit the application of this system to other pathogens. after multiple-step purification we observed similar in vivo stability for xenogenic rabbit igg and homologous mouse igg preparations in recipient mice, while rabbit f(ab′) was only detectable for day after transfer. this short half-life of f(ab′) preparations was also observed in earlier studies where a single dose of antisera resulted in better protection over a longer period compared to repeated treatment with f(ab′) fragments. until processes to stabilize f(ab′) preparations have been developed, full igg with its known and controllable risks for adverse events may be a valuable addition to the toolbox against newly emerging infectious diseases. vero e cells (atcc crl- ), mdck cells (atcc ccl- ) and hek- cells (atcc crl- ) were maintained at °c and % co in dulbecco's modified eagle's medium (dmem, sigma-aldrich) supplemented with % (v/v) fetal bovine serum (fbs, invitrogen) and mm l-glutamine (sigma-aldrich). the recombinant vesicular stomatitis expressing the gp derived from zaire ebov isolate kikwit in place of the original vsv gp (vsvΔg/ebov-gp) as well as the zaire ebov strains makona and mayinga, and the niv strain malaysia were grown on vero e cells. the viral titers of the zaire ebov and niv strains were determined by limited dilution, using cytopathic effect (cpe) as read out. vsvΔg/ebov-gp was concentrated by centrifugation for h at , × g and °c through a % (w/v) sucrose cushion and resuspended in dmem or in phosphate-buffered saline (pbs) for immunizations. viral titers were determined by plaque assay using avicel rc- overlay (fmc corporation). briefly, µl/well of -fold serial virus dilutions in dmem were added to vero e cells in well tissue culture plates. for the overlay, % avicel in pbs was mixed with an equal volume of dmem with % fbs. after incubation for h at room temperature with gentle agitation, ml/well of . % avicel overlay was added, and plates were incubated at °c for h. for visualization of the plaques, ml/well pbs containing % crystal violet, . % paraformaldehyde (pfa), and % methanol was added and incubated for min at room temperature, and titers were expressed as plaque-forming units (pfus). the mva vector vaccines delivering either the gp from zaire ebov strain guéckédou alone (mva/ebov-gp) or together with the ebov-vp matrix protein (mva/ebov-vp /gp) were produced by virus amplification in secondary chicken embryo fibroblasts, purified by ultracentrifugation through sucrose, and reconstituted in tris-buffered saline (ph . ). the vaccine preparations were stored at − °c before use. all quality control experiments were essentially performed as described previously. vaccine preparations were titrated by plaque assay using mva-specific immunostaining, and titers were indicated in focus-forming units (ffus). the construction of mammalian expression plasmids encoding the vp matrix protein (pcaggs-ebov/vp ) or the gp (pcaggs-ebov/gp) of the zaire ebov strain mayinga have been described previously. , expression plasmids encoding the matrix protein m (pcg-niv/m and pcg-niv/m myc ), the gp for fusion f (pcg-niv/f and pcg-niv/f ha ) or attachment g (pcg-niv/g and pcg-niv/g flag ) of the niv strain malaysia have also been reported. for production of ebov vlps, hek- cells at approximately % confluency were seeded in cm tissue culture flasks and cotransfected with µg of each of the plasmids mixed with four volumes of mm polyethylenimine reagent (sigma-aldrich). for production of niv vlps, cells were transfected with µg of plasmid dna encoding for the m or m myc protein and µg of plasmid dna encoding for either the f, f ha , g, or g flag proteins. transfection mixtures were prepared in dmem without additives. after incubation at °c for h, the medium was changed, and the cells were incubated for another h. culture supernatants were harvested and clarified by centrifugation at × g for min at °c. vlps were then pelleted by ultracentrifugation through a % (w/v) sucrose cushion for h for ebov vlps or h for niv vlps at , x g and °c, and pellets were resuspended in pbs and stored at °c. total antibody responses were determined by immunoperoxidase monolayer assay (ipma). briefly, for determination of ebov-gp-specific antibodies × vero e cells were seeded in -well plates and infected with pfu vsvΔg/ebov-gp. for determination of niv-f-specific and niv-g-specific antibody responses, mdck cells were transfected with µg dna of either pcg-niv/f ha or pcg-niv/g flag plasmids. cells were incubated for h at °c before fixation with % pfa. serial -fold dilutions of antisera were added to the fixed cells and incubated for h at room temperature, followed by incubation with horseradish peroxidase (hrp)-coupled secondary antibody, and visualized by staining with amino- -ethylcarbazole (sigma-aldrich). total antibody titers were calculated as geometric mean of the reciprocal of the last serum dilution at which positive staining was detected. total antibody responses against whole ebov virus were determined by elisa as described previously. , briefly, particles of the zaire ebov isolate makona were purified and concentrated by ultracentrifugation through a % sucrose cushion, and the pellet was resuspended in pbs containing % sds and inactivated by boiling for min. elisa plates were then coated with inactivated zaire ebov isolate makona, and rabbit antisera were added at the respective dilution and detected by a polyclonal hrp-coupled secondary antibody. after staining with tmb generation of therapeutic antisera for emerging viraly r schmidt et al. (kpl inc.) and addition of the stop solution (kpl inc.), the od was measured at - nm. each sample was analyzed in duplicate and the mean od values were corrected by subtracting mock od values from values obtained by incubating the same serum with ebov antigen. for endpoint titrations, serial -fold serum dilutions were added to coated elisa plates. each sample was analyzed in duplicate and the mean od values were corrected as described above. the cut-off was calculated as the mean plus standard deviation (sd) multiplied with (mean ± sd × ) of negative rabbit sera and the total antibody titers were expressed as the reciprocal of the highest serum dilution yielding an od value greater than the cut-off od of . . to measure neutralizing antibodies, a vsvΔg/ebov-gp-based prnt assay or a virus neutralization assay with zaire ebov isolate mayinga was used. for prnt, -fold serial dilutions of heat-inactivated antisera in dmem were mixed with the appropriate volume of pfu/ml vsvΔg/ebov-gp to result in a final concentration of pfu/well. the mixture was incubated at room temperature for min, and then µl/well was added to confluent -well dishes of vero e cells. after incubation for h at room temperature with gentle agitation, the inoculum was removed and replaced with ml . % avicel overlay. plates were incubated at °c and for h, and plaques were visualized using crystal violet staining. the prnt titer was calculated by averaging the reciprocal of the highest serum dilution reducing the number of plaques by % relative to the average number of plaques in positive control wells. the virus neutralization assay for ebov was performed as described previously. briefly, serial -fold dilutions of heat-inactivated rabbit antisera preparations were incubated with % tissue culture infection doses (tcid ) of zaire ebov isolate mayinga, and then added to vero e cells. the cpe was evaluated after days and titers are expressed as the geometric mean of the reciprocal of the last serum dilution at which cpe was observed. for the virus neutralization assay of niv antisera -fold serial dilutions of the rabbit sera in -well plates were mixed with tcid niv strain malaysia. the virus and serum dilutions were incubated for h at °c, after which vero e cells were added to each well. the cpe was assessed after days, and neutralizing titers are reported as the geometric mean of four replicates. all experiments with infectious ebov or niv were performed in the bsl facility of the institute of virology, philipps-university marburg, in compliance with german regulations. all animal experiments were carried out in compliance with the regulations of german animal protection laws and authorized by the regierungspräsidium darmstadt, germany. balb/c mice were purchased from janvier s.a.s. and new zealand white rabbits from charles river germany. to generate mouse antisera, groups of -week-old balb/c mice were immunized intramuscularly with either × pfu of vsvΔg/ebov-gp, . × pfu of either mva/ebov-vp /gp or mva/ebov-gp, or µg of ebov vlps mixed in a : ratio (v/v) with either sigma adjuvant system ( . mg monophosphoryl lipid a (detoxified endotoxin) from salmonella minnesota and . mg synthetic trehalose dicorynomycolate in % squalene-tween- -water, sigma), or titermax gold (block polymer crl- , squalene, and sorbitan monooleate, sigma), or % alhydrogel (invivogen). all animals were boosted two and weeks after the first immunization. three mice from each immunization approach were sacrificed at , , , and days post-immunization and blood was collected by cardiac puncture. for generation of rabbit ebov antisera, new zealand white rabbits were immunized intramuscularly with either × pfu of vsvΔg/ebov-gp, × pfu mva/ebov-gp, or µg of ebov vlps mixed in a : ratio (v/ v) with sigma adjuvant system, and boosted and weeks later. blood samples were collected from the marginal ear vein on days , , and , and animals were sacrificed days post-immunization by exsanguination. for generation of niv-f or niv-g antisera, new zealand white rabbits were immunized intramuscularly with µg of niv vlps containing either niv-m/f or niv-m/g mixed in a : ratio (v/v) with sigma adjuvant system, and boosted and weeks later. blood samples were collected from the marginal ear vein on days , , and , and animals were sacrificed days post-immunization by exsanguination. to test the stability of the respective antisera in a xenogenic transfer model, -week-old balb/c mice were administered µl of purified mouse or rabbit igg, or rabbit f(ab′) fragment by intraperitoneal injection. three mice from each antiserum transfer group were sacrificed at days , , , and after transfer and blood was collected by exsanguination. the rabbit sera were tested for total antibodies against ebov-gp by ipma. igg was purified from rabbit antisera using a multi-step process. first, animal sera were treated with . m ( % saturation) ammonium sulfate (pierce) at °c for h to remove serum proteins that are less soluble than igg. precipitated proteins were removed by centrifugation at × g for min at °c. next, the ammonium sulfate concentration of the supernatant was adjusted to . m ( % saturation) to remove serum proteins that are more soluble than igg. after centrifugation, the supernatant was removed and the igg-containing precipitate was dissolved in the original volume of the serum sample using . m tris-hcl. the dissolved precipitate was dialyzed against tris-buffered saline through a kda molecular weight cut-off (mwco) cassettes (thermo scientific) before igg was purified by affinity chromatography using protein a columns (vivapure kit, sartorius) according to the manufacturer's recommendations with some modifications. briefly, ml precipitated sample was mixed with two volumes of . m glycine/naoh, m nacl, ph . , binding buffer and centrifuged at × g for min at room temperature to let the igg bind to protein a. this step was repeated with the flow through before the column was washed eight times with binding buffer and centrifuged at × g for min at room temperature. igg was eluted in ml . m glycine/hcl elution buffer with ph . immediately into . ml previously prepared m tris-hcl, ph . , neutralization buffer to bring the samples' ph to approximately . . for concentrating to the original volume and buffer exchange of the igg sample, kda mwco centrifugal column (vivaspin, sartorius) were used according to the manufacturer's recommendations. concentrated igg was dissolved in pbs for immunization, or mm sodium acetate buffer, ph for f(ab′) preparation. to generate f(ab′) fragments, purified igg in acetate buffer was mixed with pepsin (> u/mg, sigma) in a : ratio and incubated at °c for h. the digestion was stopped with the addition of tris base (ph . ) to adjust the final ph to . . f(ab′) fragments were separated from undigested igg by separation through a kda mwco centrifugal concentrator and from remaining peptides and pepsin by separation through a kda mwco centrifugal concentrator before dissolving in pbs. the protein concentration of purified vlps was determined by bradford assay (bio-rad), and the bca assay (thermo fisher) was used for samples of the serum purification process. for verification of sample quality and purity, ebov vlp samples containing µg total protein or niv vlp samples containing µg total protein were separated by % reducing sds-page and antiserum samples containing µg total protein were separated by % non-reducing sds-page and stained with coomassie blue (fisher bioreagents) or transferred to polyvinylidene difluoride (pvdf) membranes (millipore) for western blot analysis. blots were stained with a polyclonal goat antiserum against ebov virus at a working solution of : or monoclonal antibodies against flag-tag, ha-tag, or c-myc-tag (all sigma) at the recommended dilution to detect the respective epitopetagged niv proteins. statistical analyses were performed using graphpad prism on logtransformed data using multiple 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of orphan medicines in europe from virus-like particles for the prevention of human papillomavirus-associated malignancies zika virus-like particle (vlp) based vaccine construction and characterization of virus-like particles: a review virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives production and purification of filovirus glycoproteins in insect and mammalian cell lines easy and efficient protocols for working with recombinant vaccinia virus mva vp octamers are essential for ebola virus replication the cytoplasmic domain of marburg virus gp modulates early steps of viral infection the ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo we thank all laboratory members for continuing support and lively discussions, and yvonne krebs, astrid herwig, and dirk becker for excellent technical support. this generation of therapeutic antisera for emerging viraly r schmidt et al. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, 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