key: cord- -cgiok ce authors: binjawadagi, basavaraj; dwivedi, varun; manickam, cordelia; ouyang, kang; torrelles, jordi b; renukaradhya, gourapura j title: an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination date: - - journal: int j nanomedicine doi: . /ijn.s sha: doc_id: cord_uid: cgiok ce porcine reproductive and respiratory syndrome (prrs) is an economically devastating respiratory disease of pigs. the disease is caused by the prrs virus (prrsv), an arterivirus which is a highly mutating rna virus. widely used modified live prrsv vaccines have failed to prevent prrs outbreaks and reinfections; moreover, safety of the live virus vaccines is questionable. though poorly immunogenic, inactivated prrsv vaccine is safe. the prrsv infects primarily the lung macrophages. therefore, we attempted to strengthen the immunogenicity of inactivated/killed prrsv vaccine antigens (kag), especially in the pig respiratory system, through: ) entrapping the kag in biodegradable poly(lactic-co-glycolic acid) nanoparticles (np-kag); ) coupling the np-kag with a potent mucosal adjuvant, whole cell lysate of mycobacterium tuberculosis (m. tb wcl); and ) delivering the vaccine formulation twice intranasally to growing pigs. we have previously shown that a single dose of np-kag partially cleared the challenged heterologous prrsv. recently, we reported that np-kag coupled with unentrapped m. tb wcl significantly cleared the viremia of challenged heterologous prrsv. since prrsv is primarily a lung disease, our goal in this study was to investigate lung viral load and various immune correlates of protection at the lung mucosal surfaces and its parenchyma in vaccinated heterologous prrsv-challenged pigs. our results indicated that out of five different vaccine-adjuvant formulations, the combination of np-kag and unentrapped m. tb wcl significantly cleared detectable replicating infective prrsv with a tenfold reduction in viral rna load in the lungs, associated with substantially reduced gross and microscopic lung pathology. immunologically, strong humoral (enhanced virus neutralization titers by high avidity antibodies) and cell-mediated immune responses (augmented population of interferon-γ secreting cd (+) and cd (+) lymphocytes and reduced secretion of immunosuppressive cytokines) in the lungs were observed. in conclusion, combination of np-kag and soluble m. tb wcl elicits broadly cross-protective anti-prrsv immunity in the pig respiratory system. porcine reproductive and respiratory syndrome (prrs) is an economically devastating disease in pigs causing an estimated direct loss of greater than $ million annually to the us pork industry. prrs is caused by prrs virus (prrsv), an enveloped positive-sense rna virus belongs to the family arteriviridae. there are broadly two distinct prrsv genotypes, the european (type i) and the north american (type ii), dovepress dovepress binjawadagi et al which possess a wide range of intra-and intergenotypic, genetic, and antigenic diversity. therefore, developing preventive measures to control prrs outbreaks has been a challenge to the global swine industry. though both modified live virus (mlv) and inactivated prrsv vaccines have been in use since , control of disease outbreaks has remained unsuccessful. live virus vaccines are successful in reducing the clinical disease, but are invariably implicated in spreading the mutated viruses to susceptible pigs. in contrast, available inactivated prrsv vaccines are safe, but they have failed to elicit protective immunity even against homologous infections. in addition, killed vaccine antigens do not undergo intracellular antigen presentation pathways to induce a strong cytotoxic t-cell (ctl) response, which is necessary for clearance of intracellular pathogens like viruses. [ ] [ ] [ ] thus, research aimed at developing better cross-protective inactivated prrsv vaccines is warranted. therefore, several innovative strategies should be adopted to strengthen potency and efficacy of inactivated/killed prrsv vaccine antigens (kag), with respect to suitable methods of viral inactivation and purification, use of potent adjuvants, route, and efficient delivery of vaccine to protect ags from rapid enzymatic degradation in the body. since prrsv infects primarily the pig respiratory tract and the target cells are lung alveolar and interstitial macrophages, induction of strong local mucosal immunity in the respiratory tract is important. the intranasal route of delivery of vaccines to control primary respiratory infections has shown great promise in induction of protective mucosal (ie, local) as well as systemic immunity. , , poly(lactide-co-glycolide) (plga) is a synthetic biodegradable polymer used successfully in particulate delivery of inactivated vaccines. [ ] [ ] [ ] the adjuvant mycobacterium tuberculosis whole cell lysate (m. tb wcl) was shown to augment immunogenicity of both live prrsv vaccine and plga-nanoparticles entrapped with killed prrsv antigens (np-kag) without causing any side effects in pigs [ ] [ ] [ ] [ ] and with other vaccines in rodents, guinea pigs and rabbits. , unlike complete freund's adjuvant (cfa), m. tb wcl is free from water-insoluble toxic cell wall components of the bacterium, , and it is endotoxin free and contains only water-soluble components. therefore, unlike cfa, m. tb wcl does not cause any toxicity or granulomatous lesions at the site of inoculation. previously, we have shown that a single dose of prrsv kag-entrapped in plga ( : ) nanoparticle (np-kag) elicits both mucosal and systemic immune responses. , recently, np-kag coadministered intranasally twice with m. tb wcl induced cross-protective anti-prrsv immune response in blood to a challenged heterologous prrsv, associated with a significant reduction in viremia. in this report, we made use of various types of the lung samples of that recent study to evaluate viral load and local mucosal immunity both at airway surfaces and in the lung parenchyma, and also microscopic lung histopathology in vaccinated, heterologous prrsv-challenged pigs. killed prrsv vaccine antigens (kag) were prepared as described earlier. briefly, north american prototype prrsv strain vr was grown in marc cells, freeze-thawed three times, and the harvested cell culture supernatant was subjected for clarification followed by ultracentrifugation to pellet the virus. resuspended pellet in sterile phosphate-buffered saline (pbs) was subjected to ultraviolet inactivation ( nm for hour) to prepare kag for use in vaccine preparation. for restimulation experiments, similarly prepared kag of the challenge virus, prrsv strain mn , was used. preparation of whole cell lysate of m. tb m. tb was grown in agar medium and wcl was prepared as described previously. briefly, h rv strain of m. tb was grown in oleic acid-albumine-dextrose-catalase enriched h (difco) agar plates (becton, dickinson and company, franklin lakes, nj, usa). the bacterial cells were harvested by centrifugation of colony scrapings at ×g and washed with pbs (ph . ). live bacterial cells were suspended [ g (wet weight)/ml] in pbs containing mm ethylenediaminetetraacetic acid (edta) (becton, dickinson and company), proteinase inhibitors (emd millipore, billerica, ma, usa), dnase and rnase (sigma-aldrich, st louis, mo, usa), and the bacterial cell wall was disrupted by bead beater until . % cell breakage was obtained (confirmed by acid fast staining). the cell lysate was centrifuged at ×g to pellet unbroken cells and insoluble broken cell wall components. the clear supernatant-containing water soluble fraction of the bacterium was harvested and sterilized through . µm low protein binding membrane filter. further, endotoxin levels in every batch of m. tb wcl was confirmed to be less than the acceptable levels (, . µg/mg conventional large white-duroc crossbred - weeks old weaned pigs were procured from a swine herd seronegative for prrsv, porcine respiratory coro navirus, transmissible gastroenteritis virus, and porcine circo virus -specific antibodies. a total of pigs were randomly divided into one of the ten groups (n= pigs/group) and vaccinated with the indicated vaccine formulation, intranasally ( ml/pig), twice at -week intervals ( table ). all the vaccinated pigs were intranasally challenged on postvaccination day with a virulent heterologous north american prrsv (type ii) strain mn ( × median tissue culture infective dose [tcid ]/pig). adjuvant and vaccine were entrapped separately and combined before administering to pigs, and the dose of adjuvant ( mg/dose/pig) and kag were either (low dose) or (high dose) µg/dose/pig of semipurified viral protein containing ∼ . × or . × tcid of killed prrsv, respectively, either entrapped in nps or unentrapped. the vaccine and adjuvant doses were tested to be efficacious in pigs earlier. , pigs were monitored daily for the respiratory symptoms, and rectal temperatures and body weights were recorded every third day postchallenge (pc); animals were euthanized on day pc as per the approved protocol of the institutional animal care and use committee, the ohio state university, and indicated samples were collected during the necropsy. the lungs were examined for gross lesions in all the lobes. the lung samples collected from the right cranial lobe were fixed in % neutral buffered formalin and sections ( µm) were made and stained with hematoxylin and eosin as described previously. the slides were examined by an unbiased certified veterinary pathologist to score prrsvinduced inflammation. collection of bronchoalveolar lavage fluid, preparation of lung homogenate, and isolation of lung mononuclear cells during necropsy, the lungs were harvested and the bronchoalveolar lavage (bal) fluid was collected by washing the airways using - ml of cold pbs containing antibiotics and edta; the harvested fluid was centrifuged at , ×g for minutes at °c and the clarified bal fluid was aliquoted and stored at - °c. the lung homogenate was prepared as described previously. briefly, gram of lung tissue from the right cranial lobe of every pig was collected in ml ice-cold dulbecco's modified eagle's medium and minced and homogenized using a stomacher laboratory blender (seward limited, worthing, west sussex, uk) for minutes, and the clarified supernatant (lung homogenate/lung lysate) was aliquoted and stored at - °c. the lung mononuclear cells (lmncs) from the individual pig lungs were isolated by treating the perfused and minced lung tissue using collagenase and dnase as described previously. estimation of total immunoglobulin (ig) total isotype specific pig ig levels were estimated by elisa as described previously, with a few modifications. briefly, -well enzyme-linked immunosorbent assay (elisa) plates were coated with pretitrated dilution of ( : , ) of goat the assay was performed as previously described. avidity of the prrsv-specific antibodies in the lungs prrsv-specific antibody avidity was determined as described previously, , with a few modifications. briefly, bal fluid ( : ) and lung homogenate ( : ) samples were added to prrsv-ag-coated and -blocked plates. after hours incubation of test samples at room temperature, washed plates ( ×) were treated with serially twofold diluted ammonium thiocyanate (nh cn) ( µl/well) solution at to . mol concentration and incubated at room temperature for minutes. the plates were washed ( ×) and the remaining steps of elisa carried out as described above. for calculation purpose, od value of the test sample from nh cn-untreated ( m) well was considered as % absorbance (contributed by prrsvspecific antigen-antibody interaction), and the od value of a test sample at different molar concentration of nh cn was used to calculate the percent remained antigen-antibody interaction compared to its % absorbance value. estimation of prrsv-specific igg and igg antibody subtypes total prrsv-specific igg and igg subtypes in the lung homogenate samples were analyzed as described previously, with a few modifications. briefly, kag-coated plates were blocked and a serial tenfold diluted lung homogenate was added; mouse antipig igg or igg (abd serotec, raleigh, nc, usa) ( : dilution) secondary antibody was added to detect virus-specific igg or igg subtype, respectively. further, the washed plate was treated with goat antimouse igg-hrp (sigma-aldrich) ( : , dilution). the reaction was developed using tmb ( , ', , '-tetramethylbenzidine) substrate (kpl, gaithersburg, md, usa), stopped using m phosphoric acid, and the plates were read at od nm . for calculation of the prrsv-specific igg and igg antibody levels of test samples, the od values obtained at : dilution was considered. determination of prrsv-specific inter feron gamma (ifn-γ ) secreting cells by enzyme-linked immunospot (elispot) assay the elispot assay was performed as described previously. adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs lated were included as positive and negative controls, respectively. lung homogenates were analyzed for pig cytokines, th (ifn-γ and interleukin [il- ]), proinflammatory (il- ), and immunosuppressive (il- and transforming growth factor beta [tgf-β]) by elisa as described previously. flow-cytometric analyses the phenotypes and frequencies of lymphoid and myeloid cells populations from , events of immunostained lmncs were determined by flow cytometry as described previously. for intracellular ifn-γ staining, monensin (golgiplug, bd biosciences) was added during the last hours of the -hour incubation of lmncs that were unstimulated or stimulated with prrsv mn kag as described above. lmncs were first immunostained using pig lymphocyte-specific monoclonal antibodies (cd ε, cd α, cd α, cd , and tcr n ) conjugated with different fluorochromes. cells were fixed with % paraformaldehyde and permeabilized with a cell-permeabilization buffer ( . % deionized water, % pbs with no ca or mg, % formaldehyde solution, and . % saponin) overnight at °c. cells were washed and stained with a fluorochrome-conjugated antipig ifn-γ or its isotype control monoclonal antibodies (bd biosciences) in . % saponin containing fluorescence activated cell sorting (facs) buffer. immunostained lmncs were acquired using a facs aria ii (bd biosciences) flow cytometer and analyzed using the flowjo software (treestar inc., ashland, or, usa). all the specific immune cell frequencies were presented as the percent of total lymphocytes or myeloid cells. determination of prrsv load, virus-neutralizing antibody titer, and rna copies prrsv titer and virus-neutralizing (vn) titer in bal fluid and lung homogenate samples were analyzed by indirect immunofluorescence assay as previously described. extracted rna was reverse transcribed into complimentary deoxyribonucleic acid (cdna) using quantitect reverse transcription kit (qiagen, venlo, the netherlands). the cdna was subjected to quantitative real-time polymerase chain reaction (qpcr) using primers against prrsv orf in perfecta sybr green fast mix (quanta biosciences, gaithersburg, md, usa) with forward primer (gataac-cacgcatttgtcgtc) and reverse primer (tgccgtt-gttatttggcata). standard curve was generated using serial tenfold dilution of prrsv vr stock virus starting at tcid per µl for viral rna quantification. the data were expressed as the mean ± standard error of mean of three pigs. statistical analyses were performed by one-way analysis of variance followed by tukey's multiple comparison test (or unpaired t-test for figure binjawadagi et al cated that sham or entrapped np particles were spherical with a smooth surface. dynamic light scattering of nps indicated the mean diameter ± standard deviation was ± nm for sham, ± nm for np-kag, and ± nm for np-m. tb wcl, respectively. however, all nps possessed a uniform surface electrostatic potential of - mv. other in vitro studies revealed that the entrapped antigen was pulse-released over a period of several weeks in normal physiological conditions, and was efficiently uptaken by porcine alveolar macrophages. a detailed physical and biological characterization of np-kag vaccine used in this study was published recently. in the serum of all the virus-challenged pigs (groups to ), irrespective of vaccination history at weeks postchallenge, a twofold to threefold increase in total igg amount was observed (data not shown). the total igm and iga amounts in the lung homogenate were comparable in most of the prrsvchallenged pig groups ( figure a and c). however, the levels of igg (both in lung homogenate and bal fluid) ( figure b and e), and igm and iga (in bal fluid) ( figure d and f) in pig groups to were four-to fivefold higher compared to mock enhanced prrsv-specific antibody response in adjuvanted np-kag-vaccinated pigs prrsv-specific iga response was significantly higher both in the bal fluid and lung homogenate of both low ( µg/pig) and high ( µg/pig) doses of adjuvanted np-kag-vaccinated pigs, compared to most of the vaccine trial groups ( figure g and h). in contrast, levels of virusspecific igg were comparable in the bal fluid of all the virus-challenged pig groups ( figure i) . surprisingly, in the lung homogenate, significantly higher levels of virusspecific igg was observed in group and pigs vaccinated with either dose ( figure j ). this suggests that in adjuvanted np-kag-vaccinated pigs (group ), virus-specific iga is the major antibody isotype in the airway surfaces (bal fluid), while both iga and igg isotypes appear to play important roles in the lung parenchyma. the binding strength of heterogeneous antibodies to their cognate ag is defined as avidity. in adjuvanted np-kagvaccinated pigs, increased avidity of virus-specific iga was detected in both bal fluid (both low and high doses) and lung homogenate (only low dose) samples compared to other tested groups (figure a-c) . interestingly, the virus-specific iga avidity in lung homogenate of pigs receiving the highdose vaccine was comparable among all the tested pig groups ( figure d ). comparable levels of avidity of prrsv-specific igg antibody response was observed in lung homogenate of all the tested groups (data not shown). these data suggested that enhanced avidity of prrsv-specific iga antibody persisted for a relatively longer period at the airway surfaces of adjuvanted np-kag-vaccinated pigs. balanced th and th antibody responses in adjuvanted np-kag-vaccinated pigs t helper type (th) -or th -biased immune response is measured by quantifying antigen-specific igg antibody subtypes. in case of pigs, higher levels of igg and igg indicate th and th -biased responses, respectively. our results indicated that in group pigs, comparable levels of both igg and igg subtypes were secreted in lung homogenate ( figure a and c). the ratio of igg :igg , if greater or less than one, indicates th -or th -biased response, respectively. in the lungs of adjuvanted np-kag-vaccinated pigs at day pc a balanced th and th response (ratio close to one) was detected ( figure b and d). in pig groups and , although the ratio was close to one, the detected amount of virus-specific igg and igg subtypes were low ( figure a-d) . enhanced prrsv-neutralizing antibody response in adjuvanted np-kag-vaccinated pigs except group (mock), all the other pigs were challenged using a heterologous prrsv (strain mn ), which is genetically highly divergent (∼ %) compared to the vaccine strain vr . prrsv-specific vn titers were analyzed both in the bal fluid and lung homogenate samples against mn strain, and against another variant type strain, prrsv - - (accession # - ), is genetically distinct from both mn and vr strains. in addition, vn titers were also analyzed against an antigenically highly divergent (∼ %) heterogenotypic (type ) prrsv strain, sd - ( figure e -j). in group pigs, the lung vn titers against mn were significantly higher with mean titers of and with low and high vaccine doses, respectively, compared to other tested groups ( figure e and f). the vn titers against both prrsv - - and sd - strains were significantly greater in group pigs receiving the high-dose vaccine compared to group and group , respectively ( figure h and j). the kag and soluble wcl-vaccinated pigs (group ) also had significantly higher vn titer against only prrsv - - strain compared to group animals ( figure h ). these results indicated that soluble m. tb wcl-adjuvanted np-kag vaccine elicits broadly cross-reactive vn titers. the vn titers observed in bal fluid were low (data not shown). a standard reference for measuring cmi response against prrsv is by determining the frequency of virus-specific iscs by elispot assay. in low-dose vaccinated group pigs, significantly increased iscs in lmncs was detected compared to three other tested groups ( figure a ) and compared to all the tested groups in the high-dose category ( figure g ). the quantity of ifn-γ detected in the lung homogenate was significantly higher in group pigs compared to group (low dose) and groups and (high dose) categories ( figure b and h) . another important th cytokine, il- , was not significantly modulated among the tested pig groups in the high vaccine dose category ( figure j ), but in low-dose groups and , significantly higher levels of il- were detected compared to groups , , and ( figure d ). one of the important proinflammatory cytokines, il- , was significantly reduced in the lungs of all the vaccine trial groups compared to mock-challenged animals ( figure c and i). cytokines il- and tgf-β are immunosuppressive in nature, and they play a vital role in prrsv pathogenesis. , the quantity of tgf-β was significantly reduced in group pigs compared to group ( figure e and k) , and the quantity of il- among all the tested vaccine trial pig groups was comparable ( figure f and l). figure a and b) . the frequencies of intracellular ifn-γ + cells in lmncs either unstimulated or stimulated with mn ags identified the virus-specific memory lymphocyte response. significantly increased prrsvspecific recall ifn-γ response was detected both in cd + and cd + lymphocyte subsets of group pigs vaccinated with a high dose of vaccine ( figure c and d) . these data suggest that both t-helper and ctls were potentially primed in the lungs of only group pigs. when prrsv-specific ifn-γ secreting lymphocyte subsets in only restimulated cells were compared among different vaccine trial groups, only in group animals (both vaccines doses) significantly increased cd + cd -ifn-γ + cells compared to group animals ( figure f and n) , while cd -cd + ifn-γ + cell frequency was significantly enhanced in group pigs were present compared to groups , , and with low vaccine dose and groups to with high vaccine dose ( figure g and o). similarly, cd + cd + ifn-γ + and ifn-γ + γδ t cells were significantly higher in group pigs compared to pigs in groups , , and ( figure h -i, p-q). an increased frequency of activated γδ t cells was detected in group pigs compared to other groups (table a and b) . although there was no significant difference in total natural killer (nk) (cd + ) cell frequency ( figure j and r) , an increase in ifn-γ + nk cell frequency was significant in group pigs compared to other tested groups ( figure k and s) . in addition, macrophage (cd + cd + slaii + ) and dendritic cells (cd + cd c + slaii + ) rich apc populations were significantly higher in group and pigs in the high-dose vaccine category compared to group animals ( figure t and u); in the low-dose category, though a similar trend was detected, the data was not statistically significant ( figure l and m). in the bal fluid of pig groups and (low vaccine dose), detectable replicating prrsv was relatively reduced (but not significant) compared to other vaccine groups ( figure a ); in the same pig groups with the high vaccine dose, detectable replicating virus was absent, and the data was statistically significant compared to pig group ( figure b ). in the lung homogenate of group pigs receiving the low-dose vaccine, detectable replicating virus load was significantly reduced compared to mock-challenged pigs ( figure e) ; detectable virus was absent in the high-dose group pigs, and the data was statistically significant compared to pig groups and ( figure f ). further, prrsv rna copy numbers in bal fluid and lung homogenate were quantified by qpcr. in both the lung sample adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs types of group and pigs in the low-dose vaccine category, reduction (but not significant) in viral rna copy numbers was observed compared to other vaccine trial groups ( figure c and g); and in the same pig groups vaccinated with a high dose, a significant reduction ( - fold) in rna load was detected compared to group animals ( figure d and h). gross lung lesions revealed marked consolidation in pig groups , , and , while the group pig lungs were comparable to mock animals and the group pig lungs were in between mock and the group animals. microscopic lmncs isolated on the day of necropsy were unstimulated or stimulated with killed prrsv mn ags and immunostained using a combination of indicated pig lymphocytespecific cell surface markers followed by intracellular ifn-γ and analyzed by flow cytometry. representative histograms showing stimulated total lymphocytes in lmncs with intracellular ifn-γ + (a and b) . the dotted line: isotype control and solid line: ifn-γ + -specific staining. unstimulated (clear bars) or stimulated (black bars) lmncs with killed prrsv mn ags were analyzed for total ifn-γ + cd -, cd -, and cd -expressing lymphocytes (c-e). only stimulated lmncs were compared for indicated ifn-γ + lymphocyte subsets: cd + cd -ifn-γ + (f and n) ; cd -cd + ifn-γ + (g and o); cd + cd + ifn-γ + (h and p); γδ + ifn-γ + (i and q); and total nk (cd + ) (j and r); cd + ifn-γ + cells (k and s). also immunostained for potential apcs; macrophage-rich (cd + cd + sla-ii + ) (l and t) and dendritic cell-rich (cd + cd c + sla-ii + ) (m and u) populations. each bar indicates the average frequency of indicated cells from three pigs ± standard error of mean. asterisk indicates statistically significant (p, . ) difference between the two indicated pig groups. the unpaired t-test was applied to compare the data of only (c-e); for the other data, one-way anova followed by tukey's t-test was used. figure i ii, iii, and v). these data were consistent with the gross lung lesions, especially in pig groups and ; also, these pig groups had irregular fever with reduced feed intake during first week postchallenge. in contrast, the absence of any clinical prrs disease and gross lung lesions in group pigs was associated with the absence of detectable microscopic lung pathology, and the lung architecture was comparable to mock pigs ( figure i vi) . the other pig group which had moderately reduced lung lesions was group (kag + m. tb wcl) ( figure i iv), suggesting the adjuvant mediated protective immune response in pigs. similar but severe lung pathology was observed in pigs receiving the low vaccine dose in groups , , and (data not shown). potent mucosal vaccines against pathogens which predominantly cause disease at mucosal sites have been proven efficacious, with vaccines against influenza, parainfluenza, adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs respiratory syncytial virus, rotavirus, and hiv/siv. [ ] [ ] [ ] [ ] [ ] since prrsv primarily infects the lungs of pigs, its effective control appears to be possible by induction of strong respiratory mucosal immunity. thus, development of a novel prrsv mucosal vaccine is warranted. further, among the mucosal routes, intranasal delivery of np-based vaccine elicits a higher and longer duration of igg and iga antibody responses compared to rectal, oral, or intramuscular routes. in the lungs, prrsv infects both alveolar and interstitial macrophages, and greater than % of bal cells are macrophages. we investigated anti-prrsv response both in bal cells and lmncs. cd c + apcs are richly present in the lungs in both bal cells and lmncs, but they differ in their antigen presentation potential. apcs present in the lung mucosal surfaces (represented by bal cells) activate only antigen-primed t-cells, while apcs in the lung parenchyma (represented by lmncs) activate both naïve and antigen-primed t-cells. therefore, our study comprehensively investigated overall immune responses and viral load in the lungs of pigs vaccinated and challenged through the intranasal route. a couple of earlier studies showed hypergammaglobulinemia in the serum of prrsv-infected pigs, indicated by a four-to fivefold increase in total igg levels at - weeks postinfection. , consistent with that observation, in our study, an approximately fourfold increase in total igg amounts was observed both in the serum and lung samples (bal fluid and lung homogenate); however, in the lung homogenate of adjuvanted np-kag-vaccinated pigs, the total igg levels were only twofold more compared to mock animals. these data suggest that adjuvanted np-kag has a strong positive influence on humoral response, further indicated by a significant increase in production of anti-prrsv antibodies and virusspecific high avidity vn antibody titers. inactivated vaccines are safe, but suitable adjuvant and delivery system are critical to boost their efficacy. several unsuccessful attempts were made to develop protective killed prrsv vaccines. , a recent study using uv-or bei-inactivated prrsv ( × tcid per dose) coadministered with either freund's incomplete or suvaxyn oil/water adjuvant elicited the vn titer of greater than associated with partial clearance of homologous viral challenge. in that study, cmi response was not investigated. plga is the food and drug administration (fda)-approved agent, and plga-based intranasal delivery of prrsv vaccine was found safe in pigs. , , one of the strategies to augment uptake of particulate antigens by apcs is by increasing its surface hydrophilic nature of the particles by coating with a nonionic surfactant, polaxamer . , efficient th -immunity-inducing adjuvants are critical to promote a strong cmi response to subunit and inactivated virus vaccines. the water-soluble components of m. tb wcl, such as heat shock protein- and pro-glu/ppe, are potent adjuvants. in addition, four other water-soluble components in m. tb wcl, such as short-and long-chain poly-peptidoglycans, acetylated peptidoglycans, and tetrasaccharide-heptapeptide, have potent adjuvant effects comparable to cfa in inducing production of antibodies in rabbits coadministered with an inactivated influenza virus vaccine. plga-np vaccine coadministered with a potent adjuvant elicits a protective immune response. we demonstrated potent adjuvant effects of m. tb wcl to prrs-mlv, [ ] [ ] [ ] , and also to np-kag, with no side effects. our initial studies using a single dose of np-kag elicited partial cross-protective immune response in pigs. , to potentiate the efficacy of np-kag vaccine, in a recent study np-kag was evaluated by coadministering intranasally twice with either entrapped or unentrapped m. tb wcl. our results suggested that combination of np-kag with unentrapped m. tb wcl significantly cleared the challenged heterologous virus from the circulation, supported with strong humoral and cmi responses in the blood. the enhanced t-and b-cell responses in adjuvanted np-kag-vaccinated pigs were attributed to concerted effects of both plga and m. tb wcl. induction of strong local mucosal immunity and clearance of heterologous prrsv from vaccinated pig lungs is important for effective control of prrs. therefore, in this study we investigated the immune responses exclusively both at mucosal surfaces and parenchyma of pig lungs. our results indicated that adjuvanted np-kag (group pigs) did potentiate anti-prrsv immune response in the lungs, as indicated by the following parameters: ) increased prrsv-specific igg and iga response with enhanced antibody avidity and vn titers, and balanced th and th immune responses; ) upregulated secretion of th (il- and ifn-γ) and downregulated immunosuppressive (tgf-β and il- ) cytokines; ) enhanced frequency of iscs and ifn-γ producing cd + , cd + , cd + cd + t cells, γδ + t cells, and nk cells, and expanded frequency of apcs; and most importantly, ) complete clearance of detectable replicating challenged heterologous prrsv and tenfold reduction in viral rna load in the lungs. further, the microscopic lung lesions ( figure i ) strongly supported the observed virus clearance and immunological responses. in our previous study, in pigs immunized with the adjuvanted np-kag, increased frequency of only cd -cd + ifn-γ + cells in restimulated pbmcs was detected, but in stimulated lmncs, increased populations of both cd + cd -ifn-γ + and cd -cd + ifn-γ + cells was observed, perhaps indicating the induction of both t-helper and ctl memory responses in the lungs. like in pbmcs, increased but comparable frequency of other ifn-γ + lymphocyte subsets in both restimulated and unstimulated cells was observed in lmncs of group pigs, suggesting that other t-effector and nk cells were actively secreting ifn-γ in the lungs to prrsv-challenge infection. due to lack of similar data on ifn-γ + lymphocytes in vaccinated pigs isolated prior to challenge, it is difficult to demarcate the vaccine-alone induced response. np-based delivery of vaccine facilitates affinity maturation and activation of b cells, leading to high avidity antibody production and also cmi response. , avidity of prrsvspecific iga antibody isotype was significantly higher in the bal fluid of adjuvanted np-kag-vaccinated pigs. in the lung homogenates of low-dose (but not high-dose) adjuvanted np-kag-vaccinated pigs, high avidity iga response was observed; the reason for this discrepancy could be the time of lung sample collection. overall, our results suggested that virus-specific functional iga response at mucosal tissues (lungs) was enhanced in adjuvanted np-kag-vaccinated pigs. further, our results indicated that in the lung mucosal surfaces, iga isotype appears to play an important role, while in the lung parenchyma both iga and igg isotypes contribute to protective immune response. vn antibodies against putative neutralizing epitopes on prrsv gp and m glycoproteins play an important role in prrsv clearance. in group pigs, high levels of cross-reactive vn titers were detected against a challenged prrsv mn strain, another heterologous type ii viral strain, and most importantly-even against a highly variant heterogenotypic strain (prrsv sd - ), confirming the broadly cross-protective nature of adjuvanted np-kag vaccine formulation. further, prrsv antibody avidity results were positively correlated with vn titers, in agreement with a previous report. typically, killed vaccines elicit a predominantly th response, but np-based vaccines drive either a balanced or th -biased response, required for efficient clearance of virus. in group pigs, enhanced and balanced th -th humoral and cmi responses were detected. a robust cmi response is essential for complete protection against prrsv. a crucial th cytokine, ifn-γ, is produced by nk cells, γδ t cells, cd + and cd + t cells, and cd + cd + t cells. enhanced secretion of ifn-γ and the presence of increased frequencies of ifn-γ + memory cd + and cd + cells in the lungs of adjuvanted np-kag-vaccinated pigs have confirmed the additive effect of plga-mediated delivery and adjuvanticity of m. tb wcl. a possible mechanism of improved cmi response in group pigs was mediated by cross-presentation of plga-entrapped ags to cd + t cells through mhc class i molecules, of dendritic cells and macrophages. thus, our results were consistent with the previous reports on plga np-based vaccines, which elicited strong effector and memory cmi responses. , in addition, increased levels of another important th cytokine, il- , was detected in the lungs of adjuvanted np-kag-vaccinated pigs, associated with reduced production of immunosuppressive cytokines, il- and tgf-β, which play a vital role in prrsv pathogenesis. the γδ t cells are present in high frequency in pigs and are involved in both innate and adaptive immunity at mucosal tissues. enhanced frequency of activated γδ t cells in the lungs of group pigs was observed. significantly reduced production of il- in group pigs indicated the absence of inflammatory response in the lungs of group pigs. importance of immediate availability of unentrapped potent adjuvant to intranasally delivered np-kag vaccine was critical in our study, because inadequate immune response and incomplete viral clearance was observed in other vaccine formulations pigs received. plga-entrapped hepatitis b subunit vaccine coadministered with an encapsulated adjuvant failed to induce strong antibody response, consistent with our results observed in group pigs (np-kag + np-wcl). further, pigs vaccinated with both adjuvant and kag unentrapped formulation (group ) exhibited th -biased and weak ifn-γ response, associated with partial clearance of replicating prrsv. the presence of - fold fewer prrsv rna copies in the lungs of adjuvanted np-kag-vaccinated pigs and the absence of detectable replicating challenged virus is consistent with a previous report, wherein qpcr failed to differentiate infectious and noninfectious virus, and inactivated prrsv is relatively stable in the environment. further, in samples with low levels of prrsv rna copies, cell culture method has failed to detect the replicating virus. plga-based vaccine delivery is shown to dramatically reduce the required vaccine dose by up to times. plga-np-based vaccine delivery is getting global recognition for effective delivery of subunit/inactivated mucosal vaccines, because their size, contents, and cell targeting properties can be engineered. our study has demonstrated that plga-based adjuvanted np-kag vaccine induced strong anti-prrsv immunity both systemically and locally at the lungs. in conclusion, intranasal coadministration of plga-np-entrapped inactivated prrsv vaccine with a potent adjuvant has the potential to induce superior cross-protective international journal of nanomedicine : submit your manuscript | www.dovepress.com adjuvanted plga nanoparticle prrsv vaccine immunity in the pig lungs immunity in pigs. future studies should aim to fractionate m. tb wcl to identify adjuvant components to np-kag and to identify alternate adjuvants to reduce the cost of vaccine formulation, and perform field trials to validate efficacy of this innovative vaccine delivery approach. our study not only establishes the utility of nanotechnology-based vaccines in large animals, it also envisages its potential application against important human respiratory pathogens. prrs costs industry $ million annually porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology nanotechnology solutions for mucosal immunization role of nanotechnology in pharmaceutical product development vaccine manufacturing: challenges and solutions immunological responses of swine to porcine reproductive and respiratory syndrome virus infection polymeric nano-and microparticle technologies for oral gene delivery is intranasal vaccination a feasible solution for tuberculosis? nanotechnology for the biologist biodegradable nanoparticles for drug and gene delivery to cells and tissue mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymers adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) symposium on relationship of structure of microorganisms to their immunological properties. ii. host-reactive properties of cell walls and protoplasm from mycobacteria the influence of components of m. tuberculosis and other mycobacteria upon antibody production to ovalbumin biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection site of inhibitory action of isoniazid in the synthesis of mycolic acids in mycobacterium tuberculosis altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs measurement of immunoglobulin g, a and m concentrations in boar seminal plasma intranasal administration of cpg oligonucleotides induces mucosal and systemic type immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo antigen-specific b-cell responses to porcine reproductive and respiratory syndrome virus infection maximal adjuvant activity of nasally delivered il- α requires adjuvant-responsive cd c(+) cells and does not correlate with adjuvant-induced in vivo cytokine production the assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach protection of pigs against taenia solium cysticercosis using recombinant antigen or in combination with dna vaccine isolation and properties of a macromolecular, water-soluble, immuno-adjuvant fraction from the cell wall of mycobacterium smegmatis cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from ontario farms heterogeneity in nsp of european-like porcine reproductive and respiratory syndrome viruses isolated in the united states porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology mechanism of nkt cell activation by intranasal coadministration of alpha-galactosylceramide, which can induce cross-protection against influenza viruses combined nkt cell activation and influenza virus vaccination boosts memory ctl generation and protective immunity mucosal immune response to poliovirus vaccines in childhood mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups a and b and human parainfluenza virus type by using a live cdna-derived vaccine based on a host range-attenuated bovine parainfluenza virus type vector backbone nasal immunization of nonhuman primates with simian immunodeficiency virus p gag and cholera toxin adjuvant induces th /th help for virus-specific immune responses in reproductive tissues enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs cellular variables in bronchoalveolar lavage fluids (balf) in selected healthy pigs cd c+ antigen presenting cells from the alveolar space, lung parenchyma and spleen differ in their phenotype and capabilities to activate naïve and antigen-primed t cells polyclonal hypergammaglobulinemia and formation of hydrophobic immune complexes in porcine reproductive and respiratory syndrome virus-infected and uninfected pigs porcine reproductive and respiratory syndrome virus subverts repertoire development by proliferation of germline-encoded b cells of all isotypes bearing hydrophobic heavy chain cdr the assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies intranasal m cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength influence of the surfactant concentration on the body distribution of nanoparticles adjuvants and delivery systems in veterinary vaccinology: current state and future developments the adjuvant effects of mycobacterium tuberculosis heat shock protein result from the rapid and prolonged activation of antigen-specific cd + t cells in vivo pe_pgrs antigens of mycobacterium tuberculosis induce maturation and activation of human dendritic cells recent progress in mucosal vaccine development: potential and limitations intranasal delivery of an adjuvanted modified live porcine reproductive and respiratory syndrome virus vaccine reduces ros production evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions mycobacterium tuberculosis whole cell lysate enhances proliferation of cd positive lymphocytes and nitric oxide secretion in the lungs of live porcine respiratory and reproductive syndrome virus vaccinated pigs towards tailored vaccine delivery: needs, challenges and perspectives vaccine delivery: a matter of size, geometry, kinetics and molecular patterns role of neutralizing antibodies in prrsv protective immunity type /type immunity in infectious diseases international-journal-of-nanomedicine-journal the international journal of nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype plga nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses debugging how bacteria manipulate the immune response gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus enhancement of t helper type immune responses against hepatitis b virus core antigen by plga nanoparticle vaccine delivery porcine reproductive and respiratory syndrome virus (porcine arterivirus) porcine reproductive and respiratory syndrome virus: a persistent infection the authors report no conflicts of interest in this work. international journal of nanomedicine : submit your manuscript | www.dovepress.com key: cord- -wf vonkn authors: xiao, shuqi; chen, yaosheng; wang, liangliang; gao, jintao; mo, delin; he, zuyong; liu, xiaohong title: simultaneous detection and differentiation of highly virulent and classical chinese-type isolation of prrsv by real-time rt-pcr date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: wf vonkn porcine reproductive and respiratory syndrome (prrs) is a leading disease in pig industry worldwide and can result in serious economic losses each year. the prrs epidemic situation in china has been very complicated since the unprecedented large-scale highly pathogenic prrs (hp-prrs) outbreaks in . and now the hp-prrs virus (hp-prrsv) and classical north american type prrsv strains have coexisted in china. rapid differential detection of the two strains of prrsv is very important for effective prrs control. the real-time rt-pcr for simultaneous detection and differentiation of hp-prrsv and prrsv by using both sybr green and taqman probes was developed and validated. both assays can be used for rapid detection and strain-specific identification of hp-prrsv and prrsv. however, the taqman probe method had the highest detection rate whereas the conventional rt-pcr was the lowest. the real-time rt-pcr developed based on sybr green and taqman probe could be used for simultaneous detection and differentiation of hp-prrsv and prrsv in china, which will benefit much the prrs control and research. porcine reproductive and respiratory syndrome (prrs) is widely accepted as being one of the most economically important diseases affecting swine industry [ ] . in there was an unparalleled large-scale outbreak of the so-called high fever disease in most provinces of china that affected more than , , pigs, leading to concerns within the global swine industry and in relation to public health [ ] [ ] [ ] . in march the disease was identified in the hai duong province of vietnam and it spread countrywide affecting more than , pigs [ , ] . the outbreaks caused extensive concern worldwide [ ] . studies demonstrated that highly virulent porcine reproductive and respiratory syndrome virus (hp-prrsv) was the major causative pathogen of the socalled high fever disease [ ] . genetic analysis indicated that the hp-prrsvs isolated from china and vietnam shared a discontinuous deletion of aa in nonstructural protein (nsp ), as compared with the north american type prrsv strains (na prrsv) [ , , ] . since , the hp-prrsv and classical north american type prrsv strains coexist in china. now prrs epidemic situation is very complicated in china, of which the predominant form is the hp-prrsv. rapid differential detection of the two strains of prrsv is very important for effective prrs control. therefore, it is imperative to develop an assay for simultaneous detection and strain identification of hp-prrsv and prrsv. the current immunoassay, such as immunohistochemistry and serological methods, cannot differentiate between the two strains of prrsv. conventional rt-pcr is timeconsuming, lowly sensitive, and also prone to contamination. the development of real-time rt-pcr technology offers the opportunity for more rapid, sensitive, and specific detection of virus. the current two major genotypes, the european (eu) and the north american (us) strains, have been rapidly identified by sybr green-based or taqman probe-based real-time rt-pcr assay [ ] [ ] [ ] . a specific taqman probe realtime rt-pcr has been developed for assaying the hp-prrsv journal of immunology research [ ] , but it is not able to differentially detect the hp-prrsv and prrsv. in this research, the real-time rt-pcr for simultaneous detection and differentiation of hp-prrsv and prrsv by using both sybr green and taqman probe was developed and validated. these two methods provided alternative diagnostic assays in diverse prrsv epidemiological circumstances. samples. hp-prrsv (gd and xh) and prrsv (ch- a) virus strains were kindly supplied by dr. guihong zhang (south china agricultural university, china). prrsv (cc), prv, fpv, and fcv were kindly provided by laboratory animal center in jilin university, china. and serum samples were obtained from pig farms in south china in and , respectively. sera as described previously were from pigs experimentally infected with hp-prrsv and prrsv [ ] . the viral rna of the virus-infected cell culture and serum was extracted by using qiaamp viral rna mini kit according to the manufacturer's instruction (qiagen). first-strand cdna was synthesized using the extracted total rna and amv reverse transcriptase from reverse transcription system of promega according to the manufacturer's instruction (promega). the difference of genome sequence between the hp-prrsv and prrsv was the base deletion in the fixed site in nsp gene [ , ] . after aligning hp-prrsv and prrsv strains isolated from china and the us strain (vr- ) sequences obtained from the ncbi database, the nsp region was selected to design an assay for discriminating between hp-prrsv and prrsv strains. the differential detection based on real-time rt-pcr using sybr green i and taqman probes was performed employing the same primer pair (table ). real-time rt-pcr for prrsv detection based on dual-colour taqman probes was performed using strain-specific probes including a pb-h (only detecting hp-prrsv strain) [ ] , pb-n (only detecting prrsv strain), and pb-all (simultaneously detecting both hp-prrsv and prrsv strains) ( table ) . sybr green i real-time pcr was carried out using sybr premix ex taq (takara) and the lightcycler real-time pcr system (roche applied science). amplification was performed in a l reaction mixture containing . l sybr premix ex taq ( ×), . l of each forward (nsp -qf) and reverse (nsp -qr) primer ( m), . l cdna or plasmid dna, and . l h o. the amplification conditions were ∘ c for s, followed by cycles of ∘ c for s and ∘ c for s. fluorescent signal was detected for each cycle at the end of the ∘ c extension step. for each assay, a standard curve was generated with -fold serially diluted plasmid standards of - copies/ l. meanwhile positive and negative reference samples were detected along with unknown samples. after amplification cycles, melting curve analysis was carried out with the conditions of ∘ c for s and ∘ c for s and then increased to ∘ c while continuously collecting the fluorescent signal. the melting temperature (tm) of each strain was analyzed to verify the prrsv type. the l duplex taqman probe real-time pcr reaction mixtures contained . l premix ex taq ( ×) (takara), . l of each forward (nsp -qf) and reverse (nsp -qr) primer ( m), . l of each probe (pb-h and pb-n or pb-all and pb-n, m), . l cdna or plasmid dna, and . l h o. the amplification conditions were ∘ c for s, followed by cycles of ∘ c for s and ∘ c for s. for each assay, a standard curve was generated with fold serially diluted plasmid standards of - copies/ l. the fam ( -carboxyfluorescein) and hex (hexachloro- carboxyfluorescein) signals were detected for each cycle at the end of the ∘ c extension step. plasmid dna. the conventional rt-pcr was performed by using the nsp -f and nsp -r primers described in table . l reaction mixture contains . l cdna, . l × pcr reaction mix, . l nsp -f ( m) primer, . l nsp -r ( m) primer, . l taq dna polymerase ( . u/ l), and . l h o. the negative controls included the reagents without cdna template. the reaction mixtures were performed at the amplification condition: ∘ c for min, followed by cycles of ∘ c for s, ∘ c for s, and ∘ c for min, and a final extension step of min at ∘ c. the pcr products were detected by . % agarose gel electrophoresis in × tae. then the pcr products were cloned into the plasmid pmd -t (takara) and propagated in competent escherichia coli dh cells according to the manufacturer's instructions. plasmid dna was purified using the e.z.n.a. plasmid mini kit i (omega) and quantified by measuring od using spectrophotometer nd- (wilmington, usa). . . sybr green i real-time pcr. -fold serial plasmid dilutions were tested and used to construct the standard curve. the generated standard curve covered a linear range of . × to . × copies/ l for hp-prrsv and . × to . × copies/ l for prrsv. both standard curves had a slope of − . to − . and an efficiency of . to . , which indicate a high pcr efficiency of the experiment (figures (a) and (b) ). the amplification with primers nsp -qf and nsp -qr yielded bp and bp amplified product within nsp of both hp-prrsv (gd) and prrsv (ch- a), respectively (figure ) , which was sufficient to discriminate between melting peaks of the two prrsv strains. the mean and standard deviation of tm of hp-prrsv and prrsv were . ± . ∘ c and . ± . ∘ c, respectively (figure (b) ). gd hp-prrsv strain, and only the hex fluorescent signal could be observed in the template of ch- a prrsv strain ( figure ) . however, when pb-n (hex) and pb-all (fam) were combined in a duplex real-time pcr system, only hex fluorescent signal could be observed when the template was ch- a prrsv strain, and fam fluorescent signal could be observed when the templates were gd and ch- a strains (figure ). specificity of realtime pcr using sybr green i and taqman probe was determined by analyzing nucleic acid extracts of other viruses (prv, fpv, and fcv), host cells (marc , pk ), and h o. the results of the specificity test of the two methods showed that there were no cross-amplifications from other viruses or host cells (figures (a) and ) , which confirmed that the primers and probes used in this study were highly specific for both hp-prrsv and prrsv. -fold serially diluted plasmid standards of hp-prrsv (pmd -gd) and prrsv (pmd -ch a) were used as templates for sensitivity tests in both conventional pcr and real-time pcr using sybr green i and taqman probe. the results showed that real-time pcr using both sybr green i ( figure ) and taqman probe ( figure ) can be used to detect concentrations at least copies/ l of plasmid standards whereas the sensitivity of conventional pcr was only copies/ l. the intra-and interassay reproducibility were evaluated using three replicates of , , and copies/ l plasmid standards of both pmd -gd and pmd -ch a. mean and coefficient of variation (cv) for the value were calculated. the results showed that neither the cvs of intra-assay nor the cvs of interassay were more than % (table ) , indicating the reproducibility of the two assays. our results showed that real-time pcr using both sybr green i and taqman probe could be used to simultaneously detect and differentiate hp-prrsv and prrsv in china. but the taqman probe method had the highest detection rate, whereas the conventional rt-pcr was the lowest. the sybr green i real-time pcr assay is timesaving, easy to handle, and highly sensitive. yang et al. detected the prrsv and csfv rna by sybr green i-based quantitative pcr and found that both sensitivity and specificity were equal or superior to conventional rt-pcr [ ] . although tian et al. developed a rapid sybr one step real-time rt-pcr for detection of prrsv [ ] , it could not be used for simultaneous detection and differentiation of hp-prrsv and classical north american type prrsv (prrsv). kleiboeker et al. developed dual labeled probes quantitative pcr, which could simultaneously detect na-and eu-prrsv [ ] . however, this assay could not simultaneously detect and differentiate between both hp-prrsv and classical north american type prrsv (prrsv) strains in china. the taqman probe method provided more accurate results than sybr green i with melting curve analysis. sybr green i real-time pcr assay was simpler, rapider, and lower in cost than taqman probe method. in addition to the high specificity, sensitivity, and reproducibility, the realtime pcr assay based on both sybr green i and taqman probe established by us could recognize coinfection of hp-prrsv and prrsv. because the two types of prrsv isolates coexist in chinese swine herds, recombination could occur. therefore, the results provided alternative diagnostic assays in diverse prrsv epidemiological circumstances. to compare and evaluate the developed real-time rt-pcr and conventional rt-pcr, reference strains of h-prrsv (gd and xh) and n-prrsv (ch- a and cc) and , , and serum samples were tested. the results were shown in table figure : specific amplification curves by duplex taqman probe real-time pcr. when pb-h (fam) and pb-n (hex) probes were combined in a duplex real-time pcr system, only the fam fluorescent signal could be observed when the template was gd hp-prrsv strain, no fam signal was detected when the templates were ch- a prrsv strain and other viruses (a), and vice versa, only the hex signal could be collected when the template was ch- a prrsv strain (b). when pb-n (hex) and pb-all (fam) were combined in a duplex real-time pcr system, the pb-n (hex signal) probe could only detect prrsv strain (c), whereas pb-all (fam signal) probe could detect both hp-prrsv and prrsv strains (d). serum samples showed that the taqman probe real-time pcr had the highest detection rate, whereas the conventional rt-pcr had the lowest detection rate. to evaluate comprehensively the practicality of this assay, clinical samples that span a broader geographical origin should be tested in the future. the real-time rt-pcr for simultaneous detection and differentiation of hp-prrsv and prrsv by using both sybr green and taqman probes was developed and validated. both assays can be used for rapid detection and strainspecific identification of hp-prrsv and prrsv. a total of samples were tested by real-time pcr and conventional rt-pcr. the results of reference strains for real-time pcr assays were consistent with that of conventional pcr method. the results of serum samples showed that the taqman probe method had the highest detection rate whereas the conventional rt-pcr was the lowest. the real-time pcr developed based on sybr green and taqman probe could be used for simultaneous detection and differentiation of hp-prrsv and prrsv in china, which provided two alternative diagnostic assays in diverse prrsv epidemiological circumstances. understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china molecular dissection of the unique hallmark emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china highly pathogenic porcine reproductive and respiratory syndrome, china porcine respiratory and reproductive syndrome virus variants, vietnam and china avian influenza virus, streptococcus suis serotype , severe acute respiratory syndrome-coronavirus and beyond: molecular epidemiology, ecology and the situation in china china, vietnam grapple with âĂIJrapidly evolvingâĂİ pig virus highly virulent porcine reproductive and respiratory syndrome virus emerged in china rapid detection and strain identification of porcine reproductive and respiratory syndrome virus (prrsv) by real-time rt-pcr simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (prrsv) by real-time rt-pcr and amplicon melting curve analysis using sybr green detection of u.s., lelystad, and european-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time pcr rapid detection of a highly virulent chinese-type isolate of porcine reproductive and respiratory syndrome virus by real-time reverse transcriptase pcr proteome changes of lungs artificially infected with h-prrsv and n-prrsv by twodimensional fluorescence difference gel electrophoresis first results of detection of prrsv and csfv rna by sybr green i-based quantitative pcr the development of a rapid sybr one step real-time rt-pcr for detection of porcine reproductive and respiratory syndrome virus simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- -wu tuvy authors: katz, jonathan b.; shafer, amy l.; eernisse, kenneth a.; landgraf, john g.; nelson, eric a. title: antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) are encoded by the carboxyterminal portion of viral open reading frame date: - - journal: vet microbiol doi: . / - ( ) -b sha: doc_id: cord_uid: wu tuvy antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) were revealed by serologic analysis of a recombinant protein derived from prrsv open reading frame (orf ). the hydrophilic carboxyterminal amino acids encoded by the orf of a european (lelystad) isolate of prrsv were expressed as a recombinant fusion protein (bp -p) in a baculovirus gene expression system. sera from gnotobiotic swine exposed to prototypic reference european and american isolates of prrsv and sera from conventionally reared european and american swine convalescing from naturally acquired prrsv infections were used to characterize the bp -p protein. sera from gnotobiotic and conventionally reared swine exposed to european isolates of prrsv were significantly more reactive (p < . ) with bp -p than were the corresponding american prrsv antisera using the indirect immunoperoxidase monolayer assay (ipma). prototypic european, but not american, prrsv antisera also recognized bp -p using western immunoblotting and radioimmunoprecipitation assay (ripa) procedures. however, gnotobiotically derived antiserum to an atypical american-origin prrsv was reactive with bp -p by both ipma and western immunoblot. despite a predicted potential for n-linked glycosylation, studies with tunicamycin and peptide-n-glycosidase f (pngase f) indicated that bp -p was not n-glycosylated in either insect cell cultures or trichoplusia ni larvae infected with the recombinant baculovirus. sera from rabbits inoculated with bp -p failed to neutralize both the european (lelystad) and american (atcc vr- ) reference isolates of prrsv and did not react by ipma with prrsv-infected cell cultures. taken together, the data suggest that the carboxyterminal portion of prrsv orf encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between european and most american isolates of prrsv. f ( pngase f) indicated that bpo -p was not n-glycosylated in either insect cell cultures or trichoplusia ni larvae infected with the recombinant baculovirus. sera from rabbits inoculated with bpo -p failed to neutralize both the european (lelystad) and american ( atcc vr- ) reference isolates of prrsv and did not react by ipma with prrsv-infected cell cultures. taken together, the data suggest that the carbox- in the few years since it was initially described (keffaber, ; lindhaus and lindhaus, ; paton et al., ) , the disease complex now known as the porcine reproductive and respiratory syndrome (prrs) has become an economically significant swine health problem throughout europe and north america (goyal, ) . the viral etiology of prrs was confirmed both in the netherlands (meulenberg et al., ; terpstra et al., ) and in the united states with the isolation of two previously unknown prrs viruses (prrsv), a prototypic european prrsv (lelystad isolate) and a prototypic american prrsv (vr- isolate). these to nm enveloped rna viruses wensvoort et al., ) share common antigenic determinants but may be differentiated using both polyclonal antisera and monoclonal antibodies (nelson et al., ) . serologic analysis of european and american swine naturally exposed to prrsv has confirmed that geographically based antigenic differences do exist between most north american and most european isolates of prrsv; these differences are consistent with those first noted between the lelystad and the vr- isolates bautista et al., ) . there is some evidence, however, that antigenically intermediate prrsv or possibly both prototypic antigenic types may exist within the north american swine population (bautista et al., ) . prrsv has been tentatively classified as an arterivirus, along with equine arteritis virus (eav) and two other proposed members of that genus: lactate dehydrogenase elevating virus (ldv) and simian hemorrhagic fever virus (shfv) (plagemann and moennig, ) . prrsv contains a to kb single strand positive polarity polyadenylated genome (plagemann and moennig, ) and utilizes a nested ' coterminal mr.na gene expression strategy (meulenberg et al., ; conzelmann et al., ) . the prrsv genome contains open reading frames (orfs), including the large ' terminal orfs la and lb encoding the putative viral polymerase and the ' terminal orf encoding the nucleocapsid protein (meulenberg et al., ; conzelmann et al., ) . prrsv virions are believed to contain two other structural, presumably envelope, proteins (nelson et al., ) . it has not been determined which of the orfs through encode these proteins or the identities and functions of the other proteins encoded by these orfs. recombinant dna technology permits the expression of individual gene segments in order to better understand the antigenicity of their protein products and their roles in the host-virus relationship. we report here the expression and serologic analysis of a markedly hydrophilic portion of prrsv orf (lelystad isolate). this segment was of additional interest because comparison of ldv, eav, and prrsv orfs through revealed that the orf protein products of these viruses would be the most divergent in size but potentially the most universally highly n-glycosylated of all the proteins encoded by those orfs (den boon et al., ; conzelmann et al., ; godeny et al., ) . these features suggested to us that prrsv orf might encode a glycoprotein mediating virus-host cell interactions; therefore, it might be a target of host defense mechanisms subjected to selective evolutionary pressure. we hypothesized that it might account in part for the geographically oriented antigenic differences between the majority of european and north american prrs viruses, and that was the basis for this study. the lelystad and vr- isolates of prrsv were provided by c. terpstra, central diergeneeskundic institut, the netherlands, and by d. chladek, boehringer ingelheim inc., usa, respectively. a british reference prrsv isolate, humberside , was a gift from s. edwards, central veterinary laboratory, united kingdom. prrsv isolates and were recovered in this laboratory from midwestem american swine tissues. antisera to the isolate had been found uniformly cross-reactive with numerous north american prrs virus isolates; isolate was of interest because it was completely fastidious for replication in swine pulmonary alveolar macrophages. all viruses except were propagated in cultures of marc- cells, which are a prrsv permissive cell line subcloned from ma- cells (kim et al., ) . the identity of all viruses was confirmed using prrsv-specific monoclonal antibodies in an indirect immunoperoxidase monolayer assay (ipma) format. gnotobiotic swine were used to prepare antisera to each prrsv isolate. each lo-dayold piglet was intranasally inoculated with lo to lo tcid,, of one virus isolate, and the resultant convalescent antiserum was collected between to days post-inoculation. twenty field origin sera derived from clinical cases of prrs in swine from northern germany were generously provided by t. blaha, tierartzliche hochschule, hanover, germany. fifteen american field origin prrsv seropositive swine sera were also obtained from d. kinker, iowa state university, ames, ia, usa. each serum originated from a separate swine herd experiencing prrs, and these sera represented samples from herds located in states in the eastern, southern, and midwestem united states. the ipma ( holm-jensen, ) was used to evaluate serial -fold serum dilutions for reactivity against both the lelystad and the prrsv isolates. these viruses had been inoculated h previously onto separate marc- cell monolayers. immunodetection was accomplished using protein g-horseradish peroxidase (hrp) conjugate and aminoethylcarbazole substrate (zymed, so. san francisco, ca, usa). a prrsv serum neutralization (sn) procedure was used to evaluate selected sera as recently described (yoon et al., ) . recombinant orf antigen, cesium chloride purified prssv (nelson et al., ) , and ultrafiltered concentrates of cell cultures infected with the lelystad isolate were used as antigens in western immunoblotting following denaturing polyacrylamide gel electrophoresis in % to % or % to % gradient gels (laemmli, ; burnett, ) . immunodetection was again accomplished with protein g-hrp conjugate and an insoluble tetramethylbenzidine (tmb) substrate (kirkegaard and perry inc., gaithersburg, md, usa). a radioimmunoprecipitation assay (ripa) recently described for prrsv (nelson et al., ) was used with the same antigens to evaluate sera from the gnotobiotic swine inoculated with prrsv isolates. a hydrophobicity/hydrophilicity plot (kyte and doolittle, ) of the putative orp protein revealed a markedly hydrophobic amino terminus followed by the remaining predominantly hydrophilic % ( of amino acids) of the protein (fig. ) . we postulated that this latter portion might be externalized and, therefore, available to interact with host cell receptors or antibodies and that the amino terminus perhaps represented a biosynthetically cleaved leader peptide or otherwise cryptic segment not directly interactive with the host. to focus our examination on the hydrophilic body of the protein, we excluded the hydrophobic segment through a subunit cloning strategy. total rna was extracted from a cm* cell monolayer infected with the lelystad isolate of prrsv. a -mer oligonucleotide complementary to the viral rna sequence (meulenberg et al., ) nucleotides downstream from the ore termination codon (positions , to , inclusive) was used to prime viral cdna synthesis in a ~ reaction (maniatis et al., ) . a base pair (bp) cdna segment was then amplified through cycles of the polymerase chain reaction (pcr) (saiki et al., ) using a ' primer located between positions , to , and a ' primer ( , to , ) to which a pst i recognition sequence had been appended (fig. ) . following xho i and pst i digestion and gel purification, the pcr product was directionally ligated into a baculovirus polyhedrin gene transfer plasmid, pacsg-his-nt (pharmingen, inc., san diego, ca, usa). the resulting plasmid contained the carboxyterminal amino acid-encoding portion of orf fused in frame to a ' vector sequence encoding amino acids including a polyhistidine domain. the resulting fusion polypeptide had a calculated mass of , daltons ( kda) . following dna sequence verification, recombinant plasmid dna ( ug) was cotransfected with bsu -digested acnpv baculoviral dna ( . ug) (pharmingen, inc.) into a spodopteru frugiperda sf- cell culture using a liposomal transfection reagent (dotap, boehringer marmheim, indianapolis, in, usa). clonally purified baculoviruses expressing prrs virus-immunoreactive material were identified by ipma. separate pcrs of cell culture fluids from monolayers infected by these cloned viruses were used to confirm the presence of the orf gene sequence and the absence of the nonrecombinant baculovirus polyhedrin gene sequence. the latter pcr employed primers spanning the polyhedrin gene insertion site (invitrogen, inc., san diego, ca, usa). one rdna baculovirus, bp , was used for recombinant protein production. recombinant protein (bpo -p) was produced by infecting both sf- cell cultures and -day-old trichoplusia ni larvae according to standard procedures ( o'reilly et al., ) . ] asparagine amidase (boehringer mannheim, inc.) in a further effort to evaluate the n-glycosylation status of the protein. the polyhistidine sequence within bp -p enabled efficient affinity purification of both cell culture and larval origin bpo -p using immobilized nickel ion chromatography as previously described (janknecht et al., ) . purified larval and cell culture origin bpo -p was adjuvanted (fatunmbi et al., ) with avridine (pfizer, inc., groton, ct, usa) and used to immunize four rabbits each on four biweekly occasions. sera from these animals were then evaluated by ipma, sn, western blotting, and ripa for anti-prrsv reactivity. bf'o -p expression was detected in the cytoplasm of infected sf- cells using gnotobiotically-derived lelystad isolate antiserum in an ipma format (fig. ) . bpo -p expression was noted within h post-infection (hpi) and persisted at least to hpi. comparison of bp infected and uninfected cell culture proteins by sds-page revealed the presence of a new protein in the infected cell cultures (fig. a) . the size of this protein was consistent with the kda mass predicted from the amino acid sequence of the fusion protein encoded by bp . the prrsv-specific identity of this protein was confirmed by western immunoblotting (fig. c ). bpo -infected insect larvae contained a similarly sized immunoreactive protein within hpi (figs. b, c). protracted incubation of affinity purified larval bp -p with pngase f did not result in a detectable reduction in apparent bpo -p molecular mass (fig. c ). there was also no detectable decrease in the molecular mass of bp -p from tunicamycin treated cell cultures relative to untreated controls (fig. c ). the ipma, sn, ripa, and western immunoblot procedures were used to evaluate rabbit antisera developed against both larval and cell culture origin bpo -p. none of these sera exhibited respectively. lane : molecular mass markers (kda) panel c: immunoblot of affinity purified larval bpo -p protein with and without prior digestion with pngase f (lanes and , respectively). immunoblot of sf- cell culture origin bpo -p protein produced in the presence or absence of tunicamycin (lanes and , respectively). lane : cell culture control infected with recombinant baculovirus containing vector-only transfer plasmid sequences. prrsv sn activity at a : dilution. all sera were ipma-reactive at : dilution using bp -infected sf- cells but failed to react at a : dilution with isolate-infected marc- cells. these sera also did not react at the : dilution with lelystad isolateinfected cell monolayers fixed between and hpi. two of the rabbit antipeptide sera were reproducibly reactive by western immunoblot with a diffuse ( to kda) band of antigen found in homogenates of marc- cells infected h previously with the lelystad isolate (fig. ) . repeated ripa and western blot attempts using these sera were unsuccessful in identifying a virus-specific protein in purified virion preparations. antisera to gnotobiotic lelystad, humberside , and virus isolates reacted specifically at : dilutions with nitrocellulose blotted bpo -p, while antisera to vr- and virus isolates were totally nonreactive at the same or lower dilutions (fig. b) . ripa results were consistent with these findings. these sera were also evaluated by ipma against lelystad and infected marc- cells and bpo -infected sf- cells (table ) . antisera to the lelystad and humberside isolates were differentially reactive with cells fig. . western immunoblot analysis of sera from two rabbits inoculated with bpo -p. lane : molecular mass markers &da). lanes and : blots of rabbit anti-(bp -p) sera, showing reactivity against to kda proteins found in cells infected with prrsv (lelystad isolate) hours previously. lane : blot of rabbit serum (same as used in lane ) against uninfected cell antigens. lanes and : blots of hyperimmune anti-bpgf-p rabbit serum and gnotobiotic anti-prrsv (lelystad) swine serum, respectively, against the bpo -p immunogen. hyperimmune rabbit serum also detects small ( - kda) breakdown products of bp -p resulting from its catabolism in infected insect cells. 'german sera significantly more reactive (p < . ) with lelystad and bp viral antigens than the corresponding american sera and significantly (p<o.ol) less reactive with antigen than with the other viral antigens. damerican sera significantly more reactive (p < . ) with viral antigen than the corresponding german sera and significantly (p < . ) more reactive with antigen than with the lelystad and bp viral antigens. knot determined. infected with the lelystad isolate and also recognized bpo -p in infected sf- cells. antisera to the vr- and isolates were differentially reactive with isolate infected cells. antiserum to the isolate was nearly equally reactive with cell cultures infected with either of the prrsv and with the bp virus (table ) . convalescent sera from field origin prrsv-infected german and american swine were differentially and antithetically reactive by ipma. a two-way analysis of variance was used to assess the serologic differences. the geometric mean titer (gmt) of the german sera was significantly greater (p < . ) against lelystad and bp viral antigens than against the viral antigen. conversely, american sera were significantly (p < . ) more reactive with viral antigens than either of the other two antigens (table ) . taken together, the data demonstrate that immunoreactive epitopes within the carboxyterminal portion of the protein encoded by lelystad prrsv orf are expressed during the course of european prrsv infections, and that this polypeptide sequence is at least partially responsible for the serologic differences observed between european and american prrsv isolates bautista et al., ) . antiserum to the macrophage-fastidious and antigenically intermediate isolate was reactive with bpo -p, further suggesting the importance of the orf protein in the serologic differentiation of prrsv isolates. the similarity between the ripa and western immunoblot reactivity patterns of bp -p using european and american prssv antigens and gnotobiotically derived convalescent swine antisera suggests that perhaps both linear and conformational orf epitopes may be involved in the antigenic differentiation of european and american prrs viruses. our data support the hypothesis (bautista et al., ) that coexisting high lelystad and atcc vr- prrs virus antibody responses found in some american swine may result from infections with a virus of mixed antigenicity as opposed to dual infections with prototypic american and european-like prrsv isolates. like the homologous predicted ldv and eav orf proteins, the prrsv orf protein contains the greatest number of sites for potential n-glycosylation of all the proteins encoded by orfs through (den boon et al., ; conzelmann et al., ; godeny et al., ) . n-linked glycosidic moieties have frequently been found important in mediating viral infectivity, in directing soluble and cell-associated host immune responses, in protecting critical viral protein epitopes from immune attack, and in interacting with viral polypeptide components to ensure correct viral glycoprotein conformational structure (rademacher et al., ; li et al., ; grigera et al., ) . attempts to produce observably deglycosylated and nonglycosylated forms of bpo -p using both tunicamycin inhibition and enzymatic deglycosylation approaches indicated that the recombinant protein did not contain n-linked glycosidic modifications. these findings must be interpreted cautiously. despite the theoretical potential for a high degree of n-linked glycosylation, the carboxyterminal portion of the native orf protein may in fact not be glycosylated. perhaps more likely however, the aminotetminal peptide sequence omitted during bp construction may have represented a signal sequence required to direct nascent orf into the er-golgi complex for glycosylation during insect larval and sf- cell infections. although insect cells and tissues are generally assumed to be glycosylation-competent, differences between mammalian and insect glycosylation patterns have been observed (rademacher et al., ; shimokawa and smith, ; o'reilly et al., ) and could also account for the data. the addition or removal of very small n-linked glycosides to the bp -p protein might have been undetectable given the molecular size resolution of the western immunoblotting, but this is unlikely because viral n-linked glycosidic moieties are usually substantial in apparent molecular mass (rademacher et al., ) . regardless of the foregoing considerations, the orp of the lelystad isolate clearly encodes epitopes recognized immunologically by the swine host during the course of both experimental and natural infections. monospecific bpo -p rabbit antipeptide antisera reacted by immunoblot but not by ripa with a diffuse band of antigen ( - kda) found repeatedly in virus-infected cell cultures. this material was specific to virus infected cells, but was apparently not of virus structural protein origin, because it was not detected by either ripa or immunoblot using purified virus preparations. the difference between the ripa and the western immunoblot performance of the rabbit antipeptide antisera using unpurified infected cell culture material may be due to the presentation of the relevant reactive linearized nonstructural virus protein epitopes under the conditions of the denaturing immunoblot but not under the milder conditions of the ripa. monospecific bp -p antisera were not neutralizing, but the functional significance of this finding is unclear because other workers have suggested that antibody responses to prrsv may not necessarily be protective and might even participate in antibody-dependent enhancement of infection (choi et al., ) . the failure of monospecific rabbit bpo -p anti-peptide antisera to react with prrsv-infected cells is consistent with the ideas that the natural orf translation product may be conformationally (epitopically ) different from the recombinant bp -p fusion protein, or that the native protein may be a nonstructural, perhaps soluble, virus gene product. lactate dehydrogenase elevating virus, eav, shiv, and apparently prrsv all possess three structural proteins including a nucleocapsid protein encoded by the ' terminal orp (nelson et al., ) . the other two presumed prrsv structural proteins are kda and kda in apparent molecular mass (nelson et al., ) and are thus far removed from the - kda band identified here. although the precise identity and functional role of the prrsv orf gene product thus remain enigmatic, a differential virus serotype-specific serologic response is clearly directed against it by swine exposed to prrsv. serologic survey for lelystad and vr- strains of porcine reproductive and respiratory syndrome (prrs) virus in u.s. swine herds characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) electrophoretic transfer of protein from sds-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein a antibody-dependent enhancement of sirs virus replication isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome (prrs) virus, a member of the arterivirus group enhancement of antibody response of turkeys to trivalent avian influenza vaccine by positively charged liposomal avridine adjuvant complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase elevating virus ( ldv) virology porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus i. detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. a comparative examination using cf, pla, and npla assays rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome ( prrs) virus in a homogenous subpopulation of ma- cell line a simple method for displaying the hydropathic character of a protein cleavage of structural proteins during assembly of the head of bacteriophage t glycosylation is necessary for the correct folding of human immunodeficiency virus gp in cd binding ratselhafte schweinkrankenheit molecular cloning: a laboratory manual differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome (pius) virus by monoclonal antibodies baculovirus expression vectors: a laboratory manual lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses primer-directed enzymatic amplification of dna with a thermostable dna polymerase expression of prostaglandin endoperoxidase synthase- in a baculovirus system experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus a modified semm neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome (prrs) virus in swine sera we thank tom glasson, gene hedberg, wayne romp, melanie harris, and nancy platter for illustrative services and careful manuscript preparation. key: cord- -tzmettky authors: lee, jung-ah; lee, nak-hyung; lee, sang-won; park, seung-yong; song, chang-seon; choi, in-soo; lee, joong-bok title: development of a chimeric strain of porcine reproductive and respiratory syndrome virus with an infectious clone and a korean dominant field strain date: - - journal: j microbiol doi: . /s - - - sha: doc_id: cord_uid: tzmettky the k chimeric virus of porcine reproductive and respiratory syndrome virus (prrsv) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of prrsv, fl , with the same region obtained from a korean dominant field strain, lmy. the k reached ( ) tcid( )/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. the chimeric clone pk can be used as a practical tool for further studying the molecular characteristics of prrsv proteins through genetic manipulation. furthermore, successful construction of the k will allow for the development of customized vaccine candidates against prrsv, which has evolved rapidly in korea. porcine reproductive and respiratory syndrome virus (prrsv), which causes a reproductive and respiratory disease in pigs, contains a positive-sense single-strand rna genome measuring approximately . kb. the virus is classified into two genotypes, a north american (na) type and a european (eu) type. the genome of prrsv contains nine open reading frames (orfs). orf a and orf b occupy approximately % of the viral genome and encode nonstructural proteins (nsps) (meulenberg et al., ) . the remaining orfs encode glycosylated structure proteins, including the glycoprotein (gp) , gp , gp , and gp , unglycosylated membrane (m) protein and nucleocapsid (n) protein (meulenberg, ) . previous studies indicated that b-cell epitopes for viral neutralization appeared to reside on structure proteins, including gp , gp , gp , and m (van nieuwstadt et al., ; yang et al., ; cancel-tirado et al., ; ansari et al., ; plagemann, ) . prrs is characterized by reproductive failure, including aborted, stillborn, mummified and weak-born piglets, and respiratory disorder, which can result in high death rates in suckling and weaned pigs. prrs is responsible for significant economic losses for the swine industry worldwide (neumann et al., ) . control methods including management of incoming replacement gilts, implementation of biosecurity protocols and vaccination have been applied to reduce the risk of prrsv outbreak. among them, vaccination has been considered as the most effective tool for preventing the disease (thanawongnuwech and suradhat, ) . however, current commercial prrsv vaccines have the drawback of featuring a high level of antigenic variation between the vaccine and field strains of prrsv (meng, ) . heterologous strains of prrsv compared to a vaccine strain possessing antigenic diversity resulting from amino acid sequence variation in gp have been constantly isolated and reported from korea (cha et al., ; yoon et al., ; kim et al., ; choi et al., ) . generally, field strains, which are the most frequently isolated from regional areas, have been used to develop vaccines in each country that has had an endemic prrsv outbreak. reverse genetics (rg) technology is a practical system to study virus characteristics, in vivo pathogenesis and viral protein function. the manipulation of viral rna genomes is generally difficult due to the instability of rna genomes and the lack of research tools for direct rna editing. however, with rg technology, the modification of infectious rna viruses can be easily achieved. in previous studies, rg technology has been applied to the genetic modification of genomes of positive-sense and negative-sense rna viruses and to rescue mutant viruses from cdna infectious clones (taniguchi et al., ; racaniello and baltimore, ; castrucci and kawaoka, ) . rg technology has widespread implications in the fields of virology and vaccinology (ito et al., ; collins and murphy, ; almazan et al., ) . using rg technology, a highly pathogenic strain of a virus can be attenuated by the construction of a chimeric virus with highly and weakly pathogenic viruses. in addition, a chimera virus can serve as a viral vector expressing heterologous antigen. the infectious clone of prrsv has been developed and used in previous studies (nielsen et al., ; truong et al., ; yoo et al., ; lee et al., ; fang et al., ) . in previous studies, diverse chimera viruses of prrsv have been generated using rg technology to manipulate viral genomes to determine the viral protein involved in the pathogenicity of field strains in pigs (kwon et al., ; zhou et al., ; ni et al., ) . however, rg technology has been used mainly to characterize the function of viral proteins but has not been used to develop recombinant vaccine candidates against prrsv. in this study, a chimeric virus was constructed using an infectious clone of prrsv containing genomes of the fl strain and a korean dominant field strain, lmy. the chimeric virus, named k , contains nsps from the fl strain and structure proteins from the lmy strain. marc- cells were used to rescue virus and to determine viral growth kinetics. the prrsv field isolate, lmy, was isolated at the animal, plant and fisheries quarantine and inspection agency (korea) from a case of prrsv infection associated with clinical disease. virus titers were calculated and expressed as tissue culture infectious doses (tcid )/ml. the prrsv infectious cdna clone, pfl , was generously provided by dr. fernando osorio and dr. asit pattnaik of the university of nebraska-lincoln (truong et al., ) . a genomic region including whole-structure genes from the lmy strain of prrsv was amplified using reverse transcription-polymerase chain reaction (rt-pcr) with a primer pair of -gtggatgctttcacggagttc- and -cacacttaattaacgtttttttttttttttttttt tttttttttttttttttttttttaatttcggccgtgt ggttc- . the genomic region containing the majority of orf and all of the other structure protein genes of the pfl plasmid was replaced with the amplified structure protein genes of the lmy strain to generate a chimeric clone pk using restriction enzymes ecorv and paci (neb, usa) (fig. a) . virus recovery was performed as previously described with a slight modification (truong et al., ) . marc- cells were resuspended in phosphate buffered saline (pbs) and transfected by gene pulser xcell (bio-rad, usa) at v and uf in a . -mm cuvette with generated rna transcripts and placed in a six-well plate with culture media. two days after transfection of marc- cells with in vitro transcribed rna, cells were examined using immunofluorescence assay (ifa) to detect the expression of the n protein of prrsv. growth kinetics of the chimeric virus and parental strains were performed by infection of marc- cells seeded in a well plate. culture supernatant was collected at , , , , and h post-infection, and viral titers in the supernatant were determined and expressed as tcid /ml in marc- cells. to investigate whether the lmy strain is a dominant strain of the na genotype of prrsv in korea, serum neutralization assays were performed using field serum samples collected from various regional areas. the serum neutralization assay was performed as previously described (truong et al., ) . the serum neutralization titers were determined using ifa and were expressed as the reciprocal of the serum dilution that produced a % or higher reduction in the wells. the chimeric full-length cdna clone, pk , was transfected into marc- cells. at h posttransfection, the infected cells were analyzed by ifa using antibody against n protein of prrsv. the chimeric virus, k expressed viral protein (fig. b) and gradually replicated into the neighboring cells (data not shown). recent studies have indicated that marc- cells (african green monkey kidney), which are a permissive cell line for prrsv, could be electroporated with rna for the recovery of infectious prrsv as efficiently as baby hamster kidney (bhk)- cells (truong et al., ; ansari et al., ) . these results showed that the chimeric virus, k , recovered and replicated well in marc- cells. upon the initial development of cpe, the supernatant from the electroporated cells was collected and infected into marc- cells to propagate recovered viruses. the once-passaged culture supernatant produced % cpe at days post-infection and yielded virus titers of tcid /ml. the multistep growth kinetics of the chimera virus was compared to those of parental strains in marc- cells (fig. ) . genetic engineering did not greatly affect viral replication. the result of the growth kinetics study demonstrated that the final titers of the chimera and parental viruses were similar, whereas the chimeric virus showed slightly delayed viral replication. the serum samples were divided into three groups depending on the k -specific neutralizing antibody titers, crossreactive, partially cross-reactive and resistant. the neutralizing antibody titers of cross-reactive group were greater than : , the titers of partially cross-reactive group were : and the titers of resistant group were less than : . the crossreactive samples against k virus, including partially crossreactive ones, accounted for . % whereas cross-reactive samples against mlv which widely used commercial live vaccine accounted for . % of the total field samples ( table ) . (cha et al., ; yoon et al., ; kim et al., ; choi et al., ) . furthermore, sequence diversity on the orf decreased serological cross-reactivity between prrsv strains belonging to the same genotype (kim et al., ) . the mlv containing a representative attenuated strain of na type virus is the most commonly used prrsv vaccine worldwide in field. the mlv virus was frequently isolated from vaccinated pigs in fields. however, only . % of the total prrsv elisa positive serum samples neutralized mlv, while . % of prrsv elisa positive serum samples neutralized the chimeric virus, k . the serological cross-reactivity against the k virus suggests that the humoral immune response induced by the chimeric virus would cross-react with approximately half percentage of the na field strain of prrsv in korea. the polyclonal antiserum against the k neutralized the lmy strain, but not the fl and mlv strain of prrsv (lee et al., manuscript in preparation) . these results supported that the chimeric virus would be more effective as a potential regional vaccine candidate to protect pigs than the mlv vaccine. to generate customized vaccine candidate, the genomic region of established prrsv infectious clone encoding structure proteins that play a critical role in the virus neutralizing response were replaced with the same genomic region from a korean dominant field strain of prrsv. it is highly possible that k would induce a host immune response, which can broadly cross-protect most of the na genotype of korean field strains of prrsv, because the virus expresses all structure proteins from one of the current dominant field strains of prrsv. further studies are in progress to determine a possible application of k as a vaccine candidate in korea and to evaluate the efficacy of other genetically modified k viruses. the control strategies and vaccine design a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp , and gp proteins and susceptibility to neutralization infectious cdna clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors genetic characterization of the korean porcine reproductive and respiratory syndrome viruses based on the nucleocapsid protein gene (orf ) sequences dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain we thank dr. fernando osorio, dr. asit pattnaik and dr. byungjoon kwon of the university of nebraska-lincoln for providing the prrsv infectious clone, fl .this study was supported by a grant of korea institute of planning and evaluation for technology in food, agriculture, forestry and fisheries. key: cord- -nqkw v w authors: qu, zehui; gao, fei; li, liwei; zhang, yujiao; jiang, yifeng; yu, lingxue; zhou, yanjun; zheng, hao; tong, wu; li, guoxin; tong, guangzhi title: label‐free quantitative proteomic analysis of differentially expressed membrane proteins of pulmonary alveolar macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus and its attenuated strain date: - - journal: proteomics doi: . /pmic. sha: doc_id: cord_uid: nqkw v w significant differences exist between the highly pathogenic (hp) porcine reproductive and respiratory syndrome virus (prrsv) and its attenuated pathogenic (ap) strain in the ability to infect host cells. the mechanisms by which different virulent strains invade host cells remain relatively unknown. in this study, pulmonary alveolar macrophages (pams) are infected with hp‐prrsv (hun ) and ap‐prrsv (hun ‐f ) for h, then harvested and subjected to label‐free quantitative ms. a total of proteins are identified, including that are differentially expressed. among them, proteins are located on the membrane. the most differentially expressed proteins are involved in response to stimulus, metabolic process, and immune system process, which mainly have the function of binding and catalytic activity. cluster of differentiation cd , vimentin (vim), and nmii as well as detected proteins are assessed together by string analysis, which elucidated a potentially different infection mechanism. according to the function annotations, prrsv with different virulence may mainly differ in immunology, inflammation, immune evasion as well as cell apoptosis. this is the first attempt to explore the differential characteristics between hp‐prrsv and its attenuated prrsv infected pams focusing on membrane proteins which will be of great help to further understand the different infective mechanisms of hp‐prrsv and ap‐prrsv. porcine reproductive and respiratory syndrome virus (prrsv) has been a leading economically significant viral pathogen of swine worldwide for almost years. [ ] [ ] [ ] prrsv, equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenaseelevating virus are members of the family arteriviridae. [ , ] prrsv is a positive-sense, single-stranded, rna virus with a full-length genome of kb that has a cap and a poly (a) tail. [ , [ ] [ ] [ ] prrsv was first reported in the united states in the late s. [ ] in , several large-scale, severe outbreaks of highly pathogenic (hp) atypical prrsv (hp-prrsv) were reported in china and neighboring asian countries. [ , , ] reported hp-prrsv morbidity and mortality rates were much higher than previous pandemic prrsv strains and associated with more severe clinical presentations and higher rectal temperature (> °c). [ ] the emergence of hp-prrsv has caused great economic loss to the swine industry in china and made preventing and controlling prrsv outbreaks difficult. therefore, elucidating the causes of the greater virulence of prrsv and the differences between the hp and attenuated pathogenic (ap) strains has become even more important. to this end, several trials have been conducted to identify virulence factors; these studies have resulted in some successes. [ , ] however, changes in virulence and pathogenic mechanisms are difficult to discern. other than virulence in vivo, many distinctions in the biological aspects of hp-prrsv and ap-prrsv have been noted, such as in viral binding and entry into pulmonary alveolar macrophages (pams). prrsv exhibits highly restricted cell tropism both in vivo and in vitro. [ ] the virus can be detected only in well-differentiated macrophages of lungs, lymph nodes, peyer's patches, spleen, tonsils, and thymus. pams are the main target cells of prrsv. [ ] prrsv can also replicate in vitro in the african www.advancedsciencenews.com www.proteomics-journal.com membrane proteins (mps) of pams infected by highly proteomic (hp)-and attenuated proteomic (ap)-prrsv have been elucidated by lc-ms/ms for label-free quantitative proteomics. ninety-five differentially expressed proteins were identified and characterized. the most significant difference in the biological process between pams infected with hp-and ap-prrsv is the metabolic process. most different molecular functions were classified as binding and catalytic activities. cellular component categories showed that differentially expressed proteins were confirmed as mps based on the annotation of uniprot database, such as rap a, vcl (vinculin), ifitm function in cell-cell junctions, erk signaling pathway, g protein signaling pathways, biotic stimulus, and so on. among them, vcl is a kind of f-actin-binding protein which is involved in cell-matrix adhesion and cell-cell adhesion in humans. it was demonstrated that over expression of vcl could inhibit the replication of both hp-prrsv and the attenuated prrsv in the mrna level. there were obvious differences in the inhibiting ability for hp-prrsv and its attenuated strain. this is the first attempt to explore the differential characteristics between hp-prrsv and its attenuated prrsv-infected pams focusing on mps which will be of great help to further understand the different infective mechanisms of hp-prrsv and ap-prrsv. green monkey kidney cell line ma- and its derivatives, marc- and cl- , which are considered permissive cell lines for prrsv. [ , ] reportedly, prrsv targets cellular membrane proteins (mps) and enters target cells through receptormediated endocytosis during viral infection. [ ] studies have investigated possible mechanisms employed by prrsv to infect pams. [ ] elucidating the characteristics of viruses and interactions between viruses and host cells is increasingly important. however, exploring individual proteins within the many proteins in cells is difficult. nonetheless, gradual advancements have come through use of proteomic techniques. of these, ms-based quantitative proteomic techniques offer the advantage of better accuracy and sensitivity, and have been widely used to analyze host cell responses to viral infection. among them, liquid chromatographytandem ms (lc-ms/ms) for label-free quantitative proteomics (lfqp) is an important mass spectrometric tool to detect and quantify large amounts of proteins. [ ] compared with quantitative proteomics using stable isotope labeling such as stable isotope labeling by amino acids in cell culture and isobaric tags for relative and absolute quantitation, lfqp detects greater amounts of proteins and important signaling pathways and networks. the aim of this study was to determine potentially different infection mechanisms used by the hp strain vhun [ ] and its derivative attenuated strain vhun -f [ , ] using lfqp. the animal study protocols were approved by the animal care and use committee of shanghai veterinary research institute, chinese academy of agricultural sciences. hp-prrsv strain vhun [ , ] at a titer of % tissue culture infective dose (tcid ) ml − and cell-passaged attenuated virus strain vhun -f (ap-prrsv) [ , ] at a titer of tcid ml − were stored as viral stocks. porcine circovirus , classical swine fever virus, prrsv antibody, and antigen-free -day-old piglets were used. animals were sacrificed in accordance with the ethics statement. lungs were dissected and lavaged with pbs (pbs; life technologies, inc., gibco/brl division, grand island, ny, usa) supplemented with % penicillinstreptomycin (gibco/brl), then centrifuged at × g for min, resuspended in pbs, centrifuged, and resuspended in pbs. pams were collected in roswell park memorial institute (rpmi) medium (gibco/brl) containing % fetal bovine serum (gibco/brl) [ ] and incubated in cm dishes (corning, inc., corning, ny, usa) for h at °c in a % co atmosphere. after pams were washed with pbs three times, dead and nonadherent cells were removed when confluency exceeded %. three dishes were inoculated with vhun and another three with vhun -f at multiplicity of infection . an additional three dishes were inoculated with dmem (gibco-brl) as a blank control. all dishes were incubated at °c in an atmosphere of % co , as described previously. [ ] after incubation for h, inocula were discarded and pams were washed with pbs three times. cell monolayers in all dishes were overlaid with rpmi- medium containing % fetal bovine serum and incubated at °c in a % co atmosphere for h. pams were digested with ml . % trypsinethylenediaminetetraacetic acid solution (gibco/brl), collected by gently pipetting, centrifuged at × g for min and lysed using the proteoextract transmembrane protein extraction kits (novagen, emd biosciences, inc., madison, wi, usa), [ , ] according to the manufacturer's instructions. cells were resuspended in extraction buffer and protease inhibitor cocktail, incubated for min at °c with gentle agitation, and centrifuged at × g for min at °c. after removing supernatants, pellets were resuspended in . ml extraction buffer a and protease inhibition cocktail, incubated for min at room temperature with gentle agitation, and centrifuged at www.advancedsciencenews.com www.proteomics-journal.com × g for min at °c. supernatants were precipitated with ml acetone and centrifuged at × g for min. after evaporating to dryness, μl sdt buffer ( % sodium dodecyl sulfate, mm tris/hcl at ph . , . m dithiothreitol) was added and mixtures were heated in boiling water for min. after centrifugation, supernatants were collected and quantified with a bca protein assay kit (bio-rad, usa). fig. digestion of protein ( μg for each sample) was performed according to the fasp (filter-aided sample preparation) procedure. briefly, the detergent, dtt and other low-molecular-weight components were removed using μl ua buffer ( m urea, mm tris-hcl ph . ) by repeated ultrafiltration (microcon units, kd) facilitated by centrifugation. then μl . m iodoacetamide in ua buffer was added to block reduced cysteine residues and the samples were incubated for min in darkness. the filter was washed with μl ua buffer three times and then μl mm nh hco twice. finally, the protein suspension was digested with μg trypsin (promega) in μl mm nh hco overnight at °c, and the resulting peptides were collected as a filtrate. the peptide content was estimated by uv light spectral density at nm using an extinctions coefficient of . of . % (g l − ) solution that was calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins. [ ] the peptide of each sample was desalted on c cartridges (empore spe cartridges c (standard density), bed id mm, volume ml, sigma), then concentrated by vacuum centrifugation and reconstituted in μl of . % (v/v) trifluoroacetic acid. ms experiments were performed on a q exactive mass spectrometer that was coupled to easy nlc (proxeon biosystems, now thermo fisher scientific). five microgram peptide was loaded onto a c -reversed phase column (thermo scientific easy column, cm long, μm inner diameter, μm resin) in buffer a ( % acetonitrile and . % formic acid) and separated with a linear gradient of buffer b ( % acetonitrile and . % formic acid) at a flow rate of nl min − controlled by intelliflow technology over min. ms data was acquired using a datadependent top ten method dynamically choosing the most abundant precursor ions from the survey scan ( - m/z) for hcd fragmentation. determination of the target value is based on predictive automatic gain control. dynamic exclusion duration was s. survey scans were acquired at a resolution of at m/z and resolution for hcd spectra was set to at m/z . normalized collision energy was ev and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as . %. the instrument was run with peptide recognition mode enabled. ms experiments were performed triply for each sample. [ ] the ms data were analyzed using maxquant software version . . . . ms data were searched against the uniprot sus scrofa sequence database (including sequences downloaded on / / ). an initial search was set at a precursor mass window of ppm. the search followed an enzymatic cleavage rule of trypsin/p and allowed maximal two missed cleavage sites and a mass tolerance of ppm for fragment ions. carbamidomethylation of cysteines was defined as fixed modification, while protein n-terminal acetylation and methionine oxidation were defined as variable modifications for database searching. the cutoff of global false discovery rate for peptide and protein identification was set to . . label-free quantification was carried out in maxquant as previously described. protein abundance was calculated on the basis of the normalized spectral protein intensity (lfq intensity). all statistical analyses were performed using unpaired t-tests. a p-value < . and ratio > or < . were considered to indicate significant differences. gene ontology (go) annotation and functional classification of identified proteins was with blast go ver. v . . with the current public database b g_aug (www.blast go.com). identified proteins were classified www.advancedsciencenews.com www.proteomics-journal.com using blast go steps under default parameters: blast, mapping, and annotation. protein-protein interaction networks were analyzed using (string software string-db.org/) . confidence view was assigned a score of . , indicating medium confidence. samples of prrsv-infected and dmem-inoculated pams were lysed at h post infection and protein concentrations were determined. samples ( μg) were separated by % sds-page and transferred to . μm nitrocellulose membranes (bio-rad laboratories, hercules, ca, usa). membranes were blocked with % skim milk in tris-buffered saline containing . % tween- and incubated overnight at °c with monoclonal antibodies against heat shock protein (hsp ; ab ; abcam plc, cambridge, uk) or kdel receptor (ab ; abcam plc). after washing three times, membranes were incubated at °c for min with horseradish peroxidase-conjugated anti-mouse igg or antirabbit igg (abcam plc). detection used chemiluminescence luminal reagents (pierce biotechnology, waltham, ma, usa). the porcine vinculin (vcl) were amplified from the cdna obtained from pam cells. restriction enzyme sites were incorporated into the primer sequences to facilitate molecular cloning. pcr products were cloned into the pcaggs vector to produce the porcine vcl expression vector. for transfection, cells were seeded in -well plates (corning) and transfected at - % confluency with respective constructed plasmids dna by using lipofectamine (life technologies), according to the manufacturer's instructions. the hp-hun or hun -f was infected in marc- cells at h post transfection. empty vector transfection samples served as controls in the experiment. at h post infection, total rnas of prrsv-infected cells were extracted using the rneasy mini kit (qiagen), the viral rnas in supernatants were isolated using qiaamp viral rna mini kit (qiagen), according to the instruction manual. all the isolated rnas were used as the template for synthesis of firststrand cdna by rt-pcr using rt primed by oligo (dt) primer using the primescript rt master mix (perfect real time, takara), according to the manufacturer's instructions. then the cdna templates were quantified using prrsv-specific real-time rt-qpcr. [ ] . results a total of proteins were detected by lfqp and are displayed in a heatmap (figure ) . statistical significance was determined using unpaired t-tests. for all tests, a p-value of < . and ratio of > or < . was considered to indicate a significant difference. glyceraldehyde -phosphate dehydrogenase was the internal normalization control and the ratio of glyceraldehyde -phosphate dehydrogenase between hp and ap-prrsv was . ± . . the "only one exists" group indicated that proteins were detected only in one group but not another group due to low expression level ( table ) . correlation analysis indicated good repeatability of the technology (figure ) . a total of differentially expressed proteins were identified ( table ) . a control group was used to exclude false-positive interference. data from the control group was also used to elucidate functions of target proteins. differentially expressed proteins between control and hp-prrsv-infected cells (con/hp) and the www.advancedsciencenews.com www.proteomics-journal.com p < . and ratio > or < . indicated quantitative difference between two groups. the "only one exits" group indicated that proteins were detected three times in one, but not in the other group. hp-prrsv group means proteins identified in the hp-prrsv infected pams, while ap-prrsv was proteins detected in the attenuated pathogenic prrsv infected pams. control was displayed as the mock. control and ap-prrsv-infected cells (con/ap) are in supporting information. to extend the molecular characterization of quantitative differences and only-one-exists groups, uniprot and go databases were used to characterize information about biological processes (bps), molecular functions (mf), and cellular components (cc). bps of h (vh/va > , including proteins) and a (vh/va < . , including proteins) groups are in figures a and b bp. in group h, go annotations were primarily distributed in response to stimulus ( . %), metabolic process ( . %), and immune system process ( . %). ratios were . % response to stimulus, . % metabolic process, and . % immune system process in group a. the most significant difference in bp between pams infected with hp-prrsv and ap-prrsv was seen for metabolic process, which may be the major reason for the large differences among animals challenged with different virulence of prrsv. molecular function categories of h group and a group were shown in figure a ,b mf. most different molecular functions were classified as binding ( . and . %) and catalytic activity ( . and . %). binding of h group included enzyme binding ( . %), nucleic acid binding ( . %) and protein complex binding ( . %), while group a mainly involved nucleic acid binding ( . %), nucleoside phosphate binding ( . %) and nucleotide binding ( . %) from the analysis of go distribution by level . enzyme code distribution suggested there were five transferases ( . %), one hydrolase ( . %), one lyase ( . %), and two ligases ( . %) detected in group h. while four oxidoreductases ( . %), five transferases ( . %), five hydrolases ( . %), three lyases ( . %), and three isomerases ( . %) in group a. cc categories were illustrated in figure a b cc. ninety-five detected differentially expressed proteins were annotated and categorized to ccs of macromolecular complex ( . %), membrane ( . %), membrane-enclosed lumen ( . %), and extracellular region ( . %). because of technological problems, we were unable to conclude that all the detected proteins were indeed mps. however, were confirmed based on the annotation of uniprot database and there also existed proteins partially anchored on the membrane or binding with the mps. to verify the differentially expressed proteins via lc-ms/ms for lfqp, western blots were conducted for two proteins partially located on membranes. expression of hsp and the kdel receptor from cell lysates of vhun -infected and vhun -f -infected pams, and dmem-inoculated pams were tested with antibodies to the proteins. lfqp showed that the ratios between vhun infected and vhun -f -infected pams reached . and . , respectively. western blots confirmed lfqp results ( figure ). to examine whether the differentially expressed proteins detected affects virus infection, the vcl transient overexpression vector was transfected into marc- cells, followed by the hp hun or its attenuated strain infection. the prrsv-specific rt-qpcr results showed that vcl protein could inhibit both viruses, especially for hp-hun strain. compared with the empty vector in this study, lfqp of mps of hp-prrsv-, ap-prrsv-infected pams and the control was performed. important information about target proteins related to virus infection was obtained. the hp-prrsv strain vhun and its derivative, the serially cellpassaged attenuated strain vhun -f , [ ] revealed different infection mechanisms in pams. a total of proteins were identified among the control, ap-prrsv-infected, and hp-prrsv-infected pams. of these, were detected in all three groups (figure ) . we focused on differentially expressed proteins of ap-prrsv-and hp-prrsv-infected pams; among these, were detected in only one group. prrsv has been a threat to the global pig economy for several years because of its persistent infection, immune escape, and high mortality from inflammation and high fever. [ ] the attenuated prrsv vhun -f vaccine strain attenuated from hp-prrsv vhun by serial passages, which is now used in china. [ ] we analyzed mps to identify factors associated with immunological effects and determine differences between hp-prrsv and ap-prrsv. mps classified based on go analysis are in table . a heatmap based on the uniprot database was constructed to comprehend the functions and bps of differently expressed proteins ( figure b) , which revealed clustering and abundance of the detected proteins that existed confidentially on the membrane based on uniprot database annotation. we first focused on proteins associated with immunology and inflammation with higher abundance in ap-prrsv-infected pams. ptpn (vh/va = . ), a member of the protein tyrosine phosphatase family, functions as signaling molecule that regulates cellular processes related to the jak-stat, il- , il- , and granulocyte-macrophage colony-stimulating factor (gm-csf) signaling pathways; dephosphorylation of nonreceptor kinases including jak , jak , stat , and stat ; and negative regulation of il- -, il- , il- , and ifn-mediated signaling. ptpn also functions in the response to inflammation via nf-κb. [ ] [ ] [ ] sipa (h-/a+) is a mitogen-induced gt-pase activating protein for ras-related regulatory proteins. it was related to g-protein signaling and blood-brain barrier and immune cell transmigration: vcam /cd (cluster of differentiation) signaling pathways. [ , ] c ar (h-/a+) is the receptor for the chemotactic and inflammatory peptide anaphylatoxin c a, which stimulates chemotaxis, granule enzyme release, intracellular calcium release, and superoxide anion production, participates in the innate and adaptive immune responses to www.advancedsciencenews.com www.proteomics-journal.com table . statistics analysis of proteins that existed definitely on the membrane. the lectin-induced complement pathway. [ ] [ ] [ ] [ ] [ ] [ ] polyribonucleotide -hydroxyl-kinase clp (clp ) (vh/va = . ) mainly acts as a kinase that binds atp hosting -hydroxyl-kinase activity, and functions in mrna cleavage and in sirna loading onto the rnainduced silencing complex involved in rna interference and its destruction. the kinase hclp phosphorylates and licenses synthetic sirnas to assemble into an rna-induced silencing complex for cleavage of target rna. [ ] expression of these molecules was higher in the ap-prrsv-infected group and had a similar level to the control group. therefore, we concluded that they might be related to the immune escape of hp-prrsv and that the attenuated vaccine strain would not have side effects on these immune factors expression in the host cell, and these proteins above would contribute to the resistance of prrsv. some proteins related to immunology and inflammation had higher expression in hp-prrsv-infected cells. raftlin (vh/va = . ) protein is pivotal for maintenance of lipid rafts and may be involved in regulation of b-cell antigen receptor-mediated signaling. raftlin promotes binding of double-stranded rna, activations of b cell receptors and toll-like receptor signaling pathways, and is involved in il- production to release proinflammatory cytokines. hence, the higher abundance of raftlin in the hp-prrsv group compared to the ap-prrsv and control groups may explain the more severe inflammation triggered by hp-prrsv. [ , ] iars (h+/a-), isoleucyl-trna synthetase is a target of autoantibodies in autoimmune diseases. [ , ] vcl (h+/a-), vinculin is a cytoskeletal protein associated with cellcell and cell-matrix junctions, and is related to the il- , il- , and gm-csf signaling pathways. [ ] rap a (h+/a-) is a member of the ras oncogene family, small gtp-binding protein, of which active form interacts with several effectors [ ] [ ] [ ] related to the erk and g protein signaling pathways. [ , ] it was reported that prrsv could induces prostaglandin e production through cyclooxygenase and is related to erk signaling. [ ] the differential expression of rap a may provide information for future research on the interaction between prrsv and the erk signaling pathway. the abundance of rap a was lower in ap-prrsv-infected pams than in hp-prrsv-infected pams and control cells, which had similar levels. thus, we hypothesized that rap a was associated with immunization with the attenuated vaccine. ifn-induced transmembrane protein , ifitm (vh/va = . ), responds to biotic stimulus and had the highest expression levels among these proteins in the hp-prrsv group, as compared to the proteins identified in the ap-prrsv group but it was not detected in the control group. in humans, this protein negatively regulates the entry of multiple viruses, including influenza a virus, sars coronavirus, marburg virus (marv), ebola virus, dengue virus, west nile virus (wnv), human immunodeficiency virus (hiv) type , and vesicular stomatitis virus (vsv), into host cells. [ , ] it could inhibit hemagglutinin protein -mediated entry of influenza virus, gp , two-mediated viral entry of marburg virus and ebola virus, s protein-mediated viral entry of sars coronavirus, and g protein-mediated viral entry of vsv, thereby playing a critical role in the structural stability and function of vacuolar atpase (v-atpase). establishing physical contact with the v-atpase of endosomes is critical for proper clathrin localization and is required for v-atpase to lower the ph in phagocytic endosomes, thus establishing an antiviral state. [ ] high expression of both hp-prrsv and ap-prrsv suggests that ifitm may help inhibit prrsv entry and promote resistance to hp-prrsv infection. peptidyl-prolyl cis-trans isomerase b (ppib, vh/va = . ) has peptidyl-prolyl cis-trans isomerase activity and is involved in protein folding and protein peptidyl-prolyl isomerization, which can accelerate protein folding. [ ] ppib is positively regulated by viral genome replication, is involved with the viral processes of hepatitis c virus (hcv), and interacts with and stimulates the rna-binding activity of hcv ns b. ppib is critical for efficient replication of the hcv genome. [ ] compared with the control group, ppib was downregulated in the hp-prrsv and ap-prrsv groups. the abundance of ppib was lower in the hp-prrsv group than the ap-prrsv group, suggesting an association with prrsv replication. string network analysis was used to elucidate interactions among differentially expressed proteins. the essential factors and receptors involved in the entry of prrsv are reportedly cd (q vl ), nmhc ii-a (myh , f skj ), sialoadhesin (sn, a lcj ), cd (f ryz ), and vimentin (vim) (p ). [ ] [ ] [ ] [ ] [ ] [ ] we assessed the involvement of these mps together with proteins detected by string analysis on viral entry. receptor proteins involved in prrsv entry are in figure to better understand the characteristics of pathway-like regions, the genomrnai database was analyzed to annotate detected proteins. [ ] regions containing proteins with higher abundance in hp-prrsv-infected pams are green and in ap-prrsv-infected pams are red ( figure a) . a search of the genomrnai database determined that linked proteins ik (ik cytokine), wd repeat domain , dyskerin pseudouridine synthase , and adenylosuccinate lyase (adsl) in the green region of figure a shared similar annotations and functioned to decrease expression of nf-κb or il- , [ ] which are related to inflammation. [ , ] hence, further analysis of these linked proteins may help explain differential mechanisms between hp-prrsv and ap-prrsv infection. other proteins in the green-colored region of a are reported to influence virus infection. nucleolar and coiled-body phosphoprotein increases sindbis virus infection [ ] and adsl increases human papilloma virus -gfp infection. [ ] the lower abundance of nucleolar and coiled-body phosphoprotein and adsl in the hp-prrsv-infected group may be related to the host immune response repression of prrsv and may be used by hp-prrsv during infection to self-upregulate. the red region of figure a contains some linked proteins with some figure . protein-protein interaction network determined by string software showing interactions among differentially expressed proteins between two virulent groups with cd , vim, and myh added. red, protein abundance higher in hp-prrsv than ap-prrsv; green, protein abundance in hp-prrsv lower than in ap-prrsv. line colors represent type of evidence for association: green, neighborhood; red, fusion; purple, experimental; light blue, database; black, expression; blue, co-occurrence; yellow, text mining. gene abbreviations are shown. vh, protein abundance in hp-prrsv; va protein abundance in ap-prrsv. vh/va = , detected only in ap-prrsv; vh/va = null, detected only in hp-prrsv. information. uridine monophosphate synthetase decreases hiv- infection, [ ] while programmed cell death decreases hcv replication, [ ] both downregulate nf-κb expression. [ ] the lower abundance of these proteins in the hp-prrsv group suggested that hp-prrsv escaped inhibition through an unknown mechanism. myosin- (myh ) appears to function in cytokinesis, cell shape, and specialized functions such as secretion and capping. [ ] myh is an important factor for prrsv infection and interacts with gp of prrsv, [ ] although the underlying mechanisms remain unclear because of limited research. as shown by our results, myh was related to vcl in ap-prrsv and actl in hp-prrsv. vcl is an actin filament (f-actin)binding protein involved in cell-matrix adhesion and cell-cell adhesion in humans, regulation of cell-surface e-cadherin expression, and potentiation of mechanosensing, and may be important in cell morphology and locomotion by promoting binding with actin, alpha-catenin, cadherin, dystroglycan, and ubiquitin protein ligase. [ ] in hiv research, transient overexpression of vcl reduced the susceptibility of human cells to infection with hiv- and negatively affected paxillin phosphorylation and limited retroviral infection. [ ] just like hiv- , in our study, it was demonstrated that over expression of vcl could inhibit the replication of both hp-prrsv and the attenuated prrsv in the mrna level. there were obvious differences in the inhibiting ability for hp-prrsv and its attenuated strain. actl a, which is an actin-like protein a, is involved in transcriptional www.advancedsciencenews.com www.proteomics-journal.com activation and repression of select genes by chromatin remodeling (alteration of dna-nucleosome topology) and mainly functions in chromatin binding and transcription coactivator activities. [ ] we found that myh may regulate ap-prrsv and hp-prrsv infection via different pathways. through super pathways annotation (www.genecards.org), vcl and actl a were identified as participants in the il- , il- , gm-csf, and tnf-α/nf-κb signaling pathways, where they may help with differential mechanisms of prrsv infection with different virulence. using the genomrnai database, some linked proteins were found to be related to viruses or inflammation ( figure b ). in the green region, gnas complex locus (gnas) increased human papilloma virus -gfp, [ ] and serine/arginine repetitive matrix decreased hcv infection, influenza a replication and viral numbers, and il- expression, [ , ] small nuclear ribonucleoprotein u subunit decreased influenza a replication and viral numbers. [ ] irf- decreased infection by hcv, west nile virus, and dengue virus. [ , ] in the red region, hsf decreased hcv replication. [ ] protein kinase camp-activated catalytic subunit alpha decreased vsv infection. [ ] signal transducer and activator of transcription decreased il- expression. rap a, mink , and gnas are regulatory proteins in the ras pathway. rap a and mink were detected only in the hp-prrsv group, whereas gnas was detected only in the ap-prrsv group, in accordance with a previous report that stimulation of ras increases the replication ability of hcv by reducing ifn-jak-stat pathway activity. [ ] we proposed that prrsv was also related to the ras pathway, and differed between hp-prrsv and ap-prrsv. creb-binding protein (crebbp) is composed of doublestranded rna-activated transcription factor with irf- , and double-stranded rna-activated transcription factor is activated in many virus-infected cells to promote apoptosis. [ , ] crebbp may interact with human herpes virus virf- , which could inhibit the binding of crebbp to irf- . [ ] our results showed that irf- abundance was tenfold lower in the hp-prrsv than the ap-prrsv group, which we proposed was a protective mechanism of prrsv to escape from host immunity and ensure survival after viral infection. the ability of hp-prrsv hun to induce cell apoptosis is stronger than classical prrsv (ch- a) in immune organs and lungs of piglets. [ ] we concluded that hp-prrsv might interact with crebbp and decrease irf- expression to escape from host immunity and cause severe damage to the cell, highlighting a difference from ap-prrsv. notable differences exist during the infection of ap-prrsv and hp-prrsv. hp-prrsv could inhibit host immune function and evade the immune response via unknown mechanism. [ , ] label-free ms was performed using ap-prrsv-infected and hp-prrsv-infected pams. this is the first attempt to explore the differential characteristics between hp-prrsv and its attenuated prrsv infected pams focusing on membrane proteins. by analyzing detected proteins in hp-infected, ap-prrsv-infected, and control group, proteins related to the immune response or virus replication were identified that may elucidate unique pathways used by different virulent prrsvs for cell entry, virus replication, and immune escape mechanisms. researches on these detected proteins will help with the elucidation of the identity, the expression abundance, and significance of them, future study will be focused on functions of these key membrane proteins to deepen our understanding of differential mechanisms between hp-prrsv and ap-prrsv infection. proc. natl. acad. sci proc. natl. acad. sci supporting information is available from the wiley online library or from the author. the authors declare that they have no conflicts of interest associated with this report. keywords attenuated, highly pathogenic, infection, label-free quantitative proteomics, membrane proteins key: cord- - x ro p authors: jiménez, luisa fernanda mancipe; nieto, gloria ramírez; alfonso, victor vera; correa, jairo jaime title: association of swine influenza h n pandemic virus (siv-h n p) with porcine respiratory disease complex in sows from commercial pig farms in colombia date: - - journal: virol sin doi: . /s - - - sha: doc_id: cord_uid: x ro p porcine respiratory disease complex (prdc) is a serious health problem that mainly affects growing and finishing pigs. prdc is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), mycoplasma hyopneumoniae (myh), actinobacillus pleuropneumoniae (app), pasteurella multocida and porcine circovirus (pcv ). to characterize the specific role of swine influenza virus in prdc presentation in colombia, farms from three major production regions in colombia were examined in this study. nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with siv. isolation of siv was performed in -day embryonated chicken eggs or madin-darby canine kidney (mdck) cells. positive isolates, identified via the hemagglutination inhibition test, were further analyzed using pcr. overall, of the farms were positive for siv. notably, sequencing of the gene encoding the hemagglutinin (ha) protein led to grouping of strains into circulating viruses identified during the human outbreak of , classified as pandemic h n - . serum samples from gilts and multiparous sows between and were obtained to determine antibody presence of app, myh, pcv and prrsv in both siv-h n p-negative and -positive farms, but higher levels were recorded for siv-h n p-positive farms. odds ratio (or) and p values revealed statistically significant differences (p< . ) in prdc presentation in gilts and multiparous sows of farms positive for siv-h n p. our findings indicate that positive farms have increased risk of prdc presentation, in particular, pcv , app and myh. swine infl uenza is an acute, highly contagious respiratory disease resulting from infection with type a infl uenza virus, a member of the orthomyxoviridae family. influenza a viruses have been isolated from different species, including humans, pigs, dogs, horses, sea mammals and birds (kuntz-simon g, et al., ; webster r g, et al., ) . the viruses are classified into subtypes based on antigenic properties of the external glycoproteins, hemagglutinin (ha) and neuraminidase (na). influenza a viruses have been further classified into ha and na subtypes (webster r g, et al., ; fouchier r, et al., ) . the proteins are highly variable and critical for the induction of antibody response in the host (gramer m, ) . although other subtypes have been identifi ed, infl uenza a virus h n , h n and h n subtypes are the most prevalent in pig populations worldwide (kuntz-simon g, et al., ) . in pigs, the disease is characterized by sudden onset, coughing, dyspnea, fever and prostration, followed by rapid recovery. lesions generally develop rapidly in the respiratory tract and regress quickly. the course and severity of disease are likely to vary with the strain of the virus, age and immune status of the host (easterday b c, et al., ) . morbidity is high (near %), but mortality is low (usually less than %). recovery begins to days post-infection. subclinical infections are common, and most pigs can be reinfected with other strains without showing clinical signs (reeth v k, et al., ) . porcine respiratory disease complex (prdc), a multifactorial condition, is a serious health problem in growing and finishing pigs, and poses a threat to the swine industry worldwide. prdc results from a combination of viral and bacterial agents, including porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), mycoplasma hyopneumoniae (myh), actinobacillus pleuropneumoniae (app), pasteurella multocida and porcine circovirus type (pcv ) (kim j, et al., ) . although the etiology of prdc involves multiple pathogens and varies among farms, several researchers maintain that etiology is mainly associated with myh and prrsv (dee s, ; thacker e l, et al., ) . pneumonia caused by prdc is characterized by slow growth, decreased feed effi ciency, lethargy, anorexia, fever and cough. while farms and slaughter houses in colombia have tested positive for siv over the years, the role of siv in prdc is yet to be established. there is evidence of classic h n and infl uenza a h n pandemic circulation in swine populations, but surveillance reports on prdc are scarce to date. the main goal of the current research was to generate surveillance, epidemiological, antigenic as well as phylogenetic data to ascertain the presence of swine influenza (h n ) pandemic virus and determine its association with prdc (prrsv, myh, app and pcv ) in sows from production farms in colombia. a total of gilts and multiparous sows distributed in farms from three major swine-producing areas in colombia (antioquia, valle del cauca and cundinamarca) were included in the present study. blood samples were obtained via venipuncture of the jugular vein using red top vacutainer ™ tubes. serum samples were identifi ed and stored at - °c until analysis. two hundred and forty-two nasal swabs, bronchial lavage (bl) and lung tissue samples of animals displaying symptoms compatible with siv (easterday b c, et al., ) were collected from the farms. samples were collected in brain-heart infusion medium (bhi) medium (bd®) supplemented with % antibiotic and anti-mycotic solution (sigma®), filtered through a . μm filter, and stored at - c until processing for siv isolation, either in chicken embryo eggs or the madin-darby canine kidney (mdck) cell line. briefl y, day-old spf embryonated chicken eggs were inoculated via the allantoic cavity with μl filtered sample, incubated at °c for h, and monitored daily. allantoic fluid was collected and evaluated for hemagglutination activity following standard procedures (who, ) . twenty four-well plates were seeded with × cells/well of mdck and grown in dulbecco's modifi ed eagle medium (dmem) (gibco®) supplemented with % fetal bovine serum (fbs, gibco®), % antibiotic (sigma®) and % l-glutamine (sigma®). complete growth medium was removed from confl uent monolayers and washed three times with pbs supplemented with μg/ml tpck trypsin (sigma®). each well was infected with μl of the original fi ltered sample and incubated for h at °c, % co , followed by the addition of ml/well complete dmem. cells were incubated at °c, % co , for - days and monitored daily for cytopathic effect (cpe). following the incubation period, cell culture supernatant (cs) and allantoic fl uid (af) were collected and tested with the hemagglutination assay (ha) using chicken erythrocytes according to the standard office international des epizooties (oie) protocol (swine infl uenza, ) . all ha-positive samples from egg inoculation or cell culture isolation were further analyzed for effi cient subtyping of virus. viral rna was extracted using a commercial rna extraction kit (qiamp viral rna®, qiagen, ca, usa) following the manufacturer's protocol. amplification of ha and na gene segments was performed with a duplex reverse transcriptionpolymerase chain reaction (rt-pcr) assay targeting the respective genes of h n swine infl uenza virus (choi y, et al., ) . initial reverse transcription was performed with m-mvl reverse transcriptase (invitrogen) using μl viral rna as template and the uni- primer (invitrogen, maryland, usa) to generate cdna. the conditions for reverse transcription (rt) were as follows: °c for min, °c for min, °c for min, and a fi nal step of °c. amplifi cation of bp ha (primers h f and h r) and bp na (primers n f and n r) genes of siv was performed (table ) under the following reaction conditions: °c for min, cycles of °c for min, . °c for min, °c for min, followed by °c for min, and a fi nal step of °c to terminate amplifi cation. pcr products were run on an agarose gel, and isolated and gel-purifi ed using the qiaquick gel extraction kit (qiagen ® ). gel-purified products were sequenced by macrogen ® , usa, using big-dye ® terminator cycle sequencing. dna sequences were combined and edited using the lasergene sequencing analysis software package (dnastar ® , madison, wisconsin). multiple sequence alignments were made using clustal w to identify related reference infl uenza genes. sequence comparisons and phylogenetic relationships through a bootstrap trial of were determined with the mega . program using the clustal w alignment algorithm, and evolutionary history inferred using the neighbor-joining method (saitou n, et al., ) for tree construction. gene sequences of colombian strains were compared with those of swine, avian and human infl uenza viruses, which were retrieved from the ncbi infl uenza virus resource. serum samples were subjected to the hemagglutination inhibition test to identify antibodies against siv using strain a/puerto rico / (h n ) as antigen, according to the who protocol ( ). prdc was diagnosed based on detection of antibodies against prrsv, myh, app and pcv in serum. specifi c antibodies were measured with elisa kits (table ) . data were analyzed using the statistix . program. the chi-square test was initially performed to determine the probability of presentation of respiratory disease complex with and without the presence of siv, and logistic regression analysis subsequently utilized to determine the risk factors that increase the presentation of disease when prdc farms and animals are positive or negative for siv. data were considered signifi cant at p< . , and graphs plotted using graphpad software. among the farms surveyed, were positive for swine flu pandemic virus, either from embryo chicken egg or mdck cell culture isolates (table ) . positive the hemagglutination inhibition test revealed positivity to siv-h n p in . % gilts, . % sows with - births and . % sows with multiparous (> ) births. the data suggest a trend in reactivity to siv-h n p, as the antibody response appears to decrease through time. prdc presentation was lower in siv-h n p-negative than siv-h n p-positive farms ( table ). the odds ratio value and p value revealed significant differences (p< . ) in prdc risk presentation in gilts and multiparous sows of siv-h h p-positive farms (figure ) . analysis was completed using logistic regression to de-termine the likelihood of increase in prdc disease presentation in siv-h n p-positive farms. according to our results, siv-h n p-positive farms had . , . and . times greater risk of introduction of app, myh and pcv , respectively, compared with siv-h n p-negative farms. in contrast, for prrsv, reduced risk of disease presentation (or, . ) was observed in farms positive for siv-h n p. the presence of siv-h n p in positive farms increases the risk of introduction of prcd especially pcv , app and myh. when analyzed by age group, the gilts are more susceptible to pcv , myh and app, respectively, while multiparous sows are more susceptible to pcv , app and myh. logistic regression analysis was performed to determine the risk of prdc presentation in farms positive and negative for siv-h n p. the odds ratio and p value revealed significant differences (p< . ) in risk prdc presentation per individual. in animals positive for siv-h n p, major risk of mycoplasma hyopneumoniae, pcv and app presentation was . , . and . times that of siv-negative animals, respectively. in contrast, for prrsv, the risk of presentation of disease (or, . ) was decreased in animals positive for siv-h n p. many groups have defined prdc as a multifactorial respiratory disease involving several pathogens (harms p a, et al., ; kim j, et al., ; opriessing t, et al., ; fachinger v, et al., ) , while other investigators (thacker e ,l, ) argue that prdc is enzootic pneumonia produced by mycoplasma spp. and other opportunistic bacteria, aggravated by the respiratory virus. the main goal of the present study was to conduct a systematic analysis of swine influenza virus infection and determine its role in prdc presentation in the major swine-producing areas of colombia. serological reactivity determined using hi disclosed that swine influenza virus h n is prevalent in gilts and multiparous sows from farms in colombia, although the antibody response decreased over time. we expected to fi nd isolates from infl uenza of classical h n virus origin in the fi eld. surprisingly, however, sequencing results led to grouping of viruses isolated from the farms within the original h n pandemic of , four of which shared % homology. therefore, it is important to consider swine infl uenza virus of pandemic origin as a pathogen playing an infl uential role in the presentation of prdc in commercial pig farms in the country. the pandemic h n / virus (a/ca/ / ) is a swine infl uenza a virus subtype h n strain responsible for the fl u pandemic. the virus originates from swine, but was never established as circulating in pig populations before its detection in humans (reeth v k, et al., ) . siv-h n p presentation in colombia may have been attributed to human transmission from pigs, as reported in various countries (weingartl h m, et al., ; sreta d, et al., ; pereda a, et al. ) . this hypothesis is based on the presentation of disease outbreaks in pigs and humans, as well as reports by the world organization for animal health (oie), which documented the presence of the virus in various countries, including argentina, canada, australia, ireland and united states. our results indicate that siv h n p positivity in animals increases the risk of presenting prdc, particularly pcv , app and myh. primiparous and multiparous sows are more susceptible to pvc , followed by myh and app in gilts, and app and myh in multiparous sows. a study by hansen m s, et al. ( ) in denmark revealed the presence of pcv in most lung samples from animals presenting prdc and indicated associations of this virus with the majority of viral and bacterial pathogens. the most frequently detected pathogens were in the order pcv , myc hyopneumoniae, pasteurella multocida and myc hyohinis, identified in different proportions in pigs with pneumonia in germany (palzer a, et al., ) , taiwan (chiou m t, et al., ) and the usa (choi y, et al., ) . this diversity in the presentation of pathogens involved in prdc may reflect the different diagnostic methods, health status of animals, farm management factors or simply the complex nature of pneumonia in pigs. siv-h n p-positive farms were more susceptible to prdc presentation for both gilts and multiparous sows, and interactions were mainly observed among pcv and siv. research in animals between and weeks of age showed that siv acts together with pcv in pigs under fi eld conditions. prdc induces severe clinical disease lesions presenting both infl uenza and wasting syndrome post-weaning (dorr p, et al., ; pallares f j, et al., ) . wei h, and co-workers ( ) concluded that subclinical pcv infection results in increased severity of subsequent siv-h n infection and siv can trigger the severe form of pcv , although the percentage of animals affected is comparable to that reported for other co-infections known to trigger severe pcv . myh is associated with presentation of swine enzootic pneumonia, characterized by high morbidity and low mortality in affected farms (opriessing t, et al., ) . myh acts as an immunosuppressant by increasing its pathogenicity as well as that of siv at the time of replication of the two agents (opriessing t, et al., , yazawa s, et al., . thacker e and colleagues ( ) reported that the percentage of lung lesions and clinical presentation of disease in pigs inoculated with siv days after inoculation with myh is higher than that in pigs inoculated with myh only. in our study, higher risk ( . %) of introduction of myh in siv-h n p-positive farms was observed, compared to siv-h n p-negative farms. app, a highly contagious infectious agent, causes pleuropneumonia in pigs, characterized by increased susceptibility to secondary infections (gutierrez c, et al., ) . app is considered an obligate parasite of the respiratory tract of pigs (taylor d j, ) . this report is consistent with the present study, where siv-h n ppositive farms displayed . % positivity for app. app has two different biotypes, specifically, biotype containing serotypes and biotype with six serotypes. all biotypes are capable of causing disease, with some serotypes being more aggressive than others (bossé j t, et al., ) . app is reported to increase the incidence of stress associated mainly with pleuropneumonia and interactions with viral and bacterial agents involved in the introduction of prdc (bossé j t, et al., ; kim j, et al., ) . regarding prrsv presentation in siv-h n ppositive and -negative farms, the incidence of prrsv was lower in positive ( . %) than negative farms ( . %). co-infection studies of prrsv and siv have yielded conflicting results in terms of clinical disease. in animals experiencing swine influenza infection and prrsv infection as a secondary pathogen, the disease is exacerbated, leading to prolonged fever, increased cough and weight loss ( van reeth k, et al. ; kitikoon p, et al., ). on the other hand, prior infection with prrsv does not aggravate acute siv presentation but can generate subsequent chronic infection (pol j m, et al., ) . prrsv is transmitted transplacentally through direct contact after birth, and piglets from infected mothers are infectious during subsequent stages of production, particularly the fattening and finishing stages (prieto c, et al., ) . similarly, siv is transmitted by direct contact between infected and uninfected animals via aerosol and can affect animals at any stage, particularly during fattening and fi nishing, as the pigs are raised in very close proximity to each other ( van reeth k, et al., ) . for this reason, we expected to find significant interactions between the two viruses in multiparous sows. however, this was not observed in our study. regarding tropism, prrsv has high affinity for differentiated macrophages. destruction of these cells in the lung by prrsv is suggested as the key event causing lung susceptibility to secondary pathogens (gucht s v, et al., ; van reeth k, et al., ) . prrsv and siv are the primary etiologic agents associated with respiratory disease of pigs in the united states (choi y, et al., association of siv-h n p with prdc in sows from commercial pig farms in colombia ) and co-infection could explain this role in porcine respiratory disease complex presentation. primary siv infection of epithelial cells of the airways results in cellular necrosis, production of infl ammatory mediators and rapid infiltration of phagocytic cells, including lung alveolar macrophages (pam) susceptible to prrsv infection. siv infection-infl ammation may increase the target cells for initial infection of prrsv, resulting in increased and prolonged pneumonia (kitikoon p, et al., ) . based on results from the current study, we propose that siv-h n p blocks prrsv replication or vice versa, and presentation of the disease is dependent on virulence and the time interval between infections (kitikoon p, et al., ) . prdc results from a combination of infectious agents as well as environmental stressors and challenges that affect the health of pigs, resulting in reduced performance, increased medication costs and high rates of mortality. clearly, the different forms of presentation of respiratory disease in pigs are attributed to the interactions of viral and bacterial agents, and although the etiology of prdc involves multiple pathogens with variations among farms, limited information is available on the spread of the virus in our pig population. the current study showed that pcv and app are the main viral-bacterial agents interacting with siv-h n p in positive animals. although it is difficult to establish whether swine flu virus is a pandemic primary, secondary or opportunistic agent, our data clearly indicate that siv-h n p plays an important role in the presentation of prdc, signifying the need to increase agricultural surveillance to prevent future outbreaks. this study was supported by colombia's agriculture ministry, colombian association of swine producers, cercafe and national university of colombia. we are grateful to mirela norho for statistical analysis and farms of antioquia y valle del cauca that participated in the study. the authors declare no conflicts of interest in terms of financial relationships with the industry or directly with the academy. all experiments were performed in accordance with the institutional and national guidelines for the care and use of laboratory animals. jjc, gcr, vjv designed experiments. lfm performed experiments. lfm analyzed data. lfm and jjc wrote paper. all authors read and approved the final manuscript. actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection etiological and epidemiological survey of prdc associated pathogens in taiwan detection and subtyping of swine influenza h n , h n and h n viruses in clinical samples using two multiplex rt-pcr assays retrospective analysis of etiologic agents associated with respiratory diseases in pigs the porcine respiratory disease complex: are subpopulations important? epidemio-logic assessment of porcine circovirus type coinfection with other pathogens in swine swine influenza the effect of vaccination against porcine circovirus type in pigs suffering from porcine respiratory disease complex characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gull an update on swine infl uenza ecology and diagnostics. american association of swine veterinarians the combination of prrsv virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs simultaneous serological evidence of actinobacillus pleuropneumoniae, prrsv, aujeszky's disease and infl uenza viruses in spanish fi nishing pigs an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark three cases of porcine respiratory disease complex associated with porcine circovirus type infection association of porcine circovirus with porcine respiratory disease complex vaccine effi cacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of siv vaccination genetic and antigenic evolution of swine infl uenza viruses in europe and evaluation of their zoonotic potential porcine circovirus type associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies concurrent infections are important for expression of porcine circovirus associated disease porcine circovirus type (pcv- ) coinfections in us fi eld cases of postweaning multisystemic wasting syndrome (pmws) associations between pathogens in healthy pigs and pigs with pneumonia pandemic (h n ) outbreak on pig farm dual infections of prrsvv/influenza or prrsvv/ actinobacillus pleuropneumoniae in the respiratory tract transplacental infection following exposure of gilts to porcine reproductive and respiratory syndrome virus at the onset of gestation first isolation and identifi cation of h n swine infl uenza viruses in colombian pig farms influenza in birds, pigs and humans: old theories versus current viewpoints the neighbor-joining method: a new method for reconstructing phylogenetic trees pandemic (h n ) actinobacillus pleuropneumoniae mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia immunology of the porcine respiratory disease complex mycoplasmal diseases dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine infl uenza virus: a clinical and virological study influenza virus a pathogenicity: the pivotal role of hemagglutinin infection of cesarean-derived colostrum-deprived pigs with porcine circovirus type and swine infl uenza virus genetic and pathobiologic characterization of pandemic h n infl uenza viruses from a naturally infected swine herd who, . manual on animal influenza diagnosis and surveillance experimental dual infection of pigs with an h n swine infl uenza virus (a/sw/hok/ / ) and mycoplasma hyopneumoniae key: cord- -y wjp aj authors: yuan, shishan; mickelson, daniel; murtaugh, michael p.; faaberg, kay s. title: erratum to “complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains” date: - - journal: virus res doi: . /s - ( ) - sha: doc_id: cord_uid: y wjp aj two full-length porcine reproductive and respiratory syndrome virus (prrsv) genomes, strain vr- and its cell culture passaged descendent respprrs vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. of the nucleotide changes, resulted in conservative changes and produced non-conservative changes. the results suggest that key amino acids in orf may contribute to the phenotype of respprrs, which includes increased growth rate on ma- cells and decreased virulence in swine. the results provide a genetic basis for future manipulation of a prrsv reverse genetics system. porcine reproductive and respiratory syndrome virus (prrsv) was first recognized as a 'mystery swine disease' in the united states in (keffaber, ). since the virus was identified in europe (lelystad virus; wensvoort et al., ) and in the usa (vr ; benfield et al., ; collins et al., ) , prrsv has become a serious pathogen of swine herds world-wide (rossow et al., ) . prrsv, along with lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), is a member of the family arteriviridae in the order nidovirales (cavanagh et al., ; plagemann and moennig, ) . prrsv causes respiratory disease in young swine and production of mummified, weakborn or aborted piglets in pregnant sows through an unresolved mechanism. clues to the genetic basis of pathogenicity can be gleaned from comparison of the nucleotide sequence of a virulent parent viral strain with that of the cell-passaged attenuated variant. one such pair is the prototype north american isolate, strain vr- , and resp-prrs, its ma- cell adapted attenuated descendent. in this article, we report the complete nucleotide sequence of both strains of prrsv and have deduced and characterized their nucleotide and protein differences. several key amino acid changes between parental and vaccine strains were identified using this approach, but many other nucleotide and amino acid changes also occurred in genomic regions of unknown function. the nucleotide sequences were also compared to strain b, which is claimed to be a virulent field revertant of respprrs (allende et al., ) . a full comparison of all three prrsv strains shows that the origin of b ('michelle' strain) is unclear. prrsv strains vr- and respprrs have been described in previous reports nelsen et al., ; yuan et al., ) . strain b (michelle) was described by allende et al. ( ) . for growth curve analysis, ma- cells were infected with each prrsv strain at an m.o.i. of . . after h of adsorption, the unattached virus was washed off of the cells and ml of fresh cell medium (emem/ % fbs or dmem/ % swine serum) was added. the culture was placed at °c, % co and . -ml aliquots of virus were removed and replaced with . ml fresh medium at , , , , , , , , and h. viral plaque assays of each time point were completed on ma- cells with a -ml agar overlay (an equal volume of mixture of % low gelling point agarose (fmc seaplaque) and ×mem medium). incubation at °c, % co for - h revealed the presence of plaques, which were quantitated for each time point. prrsv was harvested from infected ma- cells on day post-infection (p.i.). after pelleting of cellular debris at rev./min, the supernatants were layered onto a ml . m sucrose cushion in an sw ultracentrifuge tube and centrifuged at rev./min for h. the pelleted virions were resuspended in ste buffer ( ml; mm nacl/ mm tris ph . / mm edta, °c) and transferred to a microcentrifuge tube. another ml of ste was used to collect any remaining disrupted virions and the two fractions of viral suspension were pooled and stored at − °c. viral rna was isolated using the qiaamp viral rna kit (qiagen) and stored at − °c after quantitation by optical density and native rna agarose gel electrophoresis. approximately mg of purified viral rna in ml was added to ml of mm reverse primer / a-p ( %-ggt-cgttgacaagttggtcatctaccggttt-atcctcgga), incubated at °c for min and placed on ice. first strand cdna synthesis was then completed as described previously (yuan et al., ) . the reverse transcribed product was purified (microcon , amicon) and the cdna was eluted in ml of rnase-free water. multiple adenosine or guanosine residues were added to the %-end of purified prrsv cdna ( mg) using a terminal deoxynucleotide transferase procedure described by the manufacturer (tdt; new england biolabs), diluted to ml and stored at − °c. first round pcr was completed as described previously using mm qt ( %-ccagt-gagcagagtgacgaggactcgagctca-agcttttttttttttttttt) or qc ( %-cca-gtgagcagagtgacgaggactcgagct-caagcccccccccccccccccc) and mm qo ( %-ccagtgagcagagtg-acg) as the forward primers and mm / a-p as the reverse primer (frohman, ; yuan et al., ) . an identical second round of pcr was then completed using ml first round pcr product and qi ( %-gaggactcgagctcaagc) and / a-p ( %-ccttcggcaggcggggagta-gtgtttgaggtgctcagc; mm each) as the primer pair. automated sequencing reactions were completed with taq dyedeoxy terminator cycle sequencing kit (applied biosystems) using a pe thermocycler (perkin-elmer) at the univer-sity of minnesota advanced genetic analysis center. comparison of the parental and vaccine genomes and deduced orf amino acid comparison was completed using computer software included in the lasergene package (dnastar inc., madison, wi), wisconsin package version . and (genetics computer group (gcg), madison, wi). genbank accession numbers used for sequence analysis include the complete vr- (u , nelsen et al., ) , respprrs (af ; yuan et al., ) and b (af ) sequences. primer extension experiments had suggested that nucleotides were unaccounted for in the published sequence of strain vr- . in order to determine the unresolved bases, we derived several clones of pcr products generated from different passages of vr- and respprrs (fig. ) . initially, several standard %-race was performed on passage from the original vr- field isolate brain homogenate, passage of strain vr- and passage of respprrs vaccine strain (frohman, ) . three different %-terminal base patterns were elucidated, with added ambiguity due to the poly t tract added during the %-race procedure, immediately preceding an apparent base prrsv sequence gacguauagguguuggc. interestingly, heterogeneity at the %-end was detected early during passaging of the original prrsv field isolate, yet no heterogeneity was seen after viral plaque purification to produce strain vr- fig. . %-terminal nucleotides of strains vr- and respprrs. vr- (vr) or respprrs (r) from different passages were subjected to rapid amplification of cdna ends (race) and products were cloned and sequenced as described in section . vr- represents sequence submitted to genbank prior to elucidation of the % terminal nucleotides. strain vr- -infected brain homogenate was passaged two times on two separate occasions (vr- a, vr- b), or plaque purified and passaged (vr- ) or (vr- ) times. respprrs was passaged four (r- ) or (r- ) times. bold letters indicate previously unresolved bases and letter in italics refer to the non-viral nucleotides (t or c) arising from the race procedure. ( fig. ). further analysis of pcr clones derived from %-race experiments using poly (g) addition discriminated the remaining % terminal bases of strains vr and respprrs. only one additional base, corresponding to an adenine residue, was identified for strain vr- ( fig. ) . two additional bases, ta, were detected for the % terminal bases of four pcr clones derived from vaccine strain respprrs. the number of subsequent passages shown in fig. ( , , , , ) indicates the relative stability of the cloned virus strains. these results are discordant with a recent report with respect to the first nucleotide of vr and resp-prrs. oleksiewicz and investigators reported a %-terminal u in strain vr- and lack of such in strain respprrs (oleksiewicz et al. ) , whereas we observed a %-terminal u only in the respprrs vaccine strain. the complete leader sequence of respprrs vaccine strain shares . % identity with another prrsv vaccine strain, primepac prrs (shen et al., ) (data not shown). we have determined that the genome size for vr- is bases (u ) and that of respprrs, with an extra %-terminal u, is bases (af ). respprrs leader sequence acquired two additional mutations when passaged for another eight passages at high m.o.i. (ua base and uc at base ). from the sequence data, we cannot determine exactly when the %-terminal thymidine residue of strain respprrs was acquired during passaging. however, the possibility exists that strain respprrs has developed individual genomes of variable nucleotide sequence due to strain evolution over the course of more than passages. the respprrs sequence which we derived from -to -fold coverage of the genome (af ) is somewhat different from the sequence of re-spprrs recently reported (allende et al., ) . complete genome analysis of the parental strain vr- and its cell-culture adapted descendant, strain respprrs, revealed that nu-cleotides had changed during viral strain attenuation. the changes appeared throughout the genome ( fig. a) , except for orf and the % untranslated region (utr), and no single orf exhibited less than the % identity between parental and vaccine strains (fig. b) . of the altered nucleotides, resulted in silent mutations such that the encoded protein was not changed. one of these silent mutations resided in the %-leader, resided in orf , and one silent change was detected in orf . the rest of the nucleotide changes resulted in amino acid changes in each of the remaining identified orfs. detailed analysis of the nucleotide changes, corresponding amino acid changes and potential coding domains are listed in table . for ease of understanding, nucleotide numbering in table is based on the sequence of vr- ( - ). the %-terminal uracil residue of strain respprrs is numbered as zero, and all site designations are one less than the value of the sequence deposited in genbank. the resolution of the % terminal sequence indicated that the % ends of both of these prrsv strains coded for a aa peptide. however, no conclusive evidence has been generated to demonstrate the presence of this potential leader protein. the leader sequence of resp-prrs suggested some virus heterogeneity. in one case (table , mutation a), u a (vr- respprrs) coded for a conservative change at amino acid (f y). in the other case (table , mutation b), the c u nucleotide change, located in the third base of the st codon in this polypeptide sequence, is silent. rna folding predictions (mfold; zucker, ) suggested that each of these nucleotide changes resulted in minor alterations in nucleotide-pairing in stems of secondary structure. the %-terminal bases, an a residue in strain vr- and an at dinucleotide sequence in vaccine strain respprrs, are predicted to extend % from a long stable hairpin located immediately downstream between nucleotides and (data not shown). transversion ( ) g c a analysis of complete genome sequences and classification of amino acid changes were completed using gcg computational biology computer programs. predicted domains of viral proteins was based on hopp-woods analysis of peptide structure (jameson and wolf, ) were also completed using gcg. predicted orf non structural proteins (nsp) were derived from genome comparison of strain vr- with prrsv strain b (allende et al., ) and with equine arteritis virus van dinten et al., ) . b represent two separate sequence analyses of respprrs leader (fig. ) . full-genome schematic the nucleotide differences between vr- and respprrs reveals several changes occurred in orf a, a cluster of changes were seen in the %-terminus of orf b, and discrete changes were seen in all envelope glycoproteins (orfs - ) and in the membrane protein (orf ). when respprrs is similarly compared to strain b, many nucleotide changes are seen throughout the genome. (b) each region of the prrsv genome was analyzed for the number of nucleotide changes and the corresponding orf percent identity between vr- and respprrs. several prrsv orf replicase protein domains have been identified (fig. ; snijder and meulenberg, ; allende et al., ; . from the analysis of both vr- and respprrs genomes, it was deduced that only four of the orf specific nucleotide changes occurred in these identified domains. one mutation, a c u transition, which would alter amino acid of orf from a serine residue in strain vr- to a phenylalanine in the attenuated strain respprrs (s f), resulted in a nonconservative change located in the putative orf cleavage product, nsp (den boon et al., ; allende et al., allende et al., , nelsen et al., ) . a second recognizable mutation, a u c transition, resulted in a change from tyrosine to histidine within the helicase domain. the final orf recognizable mutation was located in the coronavirus-like domain. in this instance, the transversion from g c resulted in amino acid undergoing a non-conservative change from glycine to alanine. a glycine residue at this position, except for strain respprrs, has been shown to be conserved in all nidoviruses analyzed to date . all other amino acid changes located within orf are in regions of unknown function. however, the cluster of amino acid mutations located near the carboxyl terminal end suggests that the replicase region was altered during passage to result in a more fit virus for replication in cell culture, as evidenced by the in vitro one-step growth curve comparison shown in fig. . the growth curve comparison appears to be the reverse of virus strain replication in swine (m. roof, unpublished data). prrsv orfs - have been shown to code for four glycoproteins (gp - ), the membrane protein (m) and the nucleocapsid protein (n), respectively. sequence analysis of strains vr- and respprrs indicated that there were nucleotide changes in this region, and all but one of which resulted in amino acid alterations. there were four amino acid mutations within gp . one amino acid change (aa , lf) was located within the predicted signal sequence of gp , but the nucleotide mutation (g u ) also altered amino acid from an aspartic acid to a tyrosine of a putative orf a protein (wu, christopher-hennings, and nelson, unpublished data) . the remaining three gp changes (aa , a s; aa , k r; aa , v m) were clustered in an eight amino acid region in the middle of the protein sequence, predicted to be located on the exterior of the virion. the three amino acid fig. . viral growth curves of prrsv strains vr- () and respprrs () reveal that respprrs has enhanced growth kinetics in vitro. results are representative of three separate experiments. the number of plaques were determined in triplicate for two separate viral dilutions and were found to deviate by less than % in each individual experiment. vealed that this mutation had reverted back to a vr- -like sequence while maintaining all other orf and mutations and the avirulent viral phenotype (data not shown). the other m protein mutation was shown to be the result of two nucleotide changes within the codon for amino acid , altering the sequence from an arginine to a glycine (r g). curiously, both carboxyl terminal gp and m mutations resulted in a r g change. in , prrsv strain b was isolated from a nebraska herd and has since been shown to be virulent (allende et al., (allende et al., , . full-length genome sequencing revealed that this strain was % identical to vr- and respprrs (allende et al., ) . whether the isolate is a naturally occurring prrsv field variant, or directly related to the original vr- field isolate (isolated in ) or the respprrs vaccine strain (released in ) is not known. to address this question, a full-length genome comparison of strain resp-prrs to strain b was completed, as it has been suggested that strain b is a field revertant of respprrs (allende et al., ) (fig. a) . as can be seen in fig. a , strain b differs from respprrs at many nucleic acid residues. all three strains were analyzed in detail. we compared identical and non-identical nucleic acid residues between strains vr- and respprrs, strains vr- and b, between respprrs and b, and between all three strains. we observed instances in which all three viral genomes contained the identical base, instances in which strains vr- and respprrs were identical but different from strain b, cases in which strains vr- and b were identical but different from strain respprrs, and instances in which strains respprrs and b were identical but different from vr- . importantly, no instances were observed in which the three strains were all different from one another. if strain b were in fact derived from a field reversion of respprrs, the data would imply that for nucleotide mutations b reverted back to the exact vr- nucleotide sequence and in no modifications that occurred in gp (aa , ge; aa , g s; aa , a t) and the two in gp (aa , c y; aa , v a) also are predicted to lie on the exterior of the virion. all of these mutations but one are semi-or non-conservative changes. in the putative viral attachment protein gp , two amino acids were altered during virus attenuation. one gp change occurred within the proposed signal sequence (aa , r q) and the other change (aa , rg) is predicted to be located in the virion interior . gp has been shown to be disulfide linked to the m protein mardassi et al., ) and studies with a similar virus have revealed that this disulfide bond is critical to infectivity . while no mutations occurred in the extravirion region of gp , one mutation did occur within the m protein (aa , q e) predicted to be located on the outer surface of the virus. although this m protein mutation was conservative, mutations in this critical region of the prrsv viral attachment heterodimer could be important. however, analysis of the sixth in vivo backpassage of respprrs re-instance would it have mutated to either of the two possible remaining bases. the likelihood of such a probability is exceedingly low. we also considered the possibility that strain b resulted from a viral recombination event between strain respprrs and an unidentified but closely related field isolate. however, no region of considerable length was clearly identified as being derived from strain respprrs, which one would expect if viral recombination had taken place. therefore, the origin of b remains unclear. an attenuated prrsv strain, respprrs, was found to differ in sequence from the parental strain, vr- , by only nucleotides. this represents a . % variation during passages in ma- cells. this rate of variation is comparable to other rna viruses (steinhauer and holland, ) and suggests that prrsv is not susceptible to inherently high spontaneous mutation rates in the absence of immunological and environmental pressures. this may explain the relative stability of strain vr- when passaged alone in cell culture, as this report delineates. however, prrsv has been shown to undergo high frequency recombination in the presence of two or more virus strains (yuan et al., ) and appears to evolve rapidly in the presence of biological pressure. attenuation can result from changes in many areas of viral genomes and the nucleotide mutations described include alterations in several key prrsv regions. thus, no definitive site of prrsv attenuation can be identified simply from sequence analysis. twelve of the nucleotide changes in strain respprrs were silent. however, one or more of these silent mutations could change the secondary or tertiary structure of prrsv rna and thus alter the stability of the genome. in other viruses, such as picornaviruses, viral rna folding may play a critical role in host protein association (meerovitch et al., ) and in neurovirulence (pilipenko et al., a,b; westrop et al., ) . the % and % ends of viral sequences have been correlated with attenuation in other viruses, such as poliovirus (westrop et al., ) , and shown to be important in viral replication and transcription for coronaviruses, another member of the nidovirus order (williams et al., ) . only two mutations occurred in the %-end of prrsv during attenuation of strain vr- to respprrs, and their relative roles in attenuation must be further investigated. no change occurred in the %-end of the prrsv genome. viruses may also acquire attenuated phenotypes as a result of mutations in viral proteases (ni et al., ) , protease cleavage sites (tozser et al., ) , within the polymerase gene (pelosi et al., ; skiadopoulos et al., ) , by altering viral proteins to decrease virion stability (bailly et al., ) or to interfere with the virus/host interaction (mccright et al., ) , as well as by other mechanisms. the comparison of vr- to its vaccine correlate, resp-prrs, revealed that the possibility exists for one or more of these mechanisms to be involved in attenuation. a recent publication suggested that nine specific amino acid changes may be important in attenuation of respprrs (allende et al., ) , possibly based on the assumption that prrsv attenuation may be localized by amino acid comparison of a non-parental genome ( b) with a parental (vr- ) and its vaccine offspring (respprrs). we have determined that our sequence of the respprrs strain is somewhat different from what has been previously reported (allende et al., ) , which brings additional complexity to the derivation of potential prrsv attenuation sites. in addition, the %-terminal nucleotides for both vr- and respprrs are markedly different from those reported for strain b. in our view, prrsv attenuation sites may be genotype specific and variable, possibly resulting from a complicated interplay of genomic regions and viral and host specific factors. postulation of attenuation sites can lead to interesting hypothesisbased experiments. however, a reverse genetics system for prrsv must be produced and genetically altered at specific nucleotides in order to discern sites of attenuation. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype a recombinant human parainfluenza virus type (piv ) in which the nucleocapsid n protein has been replaced by that of bovine piv is attenuated in primates characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas, and genes of coronaviruses isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases the envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity on beyond classic race (rapid amplification of cdna ends) the antigenic index: a novel algorithm for predicting antigenic determinants reproductive failure of unknown etiology intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus theiler's viruses with mutations in loop i of vp lead to altered tropism and pathogenesis a cellular protein that binds to the %-noncoding region of poliovirus rna: implications for internal translation initiation porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents molecular basis of attenuation of neurovirulence of wild-type japanese encephalitis virus strain sa determination of %-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs a herpes simplex virus dna polymerase mutation that specifically attenuates neurovirulence in mice conservation of the secondary structure elements of the %-untranslated region of cardioand aphthovirus rnas conserved structural domains in the %-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive stranded rna viruses porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage (cp ) human parainfluenza virus candidate vaccine the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses rapid evolution of rna viruses effect of serine and tyrosine phosphorylation on retroviral proteinase substrates proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication mystery swine disease in the netherlands: the isolation of lelystad virus genetic basis of attenuation of the sabin type oral poliovirus vaccine a phylogenetically conserved hairpin-type % untranslated region pseudoknot functions in coronavirus rna replication recombination between north american strains of porcine reproductive and respiratory syndrome virus on finding all suboptimal foldings of an rna molecule the authors wish to thank chris nelsen and faith klebs for excellent technical expertise. boehringer ingelheim vetmedica, inc., provided financial support for the research. key: cord- - yxda mf authors: tian, xinsheng; feng, youjun; zhao, tiezhu; peng, hao; yan, jinghua; qi, jianxun; jiang, fan; tian, kegong; gao, feng title: molecular cloning, expression, purification and crystallographic analysis of prrsv cl protease date: - - journal: acta crystallographica section f structural biology and crystallization communications doi: . /s sha: doc_id: cord_uid: yxda mf cl protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many rna viruses, including porcine reproductive and respiratory syndrome virus (prrsv). in this study, the full-length cl protease from prrsv was cloned and overexpressed in escherichia coli. crystallization experiments yielded crystals that diffracted to . Å resolution and belong to space group c , with unit-cell parameters a = . , b = . , c = . Å, β = . °. the matthews coefficient and the solvent content were calculated to be . Å( ) da(− ) and . %, respectively, for one molecule in the asymmetric unit. porcine reproductive and respiratory syndrome (prrs), also called 'blue ear' disease, is manifested clinically by severe reproductive failure in sows and respiratory distress in pigs of various ages (paton et al., ) . this disease has become endemic in many countries and has caused great economic losses in the swine industry worldwide (tian et al., ; dea et al., ) . viruses of the order nidovirales regulate their genome expression by synthesizing two multidomain precursor polyproteins that are subsequently cleaved into functional viral proteins mainly by the cl protease (den boon et al., ; birtley et al., ; snijder et al., ; van aken et al., ) . the functional importance of the cl protease in the viral life cycle makes it an attractive target for the development of small-molecule drugs. several crystal structures of cl protease have been solved, e.g. those of human coronavirus e (hcov- e; anand et al., ) , severe acute respiratory syndrome-related coronavirus (sars-cov; yang et al., ) , equine arteritis virus (eav; barrette-ng et al., ) and transmissible gastroenteritis virus (tgev; anand et al., ) . despite the common structure of the cl proteases determined to date, consisting of two -barrels and a unique c-terminal domain, there are many significant differences between them, especially in the linker between the c-terminal domain and the c-terminal -barrel ( barrette-ng et al., ) . we have initiated a study aimed at determining the threedimensional structure of cl proteases in order to understand their function from a structural perspective and to provide a platform for the screening of new drugs. here, we report the crystallization of the cl protease from prrsv and its preliminary crystallographic analysis. viral genomic rna was extracted from marc- cell cultures which had been inoculated with the prrsv isolate (gi| ) using the rneasy plus mini kit (qiagen, germany). the reverse transcription (rt) was undertaken using the primers cl-f ( -cacatgcttgctggtgttta- ) and cl-r ( -gccac-ggtatcggcaaaagc- ). after the single-tube rt reaction, ml of pcr product was used as a template to amplify the coding sequence (cds) of prrsv cl protease with specific primers cl-f ( -cccgggcgctttcagaactcaaaa- ) and cl-r ( -ccgctcgagttattccagttcgggtttggc- ). the amplified dna fragment encoding the prrsv cl protease was inserted into the gst-fusion expression vector pgex- p- (ge healthcare) with smai/xhoi sites (introduced by pcr primers). the resultant positive clone, designated pgex:: cl, was confirmed by double-enzyme digestion and verified by direct dna sequencing. for protein expression, the plasmid was transformed into escherichia coli strain bl (de ) competent cells and the single colony was inoculated into luria-bertani (lb) medium with mg l À ampicillin (sigma, usa) at k for overnight growth. the overnight culture was then transferred into a flask containing l fresh lb medium. when the culture density (od ) reached . , the culture was induced with . mm isopropyl -d-thiogalactopyranoside (iptg; sigma, usa) and kept for $ . h at k until collection of the bacteria. the harvested culture was centrifuged at rev min À for min at k and the pellet was resuspended in chilled pbs ( mm nacl, . mm kcl, mm na hpo , . mm kh po ph . ) and homogenized by sonication. the lysate was centrifuged at rev min À for min at k and subsequently filtered through a . mm membrane for clarification. the supernatant was then loaded onto a glutathione-sepharose b column (ge healthcare). the protein-loaded column was washed with ten bed volumes of pbs and ten bed volumes of cleavage buffer ( mm tris-hcl, mm nacl, mm edta, mm dtt ph . ). to obtain gst-free protein, gst- c rhinovirus protease (kindly provided by drs j. heath and k. hudson) was added to the resin at a concentration of units per millilitre of bed volume. after the mixture had been incubated with gentle agitation overnight at k, the target protein was eluted with ml cleavage buffer (fig. ) . the harvested protein was then concentrated to a suitable concentration by ultrafiltration ( kda cutoff) and loaded onto a superdex column (ge healthcare) with an akta purifier system (ge healthcare) at a flow rate of . ml min À . the fractions containing the protein peak were collected and analyzed using % sds-page. for crystallization, the purified protein was concen- trated to approximately mg ml À and the buffer was exchanged to mm tris-hcl, mm nacl, mm edta, mm dtt ph . . the purified cl protease after gel filtration was dialyzed against cross-linking buffer ( mm hepes ph . , mm nacl) and concentrated to approximately mg ml À by ultrafiltration ( kda cutoff). subsequently, various concentrations ( , . , . , . mm) of bis-succinimidylsuccinate (egs; pierce) were added to the protein sample; the gst protein was used as a positive control (ma et al., ) . the reactions were incubated for h at room temperature and quenched with mm glycine. finally, the cross-linked samples were analyzed by % sds-page (fig. ) . crystallization trials were performed with crystal screens i and ii (hampton research) at k using the hanging-drop vapourdiffusion method. ml protein solution (at a concentration of , or mg ml À ) was mixed with ml reservoir solution and equilibrated against ml reservoir solution. x-ray diffraction data were collected in-house on a rigaku micromax rotating-anode x-ray generator operated at kv and ma (cu k; = . Å ) equipped with an r-axis vii** image-plate detector. the crystal was mounted in a nylon loop and flash-cooled in a cold nitrogen-gas stream at k using an oxford cryosystem. the cryoprotectant solution contained % glycerol and % reservoir solution. data were indexed, integrated and scaled using denzo and scalepack as implemented in hkl- (otwinowski & minor, ) . prrsv cl protease was partially generated in soluble form when expressed in e. coli. induction at k proved to increase the yield of soluble gst- cl protease (data not shown). after removing the n-terminal gst tag of the fusion protein, purified cl protease was obtained with nine additional residues (gplgspefp) at the n-terminus. with these nine extra residues, the protein product is amino acids in length, corresponding to a molecular weight of about . kda. % sds-page showed that the peak for the expressed protein occurs at the position expected for its size (fig. ) . the recombinant prrsv cl protease was also confirmed by peptide mass fingerprinting (data not shown). a subsequent chemical crosslinking experiment clearly indicated that the prrsv cl protease was in a monomeric form (fig. ) , in comparison to the dimeric form of gst. crystallization of cl protease proved to be easy under several conditions, indicating its stable tightly packed structure. a crystal grown (within d; fig. a ) from a condition consisting of . m sodium cacodylate trihydrate ph . , . m sodium acetate trihydrate diffracted x-rays to . Å (fig. b) and belongs to the monoclinic space group c , with unit-cell parameters a = . , b = . , c = . Å , = . . data-collection statistics are given in table . there is only one molecule per asymmetric unit. the matthews coefficient and the solvent content were calculated to be . Å da À and . % (matthews, ) , respectively. initial molecular-replacement calculations were performed using the program amore (navaza, ) using the crystal structure of equine arteritis virus (eav) cl protease as a search model (pdb code mbm), but no distinct peaks were obtained. this suggests that there are many differences in the three-dimensional structures of the cl proteases from prrsv and eav, despite their significant sequence homology ( % identity at the amino-acid level). ) . † r merge = p hkl p i ji i À hiij= p hkl p i i i , where i i is the intensity of an individual measurement of a reflection and hii is the mean value for all equivalent measurements of this reflection. proc. natl acad. sci. usa key: cord- -jxqi exm authors: wang, ye; chen, yihui; liang, ge; zeng, kai; chen, xiao-hui; ying, san-cheng; wang, zezhou; lv, xue-bin; gao, rong title: silence of tgf-β gene expression reduces prrsv replication and potentiates immunity of immune cells of tibetan pig date: - - journal: vet anim sci doi: . /j.vas. . sha: doc_id: cord_uid: jxqi exm transforming growth factor beta (tgf-β ) was of importance in the pathogenesis of porcine reproductive and respiratory syndrome virus (prrsv). to determine whether knockdown of tgf-β gene expression could facilitate the control of prrsv infection, specific sequences for expressing shrna targeted to porcine tgf-β gene were synthesized and cloned into psilencer . -h neovector. then they were used to transfect peripheral blood mononuclear cells of tibetan pig (tp-pbmcs) followed by prrsv inoculation. the positive recombinant plasmids were screened for inhibition of tgf-β gene expression by real-time quantitative rt-pcr. conversely, the mrna level of prrsv in shrna treated tp-pbmcs dramatically decreased, and there were significant increases of the transcription of immune genes, such as interleukin- (il- ), interleukin- (il- ), interferon-alpha (ifn-α), interferon-gamma (ifn-γ), tumor necrosis factor-alpha (tnf-α), toll-like receptor (tlr ), toll-like receptor (tlr ), myeloid differentiation primary response gene ( ) (myd ), and interleukin- p (il- p ). however, the expressions of il- and il- genes significantly reduced in comparison to the control infected cells. in addition, transfection with the shrna plasmids significantly elevated the viability of immune cells. therefore the knockdown of tgf-β gene expression by shrna not only inhibits the replication of prrsv but also improves immune responsiveness following viral infection, suggesting a novel way to facilitate the control of prrsv infection in pigs. porcine reproductive and respiratory syndrome virus (prrsv) is an enveloped, single-stranded positive-sense rna virus, belonging to the family arteriviridae, genus rodartevirus (kuhn et al., ) . this highly variant virus can cause respiratory disease, abortions, and secondary viral and/or bacterial infection of all-aged pigs, resulting in long-term infection and widespread complex disease by inhibiting immune defense of host, and have been causing major economic lose worldwide (li et al., ) . some viral proteins are multifunctional to not only maintain viral structure but also modulate immune response to facilitate viral survival by changing the numbers and functions of immune cells, including promoting the production of tgf-β and il- to impair the equilibrium of pro-inflammatory cytokines and anti-inflammatory cytokines, and summoning non-neutralizing antibody instead of neutralizing antibody (dwivedi et al., ; gómezlaguna et al., ; huang & meng, ) . tgf-β contributes greatly to maintain immune homeostasis and tolerance through regulating the differentiation, proliferation and activity of multiple leukocyte lineages, and inhibiting the production and signaling of effector cytokine in smad-dependent and -independent mechanisms (zhang, ) . actually, both prrsv- and prrsv- have been demonstrated to induce over expression of tgf-β, in order to unbalance immune system, disarm host surveillance and finally benefit viral survival (gómezlaguna et al., ; renukaradhya, alekseev, jung, fang, & &saif, ; silva-campa et al., ). among three tgf-β isoforms, tgf-β is predominant in immune system that has potent immunoregulatory properties like il- ( gómezlaguna et al., ; gorelik, ; yoshimura, wakabayashi, & mori, ) , and it is able to inhibit macrophage activation by inhibiting ifn-γ synthesis and promoting il- production (gómezlaguna et al., ) . in contrast, tgf-β and tgf-β are mainly expressed in mesenchymal tissues and bones (gorelik, ) . as a result, so we wonder whether knocking-down tgf-β gene expression can benefit immune cells to control prrsv infection. so far, unfortunately, the effect of down-regulation of tgf-β on prrsv infection is rarely observed. therefore, in this experiment, rnai was exploited to reduce tgf-β expression and subsequently evaluate its effect on prrsv replication and anti-viral immune responses peripheral blood of healthy tibetan pigs were collected from the superior vena cava in -ml tubes (kangjian, jiangsu) containing k -edta as the anti-coagulant, and tp-pbmcs were isolated by density gradient centrifugation ( g, min) in separation medium for mononuclear cells of pigs (haoyang, tianjin). after x washes in hanks' balanced salt solution without ca + /mg + and % fetal bovine serum (fbs), cells were suspended at a concentration of no more than × cells/ml in rpmi medium, supplemented with % (vol/ vol) penicillin/streptomycin mixture and % (vol/vol) heat-inactivated fbs (all from hyclone, beijing). the cells were incubated in cell repel plates (eppendorf company, germany) at °c in % co for h before use. the ratio of monocytes in the cultured cells before transfection was checked by the flow cytometry with . ug/ml mouse anti-cd monoclonal anitbody following the recommended protocol (abcam, ab , america). short interfering hairpin odns were designed based on porcine tgf-β mrna (nm_ , ) using online sirna design software (http://jura.wi. mit.edu/bioc/sirna; http://www.ambion.com/techlib/misc/sirna_finder. html). the four selected sequences were listed in table . the sequences (table ) were synthesized by invitrogen (shanghai) and cloned into the vector psilencer . -h puro (biovector science lab inc.). escherichia coli dh α were transformed with the recombinant plasmids (shil rα− ~ ), which were then amplified in lb medium supplemented with µg/ml ampicillin. the plasmids were then isolated using plasmid mini kit i (omega) and verified by restriction analysis with bamh i and hind iii (takara, dalian), and by nucleotide sequencing (bgi, beijing). as the core sequence of the vector had no target in the refseq mrna of sus scrofa (taxid: , ncbi), it was set as a negative control (pneg). all plasmids for transfection contained < . eu/ml endotoxin as assayed by tachypleus amebocyte lysate test kit (a&c biological ltd, zhanjiang). the transfection mixtures were prepared according to the manufacturer's instructions. briefly, μg individual plasmid (shtgfβ - ~ and pneg) or μl cationic liposome transfection reagent dmrie-c (invitrogen, usa) was diluted in opti-mem i reduced serum medium (invitrogen, usa) to μl. following min at ambient temperature, the plasmids were added to the diluted transfection reagent. brief mixing at ambient temperature for min formed the transfection complex completely. during this period, the tp-pbmcs were prepared for transfection. cells were centrifuged at g for min, and washed with serum-free rpmi without antibiotics. the tp-pbmcs were resuspended in serum-free rpmi without antibiotics at . × cells per ml and . ml aliquots ( × cells) were added to each well of a -well culture plate. the transfection complex was added and mixed with cells, then incubated at °c, % co for . h, ml prewarmed rpmi containing % fbs and . % penicillin/streptomycin mixture was added to the tp-pbmcs with μl prrsv(jxa strain, gene type ii, its infection dose was . tcid /ml) per ml medium. cells were again incubated at °c, % co , and tp-pbmcs were collected daily for days as followings: after centrifuge at , g for s, the cell pellets were lysed by ml rnaisoplus (takara, dalian) and frozen at − °c before rna isolation. the negative control cells transfected with only μg shtfgβ - or pneg, and blank control cells without plasmid and the virus were also cultured and processed in the same way. the viability of pbmcs was assayed by -sea cck- ( -sea, shanghai), following the manufacturer's instructions. in brief, treated tp-pbmcs were added at the concentration of × cells per μl rpmi complete medium to each well with μl cck . the cells were oscillated for s and incubated at °c, % co for h, and its absorbance was read by the microplate reader model (bio-rad) at nm. the viability was checked every h until hpi. rna was isolated following the manufacturer's instructions of rnaisoplus (takara, dalian). the rna pellet was washed with ml % ethanol, followed by brief vortex, and then was centrifuged at g for min at °c. the rna pellet was dried for ~ min, and dissolved with μl rnase-free water. absorbance at nm and nm was assayed to detect the concentration and . % agarose gel electrophoresis was used to check its integrity. after assay for rna integrity and concentration, reverse-transcription pcr was performed using transcript one-step gdna removal and cdna synthesis supermix (transgen, beijing). real-time pcr was conducted in a total reaction volume of μl, consisting μl cdna, . μl ssofast evagreen supermix (biorad, singapore), and . μl forward and reverse primers were used to detect the relative mrna level of prrsv in the rna extracted from the cultured samples, which can amplify the conserved region of glycoprotein of prrsv rna from bp to bp (genbank: ky . ) . the pcr condition was °c ( mins), and cycles of °c ( s), annealing temperature ( s), °c ( s). at the end of the pcr cycle, melt curve analysis was performed at an increment of . °c per cycle from °c to °c. tata box binding protein (tbp) and dna topoisomerase -β (top -β) were used as reference genes for normalization of gene expression. all primer sequences, their annealing temperature and amplification efficiency were listed in table , and the amplified fragments is bp. the relative expression level of gene was calculated by -ΔΔct. all the experiments were performed triplicate. data were expressed as means ± sd. statistical analysis of data was performed by two way anova and sidak multiple comparison. differences were considered significant if p< . . sequences of odns for generation of shtgfβ - ~ . wang, et al. veterinary and animal science ( ) after h culture in vitro and centrifuge washing, plenty of harvested leukocytes ( . ± . %) were proved to be monocytes with mouse anti-cd antibody (phycoerythrin);therefore, the harvested mononuclear cells are eligible for further prrsv infection experiment. the four potential interfering candidates were detailed in table . before further study, their down regulatory efficiency was evaluated; shtfgβ - was proved to be most potent in down-regulating the mrna level of tfg-β (p< . ) (fig. ) , so it was chosen for all following experiments. further study was performed to see the persistent effect of shtfgβ - on tfg-β expression. apparently, shtfgβ - inhibited the uptranscription of tfg-β gene caused by prrsv for h (p< . ) (fig. a) . in addition, the mrna levels of immune-related genes were analyzed to observe the influence of shtfgβ - and exclude the possible side effect of transfection reagent and pneg. as listed in table , h culture after transfection, no significant changes of the expressions of most immune genes were observed among the three groups (p> . ) except for tgf-β of shtfgβ - group (p< . ), indicating that the only shtfgβ - plasmid itself is not stimulatory and just exerts negligible effect on the gene expression of leukocytes except for tgf-β . the mrna level of prrsv peaked at h post infection (hpi) in tp-pbmcs, while shtfgβ - effectively inhibited the increase of prrsv ( fig. a) (p< . ) . meanwhile, cell viability of tp-pbmcs was analyzed (fig. b) , and prrsv obviously reduced cell viability (p< . ). however, the interfering plasmid evidently raised the cell viability (p< . ). the shtgfβ - ~ groups were transfected with the recombinant plasmid. the ps-prrsv group was transfected with pneg instead of recombinant interfering plasmid, while the dmrie-c group was treated with the transfection reagent only. after incubation for h, the mrna level of tgf-β is determined. all experiments are performed in triplicate. all results were shown as means ± sd. in all cases, different superscript letters indicate that there are statistically significant differences among groups (p< . ), and vice versa (p> . ). the followings are the same as here. the mrna levels of several immune genes were determined to analyze the effect of shtfgβ - on immune responses of tp-pbmcs. the shtfgβ - obviously inverted the elevation of mrna level of tgf-β gene that was provoked by prrsv (p< . ) (fig. a) . however, the expression of tfgβ gene was inhibited by prrsv (p< . ) (fig. b) , while the shtfgβ - elevated its expression for the first h (p< . ), suggesting tgf-β could possibly compensate the absent function of tgf-β in vitro. tlr mrna level was not obviously modulated by prrsv, while shtgfβ - obviously increased its expression compared to the control (p< . ) (fig. a) . unlike tlr , tlr gene was firstly activated by prrsv at first h, and its mrna level rapidly decreased thereafter with shtgfβ - incubation (fig. b) . the expression of myd and ifn-α gene were significantly inhibited by prrsv (p< . ); in contrast, the shtgfβ - dramatically elevated the mrna level of both genes (p< . ) (fig. c and d) . besides, the mrna level of tnf-α was reduced by prrsv infection at the first h, and thereafter restored to the control level (p> . ); whereas it was markedly increased by shtgfβ - (p< . ) (fig. e) . similarly, the shtgfβ - plasmid also suppressed the increased expression of il- gene, which was induced by prrsv (p< . ) (fig. f) . shtgfβ - remarkably up-regulated the transcriptional level of il- , il- and il- p gene to different extents (p< . ), all of which were slightly modified by prrsv (fig. a, b and c ). in addition, prrsv significantly reduced the expression of ifn-γ gene (p< . ), while shtgfβ - could obviously elevated its expression (fig. d) (p< . ) . meanwhile, the il- mrna level was increased by prrsv and peaked at hpi, and its transcriptional level was also down-regulated by shtgfβ - compared to prrsv group (p< . ) (fig. e ) . normally, the porcine aveolar macrophages are very sensitive and suitable target cells for prrsv. herein, instead of high-cost and trouble washing lung to obtain macrophages, we just collected monocytes/ macrophages from cultured pbmcs for prrsv infection, in which target monocytes/macrophages was proved to be plenty of leukocytes after h incubation in vitro and centrifuge in our experiment. to get rid of the possible loss of adherent monocytes/macrophages during our culture of pbmcs, we purposefully employed special cell culture plates, cell repel plates with ultra-low adherent surface from eppendorf company. also, we thoroughly washed the plates when we harvested the cultured cells for centrifuge, and carefully checked whether monocytes/macrophages still existed in the harvested cells by use of mouse anti-cd monoclonal anitbody (ab ) from abcam in flowcyt. therefore, we found that there were still plenty of monocytes/ macrophages remained in the harvested cells before infection with prrsv. furthermore, we detected the burst mrna increase of prrsv during the culture of the infected cells, which was clearly manifested in the fig. (a) , confirming that pbmcs could support the replication of prrsv in vitro and is qualified for further rnai experiment. it had been reported that the production of tgf-β could be induced by prrsv infection (gómezlaguna et al., ) , and it was the main effector of prrsv induced tregs (also known as th subset) (silva-campa et al., ; yoshimura et al., ) . over expressed tgf-β, reduced il- , ifn-γ and tnf-α expression were found in the prrsv infected group, which in turn dampened the host immune response during prrsv infection (han, zhou, ge, guo, & yang, ) . the antiviral capability of tlr could be impaired by prrsv replication to facilitate virus evading host immune responses (miller, lager, & kehrli, ) , which was in accordance with the results in this study, tlr mrna decreased to a half when the tp-pbmcs were infected with prrsv, and myd was just beyond the detectable threshold of qpcr. the slightly inhibited il- expression might hinder the recruitment of immune cells, and consequently slow down virus dissemination and infection, which was consistent with earlier report (reeth, gucht, & pensaert, ) . besides, the mrna level of il- subunit was not changed obviously by prrsv infection, implying that it was not critical factor utilized by prrsv to thwart host immune system. tgf-β was a multifunctional cytokine, dampening its expression might lead to severe damages to cells. reports had demonstrated that tgf-β , to some extent, could compensate the deficiency of tgf-β in vitro (liu et al., ; lucas, kim, melby, & gress, ) . the mrna level of tgf-β was evidently increased in the early interfering plasmid transfection compared to the infected group. however, it is not clear whether this compensation could synergize the immune suppression of tgf-β or just maintain the essential viability or growth of treated cells, which need further research later. given the indispensable status of tgf-β, we exploited rnai method to knockdown tgf-β to observe whether it could be feasible to inhibit prrsv production and alleviate immune response to some extent. as table the relative mrna levels of immune genes in tp-pbmcs transfected with shtfgβ - and pneg plasmid and cultured for h. transfection media control pneg shtfgβ - il- . ± . a . ± . a . ± . a il- . ± . a . ± . a . ± . a il- . ± . a . ± . a . ± . a ifn-γ . ± . a . ± . a . ± . a myd . ± . a . ± . a . ± . a tnf-α . ± . a . ± . a . ± . a ifn-α . ± . a . ± . a . ± . a tlr . ± . a . ± . a . ± . a il- . ± . a . ± . a . ± . a tgf-β . ± . a . ± . a . ± . b il- p . ± . a . ± . a . ± . a fig. . inhibition effect of shtgfβ - on prrsv replication (a) and its promotion of cell viability(b) of the treated cells. y. wang, et al. veterinary and animal science ( ) expected, prrsv replication could be markedly depressed in tp-pbmcs to about . %, and the cellular viability could be obviously prompted to ~ % accompanied with minimal tgf-β , indicating the essential role of tgf-β for prrsv replication again, and also implying a promising strategy for prrsv resistance. it was also confirmed that shtfgβ - just specifically down-regulated the expression of tfgβ - gene and did not provoke expressions of other immune genes during the transfection. however, tlr was obviously induced by prrsv in first h pi, following swift decrease in shtfgβ - + prrsv group to the same level of ctrl group. because prrsv was a positive ssrna virus, tlr could recognize the viral genome prior to detection of dsrna replicative intermediate by tlr , and before prrsv replication, some viral proteins which could inhibit tlr transcription might have been expressed. nevertheless, the mrna levels of these three genes were elevated after tgf-β expression was down-regulated, which suggested the fig. . mrna levels of tgf-β (a) and tgf-β (b) gene in the treated cells. the shtgfβ - +prrsv group was transfected with shtgfβ - plasmid prior to prrsv inoculation at the concentration of μl per ml. y. wang, et al. veterinary and animal science ( ) suppressed innate immune responses were restored. the levels of il- β and ifn-γ were linked to prrsv resistance and clearance (charerntantanakul, yamkanchoo, & kasinrerk, ; darwich, díaz, & mateu, ) . increased il- , ifn-γ and tnf-α expression were observed following shtfgβ - transfection, which could be indication of effective th anti-prrsv responses. in addition, the sharply increased mrna levels of il- also implied that th response was activated. obvious repression of il- expression was observed in the shrna treated group, suggesting that inhibition of tgf-β expression could lead to the reduction of il- , which probably result from the evidently increased il- , ifn-γ, ifn-α and tnf-α expression and facilitate the enhancement of the antiviral immunity to clear the infected virus. we observed this promotion of immunity at least lasted h in vitro and could maintain longer time in vivo until the shrna could not be transcribed in the infected cells, which might be continuous form to days or so depending on doses. furthermore, we just analyzed the mrna expression levels of the tlrs and cytokine genes by real time quantitative rt-pcr technique, though it is the most sensitive and economical method to analyze gene expression, our present observations could be consolidated through the detection of translation products of these cytokines and tlrs by elisa and further utilization of shtfgβ - in vivo later. our experiment firstly screened out effective specific shrna targeted to pig tgf-β gene, it could significantly knock down the mrna level of tgf-β gene and obviously increase the viability of prrsv infected cells. the knockdown of tgf-β gene expression by shrna could not only lead to significant inhibition of prrsv replication in pig immune cells, but also enhance the antiviral immune responses and reduce the prrsv yield in the infected cells. these results could inspire the development of novel promising way to prevent prrsv infection of pig and promote the immunity of animal against viral disease. the authors declare no competing financial interests. the manuscript only contains experiments using pig blood, we assured that the care and use of experimental pigs completely complied with chinese animal welfare laws, guidelines and policies. y. wang, et al. veterinary and animal science ( ) plasmids expressing porcine interferon gamma up-regulate pro-inflammatory cytokine and co-stimulatory molecule expression which are suppressed by porcine reproductive and respiratory syndrome virus certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions enhanced expression of tgfβ protein in lymphoid organs and lung, but not in serum, of pigs infected with a european field isolate of porcine reproductive and respiratory syndrome virus abrogation of tgfbeta signaling in t cells leads to spontaneous t cell differentiation and autoimmune disease pathogenesis and control of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) reorganization and expansion of the nidoviral family arteriviridae mutations in a highly conserved motif of nsp beta protein attenuate the innate immune suppression function of porcine reproductive and respiratory syndrome virus a critical function for tgf-β signaling in the development of natural cd cd foxp regulatory t cells disruption of t cell homeostasis in mice expressing a t cell-specific dominant negative transforming growth factor β ii receptor role of toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus cellular and molecular basis for the regulation of inflammation by tgf-beta non-smad pathways in tgf-beta signaling this work was financially supported by key projects of sichuan province ( nyz ), national natural science foundation of china (no. ) and national international cooperation program (no. dfa ). we sincerely thanked dr. gang wang (national engineering research center for biomaterials) for technical support. key: cord- -ejhz rra authors: kappes, matthew a.; faaberg, kay s. title: prrsv structure, replication and recombination: origin of phenotype and genotype diversity date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ejhz rra porcine reproductive and respiratory disease virus (prrsv) has the intrinsic ability to adapt and evolve. after years of study, this persistent pathogen has continued to frustrate efforts to eliminate infection of herds through vaccination or other elimination strategies. the purpose of this review is to summarize the research on the virion structure, replication and recombination properties of prrsv that have led to the extraordinary phenotype and genotype diversity that exists worldwide. porcine reproductive and respiratory syndrome virus (prrsv) is the etiological agent of a worldwide epidemic designated porcine reproductive and respiratory syndrome (prrs). prrsv is highly host and tissue restricted to swine cells of the monocyte lineage, preferentially infecting the porcine alveolar macrophage (amΦ) and is a persistent virus (duan et al., ; villarreal et al., ) . prrsv first emerged in the late s as a "mystery" disease progressing through swine populations in both europe and north america collins et al., ; hopper et al., ; morin et al., ; wensvoort et al., ) . prevailing clinical symptoms were noted to be respiratory distress in young swine and widespread reproductive failure in pregnant sows including mummified, stillborn and aborted fetuses (goyal, ) . initial characterization of circulating european (type ) and north american (type ) genotype isolates was found to be surprisingly genetically divergent. although overall disease phenotype, gross clinical symptoms, genomic organization and temporal emergence were all similar, these strains differed by $ % at the nucleotide level collins et al., ; meulenberg et al., ; nelsen et al., ; wensvoort et al., ) . the degree of genetic heterogeneity suggests a protracted period of independent evolution on the two continents . molecular clock analysis predicts the divergence of the two genotypes from a common ancestor between a decade to over a century prior to clinical recognition (forsberg et al., ; yoon et al., ) , presumably from another host species (hanada et al., ; plagemann, ) . the origin of prrsv remains unknown and no secondary animal, human, or arthropod vectors have been identified to date (otake et al., a (otake et al., , b schurrer et al., schurrer et al., , zimmerman et al., ) . in the $ years since the first emergence of prrsv, a global near worldwide epidemic has been sustained by a set of emerging and re-emerging strains supported by high frequency mutation and recombination (goldberg et al., ; meng, ; murtaugh et al., ) . prrsv remains the most economically devastating disease of swine and contributes to the deterioration of animal health through disease and the continual emergence of increasingly divergent and often virulent strains (gauger et al., ; han et al., ; holtkamp, ; tian et al., ) . other reviews have recently assessed the nidovirus family in broad terms, concentrating on what is similar between the family members and choosing to concentrate on cellular pathogenesis, but often leaving critical prrsv-specific details out of their discussions. therefore, the intent of this review is to examine what is known about prrsv virion structure and replication mechanisms that contribute to the highfrequency mutation and recombination observed, resulting in a vast array of phenotypically and genotypically divergrent strains. prrsv is a member of the arteriviridae family within the order nidovirales, which also includes the coronaviridae and roniviridae families. the nidovirus order constitutes a group of singlestranded positive-sense rna viruses which share a hallmark replication/transcription strategy, similar genomic organization, and a defining set of genetic elements, but differ in host species and range, disease phenotype, virion morphology, cellular tropism, genomic size and encoded content . the arteriviridae family is composed of five viruses which share similar genetic and biological characteristics such as genomic organization and content, morphology and a cellular tropism for the macrophage lineage . viral species include prrsv, simian hemorrhagic fever virus (shfv), lactate-dehydrogenase elevating virus (ldv), newly recognized wobbly possum disease virus (wpdv) in free-ranging australian brushtail possums (trichosurus vulpecula) and equine arteritis virus (eav; arterivirus prototype species) dunowska et al., ; plagemann and moennig, ) . the similar genomic organization, characteristic genetic elements and common functionality of orthologous proteins however have led to the acceptance of many putative functions for prrsv proteins, often derived from studies of the arterivirus prototype species eav and the more distantly related nidoviruses. each arterivirus infects only one animal species, in contrast to other nidoviruses such as some coronaviruses, which have been shown to transmit between species (hilgenfeld and peiris, ) . the prrsv genome varies from . kb to . kb in length and expresses a range of accessory and structural proteins through two distinct transcription mechanisms. the genomic organization and associated expression profiles are depicted in fig. . the prrsv genome encodes a proximal noncoding element ( -untranslated region; utr) of - nucleotides (nt; type ) and - nt (type ) in length yun and lee, ) . the utr functions will be discussed below. directly downstream of the utr has the large overlapping replicase open reading frames (orf). the orf a/b share a single translational start site but are augmented by two ribosomal frameshift (rfs) sites at genomic positions nt (rfs ; nonstructural protein (nsp) ) and nt (rfs ; nsp / ) [vr- (u ) reference sequence] meulenberg et al., b; nelsen et al., ; li et al., ) . two products are generated from the rfs site; a - rfs occurs approximately % of the time and results in an immediate termination of translation (nsp n) , and a - rfs event occurs at $ % efficiency and yields a translational extinction in the - coding frame through the putative transmembrane domain of nonstructural protein (nsp ) . the large replicase polyproteins pp a, pp a-nsp n, pp a-nsp tf, and pp ab are generated from full-length genomic rna. using the type prototype strain vr- (u ) for a reference, orf a/b is encoded by a proximal segment of approximately kb [ nt orf a, nt orf b] yielding four distinct polyproteins including pp a-nsp n ( amino acids (aa); - rfs at rfs ), pp a-nsp tf ( , aa; - rfs at rfs ), pp a ( , aa), and pp ab ( , aa; - rfs at rfs ) (fig. ) . the replicase polyproteins are co-translationally and posttranslationally processed into at least distinct nonstructural proteins (nsp) via the rfss and four virally encoded proteinases including papain-like cysteine proteinases α (plp α; nsp α), plp β (nsp β) and plp (nsp ), and the main serine proteinase (sp; nsp ) snijder et al., ) . plp α and plpβ function to cleave the nsp α ↓ nsp β and the nsp β ↓ nsp junction, respectively; plp is responsible for the cleavage of the nsp ↓ nsp junction and the main sp processes all remaining nsp products (nsp - ) (han et al., ; snijder et al., ) . orf a encodes pp a encompassing nsps - and orf ab encodes pp ab composed of all known nsps (nsp α/β, nsp - , nsp α/β, nsp - ) whereas nsp is a translational extension of nsp via a programmed - rfs at position vr- nt (rfs ) ( fig. ) (meulenberg et al., b; nelsen et al., ) . in contrast, the previously recognized structural proteins are encoded and individually expressed by a set of subgenomic rnas (sgrna) generated through a negative-strand intermediate (sgrna - ; fig. ) (van marle et al., a) . sgrnas are genetically polycistronic (except rna ) but are assumed to be functionally monocistronic/bicistronic, where only the terminal orf(s) is expressed (fig. ) . sgrna encodes orf a/b which is translated to yield glycoprotein (gp ) and a small unglycosylated envelope protein (e); orf is expressed from sgrna to yield gp ; and sgrna encodes orf yielding gp . together gp , gp , and gp form a trimeric complex resulting in the minor glycoprotein complex which functions in viral entry and is heavily nglycosylated (das et al., ; wissink et al., ) . sgrna encodes orf and orf a. orf a codes for the orf a protein, a small unglycosylated protein that is required for virus viability and orf codes for gp , the major glycoprotein with a variable number of n-glycan residues surrounding the cell attachment domain (johnson et al., ; mardassi et al., ; robinson et al., ) . orf is expressed from sgrna , resulting in the generation of the membrane protein (m). gp and m form a disulfide-linked heterodimer and together constitute the major glycoprotein complex on the virion, as was first shown for ldv (faaberg et al., ; mardassi et al., ) . finally, the nucleocapsid protein (n) is encoded by orf and is expressed from sgrna . n is the major structural element within the prrsv virion which forms disulfide-linked homodimers, functions to package the viral genomic rna (grna), and is the only known structural protein which does not encode a transmembrane domain or to not have an ectodomain upon the prrsv virion (bautista et al., ; dea et al., ; doan and dokland, ; loemba et al., ; spilman et al., ; wissink et al., ; wootton and yoo, ) . recently, the nsp protein, coded for by the most variable region of the genome with insertions and deletions, was also shown to be incorporated into or onto ultrapurified virions of several prrsv strains as a set of differently sized protein isomers, presumably through its four to five membrane spanning regions near the c-terminal end (kappes et al., ) (han et al., ; kappes et al., ) . this surprising result increases the number of viral proteins, or more (full-length nsp and its isomers, nsp tf, gps - , e, m, n, orf a), that are exposed to the porcine immune system on entry of prrsv into swine alveolar macrophages veit et al., ) . the original work suggests that only of these (gp , m, and n) make up the majority of the protein content of prrsv (drew et al., ; mardassi et al., a mardassi et al., , meulenberg et al., ; nelson et al., ) . however, the immense genetic and protein variation of all of these structural proteins, from the least conserved nsp region (han et al., ; tian et al., ) to the most conserved m protein (murtaugh et al., ; veit et al., ) , shows the complexity and the plasticity of the prrsv genome and virion structure. the and utrs flank the core protein coding regions of the prrsv genome (fig. ). both the and utrs are implicated as essential components contributing to the viral strategies imparting replicative and translational functionality; however, the exact functions of the and utrs, and the associated mechanisms of interaction, are poorly understood. both encode conserved putative rna secondary structures important to replicative function. the utr is encoded first within the prrsv genome and possesses a putative type i cap structure (sagripanti et al., ) . the utr is genetically variable, type and strains share approximately % genetic homology, and within each genotype, the pairwise identity is about % (lin et al., ; meulenberg et al., a; nelsen et al., ; oleksiewicz et al., ; tan et al., ) . detailed studies of the distantly related coronavirus species, as well as arteriviruses, have shown the utrs are regulators of genomic replication, transcription, and mrna translation, and are considered a necessary docking site for a variety of viral and host factors to complete these functions (choi et al., ; gao et al., ; liao and lai, ; lu et al., ; tahara et al., ; zhang et al., ) . the utr is located directly downstream of orf and is encoded by approximately nt excluding the polyadenylation site. the utr [ nt (type ), nt (type )] is also genetically diverse, sharing approximately % nucleotide identity between type and type sequenced isolates but about % pairwise nucleotide identity within each genotype choi et al., ; verheije et al., ; yin et al., ) . recent reviews extend upon the brief description presented here wang et al., ; yun and lee, ) . due to the unique attributes of nidovirus transcription and replication, including uncharacteristically large polycistronic rna genomes and the transcription of a nested set of , co-terminal sgrnas through a discontinuous transcription strategy, which is in itself a mechanism of recombination, nidoviral rna synthesis mechanisms have been suggested to be of unparalleled complexity among positive strand rna viruses van hemert et al., ) . prrsv replication closely ties three key features: rearrangement of host membranes to establish viral replication complexes (rc), synthesis and expression of grna, transcription of sgrna for the efficient expression of structural proteins, at the same time as the unique ability to produce aberrant prrsv sgrnas known as heteroclites (yuan et al., (yuan et al., , . genesis of grna (replication) and sgrna is inherently tied through the shared negative strand synthesis mechanism. modulation of negative-sense transcription through a non-stochastic mechanism yields either grna or one of six standard sgrnas (rna - ) through an abortive disjoining/rejoining discontinuous transcription strategy ( fig. ) (meng et al., b; nelsen et al., ; pasternak et al., ; van marle et al., a) . the characterization of the cellular entry mechanism that prrsv utilizes has been studied in detail, and will not be covered in this review (van breedam et al., ) . little is known about the establishment of prrsv infection, however, from the point postentry to the development of rc, including the formation of characteristic perinuclear double-membrane vesicles (dmvs) (knoops et al., ) . dmvs are believed to be derived from the endoplasmic reticulum (er) which are apparent sites of viral replication (pedersen et al., ) . it has been shown that the eav replicase proteins (orf a/b) are sufficient to support viral replication (molenkamp et al., b) , but that infectivity is dependent on the presence of the structural genes encoded at the -end of the arterivirus genome molenkamp et al., b; verheije et al., ) . upon entry, the grna serves as the mrna for immediate translation of the large replicase polyproteins. within orf a, three proteins are recognized putative transmembrane proteins (nsp , nsp , and nsp ). the eav nsp and nsp were shown to be sufficient to modulate host cellular membranes into structures similar to those observed during viral infection . it is believed that membrane integration and possibly protein-protein interactions of these transmembrane proteins function to torque the existing membrane structures to form the dmvs; tethering the genesis and processing of the polyprotein (s) at the site of replication. additional viral or cellular interacting partners are not well defined. the mechanism of dmv formation is unknown but may include the modulation of autophagy and/or apoptosis pathways (breckenridge et al., ; chen et al., ; costers et al., ; cottam et al., ; huo et al., ; labarque et al., ; razi et al., ; sun et al., ; wang et al., ; yin et al., ; yu et al., ) . the core replicative machinery of prrsvthe rna dependent rna polymerase (rdrp; nsp ), the zinc-binding domain (zbd; or z; nsp ), the rna helicase (nsp ), and the conserved nidovirus uridylate-specific endoribonuclease, (nendou or u; nsp )is encoded within orf b (ulferts and ziebuhr, ) . the calculated rfs efficiency of the rfs ( - % - rfs; - % - rfs) and the rfs ( $ %) (den boon et al., ) demonstrates that orf b (nsp - ) is generated approximately once out of every six translational events ( %), suggesting the stoichiometric requirements for the core replicative machinery is low compared to the encoded replicase proteins. rdrps form a characteristic right hand configuration (thumb, palm, finger(s)) with the thumb and fingers in contact to create a pocket for substrates (ferrer-orta et al., ) . comparison of single-and double-stranded rna virus rdrps show structural similarity even though there is low sequence homology between classes (ferrer-orta et al., ) . all polymerases share a core set of conserved motifs, suggesting a common ancestor (sabanadzovic et al., ) . the structure of the rdrp possesses an additional conserved motif (o'reilly and kao, ) . the rdrp of nidoviruses is phylogenetically clustered with the picorna-like virus superfamily koonin, ) but possesses a sdd (ser-asp-asp) signature, located within the active site on the palm side of the rdrp. the nidoviral sdd motif is a hallmark of the viral family that discriminates it from all other positive-sense rna virus groups that contain a gdd (gly-asp-asp) motif (den boon et al., ) . the sdd motif at this position was shown to be critical for eav replication (van dinten et al., ) ; surprisingly a s-g mutation within the prrsv rdrp was replication competent (grna) but displayed deficiencies in sgrna synthesis . another salient fact is that the arteriviral rdrp does not possess the proofreading abilities that other nidoviruses display (lauber et al., ) . the rate of random mutation introduction is therefore elevated (forsberg, ; forsberg et al., forsberg et al., , , contributing to an abnormally high evolution rate estimated at between .  and .  /synonymous site/year (hanada et al., ) . nsp encodes the prrsv helicase protein (bautista et al., ) . the prrsv multi-domain helicase (hel) is composed of the core a and a canonical domains found in super-family type helicases, a flexible accessory domain ( b), and a unique zinc-binding domain (zbd) (deng et al., ) . the prrsv hel functions to unwind dsrna in a to polarity (bautista et al., ) . both the flexible accessory domain and the zbd are critical to replicative function of eav including generation of grna and sgrna (van dinten et al., ; van marle et al., b) . the helicase is predicted to function in concert with the rdrp to facilitate replication and transcription; however, it is not understood how the to directionality of the helicase and the to rdrp synthesis coordinate these activities (fang and snijder, ) . nsp harbors the nidoviral uridylate-specific endoribonuclease (nendou) domain (ulferts and ziebuhr, ) . originally described in coronaviruses, the nendou of eav was shown to be required for genome replication, and mutation of critical residues had varying deleterious effects on replication, and particularly on sgrna synthesis (posthuma et al., ) . in examining the core nuclease region of eav and prrsv, single-stranded rna was the preferred substrate, as was shown for the coronavirus sudden acute respiratory virus (sars). however, no dependence on the divalent cation mn þ was seen. sars nsp , the coronavirus orthologue, was previously shown to mn þ dependent. eav nendou protein was also shown cleave of pyrimidines, preferring uridine over cytidine, and releasing products with , -cyclic phosphate and -oh ends. in addition, cleavage after unpaired over paired pyrimidines was preferred (nedialkova et al., ; ulferts and ziebuhr, ) . the function of nendou in nidovirus replication has not been established. how the prrsv rdrp initiates replication, either by a primer dependent mechanism or by de novo synthesis is also unknown. to assess the activity of the eav rdrp, a recombinant version of the polymerase was generated and assessed. eav rdrp activity was found in the absence of a primer with poly(u) or poly (c) templates but not with poly(a) templates, indicating a de novo initiation method in a template specific manner . introduction of primers to either the poly(u) or poly (c) templates reduced rdrp activity . incubation with non-complementary bases (i.e. for poly (u) template¼gtp and utp) did not result in isotopic labeling, which shows the rdrp did not function as a terminal transferase, and radioactively labeled primers were not incorporated into the synthetic non-viral templates . de novo polymerase activity could not be detected on viral templates however. this finding suggests the arterivirus rdrp is catalytically active without other viral factors and capable of de novo synthesis, but may require other viral or cellular co-factors to efficiently perform replication or transcription processes. similar research on prrsv has not been accomplished to date. it is generally believed that positive-sense rna viruses use conformational switches in their terminal noncoding regions in the form of higher order rna secondary structure to regulate translation, transcription of sgrnas, and genomic replication . to define the minimal cis-acting genomic element required for efficient prrsv replication, progressive deletions were introduced into self-limiting prrsv replicons encoding an internal ribosome entry site (ires)-driven luciferase (luc) reporter within the deleted region (choi et al., ) . only the smallest deletion, encoding the full m and n proteins, replicated to similar levels as the positive control. the next smallest deletion removing the m protein coding region but maintaining the complete n orf resulted in significant loss of genome replication (choi et al., ) . taken together, including the rdrp, hel, utr, utr, orf , orf , and other unknown viral proteins, these data show that key genetic elements or protein interacting partners are required for efficient prrsv replication and transcription, and are interspersed within multiple coding regions of the genome. viruses require the ability to selectively regulate transcription and translation processes both temporally and quantitatively in a highly ordered and balanced process (pasternak et al., ) . the expression mechanisms between the nonstructural replicase proteins and the structural proteins are fundamentally separated within the prrsv genome facilitating rapid expression of nsps from the grna and subsequent amplification of sgrna transcripts through a differential transcription cascade (pasternak et al., ) . sgrnas are synthesized by the viral rdrp through a highly ordered process encoded within the prrsv genome. the set of nested (nido latin: nested) sgrnas encode noncontiguous grna sequence including both the utr and the polyadenylated utr as well as one or more orfs from the region of the genome (orf - ) but lack the entire large $ kb replicase coding region (orf a/b) (fig. ) pasternak et al., ; van berlo et al., ) . it was originally hypothesized that nidoviral sgrnas could be generated through a free utr priming stage (baker and lai, ; baric et al., ) but was ultimately shown the bp untranslated of strain vr- is shown as a bar encompassing two discrete regions, the terminal sequence of bases that differs approximately % between strains of the same genotype (gray bar) and the % conserved u/guaacc hexanucleotide on the distal end that serves as the transcription regulatory sequence (trs; black bar). the prrsv replicase complex is represented by a multi-point star. (a) production of conanical sgmrna. (step ) sgrna synthesis initiates as ( À ) strand replication (blue bar) from the full-length ( þ ) sense (green bar) genome. rdrp interaction with a trs either results with a read-through and a continuation of ( À ) strand replication or, in the case of sgrna synthesis, ( step ) disassociation of the replicating strain (body aauugg; white body) and ( step ) re-joining at the leader trs (leader aauugg; leader-body junction) through sequence complementarity annealing followed by completion of ( À ) strand sgrna synthesis. all sgrnas possess identical and termini (see fig. ). (step ) ( À ) sense sgrnas serve as template for generation of ( þ ) sense sgrna synthesis, required for structural protein translation. (b) production of heteroclite sgrna at unconventional leader-body junction sites to express aberrant proteins. that nidoviruses utilize a discontinuous sgrna transcription strategy (sawicki and sawicki, ; van marle et al., a) . discontinuous replication proceeds through a replicative fusion of the viral genome utr to one of many downstream sites through base pairing interactions between sense and antisense stem-loop (sl) structures via long-range rna-rna interactions during negative strand synthesis (van marle et al., a) . specifically, an antisense transcription-regulating sequence (trs) at or near the end of each structural protein coding region (orf - ) can each individually form a kissing-loop interaction with a conserved trs sequence (uuaacc), located at the terminus of the utr. sgrnas synthesis and grna replication utilize a similar initial synthesis mechanism, whereby negative strand transcriptional extension from the termini is completed until a trs signal sequence within the body of the genome is encountered ( fig. a ) (den boon et al., ; pasternak et al., ) . the body trs is ordered into a sl structure encoding a conserved heptanucleotide primary sequence (body trs signal) (den boon et al., ) within the loop structure. this signal halts transcription of the negative strand and a "decision" is made between transcriptional readthrough and continuation of synthesis, or a disjoining of the transcriptional machinery and rejoining to the common leader trs (antisense to body-trs) by complementary base-pairing with a second sl structure within utr (pasternak et al., ; van marle et al., a) . the leader trs is located within the utr directly upstream of the aug start codon of nsp α and is part of a highly ordered and well conserved rna secondary structural motif between the utr and the nsp α coding region. transcriptional read-through of all body trs sites will result in grna synthesis. decoupling from the genomic strand at the body trs and rejoining at the leader trs results in noncontiguous transcription that lacks a large central region of the genome, yielding one of at least six sgrna products (dependent on which body trs is utilized) ( fig. a) (pasternak et al., ) . the structural integrity of the eav nsp region, composed of two papain-like protease domains and a predicted n-terminal zinc finger, was also indispensible for transcription, and has been shown to interact with the cellular cofactor p (tijms et al., (tijms et al., , tijms and snijder, ) . again, all research completed on the arterivirus mechanism of subgenomic synthesis was through the use of the model eav strain adapted for growth in tissue culture. similar research on prrsv, with strain differences in replication rates and its immense depth of variation, may yield novel findings in this field. while the mechanism of sgrna generation is conserved, the discontinuous transcription process has been shown to be able to utilize both canonical and non-canonical body trs sites and can have strain specific derivations (den boon et al., ; meng et al., b; nelsen et al., ) . alternative canonical and noncanonical body trs sites often precede the coding region of prrsv structural proteins, which function to drive sgrna synthesis to various degrees of efficiency, yielding major and minor sgrna species encoding the same structural protein. for instance, the na prrsv prototype strain (vr- ) utilizes at least two different leader-body junction sites for the generation of sgrna and sgrna subspecies, and produces a separate species, sgrna - , for the expression of a truncated gp utilizing a downstream aug . other nuances for individual prrsv strains have been noted (lin et al., ; meng et al., b) . examining mutational studies within the eav orf body trs (nucleocapsid) to abolish the generation of sgrna , the most abundantly produced sgrna (van marle et al., a) found that elimination of sgrna synthesis resulted in obvious increase in sgrna , , , and but production of sgrna remained unchanged. additionally, it was noted that a mutation within the leader trs, altering the fifth nucleotide of the conserved sequence ( -ucaag- ) eliminated synthesis of all sgrnas except sgrna ( van marle et al., a) . this effect is due to a unique non-canonical trs semi-independent generation of sgrna . , produced by both eav and prrsv (meng et al., b; van marle et al., a) . sgrna . uses a non-trs body sequence of -ucaauaccc- which lacks the terminal c residue of the canonical trs sequence but possesses an additional five nucleotides (uaccc) that match the adjoining sequence downstream of the leader trs, allowing for sense/antisense base pairing. (van marle et al., a) . data from single and double knockout mutagenesis studies of alternative (non-canonical) trs body sequences for eav showed that the expression of solely the minor sgrna species from alterative joining sites of the gp , gp , and gp structural proteins was sufficient for production of infectious progeny virus (pasternak et al., ) . it is not clear if the alternative trs body sites serve as a secondary mechanism to rescue deleterious mutations from the error-prone rdrp, or if they serve a dedicated purpose within the viral life cycle. knockout of the canonical eav trs body sequence of orf , , or resulted in infection rates at perceived wild-type levels (defined by ifa cellto-cell spread; - log pfu/ml reduction in titer) by utilizing secondary trs sequences within these coding regions (pasternak et al., ) . double knockout mutants of both the canonical and accessory body trs sequences surprisingly still resulted in generation of progeny virus, but at reduced levels (orf ¼ log reduction, orf ¼ log reduction, orf ¼ log reduction; pfu/ ml). it is presumed that even if the use of alternative trs sequences results in inefficient sgrna synthesis, the two amplification cycles (genomic rna-( À ) sense sgrna synthesis-(þ ) sense sgrna) may result in sufficient sgrna copy numbers to allow a productive infection cycle to proceed. the authors noted that the reduction in pfu titers of the single or double trs mutants corresponded "very well" to the reduction in molar ratios of each respective rna subspecies (pasternak et al., ) . prrsv eu prototype strain lelystad virus (lv) sgrnas possess a conserved six nucleotide junction sequence of ucaacc (or similar sequence), but show heterogeneity at the junction site, suggesting the joining mechanism may be "imprecise" (meulenberg et al., a) . studies on whole rna rt-pcr (cdna) of virally infected cells with na prrsv strains further identified a similar common leader-body junction sequence u(g)ua(g/c)acc (meng et al., b; nelsen et al., ; oleksiewicz et al., ) . genetic heterogeneity was also noted at these junction sites, differing by a single base to a couple of nucleotides, showing there is slight flexibility within the disjoining and reattachment of the viral rdrp during this step (meng et al., b; nelsen et al., ) . furthermore, the body trs motifs and the distance upstream from the starting aug for the expression of sgrnas differ between type and type prrsv, except for the predominant sgrna transcript (meulenberg et al., a; nelsen et al., ) . on top of this, surveying northern analyses of different prrsv strains shows that each strain has different quantities of each sgrna transcript size, and often differ in their trs motifs (gauger et al., ; guo et al., b; wang et al., ) . thus, the mechanism(s) regulating prrsv sgrna synthesis when comparing different viral strains appear to be more complex than appears when examining one viral strain in depth. defective interfering (di) rnas are a normally observed byproduct of positive-sense rna virus replication, particularly under high multiplicity of infection (m.o.i.) culturing conditions (masters, ; molenkamp et al., a; pattnaik et al., ; xiao et al., ) . di rnas are generated through nonhomologous recombination between viral genomes resulting in random internal deletions but still encode the replication elements essential for generation of defective progeny virus including the genes encoding for the polymerase, essential replicase proteins, and capsid protein(s) (yuan et al., ) . unlike dis that have been described in other nidoviruses, a group of "heteroclite" sgrnas (heteroclite¼deviating from common forms or rules) were identified within prrsv replicative products of unusual structure but containing large internal deletions (yuan et al., ) . heteroclite sgrnas species were identified within infected cells, purified virions, porcine alveolar macrophages infected with field isolates, under natural infection conditions, within plaque-purified viral infections, and are assumed to result from homologous recombination at atypical nucleotide stretches (fig. b) (yuan et al., (yuan et al., , . the essential replicative products such as the viral rdrp were found to be absent within the heteroclite rnas, discriminating them from prototypical di genomes. in addition, heteroclites do not appear to interfere with ongoing genomic rna and sgrna transcription (yuan et al., (yuan et al., , . sequencing analysis showed a short site of two to seven conserved nucleotides between the and joining regions, but these aberrant trs motifs varied in sequence (yuan et al., ) . when rt-pcr products of culture supernatants of strain vr- using a and primer pair were analyzed, at least bands were discriminated (s -s ). the result of this analysis and subsequent nucleotide sequencing showed that many similar-sized heteroclites were present in each band, but each band represented several individual heteroclites with different trs motifs. the region of the heteroclites encoded terminal orf a proteins including one or more of the papain-like proteases and joined within the downstream coding region either in-frame or within alternative reading frames, perhaps producing aberrant proteins (yuan et al., (yuan et al., , . these rna species persist in a range of experimental culturing conditions including low m.o.i. passage and plaque purification (yuan et al., ) , and were found to be packaged within the prrsv virion (yuan et al., ) . all identified heteroclites included at least the first nt of the type prototype, strain vr- , and later studies pinpointed nucleotides in nsp that bound to the n protein and therefore was an important element in viral packaging (baig and zakhartchouk, ) . additional di rna that possesses many similar genetic features has been identified, but each contains a smaller deletion (nsp - ) and encodes all structural proteins (xiao et al., ) . currently there is no known function for heteroclite sgrnas, but they have been proposed as a packaging vector to study the effect of viral factors, or the effect of exogenous elements on viral replication, translation, progeny phenotype, or immune response/modulation (yuan et al., ) . they may also allow for recombination events to occur in the background of an ongoing prrsv coinfection of two or more dissimilar strains. in this way, new viral sequences may be allowed to coexist with and interact with nascent viruses via recombination in the background, occasionally leading to new viral species with enhanced properties. hypothetical at this juncture, this concept should be evaluated by defined recombination studies. the mechanism of prrsv recombination is ill described (fig. ) . homologous recombination was first described for nidoviruses using mouse hepatitis virus (mhv), a coronavirus, and the authors posited that less than full-length rna intermediates might be generated during viral rna replication. these early studies led to the proposal that mhv replication proceeded in a discontinuous and nonprocessive manner, perhaps at sites of secondary and tertiary structure thus generating free rna intermediates, which could be used in rna recombination via a copy-choice mechanism (lai, ; lai et al., ; makino et al., ) . in the absence of selection pressure, mhv rna recombination was found to be random, but that only certain recombinants are selected over passage in tissue culture, leading to the conclusion that there were "hotspots" for recombination (banner and lai, ) . recombination was detected during both negative and positive strand rna transcription, and took place not only between two different viral strains but also between one replicating viral rna and transfected non-replicating mhv rna fragments. furthermore, the recombinants could be detected after viral growth in cell culture and in animals, and successful recombination occurred more frequently within a hypervariable region which was also subject to deletion (liao and lai, ) . these results were shown to be representative of other coronaviruses, most notably infectious bronchitis virus (ibv), where numerous reports detail the ability of the virus to recombine in the field (kottier et al., ; toro et al., ; wang et al., ) . in the case of ibv, a pathogen of poultry, recombination has been shown to be robust, perhaps due to housing poultry in large flocks (cavanagh and davis, ) . most of these early conclusions mirror what has been shown to occur in prrsv. kapur et al. ( ) provided strong statistical evidence for intragenic recombination or gene conversion in orfs , , , and , but not in orf . the first laboratory examination of prrsv recombination was done using two different prrsv strains to infect ma- cells. differential primer pairs were used in rt-pcr studies to examine cloned cell culture progeny for recombination events over an nucleotide span encompassing part of orf to orf of type prrsv. five clones were selected for sequence analysis, which revealed that four clones each represented a single unique crossover, and one clone appeared to be a triple crossover recombinant. rnase treatment of the cell supernatant before rt-pcr analysis proved that the recombinant rnas were protected from degradation and therefore represented packaged viral rnas. finally, the investigators showed that the recombinants could be detected for up to three passages, but eventually were overtaken by one parental strain that had increased replication kinetics in the cultured cell line. recombination frequencies of up to % were estimated and recombinants could also be found in animals (murtaugh et al., ; yuan et al., ) . a similar experiment was completed with type prrsv, reporting frequencies of only . and . % rna recombination occurring within a bp fragment, but also noting that recombination events are correlated with the size of the fragment analyzed (van vugt et al., ) . there are many reported algorithmically detected instances of recombination occurring between prrsv field strains of the same genotype, as well as defined coinfection studies in swine (li et al., ; liu et al., ; martin-valls et al., ; shi et al., a) . recombination hotspots have been observed to take place within the end structural genes, as well as in nsp , and nsp (li et al., ; liu et al., ) . a more thorough analysis has shown that multiple breakpoints of recombination were detected by genetic algorithm recombination detection (gard) software all along the genome of both type and type isolates. gard analysis of type genomes produced statistical breakpoints. similarly, type genomes led to the identification of breakpoints (martin-valls et al., ) . rare evidence exists for nonhomologous recombination between the prrsv genome and other rna segments. a survey of field viruses ( type ) sequenced in the gp region ( - bp) by the university of minnesota veterinary diagnostic laboratory showed that a key segment coding for gp hypervariable region was subject to insertions and deletions (faaberg, ) . this region contains different patterns for an amino acid stretch in hypervariable region , inducing non-neutralizing antibodies (ostrowski et al., ; plagemann et al., ) , but was also seen to have additions (up to amino acids) or deletions ( amino acid). two particular field isolates, encoding an extra amino acids in the hypervariable region (nggmrtaansnsss), were found to be identical in nucleotide sequence (ggggggau-gaggaccgcc) in that amino acid stretch to prrsv orf sequence, as well as many other swine host transcripts and other pathogens (unpublished data). although breakpoints and recombinants are valuable tools to understand viral evolution, there is a paucity of research directed toward understanding the viral and host machinery that prrsv utilizes to successfully carry out recombination. except for the illustration that mutations in the sdd motif of nsp of prrsv results in viral replication without sgrna transcription, and the hel and nendou domains being critical for both genomic and sgrna replication in eav and prrsv, no detailed mechanism for arterivirus homologous and nonhomologous recombination has been put forward. the molecular evolution of prrsv has been examined by many investigators in detail and will not be addressed in this review (frossard et al., ; shi et al., a shi et al., , a shi et al., , b shi et al., , b stadejek et al., ) . the cause of such rapid evolution may be primarily due to the lack of prrsv rdrp proofreading and tremendous viral recombination, resulting in an extraordinary diverse composition of isolates with varying pathogenicity. from the emergence of prrs in the united states in (keffaber, ) , it was apparent that there were several circulating viruses besides the usa prototype virus, vr- . the disease was first recognized as mostly a reproductive disease, causing anorexia, late term abortions, and delayed return to estrus in sows. the infection of sows also led to increased preweaning mortality in young pigs that survived. histologically, interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in piglets and focal vasculitis in the brain of the sow were seen. in nursing, growing, and finishing pigs, mild flu-like symptoms are evident, with pronounced hyperpnea, fever, and interstitial pneumonitis . in europe, similar disease phenotypes were observed, with the additional finding that sows sometimes had blue ears, but antigenic differences were seen between european virus isolates from different countries and these were radically dissimilar from united states and canadian isolates drew et al., ; hopper et al., ; paton et al., ; plana et al., ; wensvoort et al., wensvoort et al., , . similar findings based on herd clinical symptoms as well as seroconversion were also reported in the usa and canada (dea et al., ; morrison et al., ) . the first nucleotide sequencing efforts directed at the prrsv -end of the genome confirmed these antigenic findings, revealing approximately % nucleotide differences between type isolates and % between type and type isolates (drew et al., ; kapur et al., ; mardassi et al., b; meng et al., ; meulenberg et al., ; pesch et al., ) . further evolution now places the divergence within both genotypes at % when comparing whole genomes (han et al., ; van doorsselaere et al., ) . investigators soon found that these differences were also reflected in the degree of pathogenicity caused by different viral isolates (halbur et al., (halbur et al., , . key events in prrsv diversity were the emergence of an "atypical" or "acute" variant that appeared in iowa, usa in the mid s (meng et al., a; mengeling et al., ) , the sudden appearance in of a novel class of prrsv named mn in minnesota, usa (han et al., ) , the notable type highlypathogenic prrsv (hp-prrsv) in in china and subsequently most of asia (tian et al., ) , and the recent demonstration of enhanced pathogenicity of the type lena strain (karniychuk et al., ) . some prrsv isolates have been shown to be neurovirulent (rossow et al., ; tian et al., ) , and most recently the ability of particular strains to depress the swine immune response has been shown to vary (brockmeier et al., ; guo et al., a guo et al., , b wang et al., ) . since the beginning, instances of increased virulence have emerged episodically in different regions of the world. in each case, however, the appearance of the novel isolates is sudden and the result of a significantly divergent prrsv genome sometimes accompanied by insertions or deletions in most often nsp (brockmeier et al., ) , and often containing detectable recombination breakpoints (shi et al., a) . the remarkable phenotypic and genetic diversity is most likely the result of the innate attribute of prrsv persistence, replication, and recombination to yield an extraordinarily flexible viral genome, attempting to circumvent attempts to eradicate the pathogen from swine by the host response, vaccination or other means. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. an enzyme linked immunosorbent assay (elisa) for the detection of antibodies to the porcine reproductive and respiratory syndrome (prrs) virus new insights into rna packaging in porcine reproductive and respiratory syndrome virus nidovirales, virus taxonomy: ninth report of the international committee on taxonomy of viruses an in vitro system for the leader-primed transcription of coronavirus mrnas random nature of coronavirus rna recombination in the absence of selection pressure characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains report on the first outbreaks of the porcine reproductive and respiratory syndrome (prrs) in france. diagnosis and viral isolation functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus structural polypeptides of the american (vr- ) strain of porcine reproductive and respiratory syndrome virus de novo initiation of rna synthesis by the arterivirus rnadependent rna polymerase an rna pseudoknot in the end of the arterivirus genome has a critical role in regulating viral rna synthesis characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) regulation of apoptosis by endoplasmic reticulum pathways genomic sequence and virulence comparison of four type porcine reproductive and respiratory syndrome virus strains sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the s in the u.k induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication identification of and cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel au-rich sequences restored replication of a -proximal -nucleotide deletion mutant isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages coronavirus nsp restricts autophagosome expansion the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd prrs syndrome in quebec: isolation of a virus serologically related to lelystad virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicative-form rnas equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily structural basis for the regulatory function of a complex zincbinding domain in a replicative arterivirus helicase resembling a nonsensemediated mrna decay helicase structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus variation in open reading frames , and among porcine reproductive and respiratory syndrome virus isolates in the uk production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) identification of a novel nidovirus associated with a neurological disease of the australian brushtail possum (trichosurus vulpecula) arterivirus structural proteins and assembly family arteriviridae disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins efficient- frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein a comparison of viral rna-dependent rna polymerases divergence time of porcine reproductive and respiratory syndrome virus subtypes a molecular clock dates the common ancestor of european-type porcine reproductive and respiratory syndrome virus at more than years before the emergence of disease the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe porcine reproductive and respiratory syndrome virus: genetic diversity of recent british isolates cis-acting structural element in utr is essential for infectivity of porcine reproductive and respiratory syndrome virus genetic and phenotypic characterization of a united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection nidovirales: evolving the largest rna virus genome porcine reproductive and respiratory syndrome experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus chinese and vietnamese strains of hp-prrsv cause different pathogenic outcomes in united states high health swine comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model the porcine reproductive and respiratory syndrome virus nsp cysteine protease domain possesses both trans-and cis-cleavage activities proteolytic products of the porcine reproductive and respiratory syndrome virus nsp replicase protein complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus the origin and evolution of porcine reproductive and respiratory syndrome viruses from sars to mers: years of research on highly pathogenic human coronaviruses industry study: assessment of the economic impact of porcine reproductive and respiratory syndrome virus on an outbreak of blue-eared pig disease (porcine reproductive and respiratory syndrome) in four pig herds in great britain involvement of unfolded protein response, p and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein into virions porcine reproductive and respiratory syndrome virus nonstructural protein (nsp ) topology and selective isoform integration in artificial membranes genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate reproductive failure of unknown etiology ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral rna synthesis the phylogeny of rna-dependent rna polymerases of positivestrand rna viruses experimental evidence of recombination in coronavirus infectious bronchitis virus apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines genetic recombination in rna viruses recombination between nonsegmented rna genomes of murine coronaviruses the footprint of genome architecture in the largest genome expansion in rna viruses recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase transactivation of programmed ribosomal frameshifting by a viral protein rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments requirement of the -end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mrna transcription leader-body junction sequence of the viral subgenomic mrnas of porcine reproductive and respiratory syndrome virus isolated in taiwan recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus a -proximal stem-loop structure of untranslated region of porcine reproductive and respiratory syndrome virus genome is key for virus replication high-frequency rna recombination of murine coronaviruses porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus identification of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus molecular analysis of the orfs to of porcine reproductive and respiratory syndrome virus, quebec reference strain analysis of orf and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes and retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates the molecular biology of coronaviruses spread like a wildfire-the omnipresence of porcine circovirus type (pcv ) and its ever-expanding association with diseases in pigs molecular cloning and nucleotide sequencing of the -terminal genomic rna of the porcine reproductive and respiratory syndrome virus characterization of a highvirulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus characterization of proteins encoded by orfs to of lelystad virus isolation and characterization of an arterivirus defective interfering rna genome the arterivirus replicase is the only viral protein required for genome replication and subgenomic mrna transcription porcine reproductive and respiratory syndrome in quebec serologic evidence incriminating a recently isolated virus (atcc vr- ) as the cause of swine infertility and respiratory syndrome (sirs) comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus the everexpanding diversity of porcine reproductive and respiratory syndrome virus genetic interaction between porcine reproductive and respiratory syndrome virus (prrsv) strains in cell culture and in animals biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus analysis of rna-dependent rna polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure determination of -leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain survival of porcine reproductive and respiratory syndrome virus in houseflies evaluation of mosquitoes, aedes vexans, as biological vectors of porcine reproductive and respiratory syndrome virus genetic manipulation of arterivirus alternative mrna leader-body junction sites reveals tight regulation of structural protein expression regulation of relative abundance of arterivirus subgenomic mrnas nidovirus transcription: how to make sense…? sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis the stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mrna synthesis blue ear' disease of pigs infectious defective interfering particles of vsv from transcripts of a cdna clone open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex new insights into the genetic diversity of european porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus: origin hypothesis lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positivestrand rna viruses the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain porcine epidemic abortion and respiratory syndrome (mystery swine disease). isolation in spain of the causative agent and experimental reproduction of the disease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle early endosomes and endosomal coatomer are required for autophagy purifying selection in porcine reproductive and respiratory syndrome virus orf a protein influences variation in envelope glycoprotein glycosylation porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence permutation of the active site of putative rna-dependent rna polymerase in a newly identified species of plant alpha-like virus the cap structure of simian hemorrhagic fever virion rna coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands retention of ingested porcine reproductive and respiratory syndrome virus in houseflies spatial dispersal of porcine reproductive and respiratory syndrome virus-contaminated flies after contact with experimentally infected pigs recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china molecular epidemiology of prrsv: a phylogenetic perspective phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses the spread of type porcine reproductive and respiratory syndrome virus (prrsv) in north america: a phylogeographic approach arterivirus molecular biology and pathogenesis non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid molecular evolution of prrsv in europe: current state of play porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication coronavirus translational regulation: leader affects mrna efficiency comparison of the leader sequences of north american isolates of reference and field strains of porcine reproductive and respiratory syndrome virus (prrsv) emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp autoprotease equine arteritis virus non-structural protein , an essential factor for viral subgenomic mrna synthesis, interacts with the cellular transcription co-factor p a zinc fingercontaining papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus genetic diversity and selection regulates evolution of infectious bronchitis virus nidovirus ribonucleases: structures and functions in viral replication equine arteritis virusinfected cells contain six polyadenylated virus-specific rnas porcine reproductive and respiratory syndrome virus entry into the porcine macrophage proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity membrane proteins of arterivirus particles: structure, topology, processing and function kissing interaction between noncoding and coding sequences is essential for porcine arterivirus rna replication acute and persistent viral life strategies and their relationship to emerging diseases comparative analysis of apoptotic changes in peripheral immune organs and lungs following experimental infection of piglets with highly pathogenic and classical porcine reproductive and respiratory syndrome virus evidence of natural recombination within the s gene of infectious bronchitis virus enhancing neutralizing antibody production by an interferoninducing porcine reproductive and respiratory syndrome virus strain attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mystery swine disease in the netherlands: the isolation of lelystad virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages identification of new defective interfering rna species associated with porcine reproductive and respiratory syndrome virus infection activation of c-jun nh( )-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication conserved nucleotides in the terminus of the utr region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus complete genome sequences of porcine reproductive and respiratory syndrome viruses: perspectives on their temporal and spatial dynamics the endoplasmic reticulum stressresponsive protein grp protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus overview: replication of porcine reproductive and respiratory syndrome virus coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase studies of porcine reproductive and respiratory syndrome (prrs) virus infection in avian species this work builds upon the doctoral thesis of matthew kappes, department of veterinary microbiology and preventive medicine, iowa state university, ames, ia, , entitled "identification and characterization of a novel structural protein of porcine reproductive and respiratory syndrome virus, the replicase nonstructural protein ". project - - - d of the usda agricultural research service provided support for dr. faaberg. dr. kappes was supported by bi vetmedica, inc., and by the usda agricultural research service. usda is an equal opportunity provider and employer. key: cord- -yy etfek authors: dwivedi, varun; manickam, cordelia; patterson, ruthi; dodson, katie; murtaugh, michael; torrelles, jordi b.; schlesinger, larry s.; renukaradhya, gourapura j. title: cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: yy etfek abstract porcine reproductive and respiratory syndrome (prrs) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. the efficacy of parenteral administration of widely used modified live virus prrs vaccine (prrs-mlv) against genetically divergent prrsv strains remains questionable. therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of prrs-mlv (strain vr ) with a potent adjuvant to elicit cross-protective immunity against a heterologous prrsv (strain mn ). mycobacterium tuberculosis whole cell lysate (mtb wcl) was chosen as a potent mucosal adjuvant due to its th biased immune response to prrs-mlv. unvaccinated pigs challenged with mn had clinical prrs with severe lung pathology; however, vaccinated (prrs-mlv+ mtb wcl) pigs challenged with mn were apparently healthy. there was a significant increase in the body weight gain in vaccinated compared to unvaccinated prrsv challenged pigs. vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced prrsv neutralizing antibody titers and reduced viremia. immunologically, an increased frequency of th cells, th/memory cells, γδ t cells, dendritic cells, and activated th cells and a reduced frequency of t-regulatory cells were detected at both mucosal and systemic sites. further, reduced secretion of immunosuppressive cytokines (il- and tgf-β) and upregulation of the th cytokine ifn-γ in blood and lungs were detected in mucosally vaccinated, prrsv-challenged pigs. in conclusion, intranasal immunization of pigs with prrs-mlv administered with mtb wcl generated effective cross-protective immunity against prrsv. porcine reproductive and respiratory syndrome (prrs) is a chronic immunosuppressive disease of pigs responsible for huge economic loss to the swine industry worldwide. the economic impact of prrs alone to the us swine producers has been estimated to be approximately $ million annually [ ] . according to the animal and plant health inspection service (aphis) report of january , overall, . % of unvaccinated grower/finisher pigs were positive for prrsv antibodies in the us (http://www.aphis. usda.gov/animal health/nahms/swine/downloads/swine / swine is prrs.pdf). as a member of the arteriviridae family, prrsv contains a positive-sense, single-stranded rna genome [ ] . prrsv causes respiratory distress in pigs of all ages and reproductive failure in sows [ , ] . infection with this virus results in suppression of innate immune response (reduced ifn-␣ production and nk cell cytotoxicity) and a delay in the onset of adaptive immune response [ ] [ ] [ ] . different field isolates of prrsv have been found to carry variable levels of genetic diversity ranging between and % and one of the virulent strains, mn is antigenically highly divergent from the vaccine strain, vr [ ] . currently, modified live and inactivated prrsv vaccines have been licensed for use; however, these vaccines are not always efficacious in preventing prrsv reinfections and transmission. there continue to be questions regarding vaccine safety and efficacy against existing as well as emerging antigenically heterologous prrsv strains [ , ] . therefore, development of a broadly protective prrsv vaccine has been a challenge to prrsv researchers. recently, prrsv experts and researchers collectively agreed that only replicating prrsv vaccines have the most promise in the field and successful protection can be achieved by improving the efficacy of live prrsv vaccines (http://vetmed.illinois.edu/news/ prrswhitepaper.pdf). mucosal surfaces cover the largest surface area in the body and almost % of total immune cells in the body are localized in the mucosa-associated lymphoid tissues (malt) and at mucosal sites. nasopharyngeal malt contains the entire repertoire of immune cells which are strategically located to orchestrate regional immune functions against airborne infections [ ] . a majority of pathogens are transmitted through mucosal surfaces, but some of them cause disease primarily at these mucosal sites (e.g. influenza, prrsv, hiv, rotavirus, etc.). it has been demonstrated that systemic stimulation of the immune system mainly results in systemic protection with low mucosal immune responses. conversely, optimal stimulation of the mucosal immune system generates both mucosal and systemic immunity [ ] . the mucosal immune system possesses strong immunoregulatory mechanisms to dampen inflammationinduced pathology; therefore, mucosal immunization generally results in tolerance in the absence of suitable adjuvants or delivery systems [ , ] . intranasal delivery of live attenuated vaccines against bovine herpes virus- , influenza, and parainfluenza- when administered with adjuvants has proven effective [ , ] . intranasal delivery of influenza vaccine with a potent adjuvant resulted in effective cross protection due to immune responses generated against conserved internal viral proteins [ ] . thus, mucosal immunization may be an attractive method to induce prrsv specific cross-protective immunity. design of an effective mucosal vaccine is not easy because the adjuvant/delivery system used must not elicit any toxicity and should overcome the host-and/or vaccine antigens-induced immunosuppression. heat-killed mycobacterium tuberculosis (mtb) in an oil emulsion has been used extensively for experimental purposes as complete freund's adjuvant [ ] . unfortunately, adverse effects mediated by major cell wall components of mtb such as mycolic acids, peptidoglycan, and wax d preclude use of complete freund's adjuvant in humans and food animals [ , ] . adjuvanticity of various purified individual components of mtb have been investigated [ , ] , including water soluble whole cell lysate (wcl) of mtb [ ] . interestingly, a few of the mtb wcl fractions, such as heat shock protein- (hsp ) [ , ] and pe (pro-glu)/ppe (pro-pro-glu) have been identified to elicit potent adjuvant activity [ , ] . however, the knowledge related to mucosal adjuvanticity of mtb wcl to mucosal viral vaccines is limited. initially, we performed studies to choose a suitable bacterial candidate mucosal adjuvant to use with prrs-mlv. pigs were inoculated intranasally with nine different bacterial preparations belonging to mycobacterium, vibrio, and streptococcus, and selected mtb wcl due to its ability to induce increased th and reduced immunosuppressive responses; the detailed results of which will be published elsewhere. subsequently, prrsv specific immune responses to prrs-mlv+/− mtb wcl in pigs inoculated intranasally was evaluated in detail in a pre-challenge study, which resulted in satisfactory immune correlates of protection mediated through mtb wcl [ ] . the purpose of the current study was to confirm the mucosal adjuvanticity of mtb wcl to prrs-mlv in inducing effective cross-protection to challenge with a genetically divergent and virulent prrsv strain. stable mycoplasma-free marc- cells which support the growth of prrsv [ ] were used to prepare prrsv stocks and to perform immunological assays. cells were maintained in dmem with % fbs (atlanta biologicals, lawrenceville, ga) at • c with conventional large white-duroc crossbred weaned specificpathogen-free piglets at - wks of age were transported to animal facilities of the food animal health research program at the ohio agricultural research and development center, wooster, oh. the swine herd was confirmed seronegative for antibodies to prrsv, porcine respiratory corona virus, transmissible gastroenteritis virus, and porcine circo virus . piglets were bled on arrival, and the sera were tested to confirm the absence of prrsv antibodies. pigs were allowed to acclimate for an additional week before initiation of the experiment. animals were maintained in the large animal bsl- facility under the supervision of a veterinarian. throughout the duration of the study, all animals received food and water ad libitum. all inoculations such as adjuvant (mtb wcl, mg/pig), vaccine (prrs-mlv, × tcid per pig) and challenge (prrsv, × tcid per pig) were administered intranasally. adjuvant and vaccine were inoculated separately into each nostril. all pigs were maintained, samples collected, and euthanized as per the protocol approved by the institutional animal care and use committee (iacuc), wooster, and institutional biosafety committee (ibc), columbus, the ohio state university, ohio. for the primary study, pigs were randomly allocated to one of three groups: group , mock pigs (n = ) received dmem and they were unvaccinated and unchallenged; group , unvaccinated pigs (n = ); group , vaccinated (prrs-mlv+ mtb wcl) pigs (n = ). groups and were challenged with prrsv mn on day postvaccination (dpv) . three pigs each from groups and were euthanized on , , and day post-challenge (dpc). mock inoculated pigs (n = ) were euthanized separately prior to killing of any infected animals. in another study, a total of nine pigs were split into three groups and housed in three separate isolation rooms (n = per group). pigs were inoculated intranasally as follows: group , mock (dmem); group , vaccine (prrs-mlv); group , vaccine with adjuvant (prrs-mlv+ mtb wcl). at dpi- , groups and were challenged with prrsv mn on dpv . infection was allowed to proceed for days at which time all pigs were euthanized. mock inoculated pigs were euthanized prior to the euthanasia of any prrsv challenged animals. blood ( - ml) was collected on dpi and dpc , , , , , , , , , , , and , serum was separated from the clotted blood, and aliquots of serum were preserved at − • c until used in assays. serum was used for evaluation of viremia, viral titer, prrsv serum neutralizing antibody titers, and cytokine production. pigs were monitored daily for respiratory disease, and rectal temperature and body weight were recorded twice a week. blood was collected in acid citrate dextrose solution and processed for isolation of peripheral blood mononuclear cells (pbmc) as previously described [ ] . lung mononuclear cells (lung mnc) from individual pigs were isolated at necropsy as described previ-ously [ , , ] . tracheobronchial lymph nodes (tbln) draining the lungs were collected in dmem, cut into small pieces, and homogenized in stainless steel cellectors. homogenates were washed and the pellet was dissolved in rpmi containing % percoll and centrifuged for min at × g at • c, with no brake. red blood cells in the cell pellet were lysed and the mononuclear cells were washed and resuspended in enriched rpmi [rpmi- , % fetal bovine serum, gentamicin ( g/ml), ampicillin ( g/ml), mm hepes, mm l-glutamine, . mm non-essential amino acids, mm sodium pyruvate, and nm of -me]. necropsy was performed and lungs, tonsils, and tbln were examined for gross lesions. grossly evident pulmonary changes were assigned a score based upon the percent of virus-affected lesions (purple-red colored consolidation) in each lung lobe, and a total score for the entire lung was calculated as described previously [ ] . all the analyses were performed using a standard indirect immunofluorescence assay (ifa) as described previously [ , , , ] . for virus titration, a confluent monolayer of marc- cells in a -well microtiter plate was incubated with a -fold dilution of serum for h. for determining the virus neutralization (vn) titers, serum was heat treated for complement inactivation, diluted two-fold in dmem, and incubated with an equal volume of prrsv mn containing tcid per well for h at • c. one hundred microliter of that suspension was transferred into a -well microtiter plate containing a confluent monolayer of marc- cells and incubated for h at • c in a co incubator. cytopathic effects were examined following fixation with acetone water and addition of anti-pprsv nucleoprotein mab (clone, sdow ; rural technologies, inc., brookings, sd) and alexa- conjugated anti-mouse igg(h+l) secondary antibody, and observed under a fluorescent microscope after mounting with glycerol-pbs ( : ratio). five million pbmc, tbln mnc, and lung mnc were subjected to in vitro restimulation in a -well tissue culture plate in the presence of killed crude prrsv mn antigens (ags) ( g/ml), or recombinant prrsv carboxy terminal amino acid fragment of matrix protein (m ) ( g/ml) [ , ] in enriched rpmi for h at • c. the harvested culture supernatant was analyzed for cytokines by elisa. cells cultured in the absence of any antigens were included as a control, and the amount of cytokines secreted by these cells were subtracted from the respective restimulated experimental well values. the frequency of ifn-␥-secreting cells in pbmc was determined by an elispot assay as described previously [ , ] . briefly, pbmc were plated ( × cells/well) in enriched rpmi in a -well mul-tiscreen plate (millipore, billerica, ma) pre-coated overnight with a mouse anti-pig ifn-␥ mab (bd pharmingen, san diego, ca) at • c. cells were restimulated with killed crude prrsv mn ags ( g/ml) for h at • c in a co incubator. plates were incubated with biotinylated anti-pig ifn-␥ detection antibody, subsequently with streptavidin-hrp conjugate and developed using an insoluble substrate tetramethylbenzidine with h o peroxidase substrate system (kpl inc., gaithersburg, maryland). the frequency of prrsv specific ifn-␥-secreting cells was counted using an aid ® elispot reader system. the background values were subtracted from the respective counts of the unstimulated cells and the immune responses were expressed as the number of ifn-␥-secreting cells per million pbmc. cells stimulated with phytohemaglutinin-p were included as a positive control on every plate. pig sera collected at indicated dpc and culture supernatants harvested after in vitro restimulation of pbmc, tbln and lung mnc were analyzed by elisa for secretion of different interleukin (il) classes, such as th (ifn-␥ and il- ), th (il- ), pro-inflammatory (il- ), and immunosuppressive (il- and tgf-␤) cytokines as described previously [ , ] . flow cytometric analysis was performed to determine the phenotype and frequency of different immune cells in a multicolor immunoassay as described previously [ , ] . briefly, previously isolated cell types (pbmc, lung mnc, and tbln mnc) were treated with % pig serum to block fc receptors. cells were then stained with an appropriate mab which was either directly conjugated to a specific fluorochrome or biotinylated, or with a purified antibody to pig specific immune cell surface markers [cd , cd (southernbiotech, birmingham, alabama), cd ␣, cd ␣/␤, cd c (bd pharmingen), cd , sla class ii (serotec, raleigh, nc), tcr n (vmrd, pullman, wa), foxp (ebioscience, san diego, ca)] or with their respective isotype control mab and labeled cells were treated with streptavidin-conjugated fluorochrome or respective anti-species isotype specific secondary antibody conjugated with fluorochrome. finally, cells were fixed with % paraformaldehyde. for intracellular foxp staining, cells were surface stained for cd and cd as described above and overnight incubated at • c in permeabilization buffer, and stained with fluorochrome-conjugated pig foxp cross-reactive anti-rat foxp mab [ , ] .immunostained cells were acquired using a facs ariaii (bd biosciences, san jose, ca) flow cytometer. analysis was performed to determine different immune cell populations based on the cell surface marker phenotypes: natural killer (nk) cells (cd − cd − cd ␣ + ) [ ] ; t-helper cells (cd + cd + cd − ); cytotoxic t lymphocytes (ctls) (cd + cd − cd + ); cd cd double positive t cells (cd + cd + cd + ) also called as t-helper/memory cells [ , ] ; ␥␦ t cells (cd ␣ + tcr n + ); t-regulatory cells (cd + cd + foxp + ); cd + (myeloid cells); cd + cd + foxp − (activated t-helper cells); dendritic cells rich fraction (cd + cd c + slaii + ) using flowjo software (tree star, inc., or, usa). frequencies of individual lymphocyte and myeloid cell subsets were analyzed from a total , to , events. all data were expressed as the mean value of three, six, or nine pigs ± sem. statistical analyses were performed using a nonparametric "wilcoxon t-test" where functionality was compared between two study groups (unvaccinated vs. vaccinated, and in fig. and table , prrs-mlv vs. prrs-mlv+ mtb wcl), or nonparametric "kruskal-wallis test" followed by a "dunn's test" for multiple comparisons when comparing three or more groups for vn titers and body weight gain using sas software (sas institute inc., cary, nc). statistical significance was assessed as p < . . in this study, pigs were either unvaccinated or vaccinated with prrs-mlv+ mtb wcl and challenged using a virulent heterologous prrsv mn . clinically, unvaccinated pigs developed typical prrs symptoms with fever, mild cough, reduced food intake, and lethargy during first wks post-challenge. vaccinated, mn challenged pigs did not suffer from clinical prrs. the mean body temperature of unvaccinated, prrsv challenged pigs (n = ) until dpc was . • f higher compared to vaccinated pigs. pigs vaccinated and challenged with mn had significant increase in the net body weight gain compared to control challenged pigs (fig. a) . unvaccinated, mn challenged pigs had severe gross lung lesions on both ventral and dorsal surfaces (fig. b) , and the lung lesion scores were significantly higher at dpc and dpc compared to vaccinated challenged pigs (fig. c) . protection in mucosally vaccinated pigs against prrsv challenge was associated with a significantly reduced viral load ( fig. a ) and viral titer (fig. b) at dpc . the circulating prrsv in the blood was less but not statistically significant at dpc in vaccinated pigs, and it was almost cleared by dpc in both the pig groups ( fig. a) . prrsv neutralizing antibody titers in serum at different time points until dpc were analyzed. a significant increase in the vn titers in vaccinated and mn challenged pigs was detected (fig. a) . we found a significant difference in the vn titers in vaccinated pigs at dpc , , and when the titer was compared at each dpc. in mucosally vaccinated, prrsv-challenged pigs, there was a significant reduction in serum il- levels at dpc , , and (fig. b) . similarly, tgf-␤ serum levels were also significantly reduced but at a later stage of challenge (dpc and ) (fig. c) . overall, both the immunosuppressive cytokines il- and tgf-␤ were detected at reduced levels in vaccinated pigs ( fig. b and c). information regarding secretion of cytokines by pig mnc after prrsv restimulation ex vivo is important to understand virus specific memory immune responses. consistent with the reduced lung lesions and viremia, a significantly increased frequency of ifn-␥- table frequency of immune cells in pigs inoculated intranasally with mock (no vaccination and no challenge), unvaccinated (n = ) or vaccinated with prrs-mlv+ mtb wcl (n = ) and then challenged with prrsv mn . secreting cells in pbmc of vaccinated, prrsv challenged pigs at dpc was detected (fig. a) . such a trend was noted for ifn-␥-secreting cells in the lungs at dpc (data not shown) with a concomitant reduction in il- -secretion by lung mnc (fig. c) . the extended clinical protection in vaccinated prrsv challenged pigs was indicated by an increased trend in the secretion of ifn-␥ with a concomitant reduction in il- and tgf-␤ secretion by lung mnc and pbmc at both dpc and (data not shown). evaluation of the frequency of various immune cells at both mucosal (lung and tbln mnc) and systemic sites (pbmc) in vaccinated and virulent prrsv challenged pigs is important for associating cytokine responses. at dpc , a significant increase in the frequency of dendritic cells (dcs) and an increased frequency of th cells, th/memory cells, and ␥␦ t cells were detected in the lungs of vaccinated, virus challenged pigs (table a ). in addition, in both pbmc and tbln mnc, a significant increase in the frequency of activated th cells was detected at dpc (table b and c) . at dpc , a % reduction in the frequency of t-regulatory cells (tregs) in the lungs and tbln in vaccinated, virus challenged pigs was detected (table a and c). in blood and tbln, the frequency of th cells was significantly increased in vaccinated pigs (table b and c). at dpc , a significant decrease in the frequency of tregs in both lungs and blood was detected (table a and b). in addition, a significantly increased frequency of dcs in the blood was detected in vaccinated and prrsv mn challenged pigs at dpc (table b) . to strengthen our data on the superior adjuvanticity of mtb wcl to prrs-mlv, we performed challenge studies in pigs vaccinated intranasally with prrs-mlv in the presence or absence of mtb wcl. prrsv specific vn titers were detected at significantly higher levels at dpc and in pigs vaccinated with prrs-mlv+ mtb wcl compared to prrs-mlv alone (fig. a) . virus neutralizing antibody titers were present at low levels in pigs vaccinated with prrs-mlv+ mtb wcl from dpi (dpc ), but not in pig groups receiving prrs-mlv alone (fig. a) . in support of adjuvant mtb wcl mediated enhanced cell mediated immune (cmi) responses to prrsv, lung mnc of pigs vaccinated with mtb wcl secreted significantly higher amounts of ifn-␥ following restimulation using mn ags (fig. b) . another th response inducing cytokine il- was also secreted at significantly higher levels in a mtb wcl dependent manner in vaccinated pigs (fig. c ). as expected, the immunosuppressive cytokine, il- was secreted at significantly reduced levels in pigs vaccinated with mtb wcl compared those pigs vaccinated without mtb wcl following challenge with prrsv mn (fig. d) . frequency of immune cell populations was also evaluated at both mucosal and systemic sites between these two pig groups to determine the mtb wcl mediated adjuvant effects. in lung mnc, a significant increase in the frequency of th cells, activated th cells, and nk cells was detected in challenged pigs vaccinated with prrs-mlv+ mtb wcl compared to pigs vaccinated with prrs-mlv alone (table a) . in pbmc, a significant increase in the frequency of ␥␦ t cells was detected in prrs-mlv+ mtb wcl inoculated pigs (table b ). as expected, the frequency of tregs was significantly reduced in both pbmc and tbln mnc of pigs inoculated with vaccine with mtb wcl compared to vaccine alone (table b and c) . overall, data from these particular pig groups combination study demonstrated the superior adjuvanticity of the mucosal adjuvant mtb wcl to prrs-mlv. until now, as per our knowledge, no successful attempts have been made to elicit effective anti-prrsv immunity by intranasal delivery of live prrsv vaccine. viruses evade the host immunity by promoting secretion of il- and tgf-␤ which antagonize the protective th immune response [ ] . prrsv induces a strong immunosuppressive response resulting in the delayed onset of cell mediated immune responses [ , [ ] [ ] [ ] . both live and inactivated prrsv significantly increase il- gene expression [ ] . an increased concentration of il- in pig lungs was detected from prrsv-infected pigs for long periods of time [ , ] . nonetheless, expression of both il- and tgf-␤ genes was also increased in pigs vaccinated against prrsv by a systemic route [ ] . in our pre-challenge study, increased secretion of both il- and tgf-␤ in both the lungs and blood of pigs vaccinated intranasally with prrs-mlv without mtb wcl was detected [ ] . in contrast, in both pre-and post-challenge studies when prrs-mlv was inoculated intranasally with mtb wcl, secretion of both il- and tgf-␤ was suppressed. infiltration of tregs into the infected pig lungs contributes to the secretion of high levels of il- and tgf-␤ [ ] . the role of tregs in establishment of chronic persistent hiv, hepatitis c and b viruses, cytomegalovirus, and epstein-barr virus infections has been reported [ ] [ ] [ ] . like in mice and humans, foxp -expressing cd + cd + cells with comparable immunosuppressive properties have been identified in pigs [ ] . studies have been reported on the prrsv mediated proliferation of tregs in infected pigs, indicating their involvement in disease progression [ ] [ ] [ ] . in our study, a consistently reduced frequency of tregs in the lungs, blood, and tbln of pigs vaccinated intranasally with prrs-mlv+ mtb wcl was detected which was associated with reduced secretion of both the immunosuppressive cytokines, il- and tgf-␤. co-ordinated immunosuppressive functions of il- , tgf-␤, and tregs have been reported [ , ] . prrsv increases the expression of tgf-␤ from myeloid cells, and tgf-␤ is essential for the de novo induction of foxp and for the regulation of differentiation and function of tregs in mice, humans, and pigs [ , ] . all the mucosal sites are interconnected by a common mucosal immune system, and immunization at one primary site will stimulate proliferation and migration of antigen-specific lymphocytes, resulting in both mucosal and systemic immunity [ ] . virus neutralizing antibodies play an important role in the clearance of prrs viremia [ , ] . like in natural prrsv infection, the prrsv vaccine also induced delayed neutralizing antibody and cmi responses in pigs [ , ] . however, an increase in the prrsv-vn titers with a concomitant reduction in prrsv load in pigs vaccinated with prrs-mlv+ mtb wcl and challenged with mn was detected in our study. this suggests that reduced viremia detected from vaccinated pigs correlates with increased prrsv specific vn titers. apart from an effective humoral response, a potent cmi response is essential for complete viral clearance. the key cytokine associated with a host cmi response is ifn-␥ produced by nk cells, ␥␦ t cells, th cells, ctls, and th/memory cells [ ] . in our study, reduced clinical prrs and viremia in pigs vaccinated with prrs-mlv+ mtb wcl were associated with increased frequency of ifn-␥-secreting cells and increased levels of activated th cells, memory/th cells, and nk cells in both the lungs and blood. cd cd double positive t cells possess memory, t-helper, and cytolytic properties. they are generally called th/memory cells, and they also secrete ifn-␥ [ , ] . this important t cell subset was associated with protection in pigs vaccinated against pseudorabies virus [ , ] . recently, recombinant bcg based respiratory syncitial virus vaccine induced enhanced recruitment of cd + and cd + t cells into the lungs resulting in increased th cytokine responses and protection [ ] . the in vivo adjuvant effects of the water soluble fraction of mtb wcl containing hsp resulted in rapid and prolonged activation of antigen-specific cd + t cells [ ] . consistent with that in our study, mtb wcl-mediated, increased frequency of cd + and cd + t cells with reduced frequency of tregs was detected. importantly, this was associated with enhanced secretion of th cytokines (ifn-␥) and downregulated secretion of immunosuppressive (il- and tgf-␤) cytokines. the pro-inflammatory cytokine il- is critical for induction of specific adaptive immunity [ , ] , but excess secretion of il- results in inflammation-induced pathology [ ] . in our prechallenge study, increased secretion of il- in the lungs and blood of prrs-mlv+ mtb wcl inoculated pigs at dpv and was associated with enhanced prrsv specific cmi responses [ ] . in contrast, in the current study, reduced gross lung lesions observed in vaccinated, mn challenged pigs were associated with reduced secretion of il- . these data suggest that adjuvant mtb wcl mediated regulated secretion of il- might play a role in inducing a prrsv-specific cmi response. pigs possess a higher frequency of ␥␦ t cells and these cells are considered to be an important innate immune cell at mucosal sites. in addition, pigs have non-mhc class i cytolytic activity against prrsv infection [ ] . in mucosally vaccinated and prrsv challenged pigs, increased frequency of ␥␦ t cells in blood, lungs, and tbln that is mediated by mtb wcl was detected, suggesting a possible protective role played by ␥␦ t cells to prrsv mn in these pigs. in summary, mucosal immunization of pigs using a live prrsv vaccine along with a potent adjuvant has the potential to induce heightened innate and adaptive immune responses, with a concomitant reduction in immunosuppressive responses. thus, based on immune correlates of protection detected against virulent heterologous prrsv mn in pigs intranasally vaccinated with prrs-mlv+ mtb wcl, it may be possible to achieve an effective cross-protective immunity against pprs. our future aim will be to conduct field trials to evaluate the efficacy of our mucosal immunization approach to control prrs. assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology delivery systems: a vaccine strategy for overcoming mucosal tolerance? mucosal and systemic immune responses to measles virus haemagglutinin in mice immunized with a recombinant vaccinia virus the natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (mhc) class i-restricted cd + t cell-mediated but mhc class ii-restricted cd + t cell-dependent immune deviation resulting in selective suppression of immunoglobulin e production nasal administration of glutamate decarboxylase (gad ) peptides induces th responses and prevents murine insulin-dependent diabetes persistence of bovine herpesvirus- -specific antibodies in cattle after intranasal vaccination with a live virus vaccine a live attenuated bovine parainfluenza virus type vaccine is safe, infectious, immunogenic, and phenotypically stable in infants and children combined nkt cell activation and influenza virus vaccination boosts memory ctl generation and protective immunity effect of immunization with freund's adjuvant and pneumolysin on histologic features of pneumococcal infection in the rat lung in vivo studies on antibody production. iv. the role of a wax fraction of mycobacterium tuberculosis in adjuvant emulsions on the production of antibody to egg albumin acute granulomatous response produced in mice by trehalose- , -dimycolate the adjuvant effects of mycobacterium tuberculosis heat shock protein result from the rapid and prolonged activation of antigen-specific cd + t cells in vivo immune response to sheep red blood cells in mice pretreated with mycobacterial fractions adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) heat shock proteins transfer peptides during antigen processing and ctl priming pe pgrs antigens of mycobacterium tuberculosis induce maturation and activation of human dendritic cells ppe antigen rv c of mycobacterium tuberculosis induces a strong b-cell response intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections a simple method of estimating fifty per cent endpoints differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model cross-reactive antibody responses to nsp and nsp of porcine reproductive and respiratory syndrome virus prairie dog (cynomys ludovicianus) is not a host for porcine reproductive and respiratory syndrome virus cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus phenotypic and functional characterisation of porcine cd (+)cd (high) regulatory t cells detection of foxp protein expression in porcine t lymphocytes porcine t lymphocytes and nk cells -an update vaccine-induced, pseudorabies virus-specific, extrathymic cd +cd + memory t-helper cells in swine cytolytic function for pseudorabies virus-stimulated porcine cd + cd dull+ lymphocytes cytokine mrna profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of ifn-gamma and il- after viral clearance immune responses of pigs after experimental infection with a european strain of porcine reproductive and respiratory syndrome virus upregulation of il- gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination the impact of successive infections on the lung microenvironment cd (+)cd (+) regulatory t cells from the peripheral blood of asymptomatic hiv-infected individuals regulate cd (+) and cd (+) hiv-specific t cell immune responses in vitro and are associated with favorable clinical markers of disease status circulating cd + cd + regulatory t cells correlate with chronic hepatitis b infection the role of cd +cd + regulatory t cells in viral infections european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus induction of inducible cd +cd +foxp + regulatory t lymphocytes by porcine reproductive and respiratory syndrome virus (prrsv) cytotoxic t lymphocyte-associated antigen plays an essential role in the function of cd (+)cd (+) regulatory cells that control intestinal inflammation regulatory t cells: how do they suppress immune responses? regulatory t cells and infection: a dangerous necessity the common mucosal immune system and current strategies for induction of immune responses in external secretions virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts correlation of cell-mediated immunity against porcine reproductive and respiratory syndrome virus with protection against reproductive failure in sows during outbreaks of porcine reproductive and respiratory syndrome in commercial herds immune responses and protection by vaccine and various vaccine adjuvant candidates to virulent porcine reproductive and respiratory syndrome virus adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus functional impairment of prrsv-specific peripheral cd +cd high cells efficient lung recruitment of respiratory syncytial virus-specific th cells induced by recombinant bacillus calmette-guerin promotes virus clearance and protects from infection cytokines and acute phase proteins associated with acute swine influenza infection in pigs gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus this work was supported by a national pork board award (npb # - ) and a usda-nifa prrs cap award ( - - ) to rjg. salaries and research support were provided by the state and federal funds appropriated to ohio agricultural research and development center, the ohio state university. we would like to thank drs. juliette hanson, mahesh khatri, and hadi yassine, and todd root and matthew weeman for their help in animal studies. we would like to thank drs. eric nelson, mike roof, dobos, and belisle for providing reagents. we also thank bert bishop for statistics, and dr. michele williams for editing the manuscript.role of the funding source: sponsors have no role in study design, in the writing of the report,or in the decision to submit the paper for publication. key: cord- - ljgxnhf authors: lin, chao-nan; lin, wei-hao; hung, li-ning; wang, sheng-yuan; chiou, ming-tang title: comparison of viremia of type ii porcine reproductive and respiratory syndrome virus in naturally infected pigs by zip nucleic acid probe-based real-time pcr date: - - journal: bmc vet res doi: . / - - - sha: doc_id: cord_uid: ljgxnhf background: porcine reproductive and respiratory syndrome virus (prrsv) is a rna virus with high genetic variation. this virus causes significant economic losses in most pig-producing countries. the clinical presentation of prrsv ranges from asymptomatic to devastating. in this study, we developed a sensitive and specific zip nucleic acid probe-based real-time pcr assay to evaluate the viremia of natural prrsv-infected pigs in taiwan. serum samples were collected from pigs aged – weeks. these include clinically healthy pigs and symptomatic pigs were confirmed to have porcine respiratory disease complex (prdc). results: viremia was quantified in of the ( . %) clinically healthy pigs and in of the ( . %) prdc cases. viremias were significantly more common in pigs with prdc compared with the clinically healthy pigs (p < . ). these results suggest that a high viral load is a major feature of prrsv-affected pigs. conclusions: zna probe-based real-time pcr can be a useful tool to diagnose symptomatic and asymptomatic prrsv-infected pigs. the presence of this marker in a sample of animals with high prrsv loads (> ( . ) prrsv genomes/μl of serum) seems to indicate that it correlates with the presence of prdc in pigs. porcine reproductive and respiratory syndrome (prrs) causes significant economic losses in most pig-producing countries [ ] . the causative agent, the prrs virus (prrsv), was identified in the early s [ ] . prrsv is an enveloped, positive-strand rna virus with a genome of approximately kb, and it belongs to family arteriviridae and order nidovirales [ ] . a remarkable amount of genetic variation has been observed among the prrsvs isolates worldwide, particularly in nsp [ ] [ ] [ ] [ ] , orf [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the nucleocapsid (the orf product) [ , , , ] . the genetic characteristics of the prrsv strains clearly indicate the existence of two major genotypes, the european type (eu genotype, type ) and the north american type (na genotype, type ) [ ] . during prrsv infection, clinical disease is detectable in all of ages of pigs but is usually observed in nursery-grown pigs [ ] . the clinical presentation of prrsv can range from asymptomatic to devastating, with symptoms such as listlessness, emaciation, hyperpnea, dyspnea, chemosis, abortion, stillbirth and a reduction in semen quality [ ] . however, infection with prrsv predominantly exists at a subclinical level, participating as a cofactor in porcine respiratory disease complex (prdc) and porcine circovirus associated disease (pcvad) [ ] . prrsv can be detected using molecular methods from nasal fluid, salivary, serum and tonsil specimens from naturally infected pigs [ ] . however, because prrsv is common within the swine population, no quantitative real-time pcr assays have been described using the serum samples of both symptomatic and asymptomatic prrsv-infected pigs. the diagnosis of prrsv infection has relied on probebased real-time pcr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , sybr green-based pcr [ , [ ] [ ] [ ] , rt-pcr [ ] , virus isolation [ ] , immunohistochemistry [ ] and serological methods [ ] . zip nucleic acids (zna) are oligonucleotide-oligocation conjugates with multiple cationic spermine moieties attached to the nucleic acid oligomer [ ] . the melting temperature of a hybridized zna is easily predictable and increases linearly with the length of the oligocation [ ] . zna were shown to enable specific and sensitive reactions when used as a primer for pcr and reverse transcription [ ] . the present study describes a sensitive method for detecting type prrsv using real-time fluorescent quantitative pcr with zna probes. zna probe-based real-time pcr amplification and the limit of detection tenfold serial plasmid dilutions ( to copies/μl) were tested and used to construct the standard curve by plotting the logarithm of the plasmid copy number against the measured quantification cycles (cq) values. the generated standard curve covered a linear range of six orders of magnitude of the standard plasmid dna. the linear correlation (r ) between the cq and the logarithm of the plasmid copy number was . (slope = − . ) (figure ). to assess the limit of detection of the assay, to copies/μl of standard plasmid dna per reaction were tested in replicates. at more than copies, % of the replicates were positive, and at copies, ( %) of the replicates were positive (table ) . reproducibility and specificity of the zna probe-based real-time pcr the coefficient of variation of the mean cq values in the within-run and between-run for standard plasmid dna precision experiments ranged from . to . % and from . to . %, respectively ( table ). the cv values in the within-run and between-run for clinical specimens that spanned the whole ranged from . to . % and from . to . %, respectively (table ) . we analyzed other swine viruses to test the specificity of the zna probebased real-time pcr. no specific amplifications were detected for any of these samples (data not shown). the assay was tested on serum from both healthy and prdc pigs. as determined by zna probe-based real-time pcr, ( . %) of the prdc pigs and ( . %) of the asymptomatic pigs were positive (table ). using a chi-square test, the correlation between prrsv and prdc was calculated. among the pigs that were analyzed, a positive result for prrsv appeared to be highly significantly correlated with the presence of prdc (p < . ). in addition, the prrsv load was significantly higher (p < . ) in the prdc pigs (ranging from . to . log prrsv genome/μl, median . log ) compared to that in the asymptomatic pigs (ranging from . to . log prrsv genome/μl, median . log ) ( figure ) ( table ). these data indicated that the levels of viral load correlated with the severity of the clinical presentation of the prrsv-infected pigs. the molecular diagnosis of prrsv infection has relied on probe-based real-time pcr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and sybr greenbased pcr [ , [ ] [ ] [ ] . this study first describes a new zna probe-based real-time pcr for prrsv and evaluates this method as a diagnostic tool for prrsv infection. this assay was sensitive, specific and reliable for the amplification of prrsv cdna, with a reproducible limit of detection of approximately copies of target dna per reaction. both the intra-and inter-assay cvs for standard plasmid dna and clinical specimens were satisfactorily low. a novel type of modified oligonucleotides known as zna was recently developed as a method for the clinical diagnosis of pathogens, such as the hepatitis b virus [ ] . znas are able to discriminate between complementary sequence that are a perfect match and those that are mismatched by a single base pair [ ] . znas are particularly efficient at low magnesium concentrations, low primer concentrations and high annealing temperatures [ ] . zna probes provide broad flexibility with respect to experimental design and represent an effective alternative to minor groove binder-dna and locked nucleic acid probes [ ] . the viral load is an indicator of active infection, virushost interaction and disease progression [ ] . the asymptomatic pigs are believed to serve as important reservoirs for the transmission of prrsv to uninfected pigs [ ] . seventeen point eight percent ( / ) of the asymptomatic pigs were positive in this study. similar findings ( . %, / ) were reported in a study that employed traditional rt-pcr in china [ ] . however, this is first study to report the viral load using serum samples in asymptomatic prrsv-infected pigs using zna probebased real-time pcr. four asymptomatic prrsv-infected pigs were detected at levels as high as log viral copies/ μl ( figure ). this result may be explained by differences in the susceptibility to prrsv infection. the present study also reinforces a the previous suggestion that prrsv is one of the major viral agent for prdc [ , ] . prrsv viraemia was detected in . % ( / ) of the prdc pigs, ranging from . to . log prrsv genome/μl (mean . ± . log , median . log ). in contrast to the symptomatic pigs, the viraemia of the asymptomatic pigs ranged from . to . log prrsv genome/μl (mean . ± . log , median . log ). moreover, based on the present results, it can be concluded that when pigs are infected with prrsv, the amount of prrsv in serum samples is significantly higher in prdc pigs. similar reports had been made for another major viral zna probe-based real-time pcr can be a useful tool to diagnose asymptomatic prrsv-infected pigs. the presence of high prrsv loads in a sample of animals (> . prrsv genomes/μl of serum) is correlated to the presence of prdc in pigs. serum samples were collected from pigs from middle and southern taiwan from to . these animals included healthy pigs and symptomatic pigs (aged from to weeks old) that showed a clinical history of prdc, such as listlessness, emaciation, hyperpnea or dyspnea. rna extraction and reverse transcription were performed according to the procedures outlined in chomczynski [ ] and lin [ ] , respectively. this study protocol was approved by the animal care and use committee of the national pingtung university of science and technology. a conserved region of the m gene (the orf product) was identified in nucleotide sequences from asian (n = ), european (n = ), and american (n = ) available from genbank, which were aligned using the clustal w method and the megalign program (dnastar, madison, wi). a -bp region was amplified from the orf gene of prrsv using the primer pair prrsv-m f &-m r (table ) . a dna fragment of the orf gene was amplified from the prrsv vaccine (ingelvac® prrs mlv, boehringer ingelheim) using conventional pcr. the pcr products were cloned using the t&a cloning kit (yeastern biotech co., ltd., taipei, taiwan) and sequenced. the prrsv plasmids were purified using a plasmid miniprep purification kit (gmbiolab co., ltd., taichung, taiwan) and quantified by measuring the od using a spectrophotometer (hitachi u , dallas, tx, usa). a standard curve was generated using -fold dilutions ( - copies/μl) of the standard plasmid dna. zna probe-based real-time pcr to detect prrsv the zna probe-based real-time pcr assays were performed using the lightcycler nano (roche diagnostics, mannheim, germany). each μl reaction mixture contained . μm concentrations of the forward and reverse primers and μl of the cdna. the thermocycling conditions consisted of min at °c and cycles of sec at °c, sec at °c and sec at °c. each run included serial -fold dilutions of the standard plasmid dna as a positive control and to construction the standard curve. a negative control that was missing the dna template was included to detect any cross-contamination. reproducibility and specificity of zna probe-based real-time pcr the intra-(within-run) and inter-(between runs) assay reproducibility were evaluated using -fold serial dilutions of the standard plasmid dna (from - copies per figure absolute copy number per microliter of prrsv in serum from prdc and clinically asymptomatic pigs. the serum of prdc (n = ) and asymptomatic pigs (n = ) was analyzed using zna probe-based real-time pcr. the mean percentages (long horizontal lines) of the prrsv load in the prdc and asymptomatic pigs were compared. error bars show sd. the prrsv load was significantly higher (p < . ) in the pigs with prdc compared with the asymptomatic pigs, as assessed by unpaired, -tailed student's t-tests. reaction), tested in triplicate on three different days. the coefficients of variation of the absolute copy number obtained from each dilution were calculated. the reproducibility of the method was also evaluated by repeatedly testing the clinical samples, as previously described [ ] . the specificity of the zna probe-based real-time pcr assay was assessed by testing nucleic acid extracts of porcine classical swine fever, porcine circovirus type , porcine parvovirus and porcine pseudorabies virus. student's t-test was used to compare the viral loads between the various clinical symptom groups. the correlation between prrsv and prdc was evaluated using the chi-square test with yate's correction. p values < . and < . were considered significant and highly significant, respectively. porcine reproductive and respiratory syndrome virus (porcine arterivirus) genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china molecular variation analysis of porcine reproductive and respiratory syndrome virus in china the genomic diversity of chinese porcine reproductive and respiratory syndrome virus isolates from to analysis of molecular variation of porcine reproductive and respiratory syndrome virus in central china from genetic variation analysis of porcine reproductive and respiratory syndrome virus isolated in china from to based on orf genetic diversity of the orf gene of porcine reproductive and respiratory syndrome virus isolates in china from molecular epidemiology of prrsv: a phylogenetic perspective polymorphic genetic characterization of the orf gene of porcine reproductive and respiratory syndrome virus (prrsv) in china pathogenesis of porcine reproductive and respiratory syndrome virus real-time pcr for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type in naturally-infected and challenged pigs detection of u.s., lelystad, and european-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time pcr simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr the use of endogenous and exogenous reference rnas for qualitative and quantitative detection of prrsv in porcine semen optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus simultaneous detection of classical swine fever virus and north american genotype porcine reproductive and respiratory syndrome virus using a duplex real-time rt-pcr rapid detection and strain identification of porcine reproductive and respiratory syndrome virus (prrsv) by real-time rt-pcr rapid detection of a highly virulent chinese-type isolate of porcine reproductive and respiratory syndrome virus by real-time reverse transcriptase pcr development of a onestep real-time quantitative pcr assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus rapid differential detection of classical and highly pathogenic north american porcine reproductive and respiratory syndrome virus in china by a duplex real-time rt-pcr porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type (pcv ) subtypes pcv a and pcv b by prolonging pcv viremia and shedding simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (prrsv) by real-time rt-pcr and amplicon melting curve analysis using sybr green the development of a rapid sybr one step real-time rt-pcr for detection of porcine reproductive and respiratory syndrome virus zip nucleic acids: new high affinity oligonucleotides as potent primers for pcr and reverse transcription zip nucleic acids are potent hydrolysis probes for quantitative pcr detection of hbv resistance to lamivudine in patients with chronic hepatitis b using zip nucleic acid probes in kerman, southeast of iran nitsche a: real-time pcr in virology detection of asymptomatic antigenemia in pigs infected by porcine reproductive and respiratory syndrome virus (prrsv) by a novel capture immunoassay with monoclonal antibodies against the nucleocapsid protein of prrsv three cases of porcine respiratory disease complex associated with porcine circovirus type infection study of infectious agents involved in porcine respiratory disease complex in taiwan quantitation of porcine circovirus type isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a taqman-based real-time pcr comparison of porcine circovirus type load in serum quantified by a real time pcr in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs quantification of porcine circovirus type (pcv ) dna in serum and tonsillar, nasal, tracheo-bronchial, urinary and faecal swabs of pigs with and without postweaning multisystemic wasting syndrome single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction genetic diversity and correlation with feline infectious peritonitis of feline coronavirus type i and ii: a -year study in taiwan a real-time pcr assay for rapid detection and quantitation of canine parvovirus type in the feces of dogs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • 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 the authors declare that they have no competing interests.authors' contributions cnl designed the zna probe, analyzed the experiments data and wrote the manuscript. whl participated sample collection and performed the zna probe-based real-time pcr. lnh contributed to the rna extraction and reverse transcription. syw performed serum sample separation and arranged the data for statistical analysis. mtc managed the study, provided materials and reagents, contributed to the interpretation of the data and co-wrote the manuscript. all of the authors read and approved the final manuscript.author details key: cord- - tkhjiwl authors: gómez-laguna, j.; salguero, f.j.; barranco, i.; pallarés, f.j.; rodríguez-gómez, i.m.; bernabé, a.; carrasco, l. title: cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus date: - - journal: j comp pathol doi: . /j.jcpa. . . sha: doc_id: cord_uid: tkhjiwl porcine reproductive and respiratory syndrome (prrs) is caused by a virus that predominantly replicates in alveolar macrophages. the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the prrs virus (prrsv). expression of interleukin (il) α, il- and tumour necrosis factor (tnf)-α correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. significant correlations were observed between prrsv infection and the expression of il- , between the expression of il- p and interferon (ifn)-γ, and between the expression of tnf-α and ifn-γ. these findings suggest that prrsv modulates the immune response by the up-regulation of il- , which may in turn reduce expression of cytokines involved in viral clearance (e.g. ifn-α, ifn-γ, il- p and tnf-α). the results also suggest that expression of ifn-γ is stimulated by il- p and tnf-α, but not by ifn-α. all of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. there appears to be differential activation of septal and alveolar macrophages in prrsv infection, with septal macrophages being the major source of cytokines. macrophages play a significant role in the defence against pathogens by phagocytosis following recognition by surface pattern recognition receptors (prrs), by antigen presentation involving class ii molecules of the major histocompatibility complex (mhc ii) and by production of cytokines (mitchell and kumar, ) . cytokines may also be synthesized by several other immune or non-immune cells including lymphocytes, neutrophils and fibroblasts. the expression of cytokines following engagement of prrs by pathogen-associated molecular patterns (pamps) constitutes the main pathway involved in the activation of macrophages (zhang and mosser, ) . some cyto-kines may also act as inhibitors of macrophage activation. for example, interleukin (il)- , tumour necrosis factor (tnf)-a, interferon (ifn)-a and ifn-g act as potent activators of macrophages, whereas il- inhibits activation of these cells (mitchell and kumar, ) . ifn-g and il- are classically involved in the subtype of immune response mediated by th lymphocytes, with both cytokines working in parallel (biron and sen, ) . the proinflammatory cytokines, including il- a, tnf-a and il- , are of greatest importance during the innate immune response (biron and sen, ) . ifn-a also participates in the innate response through antiviral activity, by inducing the differentiation of na€ ıve t cells into ifng secreting effector cells and by down-regulation of il- expression (biron and sen, ; tizard, ) . in contrast, il- is considered to be an immunosuppressive cytokine as it down-regulates the expression of several other cytokines including il- a, tnf-a, il- , il- itself, il- and ifn-g (biron and sen, ; moore et al., ) . porcine reproductive and respiratory syndrome (prrs) is one of the most economically significant diseases of the swine industry (neumann et al., ) . the syndrome is characterized by interstitial pneumonia in growing pigs and reproductive failure in gilts (rossow, ) . prrs is caused by a positive-strand enveloped rna virus, known as prrs virus (prrsv), which belongs to the family arteriviridae in the order nidovirales (fauquet et al., ) . prrsv replicates mainly in porcine alveolar macrophages and, to a lesser extent, in monocytes and dendritic cells (molitor et al., ; bautista and molitor, ) . several studies have examined the role of cytokines in the pathogenesis of prrs (van reeth and nauwynck, ) ; however, it is not clear how cytokines participate in macrophage activation during prrsv infection or how they regulate development of the immune response to the virus. thanawongnuwech et al. ( ) suggested that expression of ifn-g by macrophages and lymphocytes may have an inhibitory effect on the replication of prrsv. another study of a prrsv modified-live vaccine has shown that upregulation of il- expression is associated with a lower number of ifn-g secreting cells amongst peripheral blood mononuclear cells (pbmcs) (dı´az et al., ) . the role of cytokines in the interstitial pneumonia described in prrs has not yet been determined. accordingly, the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by prrsv. thirty-two specific pathogen free, -week-old pigs from a prrsv seronegative farm were used in this study. twenty eight animals were randomly assigned to groups of four and inoculated by the intramuscular route with ml of the third passage of prrsv field isolate (kindly provided by dr. e. mateu) at . tcid . the virus was initially isolated in porcine alveolar macrophages from the serum of naturally infected piglets during a respiratory outbreak of prrs affecting a spanish farm. this field isolate has an open reading frame- sequence similarity of % with lelystad virus (genbank accession number ef ). the inoculated animals were killed at , , , , , and days post-inoculation (dpi). another group of four pigs were sham-inoculated controls. these animals were injected intramuscularly with ml of sterile rpmi medium and killed at the end of the study ( dpi). all animals were sedated with tiletamine-zolazepam (zoletilÔ; virbac, barcelona, spain) followed by a lethal dose of % sodium thiopental (thiovetÔ; vet limited, leyland, lancashire). the experiment was carried out according to the guidelines of the european union (directive / /eec) and was approved by the local ethical committee of centro de investigacio´n en sanidad animal (cisa-inia; valdeolmos, madrid, spain). the pigs were monitored daily for clinical signs, including rectal temperature and a clinical respiratory score, as previously described (halbur et al., ) . during post-mortem examination, gross lung lesions were evaluated by visual inspection and each lung lobe was scored to reflect the approximate volume or percentage of the lung tissue affected (halbur et al., ) . samples from the medial lobe of the right lung were fixed in % neutral buffered formalin and in bouin's solution, processed routinely and embedded in paraffin-wax. sections ( mm) of formalinfixed tissue were stained with haematoxylin and eosin (he) for microscopical examination. since prrsv is most frequently detected in the apical and medial lung lobes (halbur et al., ) , the medial lobe was selected for immunohistochemical examination. the avidinebiotineperoxidase complex technique (abc) was used for the detection of prrsv, macrophages and cytokine proteins as described previously (hsu et al., ) . formalin-fixed tissue was used for detection of macrophages and tissue fixed in bouin's solution for all other immunohistochemical reactions. briefly, the sections were dewaxed and dehydrated through graded ethanol and the endogenous peroxidase activity was quenched in h o % in methanol for min. the sections were washed with phosphate buffered saline (pbs; ph . , . m) and incubated for min at room temperature with ml per slide of blocking solution in a humid chamber. table describes the primary antibodies and antigen retrieval methods applied. primary antibodies were incubated overnight at c in a humid chamber. in each case the corresponding biotinylated secondary antibody was incubated for min at room temperature. an avidineperoxidase complex (vector laboratories, burlingame, california) was applied for h at room temperature. labelling was 'visualized' by application of the novaredÔ substrate kit (vector laboratories). sections were counterstained with mayer's haematoxylin, dehydrated and mounted. for negative controls, the primary antibody was replaced by blocking solution, normal serum and isotypematched reagents of irrelevant specificity. the number of labelled cells was determined as described previously (salguero et al., ) . briefly, the labelled cells were counted in non-overlapping and consecutively selected high magnification fields of . mm . results are expressed as the number of cells per mm . immunolabelled cells were identified and counted morphologically as macrophages, lymphocytes or neutrophils. pulmonary intravascular macrophages and interstitial macrophages were grouped together and described as 'septal macrophages'. the numbers of macrophages, prrsv-infected and cytokine-expressing cells were expressed as a mean ae sd. these values were evaluated for approximate normality of distribution by the kolmo-gorovesmirnov test. differences between the means of control and inoculated animals were assessed by the kruskalewallis test followed by the manne whitney-u non-parametric test (graphpad instat . , san diego, california). correlation between the presence of lung lesions and the expression of virus, macrophages and cytokines was assessed by the spearman test (graphpad instat . ). p < . was considered to represent a statistically significant difference. control animals did not display clinical signs or significant gross or microscopical lung lesions. although inoculated animals displayed no significant respiratory distress, they did develop dullness, weight loss and mild hyperthermia from dpi. from dpi until the end of the study, almost % of the pulmonary parenchyma of the inoculated animals was affected by interstitial pneumonia, and this was confirmed by microscopical examination of the tissue samples (figs. a, a). mac antibody defined macrophages in sections of lung tissue. the total number of macrophages increased in the lung of inoculated animals from dpi onwards (fig. b) . this related primarily to an increase in the number of septal macrophages (figs. b and b). the number of alveolar macrophages decreased to dpi and recovered thereafter (fig. b) . the number of macrophages, as determined by expression of mac , was significantly correlated with the microscopical score of lung lesions (r ¼ . ; p < . ) ( table ) . there was no expression of prrsv antigen in the lung of control animals. prrsv antigen was detected in the lung of infected pigs from dpi until the end of the study, peaking at dpi (fig. c) . this antigen expression was detected mainly in the cytoplasm of macrophages, and was significantly higher in alveolar macrophages than in septal macrophages (p < . ) (figs. c, c and a ). immunolabelled cells were observed not only in areas of interstitial pneumonia, but also in lung parenchyma without lesions. il- a was observed in the cytoplasm of alveolar and septal macrophages and neutrophils, the latter appearing to be a significant source of this cytokine (fig. d) . expression of il- a was always higher in inoculated animals than in controls, and had a bimodal peak at and dpi (p < . ) (fig. d) . the increase in il- a at dpi was attributed primarily to neutrophils (p < . ) (figs. d and d ). prrsv, il- a, il- and tnf-a respectively. *indicates statistically significant differences (p < . ) between the inoculated and control group. **indicates statistically significant differences (p < . ) between the numbers of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. expression of il- and tnf-a also had a bimodal peak at and dpi (p < . ) (fig. e and f) . il- expression remained elevated until the end of the study (fig. e ), but the expression of tnf-a was no different to that of control animals from dpi (fig. f ). septal macrophages were the main cell population involved in the expression of both il- and tnf-a (p < . ) (figs. e, f, e and f). alveolar macrophages and lymphocytes also expressed these cytokines, but to a lesser extent ( fig. e and f) . the labelling of proinflammatory cytokines was observed mainly in areas of interstitial pneumonia with moderate to severe thickening of the alveolar septa. few immunolabelled cells were observed in areas of the lung without lesions (fig. def) . the correlation between the lung lesion score, macrophage count and expression of proinflammatory cytokines is shown in table . table shows the correlation between the expression of tnf-a and ifn-g. tissue expression of ifn-a, ifn-g, il- and il- p ifn-a was expressed in the cytoplasm of alveolar and septal macrophages and lymphocytes. septal macrophages were the main cell type involved in the expression of this cytokine, which was significantly increased at dpi (p < . ) and decreased thereafter (figs. a and f). the number of ifn-a-expressing alveolar macrophages was also increased at dpi (p < . ). ifn-a expression was always higher in inoculated animals than in controls (fig. a) . the expression of ifn-a was significantly correlated with virus expression (r ¼ . ; p < . ) ( table ) . the kinetics of labelling for ifn-g and il- p were similar throughout the study (r ¼ . ; p < . ) (table ) , with both cytokines peaking at dpi and decreasing thereafter ( fig. b and c) . these cytokines were expressed not only mainly by septal macrophages, but also by alveolar macrophages and lymphocytes (fig. c, e and g) . inoculated animals always had more ifn-g-expressing cells than controls. the expression of il- peaked at dpi and decreased thereafter (fig. d ). il- was expressed mainly in the cytoplasm of septal macrophages ( fig. b and d) . the kinetics of expression of il- were significantly correlated with that of the virus (r ¼ . ; p < . ) ( table ) . consecutive sections immunolabelled for prrsv antigen, ifn-g and il- showed co-localization of ifn-g and prrsv antigen, whereas the expression of il- occurred in areas without expression of ifn-g (fig. aec) . the number of septal macrophages expressing these cytokines was always greater than the number of labelled alveolar macrophages (fig. ) . immunolabelling for ifn-a, ifn-g, il- p and il- was associated with areas of mild to moderate interstitial pneumonia and was much less in areas of pulmonary parenchyma without lesions (fig. ) . the correlations between the expression of prrsv, ifn-a, ifn-g, il- , il- p and tnf-a in the lung of prrsv-infected pigs are shown in table . several reports have described changes in cytokine expression during prrsv infection, but these have not addressed local cytokine production within pulmonary lesions. the present study has characterized expression of cytokines by pulmonary macrophages in order to determine the role of these molecules in the pathogenesis of the respiratory form of prrs. the experimental infection did not lead to the animals developing respiratory symptoms, but dullness, weight loss, mild hyperthermia and lesions of the pulmonary parenchyma were observed. prrsv replication peaked at dpi and was mainly localized to alveolar macrophages, which are considered as the target cell for viral replication (molitor et al., ; bautista and molitor, ) . no correlation was observed between the presence of viral antigen and the severity of the microscopical lung lesions. however, the microscopical lung lesions were significantly correlated with marked inflammatory infiltration of the septa and the number of infiltrating macrophages. moreover, the lung lesions showed significant correlation with the expression of both il- a and il- , but not of tnf-a, and macrophage counts were correlated with the expression of il- a and tnf-a, but not of il- . these observations suggest that il- a may play a significant role in the development of interstitial pneumonia during prrs. nonetheless, when all the three proinflammatory cytokines were considered, a highly significant correlation was observed between both microscopical pulmonary lesions and macrophage counts. although prrsv replicated mainly in alveolar macrophages, proinflammatory cytokines were expressed mainly by septal macrophages, especially il- and tnf-a, from dpi onwards. this fact indicates activation of septal macrophages, which may be induced by the synthesis of cytokines (zhang and mosser, ) . similar findings have been reported for other porcine viral diseases, including african swine fever, which triggers activation of interstitial macrophages expressing il- a and tnf-a after viral replication (carrasco et al., ) . in the present study there was marked intra-alveolar infiltration of neutrophils expressing il- a at dpi. the earlier increase of both il- a and tnf-a may have been responsible for the induction of this infiltration and the subsequent activation of these cells, since these cytokines are considered as neutrophil-chemoattractant and stimulant agents (van reeth and nauwynck, ) . furthermore, il- a and tnfa may induce the synthesis of il- (van reeth and nauwynck, ; mitchell and kumar, ) ; however, in our study no correlation was observed between the expressions of these cytokines, although the maximum expression of il- temporally coincided with higher expression of il- a and/or tnf-a. isolate . *indicates statistically significant differences (p < . ) between the inoculated group and controls. **indicates statistically significant differences (p < . ) between the counts of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. the interferons are known to play a significant role in the host immune response against viruses (van reeth and nauwynck, ; biron and sen, ) . ifn-a participates in the innate immune response and is able to induce synthesis of ifn-g (biron and sen, ; tizard, ) . in the present study, a significant correlation was observed between prrsv replication and ifn-a expression, suggesting that prrsv directly induces expression of ifn-a by macrophages. however, prrsv induces lower levels of ifn-a when compared with other porcine respiratory viral diseases, such as those caused by swine influenza virus or porcine respiratory coronavirus (van reeth and nauwynck, ) , which indicates that ifn-a expression may be insufficient to induce clearance of prrsv. the expression of ifn-g by macrophages and lymphocytes has been previously reported in the lung of prrsv-infected pigs (thanawongnuwech et al., ) . in that study, an increase in expression of ifn-g was observed at dpi for infection with highly virulent strains, whereas strains of low virulence induced a higher expression at the end of the study ( dpi). in the present study, the expression of ifn-g was undulating, showing a peak at dpi, just when prrsv replication was maximal. ifn-g is known to protect macrophages in vitro against prrsv replication (bautista and molitor, ) ; however, that viral replication occurred throughout the period of the present study may suggest that in this experimental infection the ifn-g response was not strong enough to eliminate prrsv infection. the production of ifn-g by pulmonary macrophages is induced by the expression of other cytokines including il- , tnf-a and ifn-a (nguyen and benveniste, ; mitchell and kumar, ; tizard, ) . in the present study there was good correlation between the expression of il- p , tnf-a and ifn-g, but poor correlation between expression of ifn-a and ifn-g. therefore, il- p and tnf-a might be the most significant cytokines involved in the induction of synthesis of ifn-g in this experimental infection. royaee et al. ( ) reported correlation between virus-specific ifn-a secreting cells and virus-specific ifn-g secreting cells in pigs vaccinated with an attenuated, modified-live prrsv vaccine. high antigenic and pathogenic differences have been related to european and north american prrsv genotypes, and suggested to occur within a given genotype (halbur et al., ; mateu et al., ; stadejek et al., ) , which may be the cause of the discrepancies between the present study and that of royaee et al. ( ) . despite the expression of ifn-a, ifn-g, il- p and tnf-a, prrsv was still replicating in the lung of infected pigs at the end of the study. il- is an immunomodulatory cytokine that is able to inhibit the synthesis and release of other cytokines (biron and sen, ; moore et al., ) . therefore, the expression of il- observed in the present study might be responsible for reduced expression of cytokines such as ifn-a, ifn-g, il- p and tnf-a, that in turn may impair prolonged viral replication in the lung of infected animals. interestingly, the expression of il- was observed in areas of lung that showed no expression of ifn-g. moreover, the expression of il- was significantly correlated with prrsv replication. these results suggest that prrsv may induce the expression of il- and, therefore, the expression of il- might inhibit the expression of other cytokines, allowing a prolonged viral replication in the lung. this idea is supported by the observed immunolabelling for il- and ifn-g in consecutive sections of the lung and by the correlation observed between the expression of il- and ifn-a. in conclusion, the results of the present study indicate that activation of septal and alveolar macrophages differs throughout prrsv infection, and that the septal cells are the main source of cytokines. proinflammatory cytokines are actively expressed at the onset of the interstitial pneumonia and there is direct correlation between this expression and the infiltration of the pulmonary interstitium by macrophages. additionally, prrsv appears able to modulate the local immune response by inducing the expression of il- by macrophages, which may in turn reduce the levels of cytokines involved in viral clearance such as ifn-a, ifn-g, il- p and tnf-a. ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages interferons and other cytokines african swine fever: expression of interleukin- alpha and tumour necrosis factor-alpha by pulmonary intravascular macrophages different european-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs comparison of the antigen distribution of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus use of avidinebiotineperoxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures genetic diversity and phylogenetic analysis of glycoprotein of europeantype porcine reproductive and respiratory virus strains in spain immune diseases immunity to prrsv: double-edged sword interleukin- and the interleukin- receptor assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states critical role of tumor necrosis factor-a and nf-kb in interferon-g-induced cd expression in microglia/macrophages porcine reproductive and respiratory syndrome deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination proinflammatory cytokines induce lymphocyte apoptosis in acute african swine fever infection porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes cell signaling: cytokines and their receptors proinflammatory cytokines and viral respiratory disease in pigs macrophage activation by endogenous danger signals the authors thank dr. e. mateu (universitat auto`noma de barcelona, spain) for providing the prrsv field isolate and dr. k. van reeth (universiteit gent, belgium) for her kind gift of f mab. j.g-l. was supported by a doctoral grant from the spanish ministry of education and science (ap- - ). this work was supported financially by the spanish ministry of education and science project number agl - /gan. supplementary data associated with this article can be found, in the online version, at doi: . /j. jcpa. . . . the authors declare no competing financial interests. key: cord- - drhoz authors: tabynov, kairat; sansyzbay, abylay; tulemissova, zhanara; tabynov, kaissar; dhakal, santosh; samoltyrova, aigul; renukaradhya, gourapura j.; mambetaliyev, muratbay title: inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with montanide™ gel st elicits virus-specific cross-protective inter-genotypic response in piglets date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: drhoz the efficacy of a novel bei-inactivated porcine reproductive and respiratory syndrome virus (prrsv) candidate vaccine in pigs, developed at ribsp republic of kazakhstan and delivered with an adjuvant montanide™ gel st (d/kv/adj) was compared with a commercial killed prrsv vaccine (nvdc-jxa , c/kv/adj) used widely in swine herds of the republic of kazakhstan. clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [elisa and virus neutralizing (vn) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time pcr and cell culture assay) were assessed in vaccinated and both genotype and prrsv challenged pigs. our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, kazakh strains of prrsv type and type genotypes. in contrast, clinical protection, absence of viremia and lung lesions in d/kv/adj vaccinated pigs was associated with generation of vn antibodies in both homologous vaccine strain lkz/ (prrsv type ) and a heterogeneous type prrsv strain (cm/ ) challenged pigs. thus, our data indicated the induction of cross-protective vn antibodies by d/kv/adj vaccine, and importantly demonstrated that an inactivated prrsv vaccine could also induce cross-protective response across the viral genotype. porcine reproductive and respiratory syndrome (prrs) is an economically devastating disease in pigs that causes an estimated direct loss of greater than $ million annually to the us pork industry (chand et al., ; holtkamp and kliebenstein, ) . the causative agent, prrs virus (prrsv), belongs to the family arteriviridae, order nidovirales, and it causes respiratory problems in pigs of all ages and reproductive failure in sows, including still births and mummification, birth of weak piglets and high preweaning mortality (kimman et al., ; rossow et al., ) . genetic and antigenic analyses have revealed two distinct prrsv genotypes, european (type ) and north american (type ). marked genetic and antigenic differences of up to % are observed between the type and type prrsv genotypes (kim et al., ; nelsen et al., ) . in addition, within the genotypes up to % genetic variation for type i and % for type ii prrsv do exist (kim et al., ; zimmerman et al., ) . this implies complexity of the prrsv and difficulties to develop protective vaccines (li et al., ) . vaccination is the only viable strategy practiced to control the clinical prrs disease and transmission in pigs. two types of prrsv vaccines are commercially available, modified live virus (mlv) vaccine and a killed virus (kv) vaccine (charerntantanakul, ) . prrs mlv vaccine has been shown to provide protective efficacy against prrsv infection in the field; however, it provides limited protection against heterologous viruses. additionally, prrs mlv vaccine has the intrinsic risk of reversion to a virulent strain (kwang et al., ) . though prrs kv vaccine is safe to use in pigs, it elicits insufficient immunity in pigs (lee et al., ) . overall, commercially available prrs kv vaccine does not induce strong required immune response, and thus failed to protect pigs against viremia to homologous as well as heterologous viral challenges (nilubol et al., ; scortti et al., ; zuckermann et al., ) . interestingly, experimental studies showed that it is possible to induce production of virus neutralizing (vn) antibody response in naïve pigs by immunization with inactivated prrsv (misinzo et al., ; renukaradhya et al., a) . passive transfer studies demonstrated that the prrs vn antibody response is responsible for viral clearance from the blood and lungs, and reduce transplacental transmission of the virus, thus played a significant role in protective immune response against prrs (lopez and osorio, ; osorio et al., ) . recent research efforts aimed to improve protective immune response to prrs kv have been focused on utilizing potent vaccine adjuvants, which help to reduce viremia, lung lesions and clinical disease in homologous and heterogeneous prrsv challenged pigs. several types of vaccine adjuvants have been investigated for their ability to potentiate the immune response to prrs vaccines (charerntantanakul, ; li et al., ) , for example polymeric adjuvant montanide tm gel (deville et al., ; parker et al., ; tabynov et al., ) . previous work in our laboratory demonstrated that montanide tm gel potentiates the immune response of a candidate inactivated prrs vaccine , but at that time the efficacy was not compared with the commercial vaccine. therefore, we developed a binary ethylenimine (bei)-inactivated experimental prrsv vaccine containing the prrsv strain, arterivirus/lkz/ (lkz/ ) (type ), and coadministered intramuscularly with montanide tm gel st adjuvant. the vaccine virus was isolated in the territory of the republic of kazakhstan in . further, to elucidate efficacy of our candidate kv prrs vaccine, vaccinated piglets were challenged with the lkz/ (parental type vaccine strain) or kostanay-cm/ (type heterogeneous strain) of prrsv. prrsv strain lkz/ (national repository of especially dangerous diseases of the republic of kazakhstan, registration number m- - /d) was propagated in marc- cells to use in our experimental vaccine preparation. virus infected cell culture supernatant at fifth passage was harvested after h of infection and centrifuged at rpm for min, and filtered through . mm filter. virus titers were determined using a confluent monolayer of marc- cells cultured for h in -well tissue culture plates, and the viral titer was expressed in tcid /ml (reed and muench, ) . inactivation of prrsv with bei was performed as described (bahnemann, ) with some modifications (azanbekova et al., ) . briefly, bei . m stock solution was prepared by cyclization of -bromoethylamine in . m naoh for h at c and stored at c. virus was inactivated by incubation with mm bei for h at c, and the unutilized bei was neutralized by incubation with . mm sodium thiosulphate at c overnight. to confirm complete inactivation, the inactivated virus suspension was inoculated onto marc- cells cultured in cm tissue culture flasks in ml medium. the cells were cultivated for week at c, during which time they were passaged three times and the absence of cpe was confirmed. furthermore, ml of inactivated prrsv was injected into three month-old pigs and serum samples collected every week for weeks was assayed for viremia by real-time pcr as described in section . . after confirmation of viral inactivation, the viral antigen was mixed with a final concentration of % (w/w) gel st polymeric adjuvant (montanide tm , seppic, france) by manual shaking for min. the prrsv vaccines used in this study were: ( ) our candidate kv vaccine mixed with a polymeric adjuvant (d/kv/adj; lkz/ ; ribsp, gvardeiskiy, kazakhstan; batch ); and ( ) a commercial nvdc-jxa kv vaccine containing oil based adjuvant (c/kv/adj; guangdong dahuanong animal health products co., ltd., jinan, china; batch ). the challenge prrsv strains include, prrsv lkz/ (type ; isolated from lugovskoi konniy zavod (lkz) farm, zhambylskaya, oblast, kazakhstan) (orynbayev et al., ) and cm/ (type ; isolated from a private farm in zatobol village, kostanayskiy rayon, kazakhstan) (tabynov et al., ) , were obtained from the depository of especially dangerous infectious diseases, ribsp. the challenge virus strains lkz/ and cm/ were propagated in marc- cells, and confirmed as type and type viruses by ez-prrsv tm mpx . real time rt-pcr using target-specific reagents kit for the rapid identification & differentiation of north american and european prrs viral rna (tetracore, md, usa). fifty large white breed pigs weighing $ kg each (average months-old) were purchased from a specific-pathogen-free herd with certified records showing free from prrsv, porcine parvovirus, porcine respiratory coronavirus, aujeszky's disease, classical swine fever virus, transmissible gastroenteritis virus, swine influenza virus and mycoplasma hyopneumoniae. the negative prrsv status of the animals was confirmed by serology using a commercial elisa kit after arrival of the animals to our facility. pigs were randomly assigned into six treatment groups: groups and , mock-vaccinated negative control groups (received ml final concentration of % gel adjuvant); groups and , vaccinated three times ( , , days post-first vaccination [dpfv]) with a volume of ml d/kv/adj containing gel adjuvant. groups and were vaccinated twice ( and dpfv) with ml of commercial c/kv/adj vaccine containing oil adjuvant as per the manufacturer's recommendations. the virus titers present in the d/kv/adj and c/kv/adj vaccines before inactivation were . tcid /ml and . tcid /ml, respectively. at dpfv, the negative control groups - (n = pigs/group) and groups - (n = pigs/group) were challenged intranasally ( . tcid ) and intramuscularly ( . tcid ) with lkz/ or cm/ prrsv (table ) . serum samples were collected from pigs at , , , , , , , , , , , , , and dpfv and stored at À c until used in assays. pigs were euthanized at dpfv using the mixture of ketamine and xylazine as previously described and performed necropsy. pigs were housed in our bsl isolation animal facility at the research institute for biological safety problems science committee, ministry of education and science of the republic of kazakhstan (ribsp). animals had free access to filtered water and unmedicated sterilized feed. pigs were initially kept for acclimation period for days before started vaccination. this study was carried out in compliance with national and international laws and guidelines on animal handling. the animal use protocol was approved by the committee on the ethics of animal experiments at the ribsp (permit number: / ). serum samples collected from pigs were aliquoted and stored at À c until used in the assays. elisa for prrsv antibodies was performed using a commercial kit (bionote, inc., hwaseong, korea) according to the manufacturer's instructions. the optical density was measured at nm using the multiskan plus microplate reader (labsystems, vantaa, finland). the presence or absence of prrsv antibodies was determined by calculating the sample to positive (s/p) ratio. samples were considered positive for prrsv antibodies if the s/p ratio was > . . prrsv vn antibody titers in serum samples collected at , , , , , , , , , , , , , and dpfv were analyzed by indirect immunofluorescence assay (ifa) as previously described (christopher-hennings et al., ) . briefly, samples were subjected to uv-treatment for min to inactivate any prrsv and subjected to heat inactivation at c for min to inactivate the complement function. two-fold dilutions of test samples prepared in serum free dmem ( ml/well) were incubated with ml of prrsv (lkz/ or cm/ ) tcid per well for h at c, subsequently ml of the suspension was transferred into well microtiter plate containing confluent monolayer of marc- cells and incubated for h at c. further, ml/well of dmem containing % fetal bovine serum was added to each well and incubated for h at c in a co incubator. the contents of the wells were removed from the plate and the cell monolayer was fixed with % acetone in water for min and the plates were allowed to dry in fumehood for min. cells were rehydrated with ml/well of pbs for - min. cpe in both the plates meant for virus titration (see paragraph . ) and vn titer were examined after treatment with ml/well of mouse anti-pprsv nucleocapsid protein specific mab (sdow- ) ( : ) followed by alexa- conjugated anti-mouse igg (h + l) secondary antibody ( : ). after each treatment plates were incubated at c for h, and washed times in between the treatments and observed under an inverted fluorescent microscope after mounting the cell monolayer with glycerol-pbs in : ratio ( ml/well). the vn antibody titer was determined to be the reciprocal dilution ratio of the sample at which > % inhibition of prrsv-induced immunofluorescence was observed. rectal temperature was measured daily from to days postchallenge (dpc) and the threshold for fever was defined as . c. respiratory disorders were scored as: = normal; = mild dyspnea and/or tachypnea if the animal was stressed by handling for s; = mild dyspnea and/or tachypnea at rest; = moderate dyspnea and/or tachypnea when stressed; = moderate dyspnea and/or tachypnea at rest; = severe dyspnea and/or tachypnea when stressed; = severe tachypnea and/or dyspnea at rest. during necropsy the lungs were excised and macroscopic lung lesions were scored to estimate the percentage of the lungs affected by pneumonia as described (halbur et al., ) . one gram of lung tissue was collected from each pig into a ml conical tube containing ml of dmem, minced, and homogenized using the ika t ultra turrax high speed homogenizer (cole-parmer, il, usa) for one min on ice. the clarified supernatant was collected by centrifugation at g for min, aliquoted and stored at À c until assayed. marc- cells were seeded onto -well plates h before infection. prrsv supernatants were serially -fold diluted (from À to À ) and ml of each dilution were plated in six replicate wells. cell culture media without prrsv was used as a control. cells were incubated at c for h in a % co incubator, fixed and immunostained as described above ( . . prrsv vn titer assay). the titers were calculated as described previously (reed and muench, ) and expressed as tcid /ml. prrsv rna was extracted from serum samples and lung homogenate using magmax tm - virus isolation kit (ambion/ applied biosystems, carlsbad, california, usa) as per the manufacturer's instructions. real-time rt-pcr was performed using the ez-prrsv tm mpx . assay (tetracore ; rockville, md, usa), which covers two target regions of prrsv type and type genes using specific primers and probes. in the assay, -fam ( carboxyfluorescein) was used as a reporter dye for detection of type and type prrsv; and the alternative reporter dye cy was used for detection of extraction/inhibition control. the reaction volume per well included . ml of ez-prrsv tm mpx . reagent (includes buffer, primer and probes), . ml of enzyme blend and ml of the extracted serum sample. each pcr run included two positive controls for type and type viruses and one negative amplification control. negative and positive controls per well contained . ml of ez-prrsv tm mpx . reagent, . ml of enzyme blend, . ml of inhibition control and ml of the positive ( . ml of type and prrsv) or negative control (  te buffer). plates were briefly mixed by shaking on a vortex mixer ( s), centrifuged, and loaded into the thermal cycler ( fast real-time pcr system; applied biosystems , foster city, ca, usa). the thermal cycling conditions were as follows: one cycle at c for min, one cycle at c for min, cycles at c for s and c for s. samples with c t values < for either genotype prrsv were considered positive. quantification of samples was expressed in terms of the number of rna copies per ml for serum and copies per gram of lung tissues. these estimates were based on linear extrapolation of the cycle threshold values against a standard curve generated by serial dilutions of known amounts of in vitro transcript rna product (  to  copies per ml). all data were expressed as the mean ae standard error mean (sem) value of - pigs in each group. the differences in body temperatures, lung pathology scores, humoral responses and viremia between groups were assessed by one-way anova followed by tukey's multiple comparisons test. p-values < . were considered significant. all statistical analysis was performed using graphpad prism software, version . (graphpad software inc., san diego, ca, usa). all animals remained in good health after vaccination with d/ kv/adj or c/kv/adj vaccine and prior to viral challenge. any local or systemic vaccine side effects were absent pre-and postchallenge and none of the experimental pigs died during the entire study period. the body temperature of the animals in every group fluctuated within the normal physiological range after vaccination, but not statistically significant (data not shown). after pigs were challenged with lkz/ or cm/ , the body temperature in the d/kv/adj-vaccinated pig groups remained within the normal range ( . - . c) during the entire study period ( days). while the body temperature of the animals in the mock-vaccinated control and c/kv/adj-vaccinated pig groups were increased > . c on days - and days - post-challenge, respectively (fig. a ). there were significant differences in mean body temperature between the mock-vaccinated, c/kv/adj-vaccinated and d/kv/ adj-vaccinated pigs following the viral challenge. the magnitude of increase in body temperature was correlated with the severity of clinical disease, i.e., lower the body temperature lower the overall severity of clinical disease. severe respiratory disorders were observed after challenge with cm/ and lkz/ at and dpc, respectively, with significantly higher respiratory disease scores in the mock-vaccinated and c/kv/adj-vaccinated animals than the d/ kv/adj-vaccinated pigs (fig. b) . the bionote elisa demonstrated that all pigs were seronegative for prrsv at dpfv and remained low until dpfv ( fig. a-c) . the animals in the d/kv/adj-and c/kv/adj-vaccinated groups became prrsv seropositive as follows: at dpfv ( % and %), ( % and %), ( %), ( % and %) and ( % and %), respectively (fig. d-h) . this result indicated that the d/kv/adj vaccine induced greater prrsv specific antibody production than the commercial c/kv/adj-vaccine in pigs. all the c/kv/adj-vaccinated pigs ( %) became seropositive at dpc ( fig. i-k) . at dpc and , all the c/kv/adj vaccinated groups showed significantly higher s/p ratios compared to the control groups (p < . ). at dpc and , the s/p ratios of the d/ kv/adj-vaccinated groups were significantly higher than the c/kv/ adj-vaccinated groups (p < . ; fig. l -o). there was no significant difference between the s/p ratios of the c/kv/adjvaccinated and mock-vaccinated pig groups at dpc and ( fig. m-o) . additionally, the antibody titers of the control groups and remained negative ( fig. a-o) . serum prrs vn antibody titers in pigs were analyzed by the standard serum neutralization assay, and our results showed neither the d/kv/adj nor c/kv/adj vaccine induced vn antibodies to lkz/ (type ) or cm/ (type ) after the first or second vaccination. in the d/kv/adj-vaccinated pigs, vn titers against lkz/ were detected at dpfv (fig. a) . the vn titers in the d/ kv/adj-vaccinated groups were significantly higher compared to the c/kv/adj-vaccinated pigs at dpc and (p < . to p < . ) (fig. a) . a rapid vn antibody response against lkz/ virus was observed in the d/kv/adj vaccinated pig group indicating that the vaccine induced better protective memory immune response. to assess broad vn antibody activity, we used heterologous prrsv strain (type ) cm/ in vn assay and detected vn titers at dpfv in pig groups and vaccinated with d/kv/adj (fig. b) . the vn titers of these animals were significantly higher than those of c/kv/adj-vaccinated pigs (group ) at dpc - (p < . to p < . ) (fig. b) . the vn titers against the challenge virus strain of the pig group vaccinated with d/kv/adj and challenged with cm/ strain were similar to those of group animals challenged with the homologous virus strain lkz/ . this indicates that our experimental pigs received prrsv vaccination (strain lkz/ ) followed by a heterologous cm/ strain challenge generated antibodies with a broader neutralizing spectrum. significant difference in the macroscopic lung lesions scores were observed between pig groups and during necropsy at dpc . in a similar trend to the body temperature, less clinicallyaffected d/kv/adj-vaccinated pigs had significantly fewer lung lesions scores (p < . ) compared to severely clinically-affected mock-vaccinated and c/kv/adj-vaccinated pigs (fig. a ). all the mock-vaccinated and c/kv/adj-vaccinated pigs challenged with lkz/ or cm/ prrsv had gross lung lesions in the cranial, middle and accessory lobes, which resulted in failure of the lungs to collapse during necropsy and lung parenchyma remained firm and rubbery. no obvious lung lesions were observed in d/kv/adjvaccinated pig groups, indicating that this vaccine-adjuvant formulation enabled pigs to tolerate challenge with type and type prrsv. further, prrsv rna copy numbers in the lungs at necropsy ( dpc) was measured using the commercial quantitative pcr (qpcr) kit. in pig groups that were immunized with d/kv/adj, prrsv rna copies were undetectable in / animals (< rna copies per gram of lung tissue). the viral loads of the mock-vaccinated and c/ kv/adj-vaccinated pig groups were up to three logs ( ) above prrsv threshold detection limits; however, the viral loads of these groups were not significantly different (fig. b ). all the serum samples collected at dpfv , , , , , and were negative for prrsv type rna copies (data not shown). viremia after challenge was assessed by both qpcr and viral titers on marc- cells (fig. ) . prrsv rna could not be detected in the serum samples obtained from the pigs vaccinated with d/ . the viral rna copy numbers in the lung homogenates were quantified by qrt-pcr and its limit of detection was rna copies/ml. data is shown as mean ae sem viral rna copy numbers of or pigs in each group; ****p < . ; analyzed by two-way anova followed by tukey's multiple comparisons test. kv/adj at any time point throughout the period post-challenge, except in group pigs observed .  copies at dpc following challenge with the type viral strain cm/ . contrastingly, serum collected from pigs vaccinated with mock and c/kv/ adj had viremia with the viral copy numbers from .  to .  copies (fig. a) . the serum samples were also analyzed of virus titration using the marc- cells (fig. b) . prrsv was not detected in the serum of pig groups and collected at dpfv , , , , , and , confirming that the virus was properly inactivated (data not shown). the mock-vaccinated pig groups challenged with lkz/ or cm/ exhibited the highest mean viral titer of . and . log tcid /ml at dpc in serum samples, respectively. all the animals vaccinated with c/kv/adj remained viremic until dpc . in contrast, d/kv/adj vaccinated pigs did not show viremia at all the dpcs by both rt-pcr and viral isolation methods (fig. ) . inactivated (killed) and mlv prrsv vaccines are commercially available and licensed for use in many countries to control reproductive and respiratory forms of prrs (murtaugh and genzow, ) . however, each vaccine type possesses its own strengths and limitations. due to safety issues inactivated vaccines are preferred over attenuated vaccines; however, efficacy of current inactivated prrsv vaccines is questionable (renukaradhya et al., a; scortti et al., ; vanhee et al., ; zuckermann et al., ) . inactivated prrsv vaccine under field situations showed to elicit protective immune response under certain conditions, which was depending on the strain of infecting field virus. by employing a controlled inactivation procedure and using a suitable adjuvant, an inactivated prrsv vaccine could be developed that systematically induces prrsv specific vn antibody response after two vaccinations in naïve piglets (nilubol et al., ) . serum vn antibodies have been identified as a key component of protective immunity against prrsv infection (osorio et al., ) . researchers have previously showed that an inactivated vaccine could induce vn antibodies resulting in strong to partial virological protection in challenged pigs (misinzo et al., ; renukaradhya et al., a) . experiments of passive transfer of vn antibodies in pigs prior to prrsv infection showed vn antibodies could fully protect pigs against viremia and reproductive failure (lopez and osorio, ; osorio et al., ) . however, others observed only low to moderate degree of protection against heterologous strains of virus when pigs were immunized with inactivated prrsv vaccines (labarque et al., ; lager et al., ) . but intranasal delivery of poly(lactic-coglycolic) acid nanoparticle-entrapped uv-killed prrsv vaccine adjuvanted with soluble mycobacterium tuberculosis derived whole-cell lysate showed the induction of cross-protective immune response in pigs (binjawadagi et al., a (binjawadagi et al., , b . at present, it is generally accepted that there is a need for new and safe vaccines that protect against homologous and heterologous/heterogeneous prrsv infections. in this study, our objective was to assess the efficacy of a candidate bei-inactivated prrsv vaccine (d/kv/adj), which contains a recently isolated prrsv field strain (lkz/ ) in kazakhstan, against a homologous (type ) and heterogeneous (type ) viral challenges. a commercial killed prrsv vaccine (c/kv/adj, strain nvdc-jxa , type ) was selected as a reference vaccine, which has been in use for immunization of pigs against prrs in most of the pig farms in kazakhstan. the efficacy of our vaccine candidate was assessed by evaluating its ability to reduce viremia and prevent clinical disease, including lung lesions in vaccinated pigs challenged with a homologous or heterogeneous prrsv. elisa antibody analysis demonstrated that the commercial vaccine induced prrsv-specific antibodies; however, only % of vaccinated pigs became seropositive prior to challenge. after challenge by day all the c/kv/adj vaccinated pigs became seropositive. however, we did not observe vn titers to lkz/ or cm/ in the serum pre-challenge, and only a slightly elevated neutralizing antibody response was detected post-challenge in c/ kv/adj-vaccinated pigs. these results are consistent with other published studies (scortti et al., ; zuckermann et al., ) , wherein vaccination with inactivated prrsv candidate vaccines slightly improve vn antibody response post-challenge. moreover, comparable to mock-vaccinated pigs, c/kv/adj vaccinated animals became ill post-challenge and manifested characteristic clinical signs of prrs (severe respiratory disorders with high scores), with simultaneous detection of viremia. noticeable macroscopic lung lesions were observed at necropsy in all the mock-and c/kv/adjvaccinated pigs challenged with lkz/ (type ) or cm/ (type . data in (a) indicates the mean prrsv ae sem rna copy numbers of or pigs in each group, and data in (b) were expressed as the geometric mean ae sem log tcid /ml of or pigs in each group. serum samples of pigs were collected up to dpc ( dpfa) challenged with prrsv (lkz/ or cm/ ). the limit of detection by the assay kit was rna copies per ml. the limit of detection for the virus titration assay was . tcid /ml. ) virus, and detected high challenged viral rna copy numbers in the lungs. thus, we conclude that the commercial c/kv/adj vaccination in pigs does not protect against clinical disease, viremia and lung lesions against field variants of kazakh strains of prrsv. these results are in line with earlier studies, such as vaccination against prrs in the field provides variable degrees of protection, and reports of clinical outbreaks of prrs in vaccinated pigs have led to doubts about efficacy of current commercial prrsv vaccines (geldhof et al., ; thanawongnuwech and suradhat, ) . in contrast, elisa results demonstrated that the d/kv/adjvaccinated pigs produced prrsv-specific antibodies more rapidly than the c/kv/adj and were % seropositive. after the third immunization, vn antibodies were detected in all the animals vaccinated with d/kv/adj prior to challenge, albeit at low levels. interestingly, the vn titer did not reduce after challenge in any pig vaccinated with d/kv/adj, and the animals had consistently high vn titers against both homologous and heterogeneous prrsv. this data is in agreement with an earlier study, which showed that high vn titers are required at the time of challenge to offer full protection against high dose of virus used to infect animals (geldhof et al., ) . the detectable levels of replicating prrsv was absent post-challenge in d/kv/adj vaccinated pigs, which could be attributed to early appearance (pre-challenge) of vn antibodies targeting the putative neutralizing epitopes on the prrsv gp and m proteins (yang et al., ) , but it needs further investigation. inactivated vaccines predominantly elicit th response (spellberg and edwards, ), but we cannot exclude the possibility of th -th -balanced response in d/kv/adjvaccinated pigs, which also requires further research. in the lkz/ (type ) and cm/ (type ) prrsv strains challenged pigs vaccinated with d/kv/adj, we observed a strong association between the induction of virus-specific neutralizing antibody response and the absence of viremia, lung lesions and clinical disease; indicating that vn antibodies may have contributed significantly to cross-protection. similar results were also observed in our previous study with this candidate vaccine, but challenged with a heterologous nadc- (type ) strain of virus . these findings demonstrated that our candidate killed prrsv vaccine has a safety profile, and it is efficacious when administered three times at days , and to large-white breed of pigs aged months. the results of this study is interesting as the candidate killed prrsv vaccine provided strong heterogeneous protection, because even modified live prrsv vaccines have been shown ineffective against inter-genotypic virus and provided incomplete protection against reinfections and heterologous viruses (botner et al., ; kimman et al., ; martelli et al., ; renukaradhya et al., a,b) . the reasons beyond better protective response by our candidate vaccine could be attributed to vaccination of pigs three times with montanide tm seppic adjuvant compared to others, wherein in many vaccine trials killed or subunit prrsv vaccines were vaccinated twice using relatively weaker adjuvant than montanide adjuvant (renukaradhya et al., a) . furthermore, at this moment due to lack of genome sequence data of both the vaccine and challenge viruses, it is difficult to draw any meaningful conclusions on this interesting result. in this study, although our candidate bei-inactivated prrsv vaccine adjuvanted with montanide tm gel st provided protection against homologous (type ) and heterogeneous (type ) strains of virus, this does not confirm that this vaccine formulation would cross-protect against other heterogeneous and heterologous field strains of viruses; which needs further vaccine trials. due to lack of sequence data of vaccine and challenge viruses used in this study, it is likely that though the commercial vaccine virus and the challenge virus strain are type viruses, the level of genetic variability is not known. however, to our knowledge, this is the first report demonstrating that an inactivated prrsv vaccine could also provide cross-protective response against a different viral genotype in pigs, which offers new perspectives for the development of effective and safe prrsv vaccines. the authors declare no potential or actual conflict of interest. selection of effective activation agents for porcine reproductive and respiratory syndrome virus inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs appearance of acute prrs-like symptoms in sow herds after vaccination with a modified live prrs vaccine pathogenesis of porcine reproductive and respiratory syndrome virus adjuvants for porcine reproductive and respiratory syndrome virus vaccines detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars load reduction in live prrs vaccines using oil and polymer adjuvants comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus prrs costs industry $ million annually pork checkoff study effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology antibody and cellular immune responses of swine following immunisation with plasmid dna encoding the prrs virus orf's , , and impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenge-exposed with an antigenically distinct prrsv isolate protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein genomic analysis of two chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence montanide tm gel st adjuvant enhances prrs modified live vaccine efficacy by regulating porcine humoral and cellular immune responses role of neutralizing antibodies in prrsv protective immunity efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cellmediated immunity efficacy of an inactivated prrsv vaccine: induction of virus-neutralizing antibodies and partial virological protection upon challenge immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents the effect of a killed porcine reproductive and respiratory syndrome virus (prrsv) vaccine treatment on virus shedding in previously prrsv infected pigs identification of porcine reproductive and respiratory syndrome virus of north american genotype in republic of kazakhstan (in russian) passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity adjuvant formulation for veterinary vaccines: montanide tm gel safety profile a simple method of estimating fifty per cent endpoints inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv type /type immunity in infectious diseases isolation of the european genotype porcine reproductive and respiratory syndrome virus (prrsv) in kazakhstan the pathogenicity of swan derived h n virus in birds and mammals and its gene analysis an influenza viral vector brucella abortus vaccine induces good cross-protection against brucella melitensis infection in pregnant heifers bei-inactivated prrs vaccine adjuvanted with montanidetm gel st elicits virus specific cross-protective immune responses in piglets development of an experimental inactivated prrsv vaccine that induces virusneutralizing antibodies categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp and gp proteins and susceptibility to neutralization porcine reproductive and respiratory syndrome virus (porcine arterivirus). diseases of swine assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge key: cord- -pghnl authors: wang, hai-ming; liu, tian-xin; wang, tong-yun; wang, gang; liu, yong-gang; liu, si-guo; tang, yan-dong; cai, xue-hui title: isobavachalcone inhibits post-entry stages of the porcine reproductive and respiratory syndrome virus life cycle date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: pghnl porcine reproductive and respiratory syndrome virus (prrsv) is a pathogen of great economic significance that impacts the swine industry globally. since the first report of a porcine reproductive and respiratory syndrome (prrs) outbreak, tremendous efforts to control this disease, including various national policies and plans incorporating the use of multiple modified live-virus vaccines, have been made. however, prrsv is still a significant threat to the swine industry, and new variants continually emerge as a result of prrsv evolution. several studies have shown that pandemic prrsv strains have enormous genetic diversity and that commercial vaccines can only provide partial protection against these strains. therefore, effective anti-prrsv drugs may be more suitable and reliable for prrsv control. in this study, we observed that isobavachalcone (ibc), which was first isolated from psoralea corylifolia, had potent anti-prrsv activity in vitro. although many biological activities of ibc have been reported, this is the first report describing the antiviral activity of ibc. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. porcine reproductive and respiratory syndrome virus (prrsv) is a positive-strand rna virus that is approximately kb in length and belongs to the family arteriviridae [ ] . prrsv is one of the most important viruses hindering the development of the swine industry globally, especially with the emergence of highly pathogenic prrsv (hp-prrsv) [ , ] . recently, new prrsv mutant strains have become pandemic in china [ , , , , , ] and are named nadc -like prrsv strains for their genomic similarity to the nadc strain isolated in the us in [ ] . nadc -like prrsv strains feature different recombinations and have produced many mosaic isolates, enhancing their genetic diversity. currently, the most effective methods to control viral diseases are vaccines and antiviral agents. however, vaccination against prrsv infection has achieved limited success, primarily due to the high genetic diversity of the virus [ , , , ] . in addition to genetic mutation, prrsv genetic diversity is also generated by recombination among different strains, including vaccine strains [ , , , , , ] . importantly, commercial vaccines cannot provide complete protection against heterologous prrsv infection [ , ] . the problems outlined above indicate that the use of vaccines may not be an ideal strategy for prrsv control; however, exploring effective anti-prrsv drugs may overcome these issues. isobavachalcone (ibc) is a prenylated chalcone of the flavonoid subclass that was first isolated from psoralea corylifolia in [ ] . ibc possesses a wide spectrum of biological activities, including antibacterial, antifungal, anticancer, anti-reverse-transcriptase, antitubercular and antioxidant functions [ ] . however, whether ibc has potential antiviral activity remains unclear. in the current study, for the first time, we observed that ibc has anti-prrsv activity. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. porcine alveolar macrophages (pams) were obtained from the lungs of -week-old specific-pathogen-free (spf) piglets, and monkey kidney cells (marc ) were cultured in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (fbs). the highly pathogenic prrsv strain hun [ , ] and the nadc -like prrsv strain hen-l (isolated in our lab) were propagated and titrated in marc cells. ibc was purchased from shanghai tauto biotech company and was dissolved in ethanol and used at the concentrations indicated. the cytotoxicity of ibc was evaluated for pams and marc cells. briefly, twofold dilutions of ibc from a starting concentration of . μm were applied to % confluent cells in a -well plate. after incubation for h at °c, the cytotoxicity of ibc was evaluated using a cell counting kit- (cck , dojindo laboratories, japan) according to manufacturer's protocol. the cc was defined as the concentration of ibc that reduced the absorbance of treated cells by % relative to that of the cell control. a total of × marc cells were plated in a -well plate, and h later, the cells were infected with hun (multiplicity of infection [moi] = ) for h at °c. next, the culture medium was replaced with different concentrations of ibc diluted in dmem containing % fbs. cells were harvested at the indicated times for further western blot or immunofluorescence analysis. the total rna of all samples was extracted using an rneasy plus mini kit (qiagen, germany), and μg of total rna was reverse transcribed into cdna according to the manufacturer's protocol (primescript™ rt reagent kit with gdna eraser, takara). the real-time pcr procedure was performed using an agilent mx p real-time pcr system. the primers and probes were described in a previous report [ ] . cells were infected with hun for h and were then treated with various concentrations of ibc. after incubating for the indicated time, cells were fixed in % ethanol, permeabilized with . % saponin in pbs, and then blocked with % bovine serum albumin (bsa) for h at room temperature (rt). after blocking, the cells were incubated with a mouse anti-prrsv n protein monoclonal antibody (igg b, made in our lab) at a dilution of : for h at rt, and dsrna was stained with the mouse monoclonal antibody j (scicons, hungary) at a dilution of : for h at rt. after washing, an aliquot of : -diluted fitc-labeled antimouse igg (sigma) was used as the secondary antibody. for nuclear visualization, cells were treated with dapi (sigma). immunofluorescence was observed using a leica tcs sp confocal microscope. cells were washed twice with pbs and then prepared with ripa lysis buffer (solarbio). the cell lysates were centrifuged at , rpm for min at °c. the supernatants were collected, and protein concentrations were determined by bca assay. next, proteins from each sample were separated via % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), after which the protein bands were transferred onto a polypropylene fluoride (pvdf) membrane. the membrane was blocked with % nonfat milk for h at rt and then incubated with mouse anti-prrsv n protein monoclonal antibody ( : , made in our laboratory) or anti-β-actin antibody ( : , sigma) for . h at rt. after being washed three times with pbst ( . % tween in pbs), the membrane was incubated for h with hrp-conjugated anti-mouse igg antibody (sigma). protein bands were visualized with ecl chemiluminescence reagent (thermo scientific). western blot bands were quantified and analyzed using imagej. the relative intensity ratio of protein bands (n protein/β-actin) was calculated as an indicator of prrsv replication ability, with the value of the control group set as ; other ibc treated groups were normalized correspondingly. to further investigate the anti-prrsv activity of ibc, the drug concentration that could inhibit prrsv infections by approximately % ( % inhibitory concentration, ic ) was determined. marc cells were infected with prrsv (moi = ) for h at °c and then treated with twofold dilutions of ibc. total rna was isolated from cells at h postinfection (p.i.), and intracellular prrsv rna copy numbers were determined by rt-pcr. the percentage inhibition was calculated as [(rna copies in control cells -(rna copies in cells treated with ibc)] ÷ (rna copies of the control) × at the indicated ibc concentration, and the value for the control group was set as . the other ibc-treated groups were normalized correspondingly. a virus attachment and entry assay was performed by preincubating marc cells at °c for min, after which prrsv hun (moi = ) and μm ibc were added, and the cells were incubated for an additional h at °c to allow attachment of the virus to the cells. next, the cells were washed three times with cold pbs to remove unbound virus. the cells were then collected and either used to detect prrsv rna levels via real-time pcr (to assess viral attachment) or provided fresh medium containing μm ibc and incubated at °c for h. a temperature shift from to °c was performed to promote entry of the virus into the cells, and after a -h incubation, total rna was collected and analyzed by real-time pcr (to assess viral entry). marc cells were infected with prrsv hun (moi = ) at °c. after a -h incubation, cells were washed three times with pbs to remove unbound viruses and were replenished with % fbs. for the postinfection inhibition study, hun -infected cells were replenished with % fbs supplemented with or μm ibc at specific time points ( , , , or h). cells were harvested at h after ibc treatment, and the levels of prrsv replication were estimated by western blot analysis. origin . software was used for all graphical representations. the ic was calculated using non-linear regression. statistical significance was established using an independent t-test, and p-values less than . were considered significant. the structure of ibc is shown in figure a . to assess the anti-prrsv activity of ibc, we first evaluated the cytotoxicity of ibc on marc cells (fig. b) and pam cells (fig. c) , and the results indicated that cell viability was not significantly affected by ibc at concentrations up to . μm and that the cc of ibc for marc cells was . μm (data not show). the cytotoxic effect of the ibc solvent (alcohol) was also evaluated, the results of which indicated that the alcohol had no effect on marc cells (fig. b) and pam cells (fig. c) . next, we evaluated the anti-prrsv capabilities of ibc in marc cells that were infected with prrsv hun (moi = ) and then treated with different concentrations of ibc. we first determined progeny virus titers and found that ibc significantly inhibited prrsv replication in a dose-dependent manner (fig. d ). to confirm this finding, we also performed an indirect immunofluorescence assay by staining cells with a prrsv n protein antibody and then observed prrsv replication by fluorescence microscopy. as shown in figure e , prrsv replication was significantly blocked by ibc. western blot analysis revealed that prrsv n protein levels decreased markedly, indicating that increasing concentrations of ibc significantly inhibited prrsv replication (fig. f) . to determine whether this inhibition was strain specific, we additionally assayed a currently pandemic nadc -like strain (hen-l ) that was isolated in our lab and observed it also to be inhibited by ibc (fig. g) . thus, these data indicate that ibc inhibition of prrsv is not strain specific. furthermore, the antiviral activity of ibc against prrsv hun was both dose dependent and successful, with an ic of . μm (fig. ) and a selectivity index (si; the ratio of the cc to the ic ) of . . despite the evidence that ibc inhibits prrsv replication, the viral stage influenced by ibc remained unknown. to determine which step(s) in the viral life cycle are affected by ibc, we first performed time-of-drug-addition assays. the results indicated that ibc markedly inhibited prrsv when added early after infection and remained inhibitory when added up to h p.i., whereas prrsv inhibition was limited when ibc was added late, at h p.i. (fig. ) . these data suggest that ibc targets the early phase of the viral life cycle, as it was not able to prevent viral infection after the virus entered the post-replicative stages of infection. next, we tested whether ibc inhibited prrsv by disturbing prrsv attachment or entry. the results demonstrated that prrsv attachment was not influenced by ibc (fig. a) . interestingly, the prrsv entry assay results were the same as those of the attachment assay, demonstrating that neither prrsv attachment nor entry was influenced by ibc (fig. b ). the addition of ibc was inhibitory from to h p.i., whereas it had no effect on prrsv attachment or entry ( fig. a and b) . this inhibition at post-entry stages led us to speculate that ibc inhibits prrsv replication at the stage of viral rna synthesis. to test whether ibc blocked viral rna replication, we performed inhibition experiments using pam cells and stained for dsrnas, which are intermediates in viral replication, with the j antibody, which recognizes dsrna. the results indicated that dsrna levels were significantly decreased in ibctreated cells (fig. a and b) . these data clearly suggest currently, prrsv is a significant problem for the swine industry in china and was a veritable pandora's box for the chinese pig industry during the first outbreak [ ] . the current pandemic strains are highly diverse, and commercial vaccines can provide only partial protection against these strains [ , ] . thus, anti-prrsv drugs may be more suitable for prrsv control in the future. traditional chinese medicine (tcm) has been widely used as a source of novel drugs, and many crude tcm herbal extracts have been shown to inhibit prrsv replication [ , , ] . ibc is extracted from p. corylifolia, which itself is used in tcm [ ] . cheng et al. systematically analyzed seventeen compounds derived from tcms and tested their prrsv antiviral activity in vitro [ ] . two compounds, chlorogenic acid and scutellarin, were shown to have great anti-prrsv replication potential in their study, and the inhibition ratios of chlorogenic acid and scutellarin can reach . and . %, respectively, at the maximum noncytotoxic concentrations [ ] ; in contrast, the ibc inhibition ratio exceeded % at μm, an improvement over the maximum non-cytotoxic concentrations for the other tested compounds. this result indicated that ibc is more effective than chlorogenic acid and scutellarin. ibc has multiple biological activities; it potently abrogates akt and erk signaling pathways and exerts anti-proliferative effects on several human cancer cell lines, and it also induces apoptosis associated with the mitochondrial pathway [ , , ] . akt has been reported to play a positive role in prrsv randomly selected for statistical analysis. the mean rates of positive cells among the total cells in each group were calculated using imagej gene expression and entry, and prrsv replication may be dependent on the activation of pi k/akt [ , , , ] . the erk signaling pathway has been demonstrated to play an important role in the post-entry steps of the prrsv replication cycle and to enhance prrsv infection [ ] . the erk pathway may also contribute to hp-prrsv pathogenesis [ ] . apoptosis also influences prrsv replication, since the induction of apoptosis in marc cells increases virus replication [ ] . ibc inhibition of prrsv replication may occur through modulation of the above-mentioned pathway. ibc may be applicable as an efficacious and safe drug for the treatment of some cancers, such as neuroblastoma [ ] . ibc has been reported to inhibit sars-cov papain-like protease; however, whether ibc inhibits infectious virus was not tested in that study [ ] . this study is the first to report that ibc has potent antiviral activity, and we demonstrate here that it blocks prrsv replication at the initiation of viral rna replication in vitro. however, there are some unanswered questions that should be further explored in the future. first, whether ibc also shows anti-prrsv activity in vivo must be investigated. second, is the blockage of viral rna formation a direct or indirect effect? third, are there other viruses that are also inhibited by ibc? in conclusion, we evaluated whether ibc has antiviral potential as a prrsv infection treatment. the in vitro study established that ibc efficiently inhibited the replication of both hp-prrsv and the current chinese epidemic nadc -like strains without significant drug toxicity. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. commercial vaccines provide limited protection to nadc -like prrsv infection highly pathogenic porcine reproductive and respiratory syndrome virus induces prostaglandin e production through cyclooxygenase , which is dependent on the erk / -p-c/ebp-beta pathway genomic sequence and virulence comparison of four type porcine reproductive and respiratory syndrome virus strains in vitro screening for compounds derived from traditional chinese medicines with antiviral activities against porcine reproductive and respiratory syndrome virus the structural biology of prrsv antiviral strategies against prrsv infection observation of high recombination occurrence of porcine reproductive and respiratory syndrome virus in field condition propagation of field highly pathogenic porcine reproductive and respiratory syndrome virus in marc- cells is promoted by cell apoptosis porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) isobavachalcone induces the apoptosis of gastric cancer cells via inhibition of the akt and erk pathways abrogation of akt signaling by isobavachalcone contributes to its anti-proliferative effects towards human cancer cells phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia isobavachalcone: an overview porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway outbreak investigation of nadc -like prrsv in south-east china widespread of nadc -like prrsv in china: another pandora's box for chinese pig industry as the outbreak of highly pathogenic prrsv in recombination in jxa -r vaccine and nadc -like strain of porcine reproductive and respiratory syndrome viruses the involvement of fak-pi k-akt-rac pathway in porcine reproductive and respiratory syndrome virus entry isobavachalcone, a chalcone constituent of angelica keiskei, induces apoptosis in neuroblastoma preparation for emergence of an eastern european porcine reproductive and respiratory syndrome virus (prrsv) strain in western europe: immunization with modified live virus vaccines or a field strain confers partial protection live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china sodium tanshinone iia sulfonate inhibits porcine reproductive and respiratory syndrome virus via suppressing n gene expression and blocking virus-induced apoptosis open reading frames a and b of the porcine reproductive and respiratory syndrome virus (prrsv) collaboratively initiate viral minus-strand rna synthesis recombinant pseudorabies virus (prv) expressing firefly luciferase effectively screened for crispr/cas single guide rnas and antiviral compounds nadc -like porcine reproductive and respiratory syndrome in china development and application of taq man-mgb fluorescence quantitative rt-pcr assay for detection of porcine reproductive and respiratory syndrome virus immune responses to modified live virus vaccines developed from classical or highly pathogenic prrsv following challenge with a highly pathogenic prrsv strain molecular epidemiology of porcine reproductive and respiratory syndrome virus in central china since : the prevalence of nadc -like prrsvs efficient porcine reproductive and respiratory syndrome virus entry in marc- cells requires egfr-pi k-akt-limk -cofilin signaling pathway role of phosphatidylinositol -kinase (pi k) and akt kinase in porcine reproductive and respiratory syndrome virus (prrsv) replication complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wild-type prrsv strains a dual effect of porcine reproductive and respiratory syndrome virus replication on the phosphatidylinositol- -kinase-dependent akt pathway pathogenicity and antigenicity of a novel nadc -like strain of porcine reproductive and respiratory syndrome virus emerged in china in vitro evaluation of the antiviral activity of the synthetic epigallocatechin gallate analog-epigallocatechin gallate (egcg) palmitate against porcine reproductive and respiratory syndrome virus importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome in china nadc -like strain of porcine reproductive and respiratory syndrome virus efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus nadc -like acknowledgements this study was supported by the national key r&d program (grant number yfd ) and the national science foundation of heilongjiang (grant number zd ). key: cord- -o kffjw authors: shi, xibao; zhang, xiaozhuan; chang, yongzhe; jiang, bo; deng, ruiguang; wang, aiping; zhang, gaiping title: nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc- cells date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: o kffjw background: porcine reproductive and respiratory syndrome virus (prrsv) induces one of most important devastating disease of swine worldwide, and the current methods poorly control it. previous studies have indicated that the nonstructural protein (nsp ) of prrsv may be an important protein for the immune escape of prrsv. results: here, we firstly explored the effect of over-expression of nsp on prrsv infection and found that over-expression of nsp enhanced the prrsv titers while the small interfering rna (sirnas) specifically targeting nsp could reduce the prrsv titers in marc- cells. conclusion: in conclusion, prrsv nsp promotes prrsv infection in marc- cells and sirnas targeting nsp may be a potential therapeutic strategy to control prrsv in future. background prrsv, a positive sense and single-stranded rna virus, is a member of family arteriviridae [ ] . since it was emerged in the united states in and in europe in , prrsv has rapidly spread in the swine producing regions and became one of the most important devastating diseases of swine worldwide. it can cause severe reproductive failure in sows and respiratory distress in young growing pigs [ ] . infection with prrsv also made pigs easy to secondary infection by other pathogens [ ] . up to date, since there is no efficient method or drugs against prrsv, it is very important and urgent to develop the effective therapeutic strategies to control prrs. the prrsv genome has nine open reading frames (orfs) composed of orf a, orf b, orf a, orf b, and orf - . orf a and orf b could produce nonstructural proteins (nsp α, nsp β, nsp etc.) [ ] [ ] [ ] [ ] . previous studies have shown that the nsp of equine arteritis virus(eav), which is another member of family arteriviridae, may play a key role in viral rna synthesis and additional functions in the viral life cycle [ ] . other and our previous work also demonstrated that prrsv nsp inhibited the host innate immune responses such as the transcription of type i interferon [ ] , the rnai innate immune response [ ] and the nlr family pyrin domain-containing (nlrp )-mediated production of il- β [ ] , which indicated that prrsv nsp may play an important role in prrsv infection. so the purpose of present study is to explore the effect of over-expression of nsp on prrsv infection and whether the sirnas targeting the prrsv nsp could influence prrsv infection. marc- cells, derived from a monkey fibroblast cell line ma- [ ] , and t cells were grown in dulbecco's modified eagle medium (gibco) plus % heatinactivated fetal bovine serum (hyclone). prrsv strain bj- was a kind gift from prof. hanchun yang (china agricultural university). the pcdna . -gfp-nsp plasmid was constructed by sub-cloning from the plasmid pcdna . -flag-nsp [ ] to pcdna . -gfp [ ] using the restriction endonuclease hind iii and ecori. the plasmids pcdna . -flag, pcdna . -flag-nsp and pcdna . -flag-nsp h a have been described in our previous work [ ] . all newly-prepared plasmids were confirmed correctly by dna sequencing. transient transfection was carried out by using lipofectamine (invitrogen). cells cultured in -well plates were transfected with the indicated expression plasmid or control vector ( ng/well) in triplicate. and h (h) later, the cells were infected with prrsv at a multiplicity of infection (moi) of . , and then the cells were lysed by freezing and thawing three times after h infection. the supernatants were collected by centrifugation, and the viral titers were detected by % tissue culture infected dose (tcid ) assay using the method of reed-muench in the marc- cells. the t cells were cultured in -well plates and transfected with pcdna . -gfp-nsp or pcdna . -gfp and nsp sirna ( nm) or control sirna ( nm) in triplicate, and h later, the cells were lysed with lysing buffer ( % nonidet p- , . % sodium deoxycholate, . % sds, mm tris-hcl (ph . ), mm nacl, mm edta, mm na vo , mm naf and a protease inhibitor cocktail). the detailed procedure for immunoblots has been described in our previous work [ ] . briefly, the proteins were separated by % sds-page and transferred to polyvinylidene difluoride (pvdf) membranes (millipore company, boston, massachusetts, usa), and then the pvdf membranes were incubated with anti-gfp (clontech) or anti-actin (cell signaling technology) antibodies. subsequently the membranes were incubated with appropriate secondary antibodies and were tested by an ecl detection system (cell signaling technology, boston, usa). cells cultured in -well plates were transfected with the indicated sirna in triplicate ( nm/well) (chemical synthesis by bioneer) (table. ). and h later, the cells were infected with prrsv strain bj- at an moi of . , and h later, the cells were lysed by freezing and thawing three times. the supernatants were collected by centrifugation, and the viral titers were detected by tcid using the method of reed-muench in the marc- cells. marc- cells cultured in -well plates were transfected in triplicate with nsp -sirnas/control sirna and plasmid pcdna . -gfp-nsp ( ng/well) or pcdna . -gfp ( ng/well). and h later, the cells were infected with prrsv at an moi of . , and h later, the cellular rna was extracted with trizol (invitrogen). m-mlv reverse transcriptase was used for the primescript™ rt reagent kit with gdna eraser (takara, dalian, china). quantitative real-time rt-pcr (qrt-pcr) was performed using sybr® premix ex taq™ (takara, dalian, china) and was tested by the first real-time pcr system (applied biosystems, foster city, ca, usa). glyceraldehyde- -phosphate dehydrogenase (gapdh) was used as an internal control. the −ΔΔct -method was used to analyze the relative amount of target gene expression. marc- cells transfected with the nsp sirna ( nm) or control sirna ( nm) and the plasmid pcdna . -gfp-nsp ( ng/well) or pcdna . -gfp ( ng/well), and h later, the cells were infected by prrsv at an moi of . , the cells were frozen and thawed in three cycles and collected after h infection. then the viral titers were determined by tcid assay using the method of reed-muench in the cells of marc- . statistical analyses were performed by student's t-test, and the differences were considered as statistical significance when p < . . firstly, we successfully constructed the expression plasmid of pcdna . -gfp-nsp (gfp-nsp ) and the western blot in fig. a confirmed the successful expression of gfp-nsp (fig. ) . secondly, the marc- cells were cultured in -well plates overnight, and then the cells were transfected with the expression plasmid pcdna . -gfp-nsp or the control plasmid pcdna . -gfp. the results in fig. b showed that the prrsv titers from the marc- cells transfected with pcdna . -gfp-nsp were about one point six times to that from marc- cells transfected with control plasmid, while the rna levels of prrsv from the marc- cells transfected with pcdna . -gfp-nsp were three times to that from marc- cells transfected with control plasmid (fig. c) . now that over-expression of nsp could enhance prrsv titers, it was reasonable to design sirnas targeting nsp to investigate whether the sirnas could reduce prrsv titers. the sequences of the sirna special for nsp and the control sirna were listed in table . t cells (fig. a) or marc- cells (fig. b ) grown in -well plates were co-transfected with the nsp sirna ( nm/well) or control sirna and the plasmid gfp-nsp ( ng/well). and h later, the cells in five random fields were analyzed by fluorescence microscopy ( ×) and only one of them was shown in fig. . the results in fig. showed that all of the three sirnas targeting nsp could inhibit the expression of gfp-nsp and didn't influence on the expression of gfp. finally, we selected two sirnas (sirna and sirna ) targeting nsp to determine whether sir-nas targeting nsp could reduce prrsv titers in marc- cell. the western blots results in fig. a confirmed that sirna and sirna could efficiently reduce the nsp expression in t cells (fig. a) . the results in fig. b and c showed that sirnas targeting nucleic acid sequence of nsp could significantly reduce viral titers of prrsv (fig. b) and reduce rna levels of prrsv (fig. c) . the endoribonuclease activity of nsp was important for nsp to enhance the prrsv titers our previous work has shown that the endoribonuclease activity of nsp was important for nsp to inhibit the transcription of ifn-β [ ] and the secretion of il- β [ ] . so next we investigated whether the endoribonuclease activity of nsp was important for nsp to promote the prrsv infection. nedialkova et al. showed that his- , his- , and lys- were the catalytic centers, and mutating one of the three amino acids could abolish its enzyme activity. the results in fig. showed that inactivating the endoribonuclease activity made nsp not promote the prrsv infection. nsp was a multi-functional protein. both our and other previous studies have shown that prrsv nsp is an interferon antagonist [ , ] and that nsp plays an important role in viral rna synthesis and in the viral life cycle [ ] . our recent work also demonstrated that prrsv nsp inhibited the rnai innate immune response [ ] and nlrp -mediated production of il- β [ ] . while our present work showed that over expression of prrsv nsp could enhance the titers of prrsv (fig. ) , so our present work gave a directly evidence that nsp was an important viral component for upregulating the prrsv titers. identification of and targeting viral important components is useful for developing viral vaccine and controlling the virus. for example, both the nonstructural protein of influenza virus and the nonstructural protein of mouse hepatitis virus (mhv) were important for the viral virulence respectively, and both the modified live-viral vaccines that deletion of nonstructural protein resulted in complete protection against challenge with influenza virus infection and mhv infection respectively [ ] [ ] [ ] . rna interference (rnai) is an exciting method to silence viral genes. inhibition of specific genes by sirna has proven to be a potential therapeutic strategy against viral infection [ ] , especially for the positive single stranded rna viruses since their genomes function as both the mrna and the replication template [ , ] . up to date, rnai has been used against several viruses such as hepatitis b virus, foot-and-mouth disease virus, dengue virus and so on [ ] [ ] [ ] . in this work, we also explore whether the sirna, which targeted the nucleic acid sequence of nsp , influenced the titers of prrsv, the endoribonuclease activity of nsp was important for nsp to enhance the prrsv titers. marc- cells cultured in -well plate were transfected with pcdna . -flag-nsp (nsp ) ( ng/well), pcdna . -flag-nsp h a (nsp h a) ( ng/ well) or pcdna . -flag (con). and h later, the cells were infected with prrsv at an moi of . or mock infected, and h later, the cells were lysed by freezing and thawing three times in three cycles, then the viral titers were measured by tcid . data represented means of three replicates, and experiments were repeated three times. error bars represented the standard deviations. *: p < . compared with the results in control. moi, multiplicity of infection and the results showed that sirna targeting nsp significantly reduced the titers of prrsv (fig. ) . a recent improved live prrsv vaccine has indicated that the orf a and orf b were the virulence determinants of prrsv [ ] . in addition, our recent work also show that rnai innate immune response was an antiviral response to prrsv and prrsv inhibited this response by prrsv nsp α and nsp , which indicated that targeting nsp would be useful for rnai innate immunity against prrsv [ ] . so it is reasonable to propose that our present results gave a new clue for generating the new prrsv vaccine by targeting prrsv nsp . in conclusion, our present study has shown that nsp was an important viral component for up-regulating the titers of prrsv and that sirnas directly targeting nsp could inhibit prrsv infection, which indicated that prrsv nsp may be an interesting target for controlling prrsv in future. abbreviations eav, equine arteritis virus; gapdh, glyceraldehyde- -phosphate dehydrogenase; gfp-nsp , pcdna . -gfp-nsp ; mhv, mouse hepatitis virus; moi, multiplicity of infection; nlrp , nlr family pyrin domain-containing ; nsp , nonstructural protein ; orf, open reading frame; prrsv, porcine reproductive and respiratory syndrome virus ; pvdf, polyvinylidene difluoride; qrt-pcr, quantitative real-time rt-pcr; rnai, rna interference; sirna, small interfering rna; tcid , % tissue culture infected dose nidovirales: a new order comprising coronaviridae and arteriviridae general overview of prrsv: a perspective from the united states the challenge of prrs immunology equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily identification of two auto-cleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle porcine reproductive and respiratory syndrome virus (prrsv) inhibits rna-mediated gene silencing by targeting ago- the endoribonuclease activity essential for the nonstructural protein of porcine reproductive and respiratory syndrome virus to inhibit nlrp inflammasomemediated il- beta induction enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line the zinc-finger domain was essential for porcine reproductive and respiratory syndrome virus nonstructural protein- alpha to inhibit the production of interferon-beta dynamic balance of pstat and pstat in c bl/ mice infected with lethal or nonlethal plasmodium yoelii porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation vaccination of pigs against swine influenza viruses by using an ns -truncated modified live-virus vaccine a novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist ns coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines rna interference: the story of gene silencing in plants and humans administration of e and ns sirnas inhibit chikungunya virus replication in vitro and protects mice infected with the virus rnai: antiviral therapy against dengue virus targeted delivery of sirna against hepatitis b virus by pres peptide molecular ligand inhibition of foot-and-mouth disease virus replication by small interfering rna attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence we would like to thank prof. hanchun yang for providing prrsv strain bj- . availability of data and materials all datasets are available in the main manuscript. authors' contributions sx, zx, zg designed the study, sx, zx and cy performed the experiments and wrote the paper, and wa, jb and dr performed statistical analysis. all authors read and approved the final manuscript. the authors declare that they have no competing interests. our present work does not contain any individual persons' data, so it is not applicable.ethics approval and consent to participate our present work didn't use animals.• 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- -c y m do authors: guo, baoqing; lager, kelly m.; henningson, jamie n.; miller, laura c.; schlink, sarah n.; kappes, matthew a.; kehrli, marcus e.; brockmeier, susan l.; nicholson, tracy l.; yang, han-chun; faaberg, kay s. title: experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: c y m do the pathogenesis of type highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) in -week old swine in the united states was investigated. rjxwn , rescued from an infectious clone of chinese hp-prrsv, replicated in swine with at least -fold increased kinetics over u.s. strain vr- . rjxwn caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to hp-prrsv than to those infected with vr- . novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of hp-prrsv to rapidly transmit between animals. furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in hp-prrsv infected swine, and showed that contact swine differed in the degree of cytokine response. in , investigators from several chinese provinces reported a unique syndrome in growing swine that was highlighted by the predominant clinical signs of high fever, anorexia, listlessness, red discoloration of skin, respiratory distress and very high morbidity and mortality rates (li et al., ; tian et al., ; tong et al., ; wu et al., ; zhou et al., ) . originally known as porcine high fever disease (phfd), this syndrome spread to vietnam in and to cambodia, laos, the philippines, bhutan, myanmar, thailand, south korea and russia in later years feng et al., ) . although there was concern that this syndrome may have been caused by a new disease agent, extensive diagnostic testing revealed only known pathogens. one consistent finding was the detection of porcine reproductive and respiratory syndrome virus (prrsv) with two discontinuous deletions in the replicase polyprotein known as nonstructural protein (nsp ), and two single nucleotide deletions in the and untranslated regions (utrs) (zhou and yang, ) . experimental infection of chinese swine with the initial novel prrsv field isolates reproduced the clinical disease (li et al., ; tian et al., ; tong et al., ; wu et al., ; zhou et al., ) , providing strong evidence for its role as the causal agent of phfd. since the severity of the clinical disease was greater than expected for a typical prrsv infection, there remained the chance that an unknown agent in the prrsv isolates exacerbated the disease symptoms. this question was resolved when phfd was reproduced in chinese swine with virus derived from an infectious clone of the jx prrsv isolate (lv et al., ) . the prior studies demonstrated that prrsv isolates with a common genetic pattern had a causal role in phfd, leading to the new designation of this viral lineage as highly pathogenic prrsv (hp-prrsv). the presence of the unique nsp deletion motif was initially thought to contribute to the severity of disease (tian et al., ) . however, studies showed that the nsp region of hp-prrsv strain rjxwn containing the novel deletion could be replaced with amino acids from low virulence strain hb- / . to result in a chimeric virus with only a minor delay in mortality (zhou et al., ) . there was also concern that there may be some unique aspect that may predispose asian pigs to a severe outcome, e.g., husbandry practices, endemic infections with other pathogens, climate, or host genetics. although a number of experiments have associated genetic changes in prrsv with an attenuation phenotype using point mutations (grebennikova et al., ; nielsen et al., ; storgaard et al., ) or by the construction of chimeric viruses (ellingson et al., ; kwon et al., ; wang et al., ; zhou et al., ), there appears to be no single locus for which mutations confer a predictable change in virulence. collectively, this area of study suggests the factors that contribute to prrsv pathogenicity are complex and viral strain-specific . as part of the single-strand positive-sense rna virus order nidovirales, family arteriviridae, prrsv genomes can vary between kb to . kb, consist of at least open reading frames (orfs) and replicate through a nested set of subgenomic rnas (van hemert and snijder, ) . members of this highly variable virus have been grouped into two genotypes, type (european-like, prototype strain lelystad) or type (north american, prototype strain vr- ), which differ in nucleotide similarity by approximately % (nelsen et al., ) . both genotypes have representatives of varying pathogenicity, and intragenic nucleotide sequence variation in each can be as much as % (shi et al., ) . for this study, we imported a plasmid containing the cdna copy of the hp-prrsv strain rjxwn genome, a type prrsv genome that is , bases in length and has % pairwise nucleotide identity to the prototype genome of vr- (zhou et al., ) . to investigate the pathogenesis of hp-prrsv infection and the potential contribution of climate, host genetics, commensal bacteria, other environmental conditions and husbandry practices to the pathogenicity of hp-prrsv in u.s. swine, we compared and contrasted the pathogenicity of rescued jxwn (rjxwn ) virus to that of vr- in -week old swine. we found that this hp-prrsv strain caused extreme morbidity, as was seen in asia, but novel to this study, resulted in up to x higher abundance of circulating virus when compared to vr- , caused extremely exacerbated thymic atrophy such that the thymus was often difficult to discern, and the host response was assessed in comparison to animals infected with strain vr- for the first time by a swine protein array including innate and adaptive cytokines in serum, bronchoalveolar lavage fluid and lymph nodes. moreover, we completed bacterial speciation and loads after necropsy and specified the degree of weight loss seen. lastly, we showed that hp-prrsv readily transmitted to contact swine causing a different pattern of cytokine responses. as a result of our study, the remaining plausible contributing factors to the high virulence seen in asia were discredited, and thus hp-prrsv strains pose a serious threat to the u.s. swine industry. pigs challenged with chinese hp-prrsv strain rjxwn (group ) began exhibiting clinical signs of disease within - days post exposure (dpe). as a group, the pigs developed fevers, became listless, anorexic, and began shivering and huddling together in a pile. clinical signs became more severe over the next few days, with pigs rapidly losing body weight (fig. ) , becoming dehydrated and weak. respiratory distress, characterized by dyspnea, tachypnea and coughing was common in all pigs. erythema of the skin was present in most of the pigs, and several developed cutaneous hemorrhages and cyanotic extremities (blue ears) (fig. ). on dpe, one pig from group was found dead. two others were euthanized due to severe weakness and moribund condition on dpe and , and one was found dead on dpe . on and dpe, clinical signs of disease in some of the hp-prrsv challenged group began to decrease in severity. while pigs remained huddled in a pile with respiratory signs, approximately half of them were subjectively less listless, would move away when approached and began showing an interest in feed again. during the study, contact pigs displayed similar clinical signs as those challenged with hp-prrsv . pigs challenged with vr- (group ) were clinically normal until dpe , when they began to exhibit slightly increased respiratory rates and became a little less active than the control group. one pig from the vr- group was found dead on dpe ; however, the cause of death was attributed to gastric dilatation and volvulus, rather than to clinical disease typical of prrsv strain vr- . control pigs (group ) remained clinically normal for the duration of the study. postmortem examination revealed severe lesions in the hp-prrsv challenged pigs including: marked interstitial pneumonia, lymphadenopathy and thymic atrophy. necropsy findings in this group include: pulmonary edema, pleuritis, peritoneal and pericardial effusions, renal petechia, and fibrinous peritonitis that are delineated in table . lungs and representative lesions are shown in fig. . no pathologic lesions were identified in control pigs (group ). pigs in the vr- challenge group (group ) had diffuse interstitial pneumonia, characteristic of an uncomplicated prrsv infection. although lung lesions were subjectively more severe in the hp-prrsv and contact groups, a statistically significant difference in lung scores was only found between the rjxwn contact group and the group inoculated with vr- (groups and , po . ) (fig. a) . lymphadenopathy was present in all infected pigs (groups - ), with no statistically significant difference between hp-prrsv and vr- infected groups (fig. a ). thymic atrophy was also a common finding in infected pigs and was significantly more severe in hp-prrsv infected and contact pigs compared to vr- infected pigs (p ¼ . and po . ; fig. b ). table summarizes aspects of the clinical disease seen in this study. shown are adwg and associated standard errors for each of the four groups for days À - and days - . sham inoculated control swine (group ) and those challenged with vr- (group ) gained weight at an expected rate for their age group. group swine challenged with hp-prrsv strain rjxwn and their contacts (group ) lost body weight during the last week of the experiment. the adwg for group on days À - includes only pigs, and on days - includes only pigs. the body weights for the contact animals were taken on the same day as pigs in the other groups. significance values are shown. microscopic lung lesions consisted of histiocytic interstitial pneumonia with increased numbers of macrophages in alveolar septa and lumina (fig. a , c). type ii pneumocyte hyperplasia was also present. these lesions were seen in the hp-prrsv challenge group (group ), the hp-prrsv contact pigs (group ) and the vr- challenge group (group ). mean interstitial pneumonia lesions were . , . , and . for chinese prrsv challenge group (group ), contact chinese prrsv pigs (group ) and vr- challenge (group ), respectively (fig. b ). lesions were significantly (p¼ . ) more severe in the chinese prrsv challenge group (group ) and contact chinese prrsv pigs (group ) as compared to the vr- challenge (group ). two sham inoculated control pigs had mild lesions with a score of , while the remaining pigs had no lesions. immunohistochemistry labeling for prrsv nucleocapsid was positive in % of chinese prrsv challenge and contact pigs (groups and ) and % of vr- challenge pigs (group ), with mean ihc labeling scores of . , . , and . , respectively (fig. ) . positive labeling was present in alveolar macrophages and interstitial macrophages for all infected groups ( fig. b and c). no significant difference was present in the amount of prrsv labeling in the lung among the three treatment groups. immunohistochemistry labeling for prrsv in lung tissue was negative in all sham inoculated control pigs. sera collected at À , , and dpe were tested for prrsv antibody. most swine infected with rjxwn (group ) had seroconverted by dpe (mean s/p ¼ . , sd¼ . ), and all had seroconverted by dpe (mean s/p ¼ . , sd ¼ . ); however, the rjxwn contact animals (group ) had not yet seroconverted by dpe (mean s/p ¼ . , sd ¼ . ) but did by dpe (mean s/p¼ . , sd¼ . ). vr- infected pigs (group ) were seronegative at dpe (mean s/p ¼ . , sd ¼ . ); but positive by dpe (mean s/p¼ . , sd ¼ . ). the sham inoculated control animals (group ) remained negative for prrsv nucleocapsid antibody throughout the study. all serum and balf samples were screened on marc- cells for infectious virus, and vi positive samples were then titered. a significant increase in virus load ( - fold) for the rjxwn challenge and rjxwn contact pigs was found in the serum when compared to the vr- challenge group (fig. a ). no significant differences were found in serum virus titer between the rjxwn challenge and rjxwn contact groups at any time points even though the contact pigs were exposed to rjxwn infected pigs at dpe. at necropsy, no significant differences in balf titers were found among the rjxwn challenge, rjxwn contact, or vr- challenge groups (fig. b ). control serum and balf samples were vi negative throughout the experiment ( fig. a and b) . to examine the levels of prrsv rna and to strengthen the case for the apparent difference in viral loads between the hp-prrsv and vr- inoculated animals seen above, qrt-pcr was completed on all serum samples as well as balf and lymph node tissue (fig. ) . the level of viral rna detected in serum samples mirrors the results obtained by virus isolation on marc- cells (fig. a, pr . at all dpe). the qrt-pcr results from balf showed little or no significant difference between the infected lymph node weight (lnw) to body weight (bw) ratios showed pronounced lymphadenopathy in all infected animals (groups - ) as compared to control animals (group )(p¼ . or higher significance), but no statistical significance between prrsv infected swine. (b) thymus weight (tw) to bw ratios showed thymic atrophy in all infected animals (groups - ) as compared to control animals (p o . ), and statistically significant differences between hp-prrsv inoculated (po . ) and naturally infected (p ¼ . ) animals compared to vr- inoculated swine. groups (fig. b ). tbln samples suggested that swine inoculated with vr- had significantly less viral rna present than those inoculated (p¼ . ) or infected (p¼ . ) with hp-prrsv (fig. c ). sham inoculated control serum, balf, and tbln tissue samples were viral rna negative throughout the experiment ( fig. a -c ). bacteria were isolated from the balf of of the rjxwn inoculated pigs (group ) and of the rjxwn contact pigs (group ) but from only of pigs in each of the sham inoculated control (group ) and vr- inoculated (group ) groups. in the rjxwn challenge group pasteurella multocida was isolated from four pigs (ranging from to cfu/ ml), p. multocida ( cfu/ ml) and actinobacillus suis ( cfu/ ml) were isolated from two pigs, p. multocida, a. suis and streptococcus suis (approximately , , and cfu/ ml, respectively) from one pig, and staphylococcus aureus and klebsiella pneumoniae (approximately and cfu/ ml, respectively) were isolated from one pig. in the contact group, p. multocida and a. suis were isolated from one pig ( cfu/ ml each) and p. multocida, a. suis, and arcanobacterium pyogenes (approximately , , and cfu/ ml, respectively) were isolated from a second pig. s. aureus and escherichia coli ( cfu/ ml each) were isolated from vr- inoculated pig, and bordetella bronchiseptica ( cfu/ ml) was isolated from sham inoculated control pig. results on cytokines measured in serum, balf and tbln homogenates are presented in figs. and and the changes in cytokine levels relative to sham-inoculated controls are listed in table . pigs directly inoculated with rjxwn (group ) had significantly elevated average serum levels of ifna, il- b, il- , il- and ifng, ranging from to times the levels detected in sera of sham inoculated controls (group ). in pigs inoculated with rjxwn , serum ifna levels were significantly elevated at dpe in comparison to both controls and vr- exposed pigs (po . ) and remained significantly elevated at dpe versus both groups. serum levels of il- were elevated by dpe, with il- , il- , il- and ifng all signficantly elevated at dpe compared to both controls and vr- exposed pigs (po . ). compared with sham inoculated controls, contact pigs exposed to rjxwn (group ) developed significant serum level elevations ( to times greater) in out of innate immunity cytokines measured (ifna, tnfa and il- b) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). in pigs exposed to rjxwn by contact transmission, serum ifna levels were signifcantly elevated at , and dpe in comparison to controls (po . ). in addition, contact pigs had significant elevations in il- b at and dpe (po . ). contact pigs also had significantly elevated levels of all adaptive immunity cytokines at and dpe (p o . ) with il- also significantly elevated at dpe. in contrast, none of the cytokines measured had significant elevations in serum levels detected in pigs inoculated with the north american prototype strain vr- (group ) when compared with sham inoculated controls. similarly, when compared to sham inoculated controls, balf of swine inoculated with rjxwn (group ) had significantly elevated ( to times greater) levels of out of innate immunity cytokines measured (tnfa, il- b and il- ) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). compared with sham inoculated controls, contact swine exposed to rjxwn (group ) had significantly elevated balf cytokine levels ( to times greater) in out of innate immunity cytokines measured (ifna, tnfa and il- ) and elevated levels ( to times greater) for all cytokines measured associated with adaptive immunity (il- , il- , il- , il- and ifng). in contrast, swine inoculated with vr- (group ) had significantly elevated balf cytokine levels ( times greater) for only out of innate immunity cytokines measured (il- b and il- ) and elevated levels ( to times greater) for of cytokines measured associated with adaptive immunity (il- and ifng). in this study, we compared the pathogenicity of chinese hp-prrsv strain rjxwn to north american prototype vr- in u.s. high health swine under controlled conditions. the clinical disease induced in this study by the hp-prrsv virus was similar to what has been reported in asia for phfd and for experimental infections with the wild-type or rescued virus ( zhou et al., ; zhou et al., ) . likewise, the clinical disease induced by the vr- virus was similar to that observed in previous reports (faaberg et al., ; rossow et al., ) . clinical disease and pathology were much more severe in the rjxwn group and their contacts than in the vr- group. overall, gross pathology lesions in the rjxwn challenged and contact groups were much more extensive and not restricted solely to the respiratory tract and lymph nodes as was the case with the vr- challenged pigs. the high occurrence of bacterial co-infections in the rjxwn challenge and contact swine likely played a prominent role in the difference in pathology and clinical disease between groups. bacterial co-infections in pigs naturally infected with prrsv have been documented, with susceptibility attributed to factors including prrsv strain differences, host genetics, management practices and environmental factors (rossow, ) . in this study, we used swine of high health status in a controlled research environment that were from -and -way crosses of commercial genetic lines. we believe the incidence and magnitude of bacteria isolated from the rjxwn challenge and contact groups when compared to the vr- and control groups suggest the differences in secondary bacterial infection susceptibility are specific to viral strain. in this study, we observed a % mortality rate in rjxwn infected pigs by dpe, which is less mortality than the original report (zhou et al., ) , but similar to other hp-prrsv strains used (li et al., ; lv et al., ; zhou et al., ) . possible explanations for differences may be the route and dose of inoculation, the age of pigs, the hp-prrsv strain utilized, and the time course of study. in addition, since the hp-prrsv lineage may exacerbate subclinical bacterial infections, it is possible different endemic bacterial infections played a role in apparent different mortality rates. in natural infections with chinese hp-prrsv, pulmonary interstitial hyperplasia with hemorrhage and edema is described, which suggests an acute septicemic process due to a secondary bacterial pathogen (tian et al., ; zhou and yang, ) . upon histopathologic examination of tissues from this study, north american pigs directly inoculated with rjxwn , as well as contact pigs, had an interstitial pneumonia that was significantly more severe than the vr- inoculated group, which is consistent with the severity of disease reported in china. although pulmonary lesions were more severe in the rjxwn challenge and contact pigs, the amount of antigen labeling was not significantly different from the vr- inoculated swine. since levels of proinflammatory cytokines, including tnfa, il- b and il- , were significantly increased in the balf of rjxwn -inoculated and contact pigs, the host response to hp-prrsv may play a role in the augmented lung pathology seen. in addition, the increased incidence of secondary bacterial infections in rjxwn challenge and contact pigs may have contributed to increased cytokine production and resultant immunopathology. there are numerous reports about the interplay of prrsv with the swine immune system that describe variable responses to infection at the cellular and cytokine level (miguel et al., ; thanawongnuwech et al., ; thanawongnuwech and thacker, ; wang et al., ) . although a large part of this variability may result from differing methods, challenge viruses, outbred pigs, and experimental designs, there are consistent findings emerging among the studies of increases in levels of selected cytokines associated with both innate and adaptive immunity. here we report a comprehensive assessment of the effects of prrsv infection on levels of cytokines critical to innate (ifna/b, tnfa, il- b, il and il- ) and adaptive (il- , il- , il- , il- and ifng) immune systems in serum, balf and tbln homogenates. it was demonstrated that infection with a highly pathogenic strain of prrsv elicited a significant elevation of all adaptive immunity cytokines measured in balf, as well as a majority of these cytokines in serum and tbln homogenates of the same groups of pigs. this observation is consistent with previous reports of table changes in cytokine levels in sampled tissues relative to non-challenged control pigs (p-values in comparison to non-challenged controls). serum values are an area under the curve overall average level measured in samples collected after exposure to virus. geometric means back-calculated from log means were used to determine relative changes in tbln and balf cytokine levels compared to sham-inoculated controls. (sun et al., ) ]. in contrast, in pigs infected in this study with rjxwn , we observed significant elevations of serum ifna as the first cytokine to peak following exposure to rjxwn in either the directly challenged or contact pigs, with the zenith occurring and dpe, respectively. however, consistent with a previous study (gomez-laguna et al., ), elevated serum ifna had little to no apparent effect on virus clearance as the viremia peaked in the rjxwn challenge and contact pigs on dpe and , respectively, and remained above the serum virus levels of vr- pigs until the end of the study ( and dpe). it is well documented that prrsv infection will increase the susceptibility of swine to co-infection with various bacteria (brockmeier et al., ; thanawongnuwech et al., ; thanawongnuwech and thacker, ; thanawongnuwech et al., ; thanawongnuwech et al., ; xu et al., ) , and based on previous findings with other hp-prrsv strains (xu et al., ) and our current rjxwn findings, it is clear that exposure to hp-prrsv greatly increases the likelihood of secondary bacterial infection due to commensal or pathogenic organisms typically found in the swine upper respiratory tract. whether the significant elevations of multiple cytokines that were measured in serum, balf and tbln following exposure to rjxwn were a direct result of the virus, an indirect effect mediated by the secondary bacterial infections, from extensive host tissue damage, or a combination of all of these events cannot be determined from our experiments. however, the pattern of multiple cytokines being elevated nearly simultaneously in serum ( of cytokines in rjxwn contact pigs and of cytokines in direct inoculated pigs) has not previously been reported and was not detected in vr- infected swine. cytokines are a diverse collection of peptides that elicit a wide range of biological responses and are characteristically understood in the context of an immune response wherein inflammation and immunity are carefully orchestrated by sequential secretion of cytokines that coordinate innate and adaptive immune responses. macrophages and stressed or damaged cells typically initiate a cytokine cascade through secretion of chemokines and proinflammatory cytokines in order to initiate the innate immune response at sites of acute infection or damage. generally, they act locally at nano-to picogram levels with short half-lives and transient activity. however, inflammatory cytokine cascades classically comprise a sequential appearance and disappearance of proinflammatory cytokines (e.g., tnfa, il- and il- ) intended to activate immune cells and their recruitment to generate additional cytokines and chemokines. this initial wave of cytokine production is usually followed by anti-inflammatory cytokine production (mainly the il- family) to moderate or down regulate the pro-inflammatory cytokines. cytokine cascades are therefore usually sequential with transiently detectable levels in peripheral blood; any dysregulation of these cascades can lead to adverse immunopathological responses. while significant elevations of several cytokine levels in tissues such as balf and tbln were expected, near simultaneous elevation of several cytokines in serum was not entirely expected as part of a normal host immune response. moreover, the levels of cytokines detected were in several instances significantly elevated (usually several times more) over levels detected with the low virulence north american prototype prrsv strain, vr- . prolonged elevations of serum ifng levels have been reported in swine infected with prrsv, a finding in contrast to serum ifng in pigs infected with influenza or respiratory coronavirus, where minimal transient detectable levels are observed (wesley et al., ) . in swine infected with rjxwn , it appears the normal sequence of cytokine production (e.g., ifng, tnfa, and il- often being the first cytokines produced in response to a viral infection, followed by il- and il- ) leading to effective virus clearance and a normal immune response was dysregulated given that significant elevations of several serum cytokines levels were still evident at to dpe. two recent studies have reported hp-prrsv infection in swine results in down regulation of a key toll-like receptor adapter gene, sarm (sterile aand armadillomotif-containing protein) (zhou et al., ; zhou et al., ) . sarm normally dampens the proinflammatory immune response by attenuating nf-kb activation and decreasing expression of il , il- and tnfa (carty et al., ) . previous studies in pigs infected with strains of hp-prrsv were reported to have swollen livers and petechial hemorrhages on the kidneys as well as immunohistochemical staining evidence of viral antigen in the liver and kidneys (among other tissues) (li et al., ; tian et al., ) . impaired hepatic and renal function as a consequence of viral or secondary bacterial disease could have a significant effect on clearance of the cytokines detected in serum, and contribute to an apparent severe cytokine release syndrome. whether our findings represent a parallel condition in swine to severe cytokine release syndromes or cytokine storms reported in humans that have been attributed to various causes cannot be proven with our data (descotes and gouraud, ; tarrant, ) . however, given prrsv causes polyclonal b cell activation, autoimmunity, lymphoid hyperplasia and hypergammaglobulinemia in pigs (lemke et al., ) , and it modulates multiple intracellular signaling pathways [reviewed in (sun et al., ) ], it is not surprising to find an exaggerated immune stimulation in swine infected with a particularly virulent strain of prrsv. early studies with cytokine administration to livestock species identified potential toxicities associated with systemic cytokine administration. interferon-g was found to have beneficial activity on immune function but was too toxic for practical usage due to febrile responses observed within h of a single dose of . mg/ kg of body weight (roth and frank, ) . similarly, administration of ng of bovine il- b/kg of body weight every h for days caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis (goff et al., ) . blood cytokines have been proposed as biomarkers of in vivo toxicity associated with new drug development (tarrant, ) . under most disease scenarios where there is rapid resolution of the infection by the host immune response, much of the biological activity of cytokines will occur in the locally infected tissues and elevated levels of the cytokines may not be detected in serum. we cannot definitively state whether the elevated cytokine syndrome reported here contributes to the pathophysiology of the disease caused by this highly pathogenic prrsv isolate. however, the association between the elevated levels of multiple cytokines and the severe morbidity and high mortality reported is consistent with a multiple cytokine toxicity syndrome in humans associated with various therapeutics (tarrant, ) . adverse reactions ranging from mild-to-moderate flu-like reactions to severe cytokine release syndromes have been observed with many therapeutic proteins in current use in human medicine, and some result in severe and even potentially life-threatening syndromes (reviewed in (tarrant, ) ). macrophage activation syndrome and cytokine storm are different names for two syndromes that share many features including a massive inflammatory response, elevated serum cytokine levels, multiorgan system disease and often death (behrens et al., ) . although these syndromes may be clinically indistinguishable, the cytokines that predominate in each may differ with tnfa being dominant in bacterial sepsis and ifng predominate in the macrophage activation syndrome (behrens et al., ) . the exact pathophysiology of the systemic toxicity in these syndromes is not fully defined. in pigs infected by natural contact with rjxwn inoculated pigs, we detected significant serum elevations in ifna, tnfa, il- b, il- , il- , il- , il- and ifng. however, given that prrsv infects the macrophage cell line in pigs, the elevated serum cytokine levels in pigs infected with hp-prrsv may represent both conditions (macrophage activation syndrome and cytokine storm) and contribute to the multiorgan damage and high mortality reported here. marc- cells were cultured in minimum essential medium (mem, safc c) with % fetal bovine serum at c, % co . wild-type (wt) type prrsv strain vr- (genbank u ), passage on marc- cells, was titrated, and used for the swine study. virus (rescued rjxwn ; rjxwn ) was rescued from a cloned cdna of chinese highly pathogenic type prrsv strain jxwn in marc- cells [pwsk-jxwn; genbank ef , (zhou et al., ) ] and passaged times on marc- cells for use in the swine study. the in vivo swine study described here was performed at the national animal disease center under approval from its animal care and use committee. thirty-two -week-old cross-bred pigs were obtained from a u.s. high-health status herd and were found to be free of prrsv antibodies by herdchek elisa, influenza virus antibodies by np elisa, and negative for porcine circovirus type by quantitative real-time pcr (data not shown). one day prior to inoculation, pigs were bled, weighed and randomly assigned to one of four groups. group (n ¼ ) consisted of sham inoculated control pigs, which received an intranasal ml sham inoculum of mem on day . group pigs (n ¼ ) were challenged intranasally with ml of  % tissue culture infective dose (tcid )/ml of chinese prrsv strain rjxwn in animal biosafety level -agriculture (bsl -ag) housing, where they remained for the duration of the experiment. group consisted of naïve pigs (n ¼ ) that were placed in contact with group two days after group was inoculated (dpe). group pigs (n ¼ ) were challenged intranasally on day dpe with ml of  tcid /ml of type prototype strain vr- . groups and were housed in separate isolation rooms in an absl facility. clinical monitoring of pigs was performed daily throughout the study. specifically, observations were made regarding the pig's mental alertness, body condition, appetite, activity level, and clinical signs of respiratory or systemic disease. serum was collected on À , , , , and dpe (sera from group pigs was collected on À , , , and dpe), and pigs were weighed on À , and dpe (group were weighed on À , and dpe). necropsy was scheduled on dpe (dpe for group ), or sooner if pigs died or were euthanized due to severe disease. at necropsy, a thorough post-mortem examination was performed, and a complete set of samples was collected for evaluating disease severity. tracheobronchial lymph nodes (tbln) and thymic tissue were weighed (lnw and tw, respectively) and compared to respective body weights (bw) to derive a tissue mass index (lnw/bw, tw/bw) measuring the effect of prrsv infection on organ weight (mengeling et al., ) . upon removal, lungs were examined and extent of macroscopic lung lesions was estimated, as previously described, and reported as a percentage of lungs affected (halbur et al., ) . sections of tissues (lung, tracheobronchial lymph node, trachea, thymus, heart, tonsil, spleen, iliac lymph node, mesenteric lymph nodes, ileum, bone marrow, kidneys, liver, inguinal lymph node, cerebrum, brainstem, cerebellum and ventral midbrain) were collected into % neutral-buffered formalin for histopathology and immunohistochemistry. tbln were collected for rna extraction and cytokine protein assays. bronchoalveolar lavage fluid (balf) was collected after removing whole lungs from pigs and aseptically lavaging with ml antibiotic-free mem. tissues were processed by routine histopathologic procedures and slides were stained with hematoxylin and eosin. a boardcertified veterinary pathologist blinded to treatment groups evaluated microscopic lesions. lung sections were scored on a -point scale that accounted for distribution and severity of interstitial pneumonia: -no lesions, -mild, focal to multifocal interstitial pneumonia (o % of lung section affected), -moderate, multifocal to coalescing ( - % of lung section affected), -severe, patchy to coalescing and extensive ( - % of lung section affected), and -severe and diffuse ( % of lung section affected). immunohistochemistry prrsv-specific antigen was detected in lung tissues using a previously described immunohistochemical (ihc) method with minor modifications (halbur et al., ) . briefly, tissue sections were deparaffinized and hydrated in distilled water. slides were quenched in % hydrogen peroxide for min, rinsed three times in distilled water and treated in . % protease for min. slides were then rinsed three times in distilled water. a primary monoclonal antibody (mab) cocktail of one part : prrsv sdow (rti, brookings, sd) and one part : prrsv sr (rti, brookings, sd) was applied to the slides and were incubated at room temperature for h. bound mabs were stained with peroxidase-labeled anti-mouse igg followed by chromogen using the dako lsab -hrp detection system (dako, carpinteria, ca) according to the manufacturer's instructions. , -diaminobenzidine (dab; vector laboratories, burlingame, ca) was applied to the slides for min. the slides were rinsed in deionized water and counterstained with gill's hematoxylin. prrsv labeling was graded on a point scale of : none, : mild scattered signals ( to cells in entire section), : moderate scattered signals (less than or equal to % of high power fields (hpf) containing immunolabeling) and : abundant scattered signals (greater than % of hpf contain labeling and/or there are at least - groups of cells or more with staining). virus isolation was attempted on all serum and balf samples as described previously (faaberg et al., ) . those samples that were positive by virus isolation were then titered by serial dilution on marc- cells to determine the quantity of virus present to produce a cytopathic effect in % of inoculated tissue culture cells (tcid ). quantitative rt-pcr (qrt-pcr), as previously described (faaberg et al., ) , was used to determine the amount of viral rna per ml of serum and balf, and per gram of tbln homogenized tissue prepared as described below. serum samples were tested on study days À , and for evidence of seroconversion with the prrs xr enzyme-linked immunosorbent assay (herdchek elisa; idexx laboratories). a sample was considered positive for antibodies to prrsv nucleocapsid protein if the sample-to-positive (s/p) ratio was equal to or greater than . . levels of tnfa, il- b, il- , il- , il- , il- , il- , il- p and ifng cytokine levels (pg/ml) were measured in serum, balf and tbln samples diluted : with dilution buffer supplied by the manufacturer using a searchlight (aushon biosystems, woburn, ma, usa) customized multiplex immunoassay (m-elisa) following the manufacturer's protocol. m-elisa samples were assayed in duplicate. the elisa limits of detection for tnfa, il- b, il- , il- , il- , il- , il- , il- p and ifng were . , . , . , . , . , . , . , . , and . pg/ml respectively. approximately g of tbln was homogenized in ml of lysis buffer containing . % triton x- , mm nacl, mm tris, mm cacl , and mm mgcl , ph . , using a tissue homogenizer (biospec products, bartlesville, ok) (greenberger et al., ) . homogenates were incubated on ice for min, then centrifuged at x g for min. supernatants were collected, passed through a . micron filter (gelman sciences, ann arbor, mi), then stored at À c prior to assessment of cytokine levels. ifna protein was measured with a porcine ifna specific elisa by using f monoclonal antibody (mab) and k mab (r&d systems inc.) as previously described (miller et al., ) . mab k was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). immulux hb flat-bottomed -well plates (dynex technology, chantilly, va) were coated overnight at c with f at a concentration of mg/plate in coating buffer ( mm carbonate buffer, ph . , sigma inc., st. louis, mo). after blocking with % non-fat dried milk, . % tween in phosphate buffered saline (pbs) for h at c, the plates were washed three times with . % tween in pbs. samples ( ml) were added into each well containing ml of % non-fat dried milk, . % tween in pbs and incubated for h at c. following three washes, ml of peroxidase conjugated k was added to each well. after h incubation, at c, and three washes, ml of substrate solution, tetramethylbenzidine (kpl inc., gaithersburg, md), was added to each well. after min, the reaction was stopped with tetramethylbenzidine stop solution (kpl inc., gaithersburg, md) and the optical density was measured at nm by an elisa plate reader. quantified recombinant porcine ifna (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifna concentrations were calculated based upon a standard curve. one unit/ml of rifna is equivalent to pg/ml. bacterial culture was performed by plating ml of balf both on a casman's agar plate supplemented with . % nicotinamide adenine dinucleotide (nad) and % horse serum, and on a % sheep's blood agar plate. both agar plates were incubated for h at c. bacterial identification was performed by s rrnaspecific pcr and dna sequencing. s rrna-specific pcr and dna sequencing whole-cell bacterial lysates, used as templates, were prepared by suspending a colony, $ mm in diameter or the equivalent, in ml of sterile water. the mixture was boiled for min, placed on ice until chilled, and centrifuged at ,  g for min to pellet cell debris and stored at À c. supernatant ( ml) was used as the template in each pcr. the forward primer ( -agagtttgatcctggctcag- ), designated univ s- , is homologous to a highly conserved sequence from the end of the s rrna gene and the reverse primer ( -gcggctgctggcacg- ), designated univ s- , is homologous to a highly conserved sequence between the third and fourth variable regions of the s rrna gene. this previously described primer set generates an amplicon of approximately bp (register and yersin, ) . reactions were carried out in a volume of ml and contained u amplitaq polymerase (applied biosystems, foster city, ca), ml x buffer ii ( mm tris-hcl , ph . , mm kcl), ml dimethyl sulfoxide, . mm mgcl , . mm primers, and mm deoxynucleoside triphosphates. an initial denaturation step of min at c was followed by cycles of s at c, s at c, and min at c, with a final extension step of min at c. ml of each pcr was analyzed by agarose gel electrophoresis and pcr products were purified with spin columns (promega, madison, wi) and sequenced directly by fluorescence-based cycle sequencing with amplitaq and bigdye terminators on an abi sequencer at the national animal disease center genomics unit. sequences were analyzed using geneious . software (biomatters ltd, auckland, new zealand). quantitative virus copy numbers and serum cytokine levels were analyzed using a mixed linear model for repeated measures (proc mixed, sas . for windows, sas institute, cary, nc, usa). linear combinations of the least squares means estimates for each variable were used in a priori contrasts after testing for either a significant (po . ) effect of prrsv challenge strain (vr- , rjxwn , rjxwn contacts or sham inoculated controls). comparisons were made between groups at each time-point using a % level of significance (po . ) to assess statistical differences. log transformed virus copy numbers and cytokine levels in balf and tbln homogenates were analyzed by analysis of variance using a general linear model for unbalanced data (proc glm, sas . for windows, sas institute, cary, nc, usa). a % level of significance (po . ) was used to assess statistical differences. geometric mean back transformations were made for final data presentation in figures and tables. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. usda is an equal opportunity provider and employer. highly pathogenic porcine reproductive and respiratory syndrome virus repeated tlr stimulation results in macrophage activation syndrome-like disease in mice effects of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasteurella multocida in pigs the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling clinical immunotoxicity of therapeutic proteins immune responses of pigs after experimental infection with a european strain of porcine reproductive and respiratory syndrome virus vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras in vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp deletion mutants porcine respiratory and reproductive syndrome virus variants, vietnam and china physiological effects of exogenous administration of interleukin- b in cows cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus changes in lymphocyte subsets and cytokines during european porcine reproductive and respiratory syndrome: increased expression of il- and il- and proliferation of cd ( À )cd (high) acute phase response in porcine reproductive and respiratory syndrome virus infection genomic characterization of virulent, attenuated, and revertant passages of a north american porcine reproductive and respiratory syndrome virus strain neutralization of il- increases survival in a murine model of klebsiella pneumonia immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus expression of inflammatory cytokines and toll-like receptors in the brain and respiratory tract of pigs infected with porcine reproductive and respiratory syndrome virus role of toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations analytical verification of a pcr assay for identification of bordetella avium porcine reproductive and respiratory syndrome experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs recombinant bovine interferon-g as an immunomodulator in dexamethasone-treated and nontreated cattle molecular epidemiology of prrsv: a phylogenetic perspective examination of the selective pressures on a live prrs vaccine virus interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus blood cytokines as biomarkers of in vivo toxicity in preclinical safety assessment: considerations for their use increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae interleukin- , interleukin- , and interferon-gamma levels in the respiratory tract following mycoplasma hyopneumoniae and prrsv infection in pigs effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr- ) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the arterivirus replicase immune responses in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china secondary infection with streptococcus suis serotype increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs porcine reproductive and respiratory syndrome in china the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular characterization of porcine sarm and its role in regulating tlrs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo highly virulent porcine reproductive and respiratory syndrome virus emerged in china the authors would like to recognize ann vorwald, sarah anderson, deb adolphson and amanda burow for their excellent technical assistance, and jason huegel, brian pottebaum, and jason crabtree for their exceptional animal care. we also appreciate the suggestions on manuscript layout and data organization made by crystal loving. key: cord- -ytlnhyxr authors: zhao, kuan; li, li-wei; jiang, yi-feng; gao, fei; zhang, yu-jiao; zhao, wen-ying; li, guo-xin; yu, ling-xue; zhou, yan-jun; tong, guang-zhi title: nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim by interfering with trim -mediated rig-i ubiquitination date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ytlnhyxr porcine reproductive and respiratory syndrome (prrs) is caused by prrs virus (prrsv), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. identification of sustainable and effective measures to mitigate prrsv transmission is a pressing problem. the nucleocapsid (n) protein of prrsv plays a crucial role in inhibiting host innate immunity during prrsv infection. in the current study, a new host-restricted factor, tripartite motif protein (trim ), was identified as an inhibitor of prrsv replication. co-immunoprecipitation assay indicated that the prrsv n protein interferes with trim –rig-i interactions by competitively interacting with trim . furthermore, n protein inhibits the expression of trim and trim -mediated rig-i ubiquitination to suppress interferon β production. furthermore, with increasing trim expression, the inhibitory effect of n protein on the ubiquitination of rig-i diminished. these results indicate for the first time that trim inhibits prrsv replication and that the n protein antagonizes the antiviral activity by interfering with trim -mediated rig-i ubiquitination. this not only provides a theoretical basis for the development of drugs to control prrsv replication, but also better explains the mechanism through which the prrsv n protein inhibits innate immune responses of the host. the tripartite motif protein (trim ) is an e ubiquitin ligase involved in various cellular processes, including regulating the innate immune response against viruses (heikel et al., ) . immediately after a viral infection, pathogen-associated molecular patterns are sensed by host pattern recognition receptors (prrs), thereby allowing cells to distinguish self from non-self and activate an immune response (sparrer and gack, ) . for rna viruses, the main cytoplasmic prr is retinoic acid-inducible gene i (rig-i), which can directly recognize and bind viral ′-ppp rna and short double-stranded rna (chan and gack, ) . after recognition, the n-terminal caspase recruitment domains (cards) of rig-i are modified by ubiquitin which is mediated by trim . such modification is essential for activating a signaling cascade, ultimately resulting in the transcriptional activation of type i and iii interferons (ifns), mediating viral clearance, and inhibiting viral replication and spread (gack et al., ; martin-vicente et al., ) . various viruses are inhibited by trim , e.g., coxsackie b virus and poliovirus (schoggins et al., ) . porcine reproductive and respiratory syndrome (prrs) is endemic in most pig-producing countries (han and yoo, ) , and is caused by prrs virus (prrsv). in , a highly pathogenic prrsv (hp-prrsv) emerged in china, causing high fever, high morbidity, and high mortality . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately . kb and encodes at least open reading frames (orfs) (han and yoo, ) . orf a and orf b encode two large nonstructural polyproteins, ppla and pplb, which are processed into at least nonstructural proteins (snijder and meulenberg, ; ziebuhr et al., ) . orf a-orf encode structural proteins, including four membrane-associated glycoproteins (gp a, gp , gp , and gp ), three unglycosylated membrane proteins (e, orf a, and m), and a nucleocapsid protein (n) (dea et al., ; snijder and meulenberg, ) . among these, nsp , nsp , nsp , nsp , and been identified and characterized as ifn antagonists (lunney et al., ) . further, the n protein is the most abundant protein during infection, accounting for approximately % of virion proteins (snijder and meulenberg, ) . it plays essential roles in the virus life cycle, including encapsidation of viral rna (spilman et al., ) . currently, it is only known that n protein suppresses ifn-β induction by antagonizing irf activation (sagong and lee, ) . however, other mechanisms through which n protein inhibits ifn-β production are not clear. the nucleocapsid protein of severe acute respiratory syndrome (sars) inhibits type i ifn production by interfering with trim mediated rig-i ubiquitination (hu et al., ) . whereas prrsv and sars both belong to the nidovirales order, whether prrsv n protein inhibits the host innate immune response by interfering with trim mediated rig-i ubiquitination is not clear. herein, we show that trim plays an important role in inhibiting prrsv replication. the n protein interacts with trim and suppresses the ubiquitination of rig-i. further, trim expression is reduced upon co-transfection with the n protein or prrsv infection. together, these findings suggest a novel mechanism through which prrsv n protein inhibits host innate immunity, and provide improved understanding of the mutual regulatory mechanism between prrsv and the host. human embryonic kidney (hek t) cells and african green monkey kidney (marc- ) cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (gibco, thermo fisher scientific, waltham, ma). cells were maintained at °c with % co . the highly pathogenic prrsv strain hun (genbank no. ef ) was used for all the experiments (tong et al., ) . total rna was extracted from porcine alveolar macrophages (pams) using rneasy mini kit (qiagen, hilden, germany), following the manufacturer's instructions. reverse transcription reactions were performed at °c for min and °c for h using the m-mlv reverse transcription polymerase system (takara, dalian, china). full-length trim -and rig-i-encoding sequences were amplified with specific indicated primers. the sequences were then cloned into pcaggs vector to generate pcaggs-trim -ha, pcaggs-trim -flag, pcaggs-trim -myc, pcaggs-rig-i-ha, pcaggs-rig-i-flag, or pcaggs - card-flag. orf of prrsv hun was cloned into pcaggs to generate pcaggs-n-ha, pcaggs-n-flag, or pcaggs-n-myc. all plasmids were constructed by homologous recombination using the nebuilder® hifi dna assembly master mix (new england biolabs; ipswich, ma) according to the manufacturer's instructions. sequences of all primers used for gene amplification will be made available upon request. to investigate the effect of trim on prrsv replication, marc- cells cultured in -well plates were transfected with μg of pcaggs-trim -flag using x-treme gene hp dna reagent (roche applied science, penzberg, germany). next, h post-transfection (hpt), the cells were infected with prrsv hun (multiplicity of infection, moi, of . ). after inoculation for h at °c, the supernatants were discarded, and cells were washed three times with phosphate-buffered saline (pbs). the supernatant was harvested , , , and h post-infection (hpi), and the cells were lysed using ripa lysis buffer (thermo fisher scientific). viral titers in the supernatants were determined using a microtitration assay, according to the method of reed and muench (reed and muench, ) . the amount of the n protein was then detected in cell lysates by western blotting (wb) using a mouse anti-n polyclonal antibody ( : ) produced by the authors. small interfering rnas (sirnas) against macaque trim (genbank no. xm_ . ) were synthesized by genepharma (shanghai, china). the sirna molecules used were ccu gga gua uua cgu uaa att (sirna-trim - ) and gca ucu acc aua gca ccu utt (sirna-trim - ). the control sirna (nc) sequence was uuc ucc gaa cgu guc acg utt. marc- cells were seeded in well plates and transfected with pm sitrim or nc using lipofectamine™ rnaimax transfection reagent (cat. no. , , ; thermo fisher scientific) . the cells were lysed in ripa lysis buffer after h of transfection and the effects of sirnas were analyzed by wb using an anti-trim monoclonal antibody (cat. no. , ; cell signaling technology; danvers, ma; : ). the knockdown efficiency of sirna was analyzed by grayscale scanning with image j software. next, efficient sirna and nc were selected for transfection; hpt, the cells were infected with prrsv hun at an moi of . . the supernatant and cells were harvested , , , and hpi, and analyzed based on virus titers and by wb. cell lysates were prepared by harvesting virus-infected or plasmidtransfected cells in ripa or ip-lysate buffer( mm tris•hcl ph . , mm nacl, % np- , mm edta, % glycerol) at °c for min containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche). after centrifuging at , × g for min, the supernatants of cell lysates were mixed with × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample loading buffer (beyotime), and placed in boiling water for min. the proteins were then separated by sds-page and transfected onto a nitrocellulose membrane. the membrane was blocked in % skim milk at room temperature for h and then incubated with the indicated primary antibody at °c overnight. after washing three times with trisbuffered saline with . % tween , the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse igg(h + l) and/or goat anti-rabbit igg(h + l) secondary antibody ( : ) for h at room temperature. the target proteins were visualized by treating the membrane with pierce ecl wb substrate (thermo fisher scientific). for the quantification of target proteins, their levels were normalized to the levels of β-actin. to detect the interaction between trim and prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-trim -flag and pcaggs-n-ha ( μg each) using x-tremegene dna transfection reagent. at hpt, the cells were lysed with ice-cold ip lysis buffer containing mm phenylmethylsulfonyl fluoride and mg/ml of protease inhibitor cocktail (roche) at °c for min. approximately % of the lysate supernatant was used as an input control and the remaining lysates were incubated with anti-ha agarose beads (cat. no. a ; sigma-aldrich; st. louis, mo) or ezview™ red anti-flag ® m affinity gel (cat.no. m ; sigma-aldrich) for h at °c. the beads were washed five times with the ip lysis buffer and then analyzed by wb using rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) or mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ). the interaction between trim and rig-i was tested using the same methods with anti-ha agarose beads and wb using mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and mouse anti-myc monoclonal antibody (cat. no. ; cell signaling technology; : ). in order to investigate whether the n protein k. zhao, et al. veterinary microbiology ( ) - competitively affects the interaction between rig-i and trim , hek t cells grown in -well palate were co-transfected with pcaggs-rig-i-ha ( μg) and pcaggs-trim -myc ( μg) with or without pcaggs-n-flag ( μg) for immunoprecipitation. further, to detect the interaction between endogenous (host) trim and n protein in prrsv-infected cells, pam cells were grown in -cm-diameter dishes and infected with prrsv at an moi of . for h. the cells were lysed in μl of ip lysis buffer and the supernatants were precleared with protein g-agarose beads (cat. no. ; roche). the supernatant was incubated with μg of anti-n polyclonal antibody for h. next, μl of protein g-agarose beads was added, followed by an additional h incubation. ip pellets were washed five times with μl of ip lysis buffer, boiled in sds-page sample loading buffer, and analyzed by wb with rabbit anti-trim monoclonal antibody (cat. no. ab ; abcam; : ) and anti-n polyclonal antibody. hek t cells were co-transfected with pcaggs-n-ha and pcaggs-trim -flag, or pcaggs-rig-i-ha and pcaggs-trim -flag. at hpt, the cells were fixed in % paraformaldehyde for min, blocked with % bovine serum albumin for h, and permeabilized with . % triton x- for min. the transfected cells were incubated with mouse anti-ha monoclonal antibody (cat. no. h ; sigma-aldrich; : ) and rabbit anti-flag monoclonal antibody (cat. no. f ; sigma-aldrich; : ) for h at °c, and then washed three times with pbs. the cells were then incubated at °c for h with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor (cat. no. ; life technologies; : ) and goat anti-rabbit igg(h + l) antibody labeled with alexa fluor (cat. no. ; life technologies; : ). samples were then stained with μg/ml of ′, ′-diamidino- -phenylindole (dapi) for min and examined using a zeiss confocal system. besides, in order to investigate the colocalization of endogenous (host) trim and n protein in prrsv-infected cells, the pam cells infected with prrsv were send to colocalization detection with anti-trim and anti-n protein antibody. hek t cells seeded in a -well plate were co-transfected with control plasmids or the indicated expression plasmids ( . μg per well) together with ng of a luciferase reporter plasmid; prl-tk plasmid ( ng) was used as a control of transfection efficiency (huang et al., ) . next, hpt, cells lysates were harvested and luciferase activity in the lysates was analyzed using a dual-luciferase reporter assay kit (promega, madison, wi), following the manufacturer's instructions. the results are expressed as the means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using graphpad prism (graphpad, la jolla, ca). the measured values are expressed as the mean with sd. the statistical significance was assessed using student's t-test, with p < . considered statistically significant. to examine the effects of trim on prrsv replication, specific sirna molecules were synthesized to knockdown trim expression in marc- cells and efficiently reduce trim expression. using sirna- , the knockdown efficiency was approximately % (fig. a) . this sirna molecule was used in the subsequent interference experiments. as shown in fig. b , n protein levels increased upon transfection with sirna- , especially and hpi, compared with those in nctransfected cells. virus titers in the culture supernatants of cells transfected with sirna- were also increased, which was consistent with the expression levels of the n protein, with a significant difference hpi (p < . ; fig. c ). by contrast, when trim was overexpressed, the n protein levels of hun were lower than those in the control cells (fig. d) . furthermore, there was a significant difference in virus titers between cells transfected with pcaggs-trim -flag or pcaggs, with an approximate . log decrease in virus titers from to hpi (p < . ; fig. e ). these observations suggested that trim is a cellular antiviral factor that represses prrsv infection. as shown in fig. d , the expression of trim -flag decreased gradually upon prrsv infection. therefore, we speculated that the virus might inhibit the expression of trim . to verify this hypothesis, marc- cells infected with prrsv or uninfected cells were lysed , , , and hpi, and analyzed by wb. the analysis revealed that the expression of trim decreased in prrsv-infected cells at every time point, as compared with the uninfected cells ( fig. a) . furthermore, trim expression was significantly suppressed when pcaggs-trim -myc and pcaggs-n-ha were co-transfected into hek t cells. these findings indicated that the n protein inhibits the expression of trim . furthermore, the n protein inhibited trim expression in a dose-dependent manner (fig. b) . since we demonstrated that prrsv infection and n protein accumulation suppress the expression of trim , we next investigated whether the n protein of prrsv exerts an inhibitory effect on trim expression by interacting with it. to test this, the interaction between trim and n protein was investigated by co-ip. based on precipitation with anti-flag agarose, flag-tagged trim interacted with hatagged n protein (fig. a) . furthermore, n protein was efficiently coimmunoprecipitated with trim using anti-ha agarose (fig. b) . we also investigated whether trim co-localizes with the n protein in hek t cells co-transfected with pcaggs-trim -flag and pcaggs-n-ha. trim was mainly located in the cytoplasm ( fig. c-a) , whereas the n protein localized in the cytoplasm and nucleus ( fig. c-b) . as shown in fig. c -d, trim and n protein colocalized in the cytoplasm. to examine the interaction of n protein and endogenous trim in the context of prrsv infection, virus-infected pam cells were stained with anti-n polyclonal antibody and anti-trim monoclonal antibody or immunoprecipitated using anti-n polyclonal antibody and the pellets were detected with anti-trim and n antibody. as shown in fig. d and e, endogenous trim was co-localized with n protein and coimmunoprecipitated with the n protein. these observations confirmed that the endogenous trim indeed interacts with the n protein of prrsv in prrsv-infected cells. in the current study, we demonstrated that the n protein of prrsv could interact with trim . further, it has been reported that trim interacts with rig-i n-terminal cards and e ubiquitin-conjugating enzymes (gack et al., ) . whether the n protein can competitively regulate the interaction between trim and rig-i was unknown. we first detected the interaction between trim and rig-i by co-ip, confirming that trim could be immunoprecipitated with rig-i (fig. a ). in addition, co-localization analysis demonstrated that rig-i and trim co-localize in the cytoplasm (fig. b) . further, we k. zhao, et al. veterinary microbiology ( ) - validated the effect of n protein on the interaction between rig-i and trim . when rig-i-ha and trim -myc were co-transfected with n-flag, the interaction between trim and rig-i was markedly diminished. meanwhile, when trim -myc and rig-i-ha were co-transfected without n-flag, the interaction between trim and rig-i was not affected (fig. c) . these results suggested that the n protein inhibits the interaction between trim and rig-i via competitive binding to trim . to investigate whether trim -mediated rig-i ubiquitination is regulated by the prrsv n protein, hek t cells grown in -well plates were co-transfected with pcaggs-flag-rig-i ( . μg per well) and ha-ubiquitin ( . μg per well), and the indicated amounts of the myc-n expression plasmids. cells lysates were immunoprecipitated using mouse anti-ha monoclonal antibody or rabbit anti-flag monoclonal antibody. the experiment revealed that trim -mediated rig-i ubiquitination was potentiated by sendai virus (sev) infection but was substantially suppressed by increasing the prrsv n protein expression, in a dose-dependent manner (fig. ) . when viral rna is recognized by rig-i, this protein is modified by ubiquitin, which is mediated by the e ligase trim . hence, this enzyme is essential for activating a signaling cascade that ultimately results in the transcriptional activation of type i and iii ifns. to examine regulation of rig-i activity by the prrsv n protein, a luciferase reporter under the control of the ifn-β promoter (ifn-β-luc) was used to quantify promoter activation. consistent with the inhibition of rig-i ubiquitination by the n protein, ifn-β promoter activation induced by rig-i or rig-i card domain overexpression was significantly inhibited by prrsv n expression, in a dose-dependent manner (fig. a, b) . however, co-expression of trim with prrsv n significantly counteracted this inhibitory effect mediated by the n protein (p < . ) (fig. c ). in addition, suppression of rig-i ubiquitination by the prrsv n protein was partially rescued by trim overexpression (fig. d) . these observations indicated that the n protein inhibits rig-i ubiquitination and that ifn production can be restored by trim overexpression. prrsv has been a major threat to global industrial pig farming ever since its emergence in the late s (shi et al., ) , and especially or pcaggs ( μg) were inoculated with prrsv at an moi of . . the cells and supernatant were harvested at the indicated times, and the replication of prrsv was evaluated by wb with an anti-n polyclonal antibody (d) and tcid assay (e). the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using student's t-test (*p < . ). since the outbreak of hp-prrsv in . to date, although there is some understanding of the mechanisms through which prrsv escapes innate immunity, very little is known about how viral proteins of prrsv antagonize the host innate immune response. therefore, understanding the interactions between prrsv and host proteins will be beneficial for the development of effective therapies to control outbreaks of this disease in the future. in the current study, we demonstrated that trim functions by restricting prrsv replication, and that it could act as a host-derived inhibitor of the virus. previously, many host factors that result in cellular resistance to prrsv via different mechanisms were identified song et al., ; wang et al., ; zhao et al., zhao et al., , . for example, cholesterol -hydroxylase protects against prrsv infection by converting cholesterol to -hydroxycholesterol, which can be used as a natural antiviral agent to combat prrsv infection (song et al., ) . further, galectin- inhibits replication prrsv by interacting with viral nsp in vitro . in the current study, using marc- cells, we demonstrated that the overexpression of trim restricts the replication of prrsv, whereas knocking down this protein promotes the replication of prrsv (fig. ) . therefore, trim acts as a novel host factor that inhibits the replication of prrsv. trim is an e ubiquitin ligase enzyme that can regulate the innate immune response against viruses (martin-vicente et al., ) . trim -mediated ubiquitination of the cytosolic prr rig-i is an essential step for the initiation of an intracellular antiviral response (gack et al., ) . data presented herein revealed that upon trim overexpression, the n protein-mediated inhibition of rig-i ubiquitination and ifn-β promoter activity were diminished (fig. ) . the host innate immunity was hence activated, leading to a series of signaling cascades and thereby inhibiting prrsv replication. trim can activate the host innate immune system and simultaneously induce a series of antiviral responses by promoting the ubiquitination of rig-i and activation of ifn-β promoter activity. however, in the course of natural infection, prrsv can complete the replication cycle and efficiently spread. hence, prrsv has evolved several general strategies to evade the innate immune response. it has been reported that some viral proteins interact with trim and inhibit rig-i activation. for example, the non-structural protein (ns ) of influenza a virus interacts with the cc domain of trim preventing its dimerization and the k -linked ubiquitination of rig-i cards, thereby suppressing rig-i signal transduction (gack et al., ) . further, trim interacts with the n protein of sars-cov, thereby inhibiting the activation of rig-i (hu et al., ) . in the current study, we found that the n protein of prrsv inhibits the ubiquitination of rig-i by competitively interfering with the interaction between rig-i and trim . this might be the mechanism through which prrsv inhibits the antiviral effect of trim . furthermore, trim levels decreased when the cells were infected with prrsv. in addition, when plasmids expressing trim and the n protein of prrsv were cotransfected into cells, the expression of trim was significantly suppressed. based on this, it would be difficult for trim to exert an antiviral effect upon prrsv infection. this might represent another mechanism through which prrsv antagonizes the antiviral response of trim . besides, the n protein of pedv, another coronavirus, is also able to antagonize ifn-β production (ding et al., ) . since prrsv, sars, and pedv all belong to nidovirales, we speculate that the respective n proteins may exert a similar effect of inhibiting trim mediated ubiquitination of rig-i. however, the effect of pedv n protein on the inhibition of rig-i ubiquitination requires further research. in the present study, we confirmed that trim inhibits prrsv replication. further, prrsv can antagonize the antiviral activity of this protein by decreasing its expression and modulating the trim mediated ubiquitination of rig-i. in addition, the n protein of prrsv inhibits ifn-β production. all these mechanisms improve the understanding of the effect of trim on prrsv replication and will further co-ip experiment was performed using agarose beads conjugated with anti-flag monoclonal antibody or (b) agarose beads conjugated with anti-ha monoclonal antibody. the precipitated proteins were analyzed by wb using antibodies against the flag and ha tags. (c) co-localization of the n protein with trim . hek cells were transfected with plasmids expressing ha-n protein and flag-trim . after h, the cells were fixed and analyzed by ifa to detect tagged n protein and trim , using mouse anti-ha and rabbit anti-flag monoclonal antibodies, respectively. the nucleus is stained with dapi (blue) in the merged images. (d and e) colocalization and interaction of prrsv n and endogenous trim . pam cells infected with prrsv were fixed h after infection, and then subjected to indirect immunofluorescence to detect n protein and trim . in addition, cell lysates from prrsv-infected or mock-infected pam cells were co-ip with mouse anti-n polyclonal antibody, and the precipitates were immunoblotted with the indicated antibodies (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). k. zhao, et al. veterinary microbiology ( ) - help to understand how prrsv evades the trim -mediated innate immune response via the n protein. hence, the current study not only offers a new target for the development of drugs to control prrsv spread but also provides an explanation of the mechanism through which prrsv n protein modulates host innate immune responses. in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. . the prrsv n protein suppresses the trim -mediated rig-i ubiquitination. hek t cells were transfected with the indicated plasmids for h, and were infected (with or without sev) for h. anti-flag immunoprecipitates prepared from the cell extracts were analyzed by wb using the indicated antibodies. k. zhao, et al. veterinary microbiology ( ) - viral evasion of intracellular dna and rna sensing current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i engineering the prrs virus genome: updates and perspectives the role of trim in development, disease and rna metabolism the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-κb essential modulator galectin- inhibits replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp in vitro porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system trim in the regulation of the antiviral innate immunity. front. immunol. , . reed porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf activation in immortalized porcine alveolar macrophages pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity molecular epidemiology of prrsv: a phylogenetic perspective the molecular biology of arteriviruses cholesterol -hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection intracellular detection of viral nucleic acids cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the interferon-induced mx inhibits porcine reproductive and respiratory syndrome virus replication porcine ', '-oligoadenylate synthetase inhibits porcine reproductive and respiratory syndrome virus replication in vitro mov inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of marc- cells virus-encoded proteinases and proteolytic processing in the nidovirales a and b) hek t cells seeded in -well plates were co-transfected using the firefly luciferase reporter plasmid ifn-β-luc and the renilla luciferase control reporter plasmid prl-tk. for the experiment, pcaggs-rig-i-flag ( . μg), or pcaggs - card ( . μg), pcaggs-n-ha were co-transfected. (c) pcaggs - card-flag ( . μg), pcaggs-n-falg ( . μg) and pcaggs-trim -myc ( . μg) plasmids were cotransfected for the experiment, hpt, the cells were infected with sev, and hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of rig-i-card. the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using key: cord- -qsaewje authors: wang, pengcheng; bai, juan; liu, xuewei; wang, mi; wang, xianwei; jiang, ping title: tomatidine inhibits porcine epidemic diarrhea virus replication by targeting cl protease date: - - journal: vet res doi: . /s - - -y sha: doc_id: cord_uid: qsaewje porcine epidemic diarrhea virus (pedv) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. there is an urgent need to find new therapeutic methods to prevent and control pedv. not only is there a shortage of commercial anti-pedv drugs, but available commercial vaccines fail to protect against highly virulent pedv variants. we screened an fda-approved library of natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of pedv replication in vero and ipec-j cells in vitro. molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of pedv cl protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (itc). the inhibiting effect of tomatidine on cl protease was determined using cleavage visualization and fret assay. tomatidine-mediated blocking of cl protease activity in pedv-infected cells was examined by western blot detection of the viral polyprotein in pedv-infected cells. it indicates that tomatidine inhibits pedv replication mainly by targeting cl protease. in addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (tgev), porcine reproductive and respiratory syndrome virus (prrsv), encephalo myocarditis virus (emcv) and seneca virus a (sva) in vitro. these results may be helpful in developing a new prophylactic and therapeutic strategy against pedv and other swine disease infections. pedv, an enveloped, positive-sense, single-stranded rna virus, is a member of the coronavirinae subfamily [ , ] , which comprises viruses that cause a variety of diseases in mammals and birds, ranging from enteritis in cows and pigs, to upper respiratory disease in chickens, and potentially lethal human respiratory infections, such as severe acute respiratory syndrome (sars) [ ] , middle east respiratory syndrome (mers) [ ] , and the novel coronavirus disease (covid- ) [ ] . fecal-oral transmission is believed to be the main mode of pedv transmission [ ] . the latest research indicates that airborne transmission may also contribute to a pedv outbreak [ ] , similar to sars-cov- and mers-cov. owing to antigenic, genetic (> % amino acid variation between respective s-proteins) and phylogenetic (g vs g ) differences between vaccine and field epidemic strains, current pedv vaccines appear to have low to moderate efficacy [ ] . there is a need for alternative approaches to control this disease, such as effective antiviral drugs for pedv treatment. the interaction between pedv and pigs has been well studied. several host antiviral factors, including the bone marrow stromal cell antigen (bst ) [ ] , interleukin- (il- ) [ ] , interferon-lambda (ifn-lambda) [ ] , gtpase-activating protein-binding protein (g bp ) [ ] , and cholesterol -hydroxylase (ch h) [ ] , have been reported to show antiviral activity against pedv infection. some natural compounds and compositions have also been reported to show anti-pedv activity in vitro, such as griffithsin [ ] , coumarin [ ] , and prenylated phenolic compounds [ ] . however, the mechanism of antiviral activity is not well understood, and there are no commercial anti-pedv drugs available to the pig breeding industry. in this study, we screened a library of natural products and found that tomatidine, a steroidal alkaloid that can be extracted from the skin and leaves of tomatoes [ ] , significantly inhibited the replication of pedv by directly inhibiting clpro activity, and showed antiviral activity against other swine disease viruses as well, demonstrating excellent potential as a natural broad-spectrum antiviral product. vero cells, st cells, marc- cells, bhk- cells, and ipec-j cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco, usa) with % fetal bovine serum (lonsera, uruguay), penicillin ( u/ml), and streptomycin ( ug/ml). the cells were incubated at °c in a humidified incubator with % co . pedv ms (genbank accession no. mt ), yz (genbank accession no. mk . ), sh (genbank accession no. mk . ) [ ] , and cv (genbank accession no. af . ) strains were maintained in our laboratory and passaged in vero cells with . % trypsin. the ms, yz, and sh strains belong to variant strains; cv belongs to a classical strain. the ms strain was used for all experiments and is represented by "pedv" in this article. tgev js (genbank accession no. kt . ) passaged in st cells, the highly pathogenic prrsv strain bb (genbank accession no. hq . ) passaged in marc- cells, emcv nj (genbank accession no. hm ), and sva ch-sd (genbank accession no. mh . ) passaged in bhk- cells, were maintained in our laboratory. tomatidine with purity > % was used for in vitro experiments (selleck chemicals, usa). a library (fda approved) of natural products was purchased from selleck chemicals. these compounds were stored as mm stock solutions in dmso at − °c until use. the workflow for high-throughput screening (hts) of the library was carried out as shown in figure a and b. vero cells were seeded in -well plates at × cells per well. when approximately % confluent, the cells were treated with μm compound or dmso ( μl, . % v/v) for h and then infected with pedv ( . moi) or mock infected for h. the cells were then washed with pbs, then culture medium containing μm compound was added back to each well. at hpi, cpe and ifa were observed under a microscope. the fluorescence intensity of ifa was measured by imagej software. the percentage of inhibition of fluorescence intensity is calculated by drug treatment relative to dmso treatment. during primary screening, compounds were ruled out if they resulted in any visible cytotoxicity or demonstrated less than % reduction of cpe compared with the dmso control group. for the second round of screening, cell viability had to be % or greater, and the inhibition of pedv had to be % or greater as determined by ifa. the ic (concentration of compounds was . , . , . , . , . , and . μm) and cc (concentration of compounds was . , . , . , . , . , . , and . μm) of each remaining candidate compound were calculated using log (inhibitor) vs. response-variable slope (four parameters) method by graphpad prism . software (graphpad software, ca, usa), and those that displayed dose-dependent inhibition of pedv and selectivity index (si, si = cc /ic ) over were considered for further study. in addition, cell viability was tested using an enhanced cell counting kit- (cck- ) (beyotime, china) following the manufacturer's instructions. the cc was calculated using graphpad prism . software. dmso was used as the negative control. figure screening protocol for pedv inhibitors. a screening procedure. vero cells were treated with μm compound for h, then infected with pedv ( . moi) for h. the cells were washed with pbs, then incubated in medium containing μm compound for another h. b screening process flowchart. the criteria for passing the primary screening were that the compound must have no apparent cytotoxicity and must reduce cpe by at least % compared with the dmso treatment. the criteria for passing the secondary screening were that the compound must leave cells at least % viable and inhibit pedv by more than %. compounds that passed the third screen inhibited pedv in a dose-dependent manner and had a selective index (si) higher than . c each dot represents the percent inhibition of each compound. the dots located above the dotted line indicate % or greater inhibition. d ifa of infected cells treated with one of the four designated compounds. pedv n-protein is colored green; a bright field shows cpe. e ic and cc curves of the four designated compounds. cell viability is calculated as a percentage of the viability in the cells treated with the compounds divided by that in the dmso-treated cells. the structure of each compound is inset. f sis of the four designated compounds. (see figure on next page.) wang et al. vet res ( ) the cells were lysed with μl of ripa lysis buffer (beyotime, china) on ice for min, then resolved by sds-page and transferred to a nitrocellulose membrane. after transfer, the membrane was incubated in blocking buffer ( % non-fat milk in pbst w/v) for h at room temperature, washed three times with pbst, then probed with the following antibodies: anti-pedv n-protein ( the detection of mrna levels of tgev, prrsv, and sva was performed as described previously [ , , ] . quantitative rt-pcr was performed using aceq ® qpcr sybr ® green master mix (vazyme, china). each reaction was performed in triplicate and results are expressed as mean ± standard deviation (sd). vero cells grown in -well plates were infected with tenfold serial dilutions of pedv samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. st cells grown in -well plates were infected with tenfold serial dilutions of tgev samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. marc- cells grown in -well plates were infected with tenfold serial dilutions of prrsv samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. bhk cells grown in -well plates were infected with tenfold serial dilutions of emcv/sva samples in four replicates. after h at °c, the culture medium was replaced with fresh dmem. the plates were incubated for - h at °c. virus titers are expressed as tcid , calculated using the reed-muench method. tomatidine ( μm) or dmso was incubated with pedv ( . moi) at °c for h and h. a mixture of tomatidine ( μm) or dmso and pedv was placed into vero cells seeded in -well plates. after incubation at °c for an additional h, the culture supernatants were replaced with fresh dmem and incubated for an additional h ( figure a ). the cells were then washed with pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were pretreated with tomatidine ( μm) or dmso for h at °c, and then infected with pedv ( . moi) for the time indicated ( min, min, h) at °c ( figure b ). the cells were then washed with icecold pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were infected with pedv ( . moi) at °c for h. the supernatant was replaced with dmem containing tomatidine ( μm) or dmso, and then incubated at °c for the time indicated ( min, h, h) ( figure c ). the cells were washed with citrate buffer (ph ) to remove non-internalized virus. the mrna levels of pedv n and gapdh in the cells were then measured using qrt-pcr. vero cells were incubated with pedv ( . moi) at °c for h and washed three times with pbs to remove free virus. at hpi, the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso, and the cultures were incubated at °c ( figure d ). the mrna levels of pedv n and gapdh in the samples collected at , , hpi were measured using qrt-pcr. vero cells were infected with pedv ( . moi) for h at °c. the culture medium was then replaced with fresh dmem. at hpi, the cells were washed three times with pbs and the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso. the cultures were incubated at °c for . , , and h, at which time the supernatants were harvested ( figure e ). the mrna levels of pedv n in the released virus was quantified by absolute fluorescence quantification. the crystal structure of pedv nsp was obtained from the protein data bank ( clpro, pdb: xfq). due to lack of crystal structure, the protein sequences of nsp (plp ), nsp (rdrp), nsp (ntp), nsp (exon), nsp (nendou), and nsp ( ′-o-methyltransferase) were subjected to comparative homology modeling using swiss model, which has been widely used and cited by more than articles, to generate a putative d model. swiss model performs the sequence alignments and searches the putative template protein to generate a d model for a query sequence. all the modeling parameters were set to default. the three-dimensional structure of tomatidine was obtained from pubchem (compound cid: ). the autodock . program was used for the docking of tomatidine to the active pocket of potential replication-relevant proteins. we generated a grid map around selected active site residues with grid point spacing of . Å. the potential tomatidine-protein complex was generated by genetic algorithm using the default parameters. the estimated free energy of binding was ranked, and the top two complexes were employed for further md analysis. the docking results were visualized using pymol . . . the ligand bound to the gromos /gromos force field generated by prodrg . . simulations were conducted with the gromacs package using the gro-mos a force field. the simple point charge (spc ) model of water was used to solvate the protein in a periodic dodecahedron box extending Å from the nearest protein atom. the solvated system was then neutralized with na+ and cl− ions, minimized by the steepest descent method ( steps), and equilibrated with a -ps constant volume (nvt) simulation. the production runs were conducted in a constant pressure ensemble (npt). the temperature was set to k and controlled with v-rescale. long-range electrostatic interactions were treated with the particle-mesh ewald (pme) method. the pressure was coupled to bar using the parrinello-rahman method. all bond lengths were constrained with the linear constraint solver (lincs) algorithm. a cut-off of Å was used to calculate short-range van der waals and electrostatic interactions. the time step was fs and the total simulation time was ns. rmsd, distance, and the number of hydrogen bonds between tomatidine and active pockets of the proteins were analyzed to judge binding stability and convergence. the pet- a- clpro was transformed into e. coli strain bl , and the cells were cultured at °c in lb medium. when optical density at nm (od ) reached . , the culture was cooled to °c and supplemented with mm iptg. the cells were harvested after incubation at °c for h, resuspended in pbs and disrupted by ultrasonication. the supernatant was filtered and loaded onto ni-sepharose (ge healthcare, usa). finally, the histagged protein was eluted using a linear gradient between the binding buffer and elution buffer a ( mm tris, ph . , mm nacl, and mm imidazole). low concentration imidazole ( mm) was used to wash impurities, and high concentration imidazole ( mm) was used to elute targeted protein. the target protein was condensed and desalinated using amicon ultra- ( kda, ge healthcare, usa). the proteins were analyzed by sds-page. all of the purification procedures were performed at °c to avoid unexpected degradation. the fluorescence quenching assay was measured by a perkinelmer enspire multimode plate reader. the reaction medium ( μl) contained μl of clpro solution at the concentration of μm and μl of tomatidine with different final concentrations ( , , , , , and μm). following incubation at room temperature for min, the fluorescence spectra of clpro with the different concentrations of tomatidine were recorded in the wavelength range of - nm upon excitation at nm. the stern-volmer equation was used to describe fluorescence quenching as follows: . in this equation, f and f represent the fluorescence intensities in the absence and presence of tomatidine. τ ( − s) indicates the lifetime of the fluorophore without quencher. k q is the bimolecular quenching constant. [q] refers to the concentration of the quencher, and k sv is the stern-volmer quenching constant. hence, the above equation may be applied to determine k sv by linear regression of a plot of f /f versus [q] . each measurement was repeated three times. all measurements were performed using the microcal itc calorimeter in an itc buffer ( mm tris, ph . , mm nacl, ph . ) while stirring at rpm. the stock solution of compound and clpro protein were diluted with the itc buffer to a compound concentration of μm and a protein concentration of μm before titrations. the final concentration of dmso in the reaction buffer was less than % of the total volume. protein solution was titrated using the compound. all titrations were performed using an initial injection of . μl followed by identical injections of μl with a duration of s and an interval of s between injections. the data were analyzed by microcal itc software. the last three data points were averaged and subtracted from each titration to account for the heat of dilution. each measurement was repeated three times. for pedv clpro eukaryotic and prokaryotic expression plasmids, the coding sequence for clpro was reverse transcribed and amplified from pedv ms strains. the resulting pcr products were assembled into pcaggs-flag and pet- a. mutagenesis of pedv clpro constructs were carried out by overlap extension pcr using specific mutagenic primers. the plasmids were verified by sequencing and double enzyme digestion. for functional substrates of pedv clpro, a nucleotide sequence encoding for an -amino-acid stretch corresponding to the cleavage site of pedv clpro derived from the junction of the nsp and nsp genes (ygvnlq^sg) in the pedv genome was inserted into the coding sequence of gfp between amino acids g and d . the first pcr product (n-gfp) was amplified with a reverse primer harboring the n-terminal of the cleavage site coding sequence (ygvnlq). the second pcr product (c-gfp) was amplified with a forward primer harboring the c-terminal of the cleavage site coding sequence (vnlqsg). the first and second pcr products were assembled using overlap pcr and cloned into pcaggs. vero cells cultured in -well plates were co-transfected with ng/well of gfp nsp / encoding plasmid, ng of clpro expressing plasmid, or an empty vector. at hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations ( , , μm) of tomatidine or dmso. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. the polypeptide substrate dabsyl-ynstlq↓aglrkm-e-edans was chemically synthesized by the genscript corporation. pedv cl protease was used at final concentrations of . , , and . μm; and fret peptide was added to the protein in a black -well plate at final concentrations of μm. prrsv gp protein expressed and purified at the same time with cl protease was used as a negative control at final concentrations of μm. the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. obacunone was used as a control drug. it is an fdaapproved compound with molecular weight similar to tomatidine, but it has no antiviral activity on pedv (according the data from hts). tomatidine ( , μm), obacunone ( μm, negative control) or dmso were pre-incubated with μm clpro for min at °c, and μm fret peptide was added to the mixture in a black -well plate [ ] . the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. the mock represents fret peptides with no protease. the relative fluorescence units (rfu) of the experimental group were obtained after subtracting fluorescence readings of the mock. the percentage of inhibition was calculated as follows: percentage of inhibition (%) = × [ − rlu of the tomatidine group ( - min)/rlu of the negative control group ( - min)] ( ) . all procedures involving animals for this study were approved by and performed in accordance with the animal ethics committee and nanjing agricultural university animal experiment central guidelines, respectively. female - week-old balb/c mice were immunized with μg of purified clpro protein in . ml, emulsified in the same amount of freund's complete adjuvant. two booster injections were administered with equal amounts of immunogen and freund's incomplete adjuvant at -week intervals. ten days after the last injection, antiserum was collected. pedv was cultured and passaged by passages in ipec-j cells in the presence of tomatidine at an increasing concentration of - - μm or in dmso. the total rna was extracted from the virus cultures, tomatidine-f , dmso-f , and f . the clpro gene was amplified and sequenced by using the primers located at ~ bp upstream and downstream of clpro. to assess the sensitivity of f viruses to tomatidine, ipec-j cells were inoculated with the virus (tomatidine-f , dmso-f , and f ) at . moi, and the culture medium was replaced with fresh dmem containing tomatidine ( μm) or dmso at hpi. the mrna levels of pedv n and gapdh in the samples collected at hpi were measured using qrt-pcr. western blot, qpcr, and tcid were used to examine tomatidine's inhibition of other swine disease viruses. three designated compound concentrations were added to the culture medium (final concentrations were . , , and μm). dmso was used as the negative control. statistical analysis was performed using graphpad prism software. results are expressed as mean ± standard deviation (sd). differences between groups were examined for statistical significance using one-way or two-way analysis of variance (anova). the asterisks in the figures indicate significant differences (*p < . ; **p < . ; ***p < . ; ns = not significant). vero cells were treated with µm natural product compounds, and infected with pedv as detailed in the timeline ( figure a) . the results indicated that ( . %) compounds showed no apparent cytotoxicity, and reduced cytopathic effect (cpe) by % compared with dmso alone. these compounds were then subjected to a second round of screening ( figure b) . four of the compounds produced negligible cytotoxicity and inhibited pedv infection by over % as determined by indirect immunofluorescence assay (ifa) ( figure c and d). in addition, they inhibited pedv in a dose-dependent manner and had a selectivity index (si) greater than (figure e and f) . tomatidine was chosen for further study as it had the highest si and the lowest price. to determine the dose range of tomatidine having anti-pedv activity, vero cells were treated with . , , and μm tomatidine for h and then infected with pedv. median tissue culture infectious dose (tcid ), western blot and qrt-pcr analysis showed that the virus titers, n-protein, and mrna levels decreased in a dosedependent manner (figure a-c) . at h post-infection (hpi), ifa showed that the number of infected cells in the tomatidine-treated groups were obviously lower than those in the negative control ( figure d ). in addition, tomatidine had similar inhibitory effects on the different pedv variant strains tested (yz, ms, and sh), as well as on the classic cv strain ( figure e ). to understand the anti-viral activity of tomatidine in porcine cells, ipec-j cells were treated with . , , and μm tomatidine for h and then infected with pedv ( . moi). tcid and qrt-pcr analysis showed that the virus titers and orf mrna levels significantly decreased in a dose-dependent manner ( figures f, g) . meanwhile, the ipec-j cells indicated that the tomatidine displayed no cytotoxicity ( figure h ). to further explore the mechanism by which tomatidine inhibits pedv infection, we first tested whether tomatidine is directly virucidal and kills pedv particles. as shown in figure a , tomatidine treatment failed to directly inactivate pedv. next, the relative amount of pedv n mrna to gapdh was detected by qrt-pcr to determine the effect of tomatidine on pedv attachment, internalization, and replication. the results showed that tomatidine treatment prior to pedv infection did not significantly block virus attachment to vero cells, indicating that tomatidine does not inhibit pedv attachment to cells ( figure b ). in evaluating the effect of tomatidine on pedv internalization, as shown in figure c , we determined that tomatidine treatment accompanied by virus internalization did not significantly impede virus replication compared to treatment with dmso. we then examined the effect of tomatidine on pedv replication by adding tomatidine during the replication stage. as shown in figure d , tomatidine treatment decreased pedv n mrna levels by approximately tenfold compared to treatment with dmso, suggesting that tomatidine inhibits pedv replication. in addition, the virus release assay showed that there was no significant difference in pedv rna levels in the supernatants between the treatment with tomatidine and dmso ( figure e ). taken together, these results indicate that tomatidine inhibits pedv infection primarily by affecting viral replication. several replicative enzymes regulate virus replication [ ] . considering that tomatidine significantly inhibited infection at the virus replication stage, we speculated that tomatidine may work directly with important viral replicative enzymes. to ascertain which replicative enzymes were targeted by tomatidine, potential binding sites were analyzed in detail using autodock to dock tomatidine into the pedv replicative enzyme structures ( figure a ). the estimated binding free energies were ranked as shown in figure b , and the top two complexes (binding energy = − . kj/mol, − . kj/mol) nsp and nsp were selected for further molecular dynamics (md) analysis. md utilizes newtonian physics to simulate atomic movements in a solvated system and is an accurate computational method for simulating protein-drug interactions [ ] . to judge the findings of our docking model, a ns molecular dynamic simulation was carried out for the assessment of receptor and ligand stability via root mean square deviation (rmsd), the distance and the number of hydrogen bonds between tomatidine and the active pockets of the proteins. in tomatidine-nsp ( clpro) simulation, the rmsd values of the backbone converged at less than . nm and remained stable starting at ns. the higher and more volatility rmsd in tomatidine-nsp implied structure instability (figure c(i) ). distances between tomatidine and the active pocket of nsp are highly conserved and tight during simulation calculations, whereas those between tomatidine and nsp fluctuated wildly ( figure c(ii) ). we measured the number of hydrogen bonds during the md simulations to better capture the intermolecular polar interactions. no hydrogen bonds were detected between tomatidine and nsp . however, tomatidine did form hydrogen bonds with the active pocket of clpro, which may contribute to the stability of the tomatidine- clpro complex ( figure c(iii) ). this means that clpro is a more likely target of tomatidine than nsp . in addition, the low binding energy of tomatidine to inactive cl (see figure on next page.) figure in silico tomatidine targeted the active pocket of pedv cl protease. a docked conformations of tomatidine with pedv nsp plp , nsp clpro, nsp rdrp, nsp ntp, nsp exon, nsp nendo u, and nsp ′-o-methyltransferase. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. c overall dynamic behaviors in the md simulations. (i) rmsd of backbones of nsp (red) and nsp (blue); (ii) distance between tomatidine and active pocket of nsp (red) and nsp (blue); (iii) number of hydrogen bond interactions of tomatidine with nsp (red) and nsp (blue). d docked conformations of tomatidine with pedv clpro or inactive clpro. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. e the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. wang et al. vet res ( ) : protease in silico strengthens the possibility that clpro is indeed the target for tomatidine ( figure d and e) . to verify the binding of clpro and tomatidine, pedv clpro fused with his tag was expressed in soluble form using an e. coli expression system ( figure a(i) ). after ni-sepharose purification, a single clear target band indicated that the clpro obtained could be used for further experiments ( figure a (ii), (iii)). the purified recombinant clpro was then used for a fluorescence quenching assay, itc and fret. a fluorescence quenching assay was carried out to determine the interaction between clpro and tomatidine. the results showed that the fluorescence emission intensity of clpro decreased distinctly in a dose-dependent manner with increasing concentration of tomatidine ( figure b upper) . the stern-volmer plot is shown in figure b lower; the kq value was calculated to be . × l·mol − s − . the maximum scatter collision quenching constant of the various quenchers is × l·mol − s − . this indicates that the quenching mechanism was static and the interaction between clpro and tomatidine was strong. the binding affinities of tomatidine with pedv clpro were measured by itc, which is widely used to determine thermodynamic parameters of protein-ligand interactions such as the dissociation constant (kd) and binding stoichiometry (n). the results showed that tomatidine directly interacted with pedv clpro with a kd of . μm and n of . sites. this result further supports the premise that pedv clpro is the target protein of tomatidine. these results allowed us to speculate that tomatidine may block the activity of the clpro, thereby inhibiting pedv replication. to explore the impact of tomatidine on clpro activity, we constructed plasmids expressing nsp ( clpro), a series of inactive clpro mutants (h a, c a, h / c a), and gfp nsp / containing the nsp /nsp junction (ygvnlq^sg) of pedv. after the vero cells were co-transfected with these plasmids, western blot assay showed that cleaved fragments of gfp nsp / were detected in the presence of clpro, but not in the presence of clpro mutants h a, c a, and h /c a, indicating that pedv clpro was active ( figure a ). when vero cells were treated with increasing concentrations of tomatidine, the cleaved fragments clearly decreased in a dose-dependent manner ( figure b ), indicating that tomatidine inhibited the activity of pedv cl protease. we also used fret to confirm the effect of tomatidine on clpro activity. the dose-dependent increase of fluorescence intensity shows that the clpro obtained possessed catalytic activity ( figure c ). moreover, compared with obacunone, tomatidine significantly inhibited the activity of pedv clpro ( figure d ). the percentage of inhibition of μm and μm tomatidine was . % ± . % and . % ± . % respectively, at min incubation. each reaction was performed in triplicate and the results are expressed as mean ± standard deviation (sd). in order to explore the inhibition of viral clpro activity of tomatidine in pedv-infected cells, anti- clpro serum antibody was prepared by vaccinating mice with the purified recombinant clpro ( figure e, lines - ) . vero cells were inoculated with pedv ( . and . moi) and μm tomatidine. western blot showed that the partial virus polyprotein (nsp - complex, rather than clpro) was detected with the anti- clpro serum antibody at hpi, as previously described [ ] . the ratios of nsp - complex/n in pedv not treated with tomatidine were similar to each other between the two doses of . and . moi. but the ratios of nsp - complex/n in pedv treated with tomatidine were significantly higher than those treated with dmso ( figure e , line - ), indicating that tomatidine may block the cl protease activity in a natural situation. tomatidine strongly inhibits virus particle production of dengue virus and chikungunya virus [ , ] . to investigate whether tomatidine has an antiviral effect against other swine disease viruses, we selected several other viruses including a coronavirus, tgev, [ ], an arterivirus, prrsv [ ] , and picornaviruses emcv and sva [ , , ] . st cells were treated with . , , and μm tomatidine for h and then infected with tgev for h. the results indicated that tgev proliferation was significantly suppressed by tomatidine and almost completely cut off at the concentration of μm ( figure a-c) . marc- cells were treated with the indicated concentrations of tomatidine for h and infected with prrsv for h. these results indicated that tomatidine exhibits antiviral activity against prrsv ( figure d -f). bhk- cells were treated with the indicated concentrations of tomatidine for h and then infected with emcv or sva for h. western blot, qpcr, and tcid analysis revealed substantial inhibition activity of tomatidine against emcv and sva (figures g-i, and j-l). none of the various cells treated with the indicated concentrations of tomatidine showed any cytotoxicity ( figure m ). outbreaks of porcine epidemic diarrhea cause significant lethality rates in neonatal piglets, creating a heavy economic burden in the global swine industry. unfortunately, available commercial vaccines fail to protect against the high virulence of pedv variants [ , ] . because there is no antiviral drug available to treat this disease, we need to develop new therapeutic methods to prevent and control pedv. natural compounds and compositions have been a rich source of drugs against many viral infections. for example, griffithsin, a highmannose-specific lectin, has been shown to reduce pedv infection in vero cells by preventing viral attachment and disrupting cell-to-cell transmission [ ] . prenylated phenolic compounds from the leaves of sabia limoniacea figure tomatidine directly inhibited the activity of pedv cl protease. a vero cells cultured in -well plates were co-transfected with ng of gfp nsp / encoding plasmid, ng of clpro or inactive mutants expressing plasmid or empty vector. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. b vero cells cultured in -well plates were co-transfected with ng of gfp nsp / encoding plasmid, ng of clpro expressing plasmid or empty vector. at hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations ( , , and μm) of tomatidine or dmso. at hpi, the cells were harvested and the cleaved fragment of gfp nsp / was visualized using western blot. c pedv clpro was used at final concentrations of . , , and . μm; and μm ( clpro substrate) was added to the protein in a black -well plate. prrsv gp protein was used as a negative control. the mixtures were then further incubated at °c for min, and fluorescence was monitored at nm excitation and nm emissions every minute. the rfu were calculated by subtracting the mock from the fluorescence readings to eliminate the effect of background signals. d tomatidine ( , μm) or obacunone ( μm, negative control) or dmso was pre-incubated with μm protease for min at °c, and μm ( clpro substrate) was added to the mixture in a black -well plate. the mixtures were then further incubated at °c for min, and fluorescence intensity was monitored at nm excitation and nm emissions every minute. rfu were calculated as above. e vero cells were inoculated with pedv ( . and . moi) and μm tomatidine. cells were transfected with ng pcaggs- clpro as clpro positive control. after incubation for h, the cellular proteins were collected and the virus polyprotein was detected with western blot using anti- clpro mouse antibody, as previously described ( ) . meanwhile, pedv n-protein, gapdh, and clpro-flag were detected with western blot using molecular antibodies against pedv n-protein, gapdh, and flag. the ratio of nsp - complex/n protein levels was depicted by integrated density analysis. the arrow indicates the location of nsp - complex. all results are mean ± sd from three independent experiments performed in triplicate. exhibit promising antiviral activities against pedv replication [ ] . but the mechanisms involved in these studies are not well understood. in this study, tomatidine, screened from a library of natural products, was shown to inhibit pedv proliferation in vitro. tomatidine displayed remarkable inhibition of pedv replication by targeting cl protease, which we investigated using molecular docking and md analysis, fluorescence spectroscopy, itc, cl protease activity, and fret assays. the pedv life cycle is composed of four stages: attachment, entry, replication, and release [ ] . our results indicate that tomatidine significantly inhibits viral infection at the replication stage. several nonstructure proteins are key to viral replication, hence we speculated that tomatidine, which is shown to inhibit viral replication, may target a non-structural protein. making use of molecular docking and molecular dynamics, we speculated that tomatidine may inhibit pedv proliferation by directly targeting cl protease. fluorescence quenching is an important technique for measuring binding affinity between ligands and proteins. itc is a technique used in a wide variety of quantitative studies of biomolecular interactions. itc works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. in our study, clpro-tomatidine binding was indicated by fluorescence quenching and itc. in order to confirm the effect of tomatidine on clpro activity, we constructed a plasmid expressing gfp nsp / protein containing the nsp /nsp junction (ygvnlq^sg) of pedv. our results show that gfp nsp / was cleaved into two fragments by clpro, indicating that pedv clpro was active. tomatidine inhibited the activity of pedv cl protease in vero cells in a dose-dependent manner. the inhibition ratio of tomatidine was also shown in a fret assay. the nm fluorescence intensity remained stable until the labeled substrate was fractured by the protease, which indicates that the purified protein possesses cl protease activity. compared with the control drug obacunone, tomatidine significantly reduced the fluorescence intensity. the ratio of nsp - complex/n in virus treated with tomatidine was significantly higher than in the dmso treated virus, indicating that tomatidine blocked the cl protease activity in a natural situation. however, anti- clpro serum raised in mice detected nsp - complex rather than clpro (nsp ) only. this phenomenon underlines the possibility that there is a quantitative difference between nsp - complex and nsp of products in pedv-infected cells at hpi. the c-like protease, which is responsible for processing polyproteins of nidoviruses and picornaviruses, is an attractive target for drug therapy. it has been reported that a series of compounds such as flavonoids, chalcones, and benzothiazolium display significant antivirus activity by inhibiting clpro activity. we found that tomatidine strongly inhibits pedv replication by targeting cl protease. tomatidine has many cell biological properties. it enhances expression of nuclear factor erythroid -related factor (nrf ) [ ] , which when knocked out benefits viral propagation [ ] . ′-amp-activated protein kinase catalytic subunit alpha- (ampk) promotes innate immunity and antiviral defense through modulation of sting signaling [ ] . tomatidine also stimulated ampk phosphorylation by activating the camkkβ pathway [ ] . tomatidine inhibits dengue virus particle production partly by inhibiting cyclic amp-dependent transcription factor atf- (atf ) [ ] . in this study, we noted that the ic ( % inhibitory concentration) of tomatidine against pedv in cells was low micromolar ( . μm); the cc ( % cytotoxic concentration) was about . μm; while the inhibition of pedv clpro by tomatidine in the fret assay at μm was only %. in addition, the binding energy of tomatidine with the viral nsp was close to that of the clpro. these results suggest that there may be other antiviral mechanisms involved. clpro of pedv, as well as of other coronaviruses (porcine deltacoronavirus), picornaviruses (hepatitis a virus, foot and mouth disease virus), and arterivirus (prrsv), has been shown to inhibit production of infβ, hijacking the host's innate antiviral immune response [ ] [ ] [ ] [ ] . this effect has been attributed to the direct cleavage of nf-kappa-b essential modulator (nemo) figure tomatidine shows broad-spectrum antiviral activity against other swine disease viruses. tcid , western blot, and qpcr were used to examine the inhibition activity of tomatidine against other swine disease viruses. three designated concentrations of compounds were added to the culture medium (final concentrations were . , , μm, with no-observable cytotoxicity). dmso was used as the negative control. tgev ( . moi), prrsv (e), and emcv (h)/sva (k) were then used to infect st, marc- , and bhk- cells, respectively, and samples were harvested at h, h, and h. the tcid of tgev (a), prrsv (d), emcv (g), and sva (j) treated with μm tomatidine or dmso were calculated using the reed-muench method. the n-protein level of tgev (b), prrsv (e), emcv (h), and sva (k) were determined by western blot. relative tgev n (c), prrsv n (f), emcv vp (i), and sva vp (l) mrna levels, was determined by qrt-pcr, and expressed relative to that in dmso-treated cells. m viability of st, marc- , and bhk- cells pretreated with the indicated concentrations of tomatidine and incubated for h, h, and h, respectively in medium containing tomatidine. the results are from one of three independent experiments. the internal loading control was β-actin. error bars represent the sd. the asterisks in the figures indicate significant differences (*p < . ; **p < . ; ***p < . ; ns = not significant). (see figure on next page.) (an important substrate in the rlr cascade) [ , ] . by blocking clpro, tomatidine may also prevent clpro cleaving nemo, thereby enhancing the innate antiviral immunity, which could in turn help to inhibit virus replication. tgev, prrsv, emcv, and sva contain c or cl protease. our results showed that tomatidine effectively inhibits tgev, prrsv, emcv, and sva replication in vitro. but the antiviral activity of tomatidine against picornaviridae looks more significant than tgev and prrsv. it may be related to the different characterization of the virus replication. the molecular docking analysis demonstrated that the binding energy between the c or cl protease of the viruses with tomatidine were weak and no hydrogen bonds were detected, which being different from that of pedv clpro (additional file ). it indicates that the antiviral activity of tomatidine is likely to rely on other pathways rather than only targeting c proteases, which is worthy being further explored. tomatidine has been proved to be safe within a certain dosage in mice. unfortunately, as a dietary supplement approved by the fda, tomatidine lacks detailed safety and pharmacokinetics evaluation in pigs. the safety, pharmacokinetics, and efficacy of tomatidine for prevention, treatment, and control of pedv and perhaps other porcine nidoviruses need to be further evaluated in pigs. it would be interesting to test whether its clpro blocking activity also holds for nidoviruses infecting humans. we conclude that tomatidine effectively inhibits pedv proliferation via inhibition of clpro activity. in addition, tomatidine displays antiviral activity against tgev, prrsv, emcv, and sva. these findings offer novel and promising therapeutic possibilities for fighting infections caused by these viruses. supplementary information accompanies this paper at https ://doi. org/ . /s - - -y. additional file . the binding energy of tomatidine to cl or c protease of tgev, prrsv, emcv, and sva in silico. a docked conformations of tomatidine with cl or c protease of tgev, prrsv, emcv, and sva in silico. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains characterization of a novel coronavirus associated with severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study structural basis for the recognition of sars-cov- by full-length human ace evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs an alternative pathway of enteric pedv dissemination from nasal cavity to intestinal mucosa in swine porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus bst suppresses porcine epidemic diarrhea virus replication by targeting and degrading virus nucleocapsid protein with selective autophagy antiviral activity of interleukin- as a response to porcine epidemic diarrhea virus infection ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha gtpase-activating protein-binding protein (g bp ) plays an antiviral role against porcine epidemic diarrhea virus cholesterol -hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of -hydroxycholesterol in vitro antiviral activity of griffithsin against porcine epidemic diarrhea virus three new coumarins from saposhnikovia divaricata and their porcine epidemic diarrhea virus (pedv) inhibitory activity prenylated phenolic compounds from the leaves of sabia limoniacea and their antiviral activities against porcine epidemic diarrhea virus therapeutic potential of steroidal alkaloids in cancer and other diseases pathogenicity and immunogenicity of a new strain of porcine epidemic diarrhea virus containing a novel deletion in the n gene xanthohumol inhibits prrsv proliferation and alleviates oxidative stress induced by prrsv via the nrf -hmox axis seneca valley virus c and c inhibit type i interferon production by inducing the degradation of rig-i identification of two antiviral inhibitors targeting c-like serine/ c-like protease of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus functional and genetic analysis of coronavirus replicase-transcriptase proteins molecular dynamics simulation for all alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro efffects of electrostatic and hydrophobic interaction on the stability of the tgev main proteinase dimer structure and cleavage specificity of the chymotrypsinlike serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex seneca valley virus c(pro) abrogates the irf -and irf -mediated innate immune response by degrading irf and irf two novel porcine epidemic diarrhea virus (pedv) recombinants from a natural recombinant and distinct subtypes of pedv variants genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china coronaviruses: an overview of their replication and pathogenesis tomatidine enhances lifespan and healthspan in c elegans through mitophagy induction via the skn- /nrf pathway amp-activated kinase (ampk) promotes innate immunity and antiviral defense through modulation of stimulator of interferon genes (sting) signaling tomatidine reduces palmitate-induced lipid accumulation by activating ampk via vitamin d receptor-mediated signaling in human hepg hepatocytes aspartic acid at residue modulates the capacity of hp-prrsv nsp to antagonize ifn-i expression porcine deltacoronavirus nsp antagonizes type i interferon signaling by cleaving stat hepatitis a virus c protease cleaves nemo to impair induction of beta interferon foot-andmouth disease virus antagonizes nod -mediated antiviral effects by inhibiting nod protein expression arterivirus nsp antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank miss. huixin zhu for her generous help with the animal experiment. in addition, the critical and helpful comments from the reviewers are highly appreciated. conception of the work: pj and pw; cellular and animal experiment: pw, jb, xl and mw; analysis and interpretation of data: pj, xw and pw; preparation of the manuscript: pj and pw. all authors read and approved the final manuscript. not applicable. all animals were housed in the animal facility of nanjing agricultural university (nau), nanjing, jiangsu, china. all experimental protocols for animals were conducted following the national guidelines for housing and care of laboratory animals (china) and performed in accordance with nau institutional regulations after approval by the institutional animal care and ethics committee of nau (syxk(su) - ). not applicable. none of the authors have any possible conflicts of interest. key: cord- - opx h authors: dwivedi, varun; manickam, cordelia; binjawadagi, basavaraj; joyappa, dechamma; renukaradhya, gourapura j. title: biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: opx h biodegradable nanoparticle-based vaccine development research is unexplored in large animals and humans. in this study, we illustrated the efficacy of nanoparticle-entrapped uv-killed virus vaccine against an economically important respiratory viral disease of pigs called porcine reproductive and respiratory syndrome virus (prrsv). we entrapped plga [poly (lactide-co-glycolides)] nanoparticles with killed prrsv antigens (nano-kag) and detected its phagocytosis by pig alveolar macrophages. single doses of nano-kag vaccine administered intranasally to pigs upregulated innate and prrsv specific adaptive responses. in a virulent heterologous prrsv challenge study, nano-kag vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood. in summary, we demonstrated the benefits of intranasal delivery of nanoparticle-based viral vaccine in eliciting cross-protective immune response in pigs, a potential large animal model. microencapsulation of vaccine agents and drugs in biodegradable polymer and its delivery in humans is an innovative approach to create more robust medications and vaccines in the st century. the biocompatible and biodegradable polymer, plga [poly (lactide-co-glycolides)], is the us fda approved material in the development of nanoparticle-based controlled release delivery system [ ] . plga slowly degrades and releases the vaccine over a long-period of time, thus avoiding the need of a booster dose [ ] . being particulate in nature, the nanoparticle-mediated delivery promotes uptake of ags by professional antigen presenting cells (apcs). nanoparticle as a delivery vehicle to vaccines and drugs has been extensively evaluated in mouse models. however, there are limitations in the translation of novel rodent findings to improve human and food animal health [ ] . therefore, pig may serve as a useful large animal model for such research. moreover, due to their physiological, anatomical, and immunological similarity to humans, pigs are already in use as an animal model system to study a few viral diseases [ ] . although, the current study is focused on a respiratory pathogen which infects pigs, and the vaccine evaluation strategy may help to consider pig as a model system. among the swine diseases, porcine reproductive and respiratory syndrome (prrs) is highly devastating, causing an estimated economic loss of $ million annually in the us [ ] . this translates into $ . million losses per day annually. prrsv infects pigs of all ages and is caused by a highly mutating, positive sense, single stranded rna virus belongs to the family arteriviridae [ ] . prrs in growing pigs causes anorexia, fever, respiratory distress, and enhanced susceptibility to secondary microbial infections; while in pregnant sows it is characterized by reproductive dysfunction and abortions [ ] . primary prrsv permissive cells are alveolar macrophages (mws) [ ] . prrsv rapidly modulates the host innate immune response, such as dampens the nk cell cytotoxicity and reduces the ifn-a production, and upregulates immunosuppressive mediators from as early as two days postinfection [ ] ; which lead to poor adaptive immune response and delayed/weak virus neutralizing antibody response, resulting in prrsv persistence. nevertheless, due to high degree of constant genetic and antigenic variations, control of prrs remains a challenge to the swine industry worldwide. optimal mucosal immunization induces protective immune response at both mucosal and systemic sites compared to systemic immunization [ ] . generation of protective iga response is essential to reduce and/or prevent the entry of pathogens whose principle port of entry is through mucosal sites [ ] . mucosal immunization induces effective immune response at both local and distant effector sites. particulate antigen delivery system facilitates the passage of ags through mucosal barrier and leads to stimulation of the underlying mucosal immune cells [ ] . biodegradable microspheres made of chitosan, plga, and liposome have been in use to deliver candidate vaccines to mucosal sites [ ] . a study using nanoparticle-entrapped killed influenza virus vaccine administered with an adjuvant intranasally to mice, rabbits, and pigs elicited protective immune response, with better immunity induced in pigs by intranasal compared to intramuscular route of vaccination [ ] . a single intranasal delivery of plga nanoparticle-entrapped schistosoma mansoni ags to mice elicited protective neutralizing antibody response in the lungs and blood [ ] . since late s, modified live prrsv (prrs-mlv) and killed virus vaccines are in use to control prrs, but neither of them protects pigs completely against heterologous field viruses [ ] . like the field virus, prrs-mlv also induces immunosuppression [ , ] . moreover, there are reports of reversion of vaccine virus into virulence leading to severe disease outbreaks [ , ] . although available killed prrsv vaccines are safe, they are poorly immunogenic [ ] . thus, to control prrs outbreaks innovative vaccine strategies are required. in the current study, killed prrsv ags were encapsulated in plga nanoparticles and characterized the candidate vaccine in vitro and in a pre-and -postchallenge study in pigs. various immune correlates of protection were analyzed both at mucosal and systemic sites to show the evidences of cross-protective immunity. preparation of vaccine antigens and plga nanoparticlekilled prrsv vaccine (nano-kag) marc- cell-monolayer was infected with the prrsv vr strain [ ] at . moi (multiplicity of infection), and the harvested infected cell culture fluid was clarified and subjected to ultracentrifugation ( % sucrose overlay) at , g for hr at uc. the semi-purified viral pellet was suspended in pbs; titrated, inactivated by uv irradiation (at nm uv-irradiation [el series uv lamps, uvp, llc (ca); watt/ v - hz/ . amps] for hr), sonicated, and the protein content was estimated using the bca kit (biorad, ca). viral inactivation was confirmed by cell culture immunofluorescence assay in marc- cells. the viral pellet was aliquoted and stored at uc. control antigen was prepared similarly using uninfected marc- cells. sterile precautions were followed throughout the antigen preparation and processing procedures to avoid any bacterial contamination. nanoparticles were prepared using standard double emulsion solvent evaporation technique [ , ] . briefly, % of plga / ( mg) was dissolved in mg of killed vr proteins, homogenized at rpm for seconds, then added to aqueous solution of % polyvinyl alcohol and homogenized. finally, the preparation was stirred overnight and the washed nanoparticles were freeze-dried and stored at uc. the amount of entrapped prrsv protein in the nanoparticles was determined as described previously [ ] . the size and shape of nanoparticles was determined by scanning electron microscopy (hitachi s- n). bronchoalveolar lavage fluid (bal) collected from three - weeks old healthy spf pigs was processed to isolate mononu-clear cells (bal-mnc) [ ] . bal-mnc ( cells per ml) were plated in a well plate containing poly-l-lysine coated cover slips for hr. non-adherent cells were aspirated and the adherent cells were treated with freeze-dried nano-kag containing different concentrations of prrsv proteins suspended in dmem containing % fbs. cells uninfected or infected with prrsv (vr strain) at . moi for hr was included as control. cells were fixed in % paraformaldehyde for min, permeabilized ( . % triton x- ) and blocked (pbs containing % bsa and . % triton x- ) for hr at room temperature (rt). subsequently, treated with anti-prrsv nucleocapsid specific mab sdow (rural technologies, inc.,) and early endosome specific goat polyclonal anti-early endosome antigen (eea- ) igg (santa cruz biotechnology, santa cruz, ca), diluted in the dilution buffer (pbs containing % bsa and . % triton x- ) for hr at rt. followed by treatment with goat anti-mouse igg alexa flour and donkey anti-goat alexa flour (invitrogen), and incubated for hr at rt. cells were washed in between the treatment steps and treated with a mounting medium containing . % dabco (sigma). stained coverslips were mounted on a clean glass slide using transparent nail polish and viewed under a leica confocal microscope. the acquired images were analyzed using leica confocal software. in vitro uptake of nano-kag by pig mws and determination of cd / expression bal-mnc ( cells per ml) were seeded in a -well plate and untreated or treated with k-ag or nano-kag ( , . , and . mg/ml of prrsv protein) and incubated for hr at uc. cells uninfected or infected with prrsv (mn strain) at . moi for hr was served as control. cells were treated with anti-prrsv n' mab followed by goat anti-mouse igg alexa flour , washed, and fixed before analysis. to assess the expression of cd / on professional antigen presenting cells (apcs), bal-mnc were treated as above for hr at uc, washed and stained using biotinylated human ctla -mouse immunoglobulin fusion protein (ancell, mn) and pe-conjugated cd (southern biotech) [ ] , followed by streptavidin percpcy . . cells were fixed and analyzed using facs aria ii (bd biosciences) flow cytometer. conventional large white-duroc crossbred weaned specificpathogen-free (spf) pigs from three different litters at - wks of age were confirmed seronegative for prrsv, porcine respiratory corona virus, transmissible gastroenteritis virus, and porcine circo virus antibodies. in a pre-challenge study, pigs (n = ) were grouped randomly into three groups (n = per group). group iunvaccinated (mock) pigs inoculated with dmem and pbs; group ii -inoculated with k-ag; group iii -inoculated with nano-kag. each vaccine (nano-kag and k-ag) dose has one mg of crude viral preparation containing , tcid of inactivated virus. the vaccine was inoculated once, intranasally. all the pigs were euthanized on post-immunization day (pid) and evaluated for innate and virus specific adaptive immune responses. in a post-challenge study, pigs (n = ) were divided randomly into four groups (n = per group). group i -mock pigs; group ii -inoculated with normal saline; group iii -inoculated with k-ag; group iv -inoculated with nano-kag. each vaccine dose had same amount of ags as described above. groups ii, iii, and iv were challenged with prrsv mn ( . tcid /ml , ml per pig) on pid and euthanized on day post-challenge (dpc or pc) . all the inoculations were performed once by intranasal route. the dose of nano-kag ( mg per pig) was chosen based on the results of a dose-dependent response study performed earlier in pigs. mock-inoculated pigs were euthanized separately before sacrificing virus challenged animals. pigs received food and water ad libitum and maintained under the supervision of a veterinarian. all the pigs were maintained, samples collected, and euthanized as per the standard procedures with necessary efforts to minimize suffering of animals. the animal use protocol was approved by the committee on the ethics of animal experiments of the ohio state university. this study was carried out in strict accordance with the recommendations by public health service policy, united states department of agriculture regulations, the national research council's guide for the care and use of laboratory animals, and the federation of animal science societies' guide for the care and use of agricultural animals in agricultural research and teaching, and all relevant institutional, state, and federal regulations and policies regarding animal care and use at the ohio state university. the protocol was approved by the committee on the ethics of animal experiments of the ohio state university (protocol number: -ag ). all the pigs were maintained, samples collected, and euthanized, and all efforts were made to minimize suffering of animals. during necropsy the lungs and lymph nodes were examined grossly and histologically. macroscopic pulmonary lesions were given an estimated score based on the percentage of consolidated lesions in individual lobes as described previously [ ] . the lung tissue samples collected from the caudal lobe was fixed in % neutral buffered formalin and sections ( mm) made were stained for hematoxylin-and-eosin (h&e) as described previously [ ] . frozen lung sections were immunostained as described previously [ ] . briefly, sections were treated with prrsv nucleocapsid protein specific mab (sdow ) or isotype control mab followed by abc peroxidase staining kit (vectastain elite, vector labs) and the labeling was visualized by application of dab ( , diaminobenzidine) substrate (vector laboratories) and counterstained with hematoxylin. immunostained slides were examined by an unbiased certified veterinary pathologist to score the presence of prrsv ags. prrsv titer and virus neutralizing antibody titer in serum and in the lung homogenate was analyzed by indirect immunofluorescence assay (ifa) as previously described [ ] . prrsv specific iga and igg antibodies in serum and lung lysate (homogenate) were analyzed by elisa. briefly, elisa plates were coated with pre-titrated semi-purified killed prrsv (mn ) ags ( mg/ml) in carbonate-bicarbonate buffer (ph . ), washed and blocked ( % bsa+ . % tween in pbs). serum ( : ) and lung lysate ( . mg/ml, w/v) samples were added and incubated for hr at rt. the bound virus specific isotype antibody was detected using anti-pig iga and igg secondary antibodies conjugated with hrp (kpl). plates were developed using the chromogen tmb and read at nm. we also included non-prrsv antigen-coated plates as control and the od values obtained from experimental plate were subtracted from the control. five million pig pbmc, tbln (tracheobronchial lymph nodes) mnc, and lung mnc were subjected to ex vivo restimulation in the absence or presence of prrsv mn ags ( mg/ml) as described previously [ ] , and the harvested supernatant was analyzed to measure cytokines. cytokines secreted by immune cells cultured in the absence of prrsv ags was subtracted from the corresponding test value. serum samples, harvested culture supernatants, and lung lysates were analyzed for th (ifn-c and il- ), th (il- ), proinflammatory (il- ), and immunosuppressive (il- and tgf-b) cytokines by elisa [ ] . amount of cytokines present in the lung lysate was normalized to picogram per gram of lung tissue. flow cytometry analysis was performed to determine the phenotype and the frequency of different immune cells by a multicolor immunoassay as described previously [ ] . since mnc were isolated from different amounts (weights) of tissues (lungs and tbln) we did not assess the absolute cell numbers. in this study, we determined relative frequency of individual immune cell subset by immunostaining fixed number of mnc (one million) from each site of collection, and , events were acquired in bd facs aria ii (bd biosciences) and analyzed using flowjo software. all data were expressed as the mean of three pigs +/ sem. statistical analyses were performed using one way analysis of variance (anova) followed by post-hoc tukey's test using graphpad instat (software version . ) to establish differences among unvaccinated, k-ag and nano-kag pig groups in postchallenge trial and between k-ag and nano-kag pig groups in pre-challenge trial. statistical significance was assessed as p, . . morphology of sham and prrsv ags entrapped plga nanoparticles was determined by scanning electron microscopy which revealed the size of particles as - nm (figure , a) . the average protein content in nanoparticles or core-loading was . - . % (w/w), which represents an encapsulation efficiency of - %. upon re-dispersion of the nano-kag in pbs, prrsv proteins were released slowly in the first hr, later a gradual release profile was observed over the next -weeks (data not shown). uptake of nano-kag by apcs was studied using bal-mnc harvested from three healthy spf pigs. the confocal images of alveolar mws revealed preferential uptake of nano-kag but not unentrapped viral ags (k-ag), prrsv infected cells served as a positive control (figure , b) . engulfed nanoparticles delivered the prrsv ags to early endosomes and it was comparable to virusinfected control (figure , c & d) . further, nano-kag engulfed apcs underwent maturation as indicated by significantly increased expression of cd / (figure , e) . in addition, % of bal-mnc treated with nano-kag was positive for prrsv protein comparable to virus infected cells (figure , f ii & iv). in contrast, only % bal-mnc treated with k-ag were positive for viral protein (figure , f iii) . our results suggested that prrsv ags delivered in nanoparticles were phagocytosed by apcs and the released protein was found in the endosomes. in a pre-challenge study, intranasal delivery of nano-kag resulted in induction of innate immune response at both mucosal and systemic sites, indicated by a significant increase in the frequency of nk cells, dcs, and cd t cells in the lung mnc ( figure , a-c); and cd t cells and dcs in the pbmc compared to k-ag vaccinated pigs (figure , h & i) . immune cells involved in adaptive arm of the immune response, such as cd + cd + t cells (th/memory) and cd + t cells were increased significantly in the lung mnc of nano-kag compared to k-ag vaccinated pigs (figure , d & e) . further, lung mnc and pbmc from nano-kag immunized pigs secreted significantly reduced levels of the cytokine, il- , and higher amounts of il- in a recall response (figure , f, g, & j) . in addition, innate cytokine ifn-a was secreted at significantly higher levels in pigs vaccinated with nano-kag compared to both the control groups ( figure , k). in a post-challenge study, nano-kag vaccinated mn challenged pigs were clinically healthy with no fever or respiratory distress. in contrast, both k-ag and unvaccinated, mn challenged pigs had irregular fever with reduced feed intake during the first two-week post-challenge. microscopic examination of h&e stained lung sections of unvaccinated and k-ag vaccinated, mn challenged pigs' revealed severe pneumonic lesions with massive infiltration of mononuclear cells with large consolidated area. in contrast, significantly reduced lung lesions were observed in nano-kag vaccinated virus challenged pigs (figure , a) . significantly reduced gross lung lesion scores in nano-kag immunized group compared to other two virus challenged groups was observed (figure , c) . immunohistochemistry analysis had revealed abundant prrsv antigen positive cells in the lung sections of unvaccinated and k-ag vaccinated, mn challenged pigs compared to nano-kag received pigs (figure , b & d) . prrsv titer in serum samples indicated a reduced viral load of greater than one-log at pc with complete viral clearance by pc in nano-kag vaccinated, compared to k-ag vaccinated and unvaccinated pigs (figure , e) . similarly, prrsv load in the lungs was also reduced (although not significant) in nano-kag compared to k-ag immunized, mn virus challenged pigs (figure , f) . also the prrsv titer (tcid /ml) in both the serum and lung homogenate showed a lung homogenates of nano-kag immunized pigs contained significantly higher levels of virus specific iga and igg antibodies compared to unvaccinated and k-ag vaccinated, mn challenged pigs (figure , a & b) . in the serum samples of nano-kag vaccinated pigs increased iga antibody levels at pc (samples collected on the same day as viral challenge but before inoculating the challenge virus) with a significant increase at pc compared to either unvaccinated or k-ag vaccinated, mn challenged pigs was detected (figure , d) . the prrsv specific igg antibody levels in serum (figure , e) and both iga and igg levels in the nasal swab (figure , g & h) were significantly higher in nano-kag vaccinated, compared to unvaccinated and k-ag immunized pigs at dpc . significantly increased prrsv specific neutralizing antibody (vn) titers in serum of both k-ag and nano-kag vaccine received pig groups was observed at pc , which still remained high (although not significant) only in the nano-kag group at pc . in the lungs, a similar trend of increased (but not significant) titers of neutralizing antibodies in nano-kag immunized pigs was seen (figure , c & f) . nano-kag vaccine received mn virus challenged pigs had significantly increased innate ifn-a production in the lungs ( figure , a) . in k-ag vaccinated and unvaccinated pigs, a fourfold reduction in nk cell frequency compared to mock pigs was observed. in contrast, in nano-kag vaccinated pigs the nk cell frequency was significantly higher than the k-ag and unvaccinated virus challenged pigs ( figure , b) . further, lung nk cellcytotoxic function in unvaccinated and k-ag vaccinated, mn virus challenged pigs was completely suppressed; however, in nano-kag received pigs it was partially rescued (figure , c) . the frequency of cd t cells and cd + (but not cd + ) t cells in the lungs of nano-kag vaccinated animals were significantly increased compared to k-ag and unvaccinated, virus challenged pigs ( figure , d, e & f). in the peripheral blood of nano-kag immunized pigs a significantly increased frequency of dcs, and in tbln significantly increased frequency of both dcs and cd t cells was observed ( table ) . pigs vaccinated with nano-kag had significantly reduced foxp + t-regulatory cell (treg) population in the lungs, compared to unvaccinated and k-ag received pigs (figure , a) . immuno- suppressive cytokines (il- and tgf-b) response in the lungs was significantly reduced in nano-kag vaccinated pigs; and also their decreased secretion was detected in lung mnc restimulated with killed mn ags (figure , b, c, e & f). in contrast, a significantly increased ifn-c in the lung homogenate, and its secretion in antigen restimulated lung mnc was detected in nano-kag vaccinated pigs (figure , d & g) . in addition, pbmc secreted significantly reduced il- and tgf-b compared to k-ag vaccinated virus challenged pigs ( figure , h & i) , while in tbln-mnc, increased il- was seen compared to both virus challenged groups (figure , l) . increased ifn-c was secreted in both pbmc and tbln-mnc of nano-kag vaccine group compared to k-ag immunized pigs ( figure , j & m) . the level of proinflammatory cytokine, il- , in a restimulation response was significantly reduced in tbln-mnc of nano-kag compared to both virus challenged pigs ( figure , k & n) . nanoparticle mediated vaccine delivery has shown a great promise in mouse models against influenza, parainfluenza, hepatitis b, plasmodium, and venezuelan equine encephalitis pathogens [ , , ] . however, the knowledge related to crossprotective efficacy of such vaccines in a suitable large animal model is limited. our study has revealed the potency of nanoparticle-entrapped prrsv vaccine in pigs. prrs has been a dreadful disease causing huge economic loss in a majority of the swine producing countries in the world. nano-kag vaccine has upregulated the frequency of major innate immune players (nk cells, cd t cells, and dcs), and also enhanced the secretion of anti-viral cytokines (ifn-a and ifn-c) , which otherwise is suppressed by prrsv [ ] . suggesting that in nano-kag vaccinated virulent heterologous prrs challenged pigs, these effectors played a pivotal role in the viral clearance. in contrast, increased viral load in the lungs of k-ag vaccinated pigs was perhaps due to antibody-mediated enhancement in the uptake lungs was determined by immunofluorescence assay. each bar represents average values from three pigs sem. asterisk represents the statistical significant difference (p, . ) between k-ag and nano-kag received pig groups, and q represents the statistical significant difference (p, . ) between unvaccinated and nano-kag received pig groups. a similar trend in results was obtained in an independent second trial performed using same number of animals. doi: . /journal.pone. .g of prrsv by alveolar mws, in addition to increased immunosuppressive response. particulate ags has an inherent affinity for mucosal m cells and apcs and are phagocytosed passively by apcs [ ] . nanoparticles protect the entrapped proteins from protease-mediated degradation at mucosal surfaces, and also aid in slow sustained release of the vaccine [ ] . plga nanoparticle induces activation and maturation of human dcs by upregulating the expression of costimulatory and mhc class ii molecules, secretion of proinflammatory cytokines, and enhances the apcs allostimulatory property [ , ] . consistent with the inherent adjuvant property of plga, pig apcs treated with plga nanoparticle vaccine (nano-kag) had increased expression of a costimulatory molecule, cd / . in our study, we did not include a pig group with empty pgla nanoparticles, as our primary goal was to augment the killed prrsv vaccine induced anti-prrsv cross-protective immunity with the help of plga, a well-known adjuvant and a delivery system to vaccines. thus, at present we do not have the information on how much of post-vaccination t cell immunity detected in nano-kag vaccinated pigs was due to the adjuvant effect of the pgla nanoparticles alone, which will be considered during our future investigations. however, a rapid uptake of nano-kag by lung apcs followed by translocation of viral ags into endosomal compartment was observed. suggesting that virus specific adaptive immune response could be elicited in the respiratory tract of pigs using plga nanoparticle-based killed prrsv vaccine. differential cell counts from bal fluid harvested from healthy mice, humans, and pigs have indicated that greater than % of cells are alveolar mws [ , ] , suggesting that intranasally delivered nano-kag were phagocytosed by mws. earlier studies have demonstrated rapid uptake of chitosan nanoparticles by apcs followed by gradual release of antigen due to slow rate of degradation of chitosan by lysozymes [ ] ; resulting in increased expression of costimulatory molecules, activation of dcs, and antigen presentation through mhc class i and ii molecules [ ] . in our pre-challenge study, nano-kag vaccine significantly increased the frequency of cd + t cells, th/ memory cells, with concomitant increase in the secretion of innate (ifn-a), proinflammatory (il- ), and th (ifn-c) cytokines. immune potentiating ability of chitosan nanoparticles is mediated by the action of innate immune cells, in addition to enhanced production of il- and ifn-c [ ] . phagocytosis of polystyrene latex microspheres by mws activate the signal transduction events in innate immune cells [ ] . once activated, the apcs present the antigen through mhc class i and ii molecules to cd + and cd + t cells, respectively. in our post-challenge study using a virulent heterologous prrsv, nano-kag induced superior innate immune response was observed. studies have shown immune potentiating activity of nanoparticles in mice, pigs, and macaques; but the immune correlates were not evaluated in vaccinated virus challenged animals [ ] . immunologically, prrsv modulates innate immune function of pigs by dampening the ifn-a production and nk cell frequency as well as its cytotoxicity, leading to weak/ delayed adaptive immune response [ , ] . the nk cytotoxic function in nano-kag received pigs was partially rescued (not significant), suggesting the beginning of appearance of nk cell cytotoxic function with a complete rescue in their frequency (comparable to mock pigs). in pigs vaccinated with nano-kag, virus induced immunosuppressive responses were dampened along with a significant boost in both innate and virus specific adaptive immune response. the cd t cell is an important innate immune cell at mucosal sites and they possess non-mhc class i cytolytic activity. pigs possess relatively large population of cd t cells compared to other species and they secrete ifn-c [ ] . cd t cell plays an important role in reducing the vaccinia virus load, and also in destruction of herpes simplex type infected cells [ ] . in nano-kag vaccinated pigs increased population of cd t cells, in addition to nk cells, cd + and cd + t cells, and increased secretion of ifn-c were detected at both mucosal and systemic sites. now and earlier we have demonstrated that both k-ag and mlv-prrs vaccinated and virus challenged, as well as unvaccinated prrsv infected pigs have immunosuppressed response mediated by increase in the population of tregs and secretion of il- and tgf-b, and reduced production of ifn-c [ , , ] . in contrast, in nano-kag immunized pig lungs, tbln, and blood significantly reduced tregs frequency, associated with decreased il- and tgf-b, and increased ifn-c secretion, compared to unvaccinated and k-ag vaccinated virus challenged pigs was observed. in a pre-challenge study, at two-week post-vaccination increased secretion of proinflammatory cytokine, il- , in nano-kag vaccinated pigs appears to be involved in initiation of adaptive immune response. diminished production of il- in post-challenged pigs at six-week post-vaccination was associated with reduced inflammatory lung pathology. mucosal immunization elicits production of iga antibodies and effector response at distant tissues [ ] . the iga antibody is protective against various viral infections and they possess significant virus neutralization activity at both mucosal surfaces and blood [ ] . in nano-kag immunized pigs increased levels of prrsv specific iga, igg in the lungs, blood, and nasal wash were observed. although, these prrsv specific antibodies did not significantly increase the neutralizing titers in the lungs, there was complete clearance of viremia at pc , which was associated with various other adaptive immune correlates. whereas in the lungs, the reduced viral titer was not significant, but a significant reduction of prrsv antigen by immunohistochemistry and reduced lung pathology implicates the presence of active anti-prrsv mucosal immune response elicited by nano-kag vaccine in the lungs (fig & ) . the most important finding from our study to swine farmers and researchers is that pigs vaccinated intranasally with nano-kag vaccine completely clear the prrsv viremia of a virulent heterologous virus by two-week post-challenge. further, in yet another nano-kag vaccine study we inoculated a booster dose of the vaccine and co-administered with a potent mucosal adjuvant, intranasally, showed the complete clearance of the replicating heterologous prrsv from the lungs and clearance of viremia earlier than the current study (binjawadagi et al., manuscript submitted). in conclusion, our study has suggested that innovative strategy of intranasal delivery of prrsv nano-kag vaccine has the potential to control prrs outbreaks, and it has the potential to reduce economic losses to swine producers. considering pig a useful large animal model, our study may serve as a useful impetus to undertake intranasal plga nanoparticle-based vaccine trials against human respiratory viral infections. each bar represents average values from three pigs sem. asterisk represents the statistical significant difference (p, . ) between nano-kag and k-ag received pig groups and q represents the statistical significant difference (p, . ) between unvaccinated and nano-kag received pig groups. a similar trend in results was obtained in an independent second trial performed using same number of animals. doi: . /journal.pone. .g controlled-release vaccines-biodegradable polylactide/polyglycolide (pl/pg) microspheres as antigen vehicles degradation of poly(d,l-lactic acid) microspheres effect of molecular weight a road less travelled: large animal models in immunological research the porcine lung as a potential model for cystic fibrosis prrs costs industry $ million annually nidovirales: a new order comprising coronaviridae and arteriviridae porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of threeweek-old colostrum-deprived pigs evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions mucosal immunity: implications for vaccine development the mucosal immune system: from fundamental concepts to vaccine development routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans pla and plga nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis b vaccine a novel bioadhesive intranasal delivery system for inactivated influenza vaccines single-dose mucosal immunization with biodegradable microparticles containing a schistosoma mansoni antigen strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective a modified live prrsv vaccine and the pathogenic parent strain induce regulatory t cells in pigs naturally infected with mycoplasma hyopneumoniae recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of cd + t cells effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection incorporation of microspheres into nerve guidance channels for drug delivery purposes. applied chemistry and chemical enginnering delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders intranasal m cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus an immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant induction of protective immune responses against venezuelan equine encephalitis (vee) virus aerosol challenge with microencapsulated vee virus vaccine immunogenicity of bovine parainfluenza type virus proteins encapsulated in nanoparticle vaccines, following intranasal administration to mice interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus dendritic cell progenitors phagocytose particulates, including bacillus calmette-guerin organisms, and sensitize mice to mycobacterial antigens in vivo biodegradable polymer microspheres as vaccine adjuvants and delivery systems differential effects of agarose and poly(lactic-coglycolic acid) on dendritic cell maturation differential levels of dendritic cell maturation on different biomaterials used in combination products cellular variables in bronchoalveolar lavage fluids (balf) in selected healthy pigs metal-rich ambient particles (particulate matter . ) cause airway inflammation in healthy subjects studies on chitosan: . lysozymic hydrolysis of partially nacetylated chitosans antigen presentation and t cell stimulation by dendritic cells alveolar macrophage priming by intravenous administration of chitin particles, polymers of n-acetyl-d-glucosamine, in mice role of lipid rafts in innate immunity and phagocytosis of polystyrene latex microspheres induction of hiv-specific antibody response and protection against vaginal shiv transmission by intranasal immunization with inactivated shiv-capturing nanospheres in macaques gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus alpha beta and gamma delta t-cell networks and their roles in natural resistance to viral infections intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs distribution of poliovirus antibody in serum, nasopharynx and alimentary tract following segmental immunization of lower alimentary tract with poliovaccine cross-protection in mice infected with influenza a virus by the respiratory route is correlated with local iga antibody rather than serum antibody or cytotoxic t cell reactivity we would like to thank ms. ruthi patterson for her help in animal studies and tissue processing, and mathew weeman, drs. mahesh khatri and juliette hanson for their help in animal studies. drs. michael murtaugh and eric nelson had provided the prrsv reagents. conceived and designed the experiments: vd gjr. performed the experiments: vd cm bb dj gjr. analyzed the data: vd cm gjr. wrote the paper: vd gjr. key: cord- -s ypy hf authors: wang, dang; fan, jinxiu; fang, liurong; luo, rui; ouyang, haiping; ouyang, chao; zhang, huan; chen, huanchun; li, kui; xiao, shaobo title: the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity date: - - journal: mol immunol doi: . /j.molimm. . . sha: doc_id: cord_uid: s ypy hf since its emergence in the late s, porcine reproductive and respiratory syndrome (prrs) has been devastating the swine industry worldwide. the causative agent is an arterivirus, referred to as prrs virus (prrsv). the pathogenic mechanisms of prrs are poorly understood, but are believed to correlate with the ability of prrsv to inhibit immune responses of the host. however, precisely how the virus is capable of doing so remains obscure. in this study, we showed that prrsv infection led to reduced ubiquitination of cellular proteins. screening all of the nonstructural proteins (nsps) encoded by prrsv revealed that, apart from the nsp which contains the deubiqintinating (dub) ovarian tumor (otu) domain, nsp , which encodes a unique and conserved endoribonuclease (nendou) throughout the nidovirus order, also possesses dub activity. in vivo assay demonstrated that nsp specifically removed lysine (k )-linked polyubiquitin chains and the conserved sites c , h , d , k , and y were critical for its dub activity. remarkably, dub activity was responsible for the capacity of nsp to inhibit nuclear factor κb (nf-κb) activation. mutations abrogating the dub activity of nsp toward k -linked polyubiquitin chains of iκbα nullified the suppressive effect on nf-κb. our data add nsp to the list of dubs encoded by prrsv and uncover a novel mechanism by which prrsv cripples host innate immune responses. protein ubiquitination is a reversible process that plays a vital role in nearly every aspect of cellular physiology, including protein degradation, protein trafficking, transcription, cell-cycle control, and cell signaling; (liu et al., ; pickart, ) . not surprisingly, ubiquitination is targeted for manipulation by a wide range of microbial pathogens (randow and lehner, ). in particular, many viruses have evolved elaborate strategies to inhibit or redirect the ubiquitination machinery of the host for their survival (viswanathan et al., ) . for example, human immunodeficiency virus (hiv- ) prevents antiviral interferon response via vpr-and vif-directed, ubiquitin-mediated proteosomal degradation of interferon regulatory factor (irf- ) (okumura et al., ) ; the papain-like protease (plpro) domains of many coronaviruses, such as severe acute respiratory syndrome (sars) coronavirus (sars-cov), human coronavirus nl (hcov-nl ), and mouse hepatitis virus a (mhv-a ), have deubiquitinating (dub) activity that blocks type i interferons (ifns) induction (barretto et al., ; chen et al., b; clementz et al., ; devaraj et al., ; frieman et al., ; lindner et al., ; zheng et al., ) ; the leader proteinase (l pro ) of foot-and-mouth virus (fmdv) acts as a deubiquitinase that cleaves ubiquitin chains from retinoic acid-inducible gene i (rig-i), tank-binding kinase (tbk ), tnf receptor associated factor (traf ), and traf , thereby inhibiting the activation of type i ifn signaling ; the n-terminal protease (npro) of bovine viral diarrhea virus interacts with irf- and promotes its polyubiquitination and subsequent degradation through the proteasome (chen et al., a) ; the latency associated protein orf of murid herpesvirus- (muhv- ) associates with the host ubiquitin-ligase complex to promote poly-ubiquitination and subsequent proteasomal degradation of p /rela, which inhibits the activity of nuclear factor b (nf-b) to facilitate the establishment of muhv- latency (rodrigues et al., ) . collectively, these previous findings reveal that hijacking of the cellular ubiquitin system is an emerging, central theme around virus replication. in this regard, studying uniquitination events in virus-infected cells holds great promise to unravel important modulators of the intricate relationship between host and pathogen. not less important, understanding the mechanisms by which viral products interact with the ubiquitin system provides novel insights into viral pathogenesis and informs approaches to antiviral drug development. porcine reproductive and respiratory syndrome (prrs) is a relatively new viral infectious disease of the swine (rossow, ) . it is characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs. since it was first reported in the united states in and in europe in , prrs has devastated the swine industry worldwide, causing tremendous economic losses. as such, prrs is now considered to be one of the most important diseases in countries with intensive swine industries (meulenberg, ; murtaugh et al., ; neumann et al., ) . the causative agent, prrs virus (prrsv), is a single-stranded positive-sense rna virus classified within the order nidovirales, family arteriviridae, which also includes equine arteritis virus (eav), murine lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv) (cavanagh, ) . at least nine open reading frames (orfs) have been identified in the prrsv genome. orf a and orf b, which are situated in the -proximal three quarters of the genome, encode the viral non-structural proteins (nsps): nsp ␣, nsp ␤, and nsp to nsp . orf a, orf b, and orf to orf are located at the end of the genome and encode the viral structural proteins gp , e, gp , gp , gp , m, and n, respectively. because the nsps constitute ∼ % of the prrsv genome coding capacity, much attention has been garnered in studying the functions and immuno-modulatory roles of prrsv nsps (dokland, ) . to date, several nsps, including nsp ␣/␤, nsp , nsp , and nsp have been reported to have inhibitory effect on activation of the ifn-␤ promoter (beura et al., ; kim et al., ; li et al., ; shi et al., ; song et al., ) . more recently, the cysteine protease domain (cp) of prrsv nsp was identified as a member of the ovarian tumor domain (otu) family of deubiqintinating (dub) enzymes. it was shown that the otu domain of prrsv nsp antagonizes the induction of type i ifns by interfering with the nf-b pathway . however, it remains unclear whether other nsps encoded by prrsv have dub activity. in the present study, we screened the nsps of prrsv and found both nsp and nsp possess dub activity. interestingly, nsp specifically targeted lysine (k )-linked but not k -linked ubiquitination chains for cleavage. this attenuated ligand-induced degradation of inhibitor of nf-b alpha (ib␣), thereby inhibiting the activation of nf-b. our data identify nsp as a second dub encoded by prrsv and describe a novel mechanism by which prrsv antagonizes innate immunity of the host. marc- cells, pk- -cd cells and hek cells were maintained in dulbecco's modified eagle media (dmem, invitrogen) supplemented with % heated-inactivated fetal calf serum (fbs), u/ml penicillin, g/ml streptomycin sulfate at • c in a humidified % co incubator. pk- -cd cells, a porcine kidney cell line stably expressing the receptor cd of prrsv, were gifts of dr. en-min zhou (northwest a&f university, china) (wang et al., ) . the wuh strain of prrsv (li et al., ) , which was isolated from the brains of pigs that contracted the "high fever" syndrome in china at the end of , was used in this study. prrsv was propagated in marc- cells or pk- -cd cells, and the supernatants of infected cells were clarified and stored at − • c in aliquots. poly(i:c) (sigma-aldrich) and tumor necrosis factor ␣ (tnf-␣) (sigma-aldrich) were also used to stimulate cells. full-length ha-tagged ubiquitin (ub) plasmid (ha-ub) and ha-ub mutants in which all but one lys residue (ha-k -ub or ha-k -ub) were substituted with arg were gifts of dr. t. ohta (st. marianna university school of medicine, japan) (nishikawa et al., ) . pcdna . -flag-ub was previously described (clementz et al., ) . the luciferase report plasmid pnf-b-luc was purchased from stratagene. the hemagglutinin (ha) or v epitope tag was amplified by pcr and cloned into the pcaggs-mcs vector (niwa et al., ) to generate pcaggs-ha or pcaggs-v plasmid with n-terminally ha or v tag, respectively. for construction of the mammalian expression plasmids encoding various nonstructural proteins of prrsv, cdna fragments encoding full-length nsp ␣, nsp ␤, nsp - , nsp - of prrsv strain wuh (genbank accession no. hm ) were amplified by pcr and inserted into the pcaggs-ha or pcaggs-v plasmid. c a, c a, h a, h a, k a, d a, d a, and y a mutants of nsp were generated by overlap extension pcr in the pcaggs-v -nsp backbone. detailed sequences of the mutagenesis primers are available upon request. all mutants were validated by dna sequencing. the mammalian expression plasmid for ib␣ was constructed by pcr amplifying the cdna of ib␣ (genbank accession no. nm ) from hek cells, followed by cloning into the pcmv-tag b vector (stratagene). the monoclonal antibody (mab) a f used for detection of prrsv nsp was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant nsp protein of prrsv strain wuh . the a f mab specifically recognized prrsv nsp in western blot and indirect immunofluorescence assays (unpublished data). the anti-beta-actin (biotechnology, china), anti-flag (macgene, china), anti-ha (mbl, japan), anti-v (mbl, japan), and anti-ubiquitin (santa cruz, ca) antibodies were used to detect the indicated proteins. horseradish peroxidase-conjugated anti-mouse or antirabbit igg antibodies were obtained from beyotime institute of biotechnology (jiangsu, china). marc- cells and pk- -cd cells were infected with prrsv strain wuh at different mois or sham-infected with dmem. at different time points post infection, cell lysates were collected and subjected to western blot analysis with an anti-ubiquitin antibody to measure the abundance of ubiquitinated proteins in the cell. hek cells grown in -well plates were co-transfected with . g/well of luciferase reporter plasmid pnf-b-luc along with . g/well of prl-tk plasmid (promega, for normalization of transfection efficiency) and various viral nsp-encoding plasmids or an empty control plasmid. in some experiments, cells were further transfected with poly(i:c) ( . g/well) at h after the initial co-transfection. cells were harvested h later and firefly luciferase and renilla luciferase activities were determined using the dual-luciferase reporter assay system (promega) according to the manufacturer's protocol. data represent relative firefly luciferase activity normalized to renilla luciferase activity and are representative of three independently conducted experiments. data are presented as means ± standard deviation (sd). a p-value of less than . was considered highly statistically significant. to determine the effect of nsp on expression of il- , il- , and ccl , hek cells in -well plates were transfected with g of empty vector or a plasmid encoding v -nsp . h later, cells were mock-transfected or transfected with g of poly(i:c) for h. total rna was extracted from the cells using trizol reagent (invitrogen, u.s.a.). one microgram of this total rna was reverse transcribed to cdna using amv reverse transcriptase (toyobo, japen), which ( l of l cdna) was subsequently used in a sybr green pcr assay (applied biosystems, u.s.a.). the abundance of individual mrna transcript in each sample was assayed three times and normalized to that of porcine glyceraldehyde- phosphate dehydrogenase (gapdh) mrna (as an internal control). the primers were designed by primer express software v. . (applied biosystems, u.s.a.). detailed sequences of the primers used are available upon request. hek cells cultured in -mm dishes were cotransfected with . g of ha-ub, ha-k -ub, or ha-k -ub plus appropriate amounts of vector encoding wild-type prrsv nsp or the indicated mutant using lipofectamine (invitrogen). where applicable, the empty vector was supplemented to keep the total amount of dna transfected constant. at h post transfection, cells were harvested by adding l lysis buffer a (lba) ( mm tris-hcl [ph . ], % sodium dodecyl sulfate, % dl-dithiothreitol, and % glycerol) containing mm n-ethylmaleimide (nem) (sigma) and mm iodoacetamine (sigma). cell lysates were then analyzed for expression of ubiquitin-conjugated proteins by western blot with an anti-ha antibody ( : , ) (mbl, japan). to verify the expression levels of nsp and the mutants, an anti-v antibody (mbl, japan) was used to detect the v -tagged proteins. beta-actin was immunoblotted with anti-beta-actin mab (beyotime, china) to demonstrate equal protein sample loading. transient transfection of hek cells with the indicated plasmids was performed routinely using lipofectamine as per the manufacturer's instructions (invitrogen). transfected hek cells from each -mm dish were lysed by adding l ml of lysis buffer ( mm tris-hcl [ph . ], mm nacl, % triton x- , nm phenylmethylsulfonyl fluoride [pmsf]), and the protein concentration was measured and adjusted. for immunoprecipitation, g of total cell lysates were incubated with . g of the indicated antibody and l of protein + g-agarose (beyotime, china) overnight at • c. the agarose beads were then washed three times with ml of lysis buffer. the immunoprecipitates were subjected to % sds-page and subsequent immunoblot analysis using the indicated antibodies. to investigate the levels of ubiquitinated cellular proteins during prrsv infection, we infected marc- cells (a clone of the african green monkey kidney cell line ma- ) with prrsv strain wuh at an multiplicity of infection (moi) of . . cell lysates were collected at different time points post infection and subjected to western blot analysis with anti-ubiquitin. as shown in fig. a , while the level of ubiquitinated cellular proteins was steady in mock-infected cells (lanes - ), it varied dynamically during the course of prrsv infection. a decrease in ubiquitination was first observed at h post infection (h.p.i.) (compare lanes vs. ), reaching a plateau phase between and h.p.i. (lanes - vs. ). although there was a slight rebound at h.p.i. (lane ), the level of ubiquitinated cellular proteins remained substantially lower than that of mock-infected cells (lane ). when marc- cells were infected with prrsv at increasing mois, the levels of ubiquitinated cellular proteins were reduced in a dose-dependent manner (fig. b) . importantly, infection with uv-inactivated prrsv, which is capable of receptor binding and internalization but not viral gene synthesis, did not alter the cellular level of ubiquitinated protein conjugates (fig. b) . similar results were observed in prrsvinfected pk- -cd cells (fig. c) . these data demonstrate that the level of ubiquitinated cellular proteins is reduced in prrsvinfected cells and that this is a result of active viral replication. of note, the latter notion was also supported by the western blot data that measured expression of prrsv nsp protein at different time points post infection (fig. a) . because the decreased cellular protein ubiquitination depended on prrsv replication and it has been shown that prrsv nsp has dub activity, we sought to determine whether other nsp(s) also contributes to protein deubiquitination. to this end, all of the nsps of prrsv strain wuh , except nsp (which encodes a very short peptide of amino acids), were cloned into a mammalian expression vector pcaggs-ha, such that they would be expressed as n-terminal ha-tagged fusion proteins. these nsp constructs were transiently transfected into cells and their expression were verified by western blot using an anti-ha antibody (data not shown). subsequently, each of these nsp constructs was co-transfected with a plasmid encoding flag-tagged ubiquitin (pcdna . -flag-ub) into hek cells (human embryonic kidney epithelial cells) and western blot was performed to detect the ubiquitin-conjugated proteins. as a negative control, the empty pcaggs-ha plasmid was used in place of the nsp-encoding vectors. as shown in fig. a , of the tested nsps, ectopic expression of nsp or nsp resulted in markedly reduced levels of ubiquitinconjugated proteins. notably, the effect of nsp appeared to be stronger than that of nsp (compare lanes vs. ). since, the dub activity of nsp had been characterized in previous studies, we focused on nsp in subsequent experiments. to further confirm the dub activity of nsp , hek cells were transfected with the empty vector or increasing amounts of v -tagged nsp expression plasmid along with ha-tagged ubiquitin vector (ha-ub) and the levels of ubiquitin-conjugated proteins were monitored at h post-transfection. as shown in fig. b , compared to the control vector transfected cells (lane ), the degree of deubiquitination directly correlated with the amount of nsp expressed (lanes - ). these data strongly suggest that nsp is directly responsible for the decrease in ubiquitinated cellular proteins. protein ubiquitination is an important posttranslational modification that has an essential role in the positive and negative regulation of nf-b signal transduction pathway, among different ubiquitination types k -and k -linked polyubiquitin chains are of great significance (wertz and dixit, ) . to further identify which ub linkage type is targeted by nsp , hek cells were transfected with ha-k -ub or ha-k -ub in place of ha-ub. these constructs allow solely the formation of k -or k -linked polyubiquitin chains, respectively. as shown in fig. c and d, while the accumulation of k -linked ubiquitinated proteins was reduced by nsp in a dose-dependent manner (fig. c) , k linked ub moieties were left intact upon nsp coexpression (fig. d) . these data indicate that prrsv nsp specifically targets k -linked polyubiquitin chains. . . the c residue is a potential catalytic site for nsp dub activity there are five families of dubs characterized by specific structural domains: ubiquitin c-terminal hydrolases (uchs), ubiquitin-specific proteases (usps), ovarian tumor proteases (otus), josephins and jab /mov metalloenzymes (jamms). uchs, usps, otus and josephins function as cysteine proteases, whereas jamms are zinc dependent metalloproteases. based on the structure of their catalytic domains, the human dubs are classified into five subfamilies, most of which are cysteine proteases characterized by a cysteine (cys, c) active site located within the amino terminus (nijman et al., ; wilkinson et al., ) . to identify the potential cysteine catalytic site for nsp dub activity, the amino acid sequences of nsp from various prrsv strains were aligned using the clustal w program. sequence alignment showed that cys of nsp is highly conserved across different genotypes of prrsv. however, cys is only identical among type genotype prrsv (fig. a) . to determine whether these two cysteines are critical residues involved in the dub activity of nsp of prrsv wuh strain, we constructed c a and c a nsp mutants and compared them with wt nsp for the ability to reduce protein ubiquitination. as shown in fig. b , overexpression of wt nsp or the c a mutant significantly inhibited k -linked ubiquitination of cellular proteins (compare lanes and vs. , respectively). in contrast, the c a mutant had no such effect (compare lanes vs. ). these data suggest that the c residue is pivotal for the dub activity of prrsv nsp . . . residues d , k , d and y are also associated with the dub activity of nsp prrsv nsp encodes an endoribonuclease (nendou), which is conserved throughout the nidovirales order but has not been identified in rna viruses of other families (ivanov et al., ; nedialkova et al., ) . nedialkova et al. ( ) compared the amino acid sequences of the nendou domain of arterivirus (in nsp ) and its counterpart in nsp of sars-cov and found that at least residues, corresponding to h , h , k , d , d , and y in prrsv nsp , are highly conserved among prrsv strain vr , sars-cov strain frankfurt , and eav strain bucyrus (nedialkova et al., ) . considering that prrsv is divided into distinct genotypes and even exhibits remarkable genetic diversity within each genotype, we compared the amino acid sequences of nsp of several representative prrsv strains isolated from different geographical regions in different years. as shown in fig. a , the six residues reported by nedialkova et al. ( ) are indeed highly conserved among different genotypes of prrsv and eav. to determine whether these residues contribute to the dub activity of nsp , we performed alanine substitution at each site. each of these mutants was co-transfected with ha-ub vector into hek cells and western blot was performed to detect the expression of ubiquitin-conjugated proteins and the nsp mutant. as shown in fig. c , while mutants bearing the h a or d a substitution (lanes and , respectively) acted as effective as wt nsp (lane ) in preventing the accumulation of ubiquitination of cellular proteins, mutants harboring h a, k a ory a substitution remarkably lost the dub activity (lane , and ), as compared to vector-transfected cells (lane ). intermediate dub activity was observed for the d a mutant (lane ). collectively, these data suggest that residues h , k , d and y are also associated with the dub activity of nsp . given that mutation at h or d destroys the nendou activity (nedialkova et al., ) , our data also indicate that the dub activity of nsp is uncoupled from its nendou activity. ␤, , , , , , , , , , ) or empty vector ( . g). cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-flag antibody. western blot with anti-beta-actin serves as a protein loading control. (b) hek cells grown in -mm dishes were transfected with ha-tagged ub expression plasmids ( . g), along with increasing quantities ( , . , . , . , or . g) of plasmid encoding v -nsp , using lipofectamine . cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-ha antibody. western blot with anti-v antibody shows expression of nsp , and western blot for beta-actin serves as a protein loading control. (c-d) the experiments were performed similarly to those in panel b, except that the ha-k -ub or ha-k -ub plasmid was used in lieu of ha-ub. ubiquitination is an important regulatory mechanism of nf-b signaling (wertz and dixit, ) . the discovery that nsp has dub activity would imply that nsp may affect nf-b activity. indeed, it has been shown that ectopic expression of prrsv nsp inhibits nf-b activation through unknown mechanism(s). in agreement with these previous reports (beura et al., ) , our luciferase reporter assays showed that nsp down-regulated poly(i:c)-induced activation of a synthetic nf-b-dependent promoter in a dose-dependent fashion (fig. a) . we next asked the question whether nsp also attenuates expression of poly(i:c)induced, endogenous nf-b-responsive genes. to this end, hek cells were transfected with a plasmid encoding v -nsp or the empty control vector. twenty-four hours post-transfection, cells were further transfected with poly(i:c) or mock-transfected. total rna was extracted from the cells and analyzed for the abundance of endogenous interleukin- (il- ), il- and chemokine (c-c motif) ligand (ccl , also known as rantes), mrnas by sybr green real-time rt-pcr. while poly(i:c) robustly induced the expression of il- ( fig. b) , il- (fig. c) , and ccl (fig. d) in cells transfected with control vector, it was substantially less effective in cells expressing nsp . these results show that nsp negatively regulates the induction of nf-b-target genes following stimulation by intracellular dsrna. generally, attachment of k- linked ub chains promotes the degradation of a protein by the ub-proteasome system (ups), one g) , along with the indicated nsp expression plasmids ( . g). cell lysates were prepared at h posttransfection and analyzed for ub-conjugated proteins by western blot with an anti-ha antibody. western blot with anti-v antibody shows expression of nsp , and western blot for beta-actin serves as a protein loading control. of the most important machineries for non-lysosomal degradation of cytoplasmic and nuclear proteins, while modification by k linked ubiquitination mediates largely non-proteolytic functions such as protein trafficking or kinase and phosphatase activation (ikeda and dikic, ) . a key step that leads to nf-b activation in response to many extracellular stimuli is the degradation of ib␣, which sequesters nf-b proteins in the cytoplasm in resting cells (wertz and dixit, ) . because prrsv nsp selectively removes k -linked ubiquitin moieties (fig. c) , we hypothesized that it may inhibit nf-b activation by interfering with the ubiquitination of ib␣ and subsequent proteosomal degradation. to test our hypothesis, hek cells were transfected with an nsp -encoding vector or the empty vector (as a control), followed by stimulation by tnf-␣. at and min post-stimulation, cell lysates were collected to detect ib␣ protein by western blot. as shown in fig. a and b, tnf-␣-induced degradation of ib␣ was significantly attenuated in cells expressing nsp , compared to cells transfected with the empty vector, indicating that nsp inhibits ligand-induced (a) hek cells grown in -well plates were transfected with . g/well of × nf-b-luc reporter plasmid, along with . g/well of prl-tk plasmid and increasing quantities ( , . , . , or . g) of plasmid encoding v -nsp , using lipofectamine . twenty-four hours after the initial transfection, the cells were further treated with poly(i:c) or mock treated. luciferase assays were performed at h after infection. (b-d) hek cells were transfected with g of plasmid encoding v -nsp or an empty vector, and, h later, the cells were transfected with g of poly(i:c). twenty-four hours after the second transfection, total rna was extracted and the expression of il- (b), il- (c) and ccl (d) and gapdh genes were evaluated by quantitative real-time rt-pcr. results are expressed as increases in mrna levels relative to those in cells transfected in the absence of poly(i:c) and were normalized by using gapdh housekeeping gene expression. results are representative of those from three independent experiments. ib␣ degradation. to further test whether nsp removes k -ub from ib␣, hek cells were transiently co-transfected with an nsp expression construct, ha-k -ub, and an ib␣-encoding vector. at h post transfection, mg (a proteasome inhibitor) was added for h to retain ubiquitinated ib␣. cell lysates were (a) hek cells in -mm dishes were cotransfected with the expression plasmids encoding ha-nsp or the pcaggs-ha (vector) empty plasmid ( g). transfected cells were treated with tnf-␣ ( ng/ml) for the amounts of time ( min, min) and subsequently immunoblotted. western blots were analyzed for total ib␣. the beta-actin was detected as a loading control. (b) relative levels of ib␣ were estimated by densitometric scanning after normalization against beta-actin and are shown as bar diagrams. data represent means of three replicates. (c) hek cells grown in -mm dishes were transfected with the expression plasmids encoding v -nsp ( . g), ha-k -ub ( g), pcmv-tag-ib␣ ( . g) using lipofectamine . mg ( nm) was treated at h after transfection. cell lysates were prepared at h after treatment and immunoprecipitated with anti-flag antibody and ubiquitin conjugation of protein was verified by immunoblotting with anti-ha antibody. the input tagged proteins were verified with indicated antibodies. then collected for co-ip assay. as shown in fig. c , ib␣ was conjugated with k -ub in cells without nsp coexpression (lane ). however, in cells expressing nsp , there were very few k -ub moieties attached to ib␣, despite the latter being expressed at comparable levels (lane ). in aggregate, these results suggest that nsp inhibits nf-b through removing k -linked polyubiquitin chains from ib␣ and thus preventing subsequent ib␣ degradation. g) , and the designated nsp expression plasmids ( . g), using lipofectamine . twenty-four hours after the initial transfection, the cells were further treated with poly(i:c) or mock treated. luciferase assays were performed at h after treatment. (b-c) hek cells in -mm dishes were transfected with the expression plasmids encoding v -nsp and mutants ( . g), ha-k -ub ( g), pcmv-tag-ib␣ ( . g), using lipofectamine . mg ( nm) was treated at h after transfection. cell lysates were prepared at h after treatment and immunoprecipitated with anti-flag antibody and ubiquitin conjugation of protein was verified by immunoblotting with anti-ha antibody. the input tagged proteins were verified with indicated antibodies. to delineate the molecular determinants of nsp involved in nf-b inhibition, various nsp mutants with differing dub activities ( fig. b and c) were analyzed for their abilities to affect poly(i:c)-induced nf-b activation in hek cells by lucifease reporter gene assay. as shown in fig. a , the results showed that mutants with impaired dub activity were attenuated for the ability to suppress nf-b activation. notably, the c a, h a and d a mutants with dub activities comparable to that of wt nsp were as effective as the latter in blocking activation of nf-b, while the c a, h a, k a and y a mutants with little or no dub activity were without inhibitory effect. the d a mutant possessing intermediate dub activity had a moderate inhibition on nf-b. taken together, these data show that dub activity is proportional to the suppressive effect on nf-b activation. next, we determined the abilities of these nsp mutants to remove k linked polyubiquitin chains from ib␣. as shown in fig. b and c, the dub activity toward polyubiquitinated ib␣ of the individual nsp mutant directly correlated with the capacity to inhibit nf-b activation. collectively, these results provide further support to the notion that prrsv nsp inhibits nf-b activation by means of its dub activity toward polyubiquitinated ib␣. in this study, we attempted to elucidate the mechanism(s) employed by prrsv to reduce cellular protein polyubiquitination and accidently found that prrsv nsp possesses dub activity. we also demonstrated that nsp specifically cleave k -linked, but not k -linked polyubiquitin chains. importantly, our data showed the dub activity is responsible for the ability of nsp to inhibit nf-b activation, revealing a novel mechanism evolved by prrsv to disarm host innate immune responses. at present, we favor a model in which nsp removes k -linked ubiquitin moieties from ib␣, thereby preventing the proteasomal degradation of ib␣ and subsequent liberation of nf-b. this is based on our data that the inhibitory effects of nsp mutants on nf-b correlated with their abilities to deubiquitinate ib␣ conjugated with k -linked ubiquitin chains. however, our study do not rule out the possibility nsp may also act on other cellular targets to regulate nf-b activity. previous studies have revealed that prrsv nsp contains endoribonuclease (nendou) activity (nedialkova et al., ) . although, nendou is highly conserved throughout the nidovirales order, thus far no nendou homologs have been identified in rna viruses of other families. in this regard, nendou is considered to be a genetic marker of nidoviruses (ivanov et al., ) . adopting a catalytic mechanism resembling that of rnase a, nendou acts independent of mn + and with a preference for uridylate, as has been demonstrated for nendous encoded by eav, sars-cov and prrsv (nedialkova et al., ) . through as-yet-unknown mechanisms, nendou facilitates viral rna synthesis (kang et al., ; posthuma et al., ) . in this study, we demonstrated that prrsv nsp also has dub activity, ascribing a novel function to this enigmatic protein. in addition, our mutational analyses suggested that the dub activity of nsp is separated from its nendou activity and that the former rather than the latter mediates the suppressive effect on nf-b. it will be interesting to investigate in future studies whether the nsp counterparts encoded by other nidoviruses also possess dub and nf-b-inhibitory activities and whether these relate to the nendou activity. lee et al. ( ) firstly demonstrated that prrsv infection activates nf-b signaling in marc- cells and pams through inducing ib degradation and p nuclear translocation. our group also showed that prrsv infection triggers activation of nf-b and that the nucleocapsid (n) protein of prrsv could elicit this process in marc- cells luo et al., ) . however, activated nf-b could only be detected after h post infection. given the recent reports that several prrsv nsps, including nsp ␣, nsp ␤, nsp , and nsp , could function as negative regulators of nf-b (beura et al., ; song et al., ; sun et al., ) , it is plausible that the regulation of nf-b is a dynamic process during the course of prrsv infection, and that prrsv has developed sophisticated strategies to either activate or inhibit nf-b at various stages of the viral life cycle to facilitate viral replication and/or disrupting host innate immune responses, offering the virus a maximal survival advantage. previous studies have shown that the nsp s of prrsv strains fl and bj- inhibited ifn-␤ production in hela and marc- cells (beura et al., ; shi et al., ) . we also found that the nsp encoded by the highly pathogenic wuh strain, which is prevalent in china and surrounding countries, could inhibit activation of irf- and ifn-␤ transcription in hek cells by luciferase reporter assays (data no shown). to our surprise, all of the nsp mutants characterized in the current study were impaired for the ability to block sev-induced ifn-␤ and irf- activation (data no shown). this result contrasted with the effects on nf-b, in that only the nsp mutants losing the dub activity were attenuated (fig. ) . thus, while the dub activity of nsp mediates the inhibition on nf-b activation, other mechanisms are involved in the nsp blockage of ifn-␤ production and irf- activation. apart from regulating innate immune signaling, ubiquitination has been demonstrated to orchestrate aspects of viral life cycle (isaacson and ploegh, ; randow and lehner, ) . for instance, the assembly and release of some viruses are subjected to ubiquitination-dependent regulation. a number of viral proteins, such as hiv gag (demirov et al., ; garrus et al., ) , ebola virus vp (yasuda et al., ) , epstein-barr virus (ebv) lmp a (ikeda et al., ) , and human papillomavirus (hpv) e (kamio et al., ; oh et al., ) , can be directly modified by ubiquitin or ubiquitin-like proteins, and these modifications play an important role in virion egress. while the precise role(s) of ubiquitination and/or deubiquitination in prrsv infection remains to be established, the findings that prrsv encodes at least two dubs, i.e., nsp and nsp , signify that these posttranslational modifications may play an important part in prrsv propagation and/or pathogenesis. for example, it is possible that nsp may cleave k -linked polyubiquitin chains attached to viral replicase proteins and/or structural components to control the half-lives of these viral products, providing a fine-tuned condition that allows viral replication and/or packaging/release at maximal efficiency. considering the fact that the papain-like proteases of several coronaviruses (sars-cov, nl and mhv) also possess dub activity (barretto et al., ; barretto et al., b; lindner et al., ; zheng et al., ) , the involvement of ubiquitination-dependent regulation in viral life cycle may represent a selective survival advantage during the evolution of nidoviruses. in summary, the present study uncovered that prrsv nsp possesses dub activity and went extra miles to show that dub activity correlates with the ability of nsp to inhibit nf-b through removing k -linked polyubiquitin chains from ib␣. not only do these findings describe a novel mechanism acquired by prrsv to counteract host innate immune responses, but they open new avenues to develop novel therapeutics against prrs by targeting the interaction of nsp with host ubiquitination system. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation nidovirales: a new order comprising coronaviridae and arteriviridae ubiquitination and proteasomal degradation of interferon regulatory factor- induced by npro from a cytopathic bovine viral diarrhea virus proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases overexpression of the n-terminal domain of tsg inhibits hiv- budding by blocking late domain function regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the structural biology of prrsv severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-kappab signaling tsg and the vacuolar protein sorting pathway are essential for hiv- budding protein modifications: beyond the usual suspects' review series the epstein-barr virus latent membrane protein a py motif recruits ww domain-containing ubiquitin-protein ligases ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection major genetic marker of nidoviruses encodes a replicative endoribonuclease socs [corrected] inhibits hpv-e -mediated transformation by inducing degradation of e protein biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus gp protein encoded by a synthetic orf gene the cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein antagonizes interferon regulatory factor activation the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme immunity by ubiquitylation: a reversible process of modification activation of nf-kappab by nucleocapsid protein of the porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway prrsv, the virus the ever-expanding diversity of porcine reproductive and respiratory syndrome virus biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states a genomic and functional inventory of deubiquitinating enzymes mass spectrometric and mutational analyses reveal lys- -linked polyubiquitin chains catalyzed by brca -bard ubiquitin ligase efficient selection for high-expression transfectants with a novel eukaryotic vector the papillomavirus e oncoprotein is ubiquitinated by ubch and cullin -and skp -containing e ligase hiv- accessory proteins vpr and vif modulate antiviral response by targeting irf- for degradation mechanisms underlying ubiquitination site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle viral avoidance and exploitation of the ubiquitin system termination of nf-kappab activity through a gammaherpesvirus protein that assembles an ec s ubiquitin-ligase porcine reproductive and respiratory syndrome endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction nonstructural protein alpha subunit-based inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions viral hijacking of the host ubiquitin system to evade interferon responses the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase pk- cells transfected with porcine cd by piggybac transposon system are susceptible to porcine reproductive and respiratory syndrome virus signaling to nf-kappab: regulation by ubiquitination metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase t nedd regulates egress of ebola virus-like particles from host cells plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we thank dr. t. ohta for providing expression constructs. this work was supported by the national science fund for distinguished young scholars of china ( ), the national basic research program ( ) of china ( cb ), the national natural sciences foundation of china ( , ), and the key grant project of chinese ministry of education ( ). key: cord- -rodqdtfj authors: montaner-tarbes, sergio; del portillo, hernando a.; montoya, maría; fraile, lorenzo title: key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (prrsv) date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: rodqdtfj the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from to % in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from to % in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. keywords: porcine reproductive and respiratory syndrome virus, prrsv, virus biology, immunology, vaccinology, extracellular vesicles economic impact prrsv is responsible for respiratory disease in weaned and growing pigs, as well as reproductive failures in sows. it is considered one of the most important swine diseases worldwide, with an economic impact estimated at $ million in losses every year to u.s. producers, representing an increase of . % in the last years ( , ) . in europe, the situation is similar and economic disease models have been carried out to determine the economic burden in the best and worst case scenario combining reproductive failure and respiratory disease, estimating annual losses from a median of e , , if the farm was slightly affected during nursing and fattening, to a median of e , if a farm of , sows is severely affected in all productive phases ( ) . nevertheless, there is scarce of information about the economic impact of this disease as a consequence of multiple factors (vaccination, treatment, respiratory symptoms, reproductive failure, and other prrsv-related diseases) making a difficult task to quantify exactly this parameter under field conditions. thus, the exact economic impact of prrsv remains a key gap in the knowledge for this disease. the porcine reproductive and respiratory syndrome virus (prrsv) was first isolated in the early s in europe and north america ( , ) . it is an enveloped single-stranded positivesense rna virus of the family arteriviridae, genus porarterivirus according to the international committee of taxonomy of viruses ( ) . presently, there are four distinct species included in this genus (porarterivirus), prrsv- and prrsv- (with - % variation in nucleotide sequences), along with other two viruses that do not affect pigs (lactate dehydrogenaseelevating virus and rat arterivirus ) ( ) . the genome size of prrsv is about kb with open reading frames (orfs), with replicase genes located at the ′ -end followed by the genes encoding structural proteins toward the ′ -end ( ) . the majority of the genome (∼ - %) encodes non-structural figure | genome structure and mature viral particle of prrsv virus. (a) non-structural proteins are located in the ′ end of the genome, codifying for two different polyproteins pp a and pp ab that are cleaved into at least nsps (nsp to nsp and nsp α and nsp β, and nsp α, and nsp β). structural proteins located near the ′ end, are associated to the viral envelope and rna packaging. (b) prrsv mature viral particle, composed of a lipid bilayer envelop with viral receptor glycoproteins involved on infection and cell internalization. single stranded positive rna is associated with nucleocapsid protein in the internal layer of the virus. proteins involved in replication (orf a and orf ab), whereas orfs - encodes structural proteins (n, m, gp -gp , e) ( figures a,b) ( ) . using orf in molecular epidemiological studies, an enormous genetic variability has been described ( ) . yet, data on whole genome sequencing is scarce and constitute another important gap in the knowledge of this virus and its evolution (box ). prrsv replicase genes consist of two orfs, orf a and orf b, which occupy the ′ proximal three-quarters of the genome ( figure a) . both are expressed from the viral genome, with expression of orf b depending on a conserved ribosomal frameshifting mechanism. subsequently, extensive proteolytic processing of the resulting pp a and pp ab polyproteins yields at least functional non-structural proteins (nsps), specifically nsp to nsp , with both the nsp and nsp parts being subject to internal cleavage (giving origin to nsp α and nsp β, and nsp α, and nsp β, respectively), most of which assemble into a membrane-associated replication and transcription complex ( ) . recently, a programmed ribosomal frameshift encoding an alternative orf that generates two extra proteins, nsp tf and nsp n, was discovered in prrsv and other arteriviruses ( , ). these nsps, described for prrsv, have proven to box | gaps in knowledge in prrsv. be necessary and sufficient for the induction of membrane modifications resembling those found in infected cells ( ) . most importantly, all positive rna viruses seem to induce one of two basic morphotypes of membrane modifications: invaginations or double-membrane vesicles. prrsv also has a set of structural proteins, including a small non-glycosylated protein and a set of glycosylated ones: gp ab, gp , gp , gp , and gp a, m and n proteins ( ) . however, nsp , traditionally classified as a non-structural protein, has been found to be incorporated in multiple isoforms within the viral envelope (ovarian tumor domain protease region, hypervariable region and c-terminal region) ( ) , giving new insights into the structure of this virus ( figure b) . first, the nucleocapsid protein (n), as one of the most important parts of the mature viral particle, has been deeply characterized on prrsv, finding important features shared in most nonsegmented rna viruses. the n protein consists of amino acids for genotype and amino acids for genotype . the viral envelope glycoproteins (gp to gp ) are the first interactors with host cell receptors to initiate infection and are exposed to the immune system when viral particles are in blood and lymphoid tissue circulation (figure ). there is also another protein that contribute to virion structure, m protein, that is required during viral entry to interact with heparan sulfate cell receptor on macrophages. later, gp is thought to bind to sialoadhesin and virus internalization and uncoating is triggered by a formation of a viral heterotrimer (gp a, gp , and gp ) with scavenger receptor cd (figure ) ( , ) . gp is the most abundant glycoprotein. first, it interacts with two cell entry mediators, heparan sulfate glycosaminoglycans and sialoadhesin/cd ( , ) to favor viral entry and then possibly with the n protein and its mhc-like domain to carry n-viral rna complex to the budding site (figure ) . gp , gp , and gp are protected with glycan shields, like most prrsv membrane proteins, to avoid antibody recognition and neutralization. gp has two glycosylation sites, gp have seven and gp have four, figure | interactions between viral proteins and cell receptors for virus attachment, entry, uncoating and release of genetic ssrna to cell cytoplasm. blocking cd , cd tetraspanin or vimentin seems to inhibit viral replication or infection in the host cell, but reduced replication or no effect is seen when receptors such as heparan-sulfate or siglec- are blocked, demonstrating that some viral proteins and cell receptors are indispensable in terms of production of infectious viral progeny and dissemination in the host. three of which are directly related to virus survival, causing lethal damage in virus production when more than two of these sites are mutated ( ) (figure ). viral replication starts by interaction of viral glycoproteins with different cellular receptors (figure ) ( ) . cd and cd play a main role during infection, uncoating of the viral particle, activation of clathrin-mediated endocytosis and release of viral genome in the cytoplasm ( ) . cd has been defined as the main receptor for viral infection by evaluating the effect of prrsv on cd knockout pigs, where there is complete resistance to infection ( ) . cysteine-rich domain in this receptor seems to be necessary to establish interactions with prrsv- species, since its deletion by crispr/cas system (exon of the gene encoding this region) implies protection for a large panel of these viruses demonstrated by in vitro challenge of edited-pig macrophages and in vivo experiments with srcr animals ( ) ( ) ( ) . more important, edited pigs show no side effects when kept under standard husbandry conditions and cd seems to maintain its biological function (hemoglobin-haptoglobin scavenger) regardless the lacking cysteine-rich domain, nevertheless, other unknown functions could be impaired by this modification. in conclusion, gene-edited pigs lacking srcr region of cd could be an important asset to confront prrsv epidemics with the final goal of eradication. cd seems to be related only to co-interactions with sialic acid in the virion surface, however, knockout pigs for either exon , , or of cd were not protected from infection and viral load as well as antibody responses were similar to heterozygous (cd +/− ) or wild type pigs (cd +/+ ) ( ) . the former experiments suggested that other unknown mechanisms could be involved in prrsv infection such as other receptors, new unknown susceptible cell types different from macrophages or possible leaking of cd expression in the knockout model. other molecules are also involved in viral entry, such as cd ( ) and vimentin ( ) ; blocking of any of these four molecules (cd , cd , cd , and vimentin) had an effect on viral infection, either on internalization or complete inhibition of viral replication ( ) . after cell entry, prrsv causes a series of intracellular modifications to complete its replication cycle, which includes rearrangements of intracellular membrane organelles to generate the replication complex. these include the formation of perinuclear double membrane vesicles apparently derived from endoplasmic reticulum, synthesis of genomic rna (grna), transcription of segmented rna (sgrna) and expression of viral proteins ( , ) . at late stages of replication, the mature virions accumulate in the intracellular membrane compartments and they are then released into the extracellular space through exocytosis ( ) . a non-classical spread pathway has been detected in several viruses including prrsv where virus dissemination is mediated by cell to cell nanotubules ( ) . it was reported that almost all prrsv proteins interact with myosin and actin (especially f-actin and myosin iia) where nanotubules connected cells allowing the movement of structural proteins and rna, infecting naïve cells in a non-classical way even in the presence of neutralizing antibodies in the cell media. in addition, this non-classical pathway demonstrated that prrsv cell entry receptors were not necessary to establish infection, as nonpermissive cells became infected when were contacted by infected cells via nanotubes. this spreading strategy has been proposed as a mechanism to facilitate infection either by surfing of viral particles between adjacent cell membranes or as a receptor-independent mechanism for infection ( ) ; importantly, has been reported for other viruses such as hiv- where nanotube number on macrophages increases after infection ( ) and herpesvirus transmission between bovine fibroblasts ( ) . interestingly, although several viral proteins were detected in nanotubules (nsp β, nsp , nsp tf, nsp , nsp , and nsp , gp and n), gp was detected in only a few nanotubes. in particular, the role of gp in this nonclassical spread pathway is not fully understood and it will be interesting to further evaluate gp interaction with other cellular components to elucidate the reason why gp is not transported to new recipient naïve cells. altogether these data indicate that prrsv has evolved different pathways to spread even though, in vivo, the virus shows narrow cell tropism for monocytes and macrophages ( , ) (box ). the innate immune response is the first system any given pathogen encounters, specially to prevent viral replication and invasion into mucosal tissues (respiratory tract in the case of prrsv) and, importantly, to initiate the strong adaptive immune response to fight against intracellular infectious agents ( ). type i interferons (ifn α/β) comprise one of the most potent mechanisms against invading viruses in the first stages of infection, triggering an array of ifn-stimulated genes (isg) ( ) . generally speaking, all nucleated cells have the ability to produce ifn α/β, but plasmacytoid dc (pdc) are the most potent producers of this family of cytokines ( ) . prrsv has evolved a set of mechanisms for suppressing ifn α/β in vivo, maintaining low expression levels of this cytokines on infected pigs ( ) during almost all time-course of infection shortly after transient elevation in the lungs ( ) . suppression of ifn α/β also takes place in vitro in prrsv infected marc- and porcine alveolar macrophages ( , , ) . further studies have shown that ifn type i suppression is a major strategy of prrsv to modulate host antiviral defense. in fact, several viral proteins have been identified as ifn antagonists (nsp α, nsp β, nsp , nsp , nsp , and n) ( , ( ) ( ) ( ) . as an example for n protein, upon dsrna stimulation, ifn-β production was shown to decrease proportionally with increasing levels of n expression and additionally it was found to downregulate ifn-dependent gene production by dsrna interfering with dsrna-induced phosphorylation and nuclear translocation of irf ( ) . among prrsv non-structural proteins with type i ifn modulation capacity, nsp has been considered as the strongest antagonist of ifn-β production by acting on interferon regulatory factor (irf ) phosphorylation and nuclear translocation. almost all nsps, excepting nsp , have been related to the perinuclear region, associated with intracellular membranes, supposedly derived from the endoplasmic reticulum (er), which are modified into vesicular double-membrane structures with which the viral replication and transcription complex (rtc) is thought to be associated with ( , , ) . nsp translocates to the nucleus during the first hours of infection, where it is capable of inhibiting irf association with creb-binding protein (cbp), promoting cbp degradation by a proteasome-dependent mechanism, without which the transcription enhanceosome may not assemble the transcription machinery for the interferon expression ( , ) . recently, post-transcription protein expression of ifn β was shown to be regulated by prrsv by means of upregulating cellular mirna in porcine alveolar macrophages ( ) nsp is the largest (mature) prrsv protein and contains at least four distinct domains: the n-terminal cp/otu domain, a central hypervariable region, a putative transmembrane domain, and a c-terminal region of unknown function that is rich in conserved cysteine residues. this protein is unique in the context of prrsv due to its genetic heterogeneity, its participation in diverse roles supporting the viral replication cycle, and its packaging within the prrsv virion ( , ) . previous studies suggest that nsp has different roles related to immune evasion mechanisms. it has been determined that nsp otu domain (thiol-dependent deubiquitinating domain) inhibits the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) by interfering with the polyubiquitination process of ikbα (nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor) and, subsequently, preventing the degradation of the ikbα protein ( ) . moreover, viable deletion mutants in nsp , when infecting cells, caused a downregulation of cytokines (il- β and tnf-α) mrna expression, in comparison with that of parental virus, suggesting that certain regions of nsp might contribute to the induction of a virus-specific host immune response and that deletion of such a region could produce a more virulent virus ( ) . there are several isoforms of nsp , sharing a consistent core set between viral strains, which are integrated into mature virion at the final stage of replication (figure b) , although some of them could be strain-specific. inclusion of nsp within the prrsv virion suggests that it may function in previously unknown roles related to extracellular function, entry, or immediate-early viral replication events ( ) . truncated forms of nsp have also been identified, named nsp tf and nsp n, with apparent roles in modulation of immune evasion. when deletion mutants for those forms were used to infect cells, there was a significant change in gene expression, a strong activation of those involved in cytokine-cytokine receptor interaction, tnf signaling, toll-like receptor signaling, nodlike receptor signaling, nf-κb signaling, rig-i-like receptor signaling, chemokine signaling, jak-stat signaling, cytosolic dna-sensing, and nk cell mediated cytotoxicity ( ) , suggesting that an active role (direct or indirect) is played by these truncated forms in modulating host cells innate immune response, making prrsv infectious cycle more complicated than it was initially thought. nsp , is a nidovirus conserved endoribonuclease with an uridylate-specific endonuclease (nendou). it has been demonstrated in vitro that overexpression of nsp enhanced viral titter ( ) . moreover, nsp antagonizes type i ifn, specifically ifnβ production, activated by the retinoic acid inducible gene like receptor, showing substrate specificity toward mitochondrial antiviral signaling proteins (mavs) and rig-i (transcripts and proteins), and demonstrating that this activity was associated to the endoribonuclease activity of this protein in which transfection mutant viruses were unable to degrade mavs mrna and impair ifnβ production ( ) . another mechanism whereby this protein limits antiviral response is related to inflammasome and synthesis of il- β, due to its important role in both the innate and adaptive immune response and in pathological mechanisms. it has been shown that prrsv could activate nlrp inflammasome in early stages of infection but induce host's immunosuppression later as measured by determining the levels of pro-il- β and procaspase- mrna and the mature il- β protein in porcine alveolar macrophages (pam) ( ) . it is not surprising that nsp also interacts with the rna-silencing complex (risc), as it has been demonstrated in vitro in a marc- cell line that this protein and nsp α are responsible for inhibiting risc and downregulating argonaute- protein expression increasing viral titter significantly, which demonstrates a direct relationship between this silencing complex and viral replication at least in vitro ( ) . other non-structural proteins have been studied but there is an important gap on information about in vivo and in vitro functions and interaction in signaling pathways. additionally, the enormous variation among strains makes it difficult to characterize all protein variants and interactions with cell systems (macrophages, dendritic cells "dcs, " monocytes and others) (box ) . recently, a body of evidence associates host genetics with different outcomes following prrsv infection in the respiratory and reproductive form of the disease ( ) ( ) ( ) ( ) ( ) . although pathways and mechanisms involved in specific disease-resistance traits have not yet been fully characterized, it is clear that the genetic variation in disease resilience is polygenic, regulating aspects of both innate resistance and acquired immunity ( ) . in connection with innate response, the average daily gain (adg) after prrsv infection was associated with a single genomic region in chromosome (ssc ) which is best represented by the snp tag marker wur, located in the ′ non-coding region of the interferon-inducible guanylate-binding protein (gbp ) gene ( ) . the pig genetic resistance to prrsv infection has been historically overlooked in prrsv research probably generating a confounding factor in immune response studies. a key gap in the knowledge of prrsv is linked the pig genetic variability after prrsv infection with the enormous variability of the virus itself (box ) . in pigs, prrsv replicates in cells belonging to the innate immune system. pams are the primary cells to be infected in the lungs as well as other cells of the monocyte/macrophage lineage, which later could disseminate the virus to other tissues or support replication to release viral particles into the bloodstream ( ) (figure ) . moreover, prrsv is thought to be able to infect professional antigen presenting cells such as dcs and monocyte derived dendritic cells, (modc) impairing their normal antigen presentation ability by inducing apoptosis, down-regulating the expression of ifn-α, mhc class i, mhc class ii, cd b/c and cd , upregulating the expression of il- and inducing minimal th cytokine secretion ( ) ( ) ( ) ( ) . nevertheless, new evidence suggest by in vivo and in vitro experiments that specifically lung cdc , cdc , and modcs are not infected by prrsv- viruses from subtypes and and one possible explanation is the lower expression of cd and cd in those dc subtypes, associating previous results of infection in dcs to culture conditions of monocytes in vitro that could cause a sensibilization to infection by certain strains as lena ( ) . in addition, these findings were also tested in tonsil cdc and tracheal cdc and cdc observing that those cell populations are not infected by prrsv virus ( , ) . moreover, a new type of pam has been characterized and named porcine intravascular macrophages (pim) due to its association to endothelial lung capillaries and not to the alveoli, presenting strong capacity to phagocytised bloodrelated particles ( ) . importantly, when infected pim cells gave similar results of viral load to those derived from infected pam, but significantly upregulates of tnfα and non-significantly il- and il- expression after infection when compared to normal alveolar macrophages, indicating that these cells have an important pro-inflammatory role during prrsv infection in the lungs ( ) . new interactions between cells and the virus need to be further explored to unravel possible immunological features that leads to correlates of protection. recently, it has been shown that a domain within nsp α is able to stimulate the secretion of cd , which in turn inhibits modc function in vitro, impairing the ability of modc to stimulate t cell proliferation ( ) . production of ifn α/β and the mechanisms for cell activation by pdc are severely suppressed during prrsv infection, although these cells are not permissive to prrsv infection ( , ) . however, this phenomenon is strain dependent, as other prrsv strains are able to stimulate pdc for ifn α/β production in large quantities ( ) . again, there is an enormous variability between prrsv strains in relation with their effect on antigen presenting cells which prevent scientists from finding common mechanisms. it might be of interest to link this key gap of knowledge for prrsv with host genetics (box ) . moreover, in prrsvinfected cells, n is abundantly expressed benefiting from the discontinuous transcription mechanism ( ) . this protein is also distributed in the nucleus, induced by two nuclear localization signals called cryptic nls or nls- and functional nls or nls- (positions - and - , respectively) ( ). the effect of n protein has been examined in pams and modcs using transfection, finding a significant upregulation of il- gene expression. natural killer (nk) cells constitute another powerful arm of the innate immune system against prrsv, particularly when considering the high percentage of circulating nk cells in pigs ( ) . the cytotoxic function of nk cells is reduced in prrsv infected pigs from day after infection up to - weeks ( , , ) . initial studies using in vitro systems demonstrated that the stimulation of porcine nk cells with proinflammatory cytokines (il- and il- ) was capable of activating nk cells and inducing them to express high levels of ifn-γ and perforins to cause lysis of infected cells, but a different scenario appears if cells are evaluated post-infection, indicating that a virus such as prrsv is capable of impairing nk cell cytotoxicity ( ) . in vitro, the nk cytotoxicity against prrsv-infected pams was decreased and degranulation of nk cells inhibited ( ) . in vivo, the immune response is the same as that observed in vitro, with some studies reporting that approximately half of viremic pigs had a reduction > % in nk cell-mediated cytotoxicity and enhanced secretion of il- , il- , and il- and reduced frequency of cytotoxic t-cells (cd − cd + t) and double positive t cells (cd + cd + t) and upregulated frequency regulatory t-cells (tregs) ( ). innate immune responses against prrsv are obstructed by different mechanisms as are adaptive responses. the modest and delayed b cell mediated neutralizing antibody response is one of the main characteristics associated to prrsv acquired immune responses. even though prrsv specific antibodies appear early at - days post-infection, the efficacy of those antibodies remains unclear. neutralizing antibodies take longer, appearing nearly month after infection ( ) . however, passive transfer of these neutralizing antibodies conferred almost full protection in a prrsv reproductive model ( % of offspring alive after challenging pregnant sows with high neutralizing antibody titter). nevertheless, in another experiment using the reproductive model, when the presence of prrsv was examined after the transfer of neutralizing antibodies, lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating prrsv similar to infected controls, although pigs were apparently protected against infection. in summary, passive transfer of high neutralizing antibody titter conferred protection to gilts and offspring (not detectable viremia), but did not eliminate the presence of viral particles in peripheral tissues nor transmission to animals they were in contact with ( ) ( ) ( ) . curiously, the role of neutralizing antibodies in the protection against the respiratory form of the disease is a key gap of knowledge for prrsv. this point is critical to define precisely targets for improved vaccines based on the humoral immune response against this virus (box ). n protein is involved in several mechanisms for immune evasion and is also one of the most immunogenic structural proteins ( ) . antibodies against n appear early during acute infection, together with those against m and gp proteins, but are non-neutralizing and could be involved in antibody dependent enhancement ( , ) . there are other "antibody-related mechanisms" that do not necessarily involve neutralizing activity. antibody-dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc) and antibodydependent complement mediated virolysis (adcv) have been examined in the context of prrsv, although none of these mechanisms were evident during infection or have not been deeply investigated on in vitro and in vivo models of this virus ( ) . it is important to note that neutralizing antibodies appear late in prrsv infection and other immune mechanisms (cellular or antibody mediated immune response) might be acting to suppress viral replication in blood, causing the virus to be isolated in lymphoid tissues and maintaining suboptimal replication that will finally end in viral clearance. for type prrsv- it has been demonstrated that immunization of pigs with ectodomain peptides from gp /m complex did not induce neutralizing antibodies ( ) although those ectodomain-specific antibodies generated were capable of binding virus. an important feature that makes difficult to validate the location of neutralizing epitopes is the number of glycosylations in or around it. for prrsv- strains, up to glycosylations may be found in, or flanking the gp neutralizing epitope that is located between amino acids - ( ) , whereas for prrsv- strains there are four potential glycosylation sites ( ) . when tested, prrsv with mutations in gp glycosylation sites (either at n or in the hypervariable region, upstream the neutralizing epitope) enhanced immunogenicity with increased concentration of antibodies directed to this epitope - fold higher compared with those induced by the wild type strains ( ) . same results were obtained when administering another deglycosylation mutant (double deglycosylation in the putative glycosylation moieties on gp ) twice, which conferred better protection against homologous challenge ( ) . in addition, when this protein is expressed early during infection, it stimulates production of early neutralizing antibodies and ifn-β, two main antiviral mechanisms, demonstrating its role in induction of self-protection mechanisms from the host ( ) . available data about neutralizing antibodies induced by this protein are controversial, which may be due to the high variation among prrsv strains ( ) and, as previously commented, the host genetics. orf is also complemented by a small frameshift of the subgenomic mrna called orf a, encoding a type i membrane protein consisting primarily of alpha helix with a membrane-spanning domain (called gp a) that is incorporated into virions as a very minor component, playing a role in viral replication, as mutation in the initiation codon or premature termination related to expression for this protein leads to non-efficient viral replication and lower titter ( , ) . this protein is capable of eliciting specific antibody immune response in natural infections and after immunizations, although those are not neutralizing neither protective in a challenge trial after infection, making difficult to define the role of this particular small protein in the whole immune response and viral clearance of prrsv infection ( ) . in summary, the role of humoral immunity remains elusive in prrsv infection (neutralizing and non-neutralizing antibodies) and a better characterization will be required to overcome this relevant gap of knowledge (box ) . treg typically increase in number in chronic viral diseases to prevent a persistent inflammatory response and pathological damage associated to viral infections. conversely, tregs are described as key contributors in modulating the host immune response to viral infection. this cell population is an important component in regulating the magnitude of the immune response to infection (in viruses such as hiv and hcv), thus preventing excessive inflammation and tissue damage. however, they can also be inappropriately induced by viruses to switch the balance of the immune response in favor of maintaining viral replication ( ) . in prrsv, the role of tregs remains unclear and appears to be a consequence of il- induction of some strains as early as days post infection ( ) . in some experiments, in vitro infected dcs with prrsv- exhibited an unbalanced ability to stimulate t cell immune responses in a strain-dependent manner, but no tregs were detected, at least in vitro, as measured by expression of cd and foxp markers ( ) . when using prrsv- strains, the case seems to be different, as the virus was capable of stimulating il- production with concomitant generation of tregs ( ) which was associated to nucleocapsid protein expression in the in vitro system. this group also suggested that il- production and treg could be related to impaired gamma interferon (ifn-γ) production and altered development of protective t-cell response by inhibiting t-cell proliferation as seen in the early stage of infection with viruses such as hcv. vaccine strains currently in use in the united states do not provide adequate heterologous protection, one possible explanation could lay on their inability to induce an adequate ifn-γ response due to their ability to stimulate tregs, at least in vitro ( ). structural conformation, but not nuclear localization, of the expressed n protein was suggested as essential for the ability to induce il- that, in consequence, causes induction of tregs as measured by markers cd +cd +foxp + ( ) . it should be noted that when the role of the nuclear localization signal was evaluated using deletion mutants, results suggested that nls- was not essential for virus survival, although pigs developed a significantly shorter duration of viremia and higher neutralizing antibodies than those of wild-type prrsv-infected pigs ( ) . the role of tregs cells in the immune response against prrsv is a key gap of knowledge in order to develop more efficacious prrsv vaccines (box ) . moreover, reports have highlighted the impact of prrsv infection on thymic cellularity mainly as a loss of cd + /cd + cells in the thymus of prrsv-infected pigs. acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus are some of the mechanisms whereby prrsv suppresses the immune response. in addition, presence of prrsv antigens in the thymus could also induce tolerance and presents a mechanism that could explain the presence of tregs during prrsv infection ( ) . nevertheless, the picture is not complete and basic knowledge about the effect of prrsv on cell development in the thymus would be of great interest to understand the effect of this viruses in the host. prrsv immunology thus remains an unsolved puzzle due to complex interactions between different viral strains and the host. similar immune responses could be the key feature of this virus, such as persistence viremia, a strong inhibition of innate cytokines (ifn-α/β, tnf-α, il- β, ifn-γ), dysregulation of nk cell function (cytotoxicity and degranulation), rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibody, late and low cd + t-cell response, and induction of regulatory t cells (tregs) ( ) . as a whole, neutralizing antibodies and prrsv-specific ifn-γ secreting cells do not fully depict the immune effector functions related to protective immunity, as the viral targets related to them are unknown. as a consequence, correlates of protection remain elusive for this infection due to the laborious work in vitro and in vivo and the enormous genetic diversity that causes confusion and makes it difficult to predict how immune responses against one isolate or strain could be applied to another in a cross-protective immune prediction model ( , ) . without any doubt, the most important gap of knowledge for prrsv is the lack of correlates of protection that makes extremely difficult to have robust models to check vaccines efficacy against this disease (box ). since the beginning of prrsv outbreaks in europe and the usa, the development of efficacious prrsv vaccines has been a challenge. classical approaches are not working properly for several reasons: viral mutation can lead to more pathogenic strains, there is a lack of knowledge on how the porcine immune system interacts with all prrsv proteins, and most importantly, there is no robust parameter (surrogate marker) that can be unequivocally linked with viral clearance. thus, there is no relationship between complete homologous or heterologous protection and classic immunological parameters such as an increase/decrease in particular cell population ( ) , ifnγ production, neutralizing antibodies ( ), non-neutralizing antibodies and clinical outcome ( ) . in addition, highly divergent strains make it more difficult to develop a universal vaccine for this virus ( ) . several different vaccines against prrsv have reached the market and have been reviewed recently ( ) . most of these vaccines rely upon modified live virus (porcilis prrs from merck, ingelvac prrsflex eu from boehringer ingelheim, amervac-prrs from hypra, pyrsvac- from syva) against prrsv- , as well as some to control prrsv- (fostera prrs from zoetis, ingelvac prrs mlv/ingelvac prrsatp from boehringer ingelheim). there is also evidence that most mlv vaccines of both prrsv- and prrsv- species elicit specific humoral and cell-mediated immune (cmi) responses, as they confer protection to homologous parental strains and partial protection to heterologous strains. although it is possible to control some prrsv outbreaks by use of mlv in combination with good practices, there are major safety issues such as a high mutation rate leading to reversion to virulence and recombination among vaccine and wild type strains. cases have been reported in which new viruses have been introduced as a consequence of mlv vaccines. for example, nucleotide sequence identities of atypical danish isolates were between . and . % with the vaccine virus respprrs and . - . % with vr , which is the parental virus to the vaccine virus, supporting the conclusion that the introduction of prrsv- in denmark was due to the spread of vaccine virus ( ) . in china a recombination event was reported in which a prrsv variant with nucleotide deletions and insertions in the non-structural protein (nsp ) gene also showed a possible recombination event between a mlv strain and a prototype chinese field strain ( ) . current inactivated vaccine approaches are not highly effective since elicited immune responses are not enough to prevent spreading of the virus. however, this type of vaccine can augment anamnestic virus neutralizing antibodies and virusspecific ifn-γ responses following a wild-type virus infection or prrsv-mlv vaccination which can contribute to viral clearance ( , ) . thus, the combination of modified live vaccines with inactivated ones can be a reasonable approach to control the disease under field conditions ( ) but unfortunately, there is no robust data comparing this approach with other options available on the market. on the other hand, most inactivated vaccines are not approved for use in the united states due to the poor efficacy showed in challenge trials ( ) as measured by production of prrsv specific neutralizing and non-neutralizing antibodies and low cellular immune responses leading to their failure in the porcine market. according to the centre for food security and public health of iowa state university, only box | exosomes and therapeutic applications in prrsv. frontiers in veterinary science | www.frontiersin.org "biosuis prrs inact eu+am" is approved to be used in the us. however, new strategies are being evaluated to overcome these problems ( ) , including nanoparticle entrapped antigens ( ) ( ) ( ) ( ) , plant based approaches ( ) or vectored vaccines ( ) . several attempts have been made to use structural proteins to develop vaccines against prrsv because they are specific targets of neutralizing antibodies. for this reason, one may hypothesize that antibodies against those proteins could be the main key to inhibit viral replication and spread as it is common for many viruses. approaches such as vlps combining different structural proteins have been tested ( ) ( ) ( ) , finding that anamnestic response is possible (boosted igg and ifn-γ producing cells) in previously vaccinated or infected pigs but not in the prechallenge period. these structural proteins are able to prime the immune system, but no reduction of viremia was observed after challenge ( ) . those results suggest that other viral proteins may be targeted to induce a protective response in pigs. a plausible explanation for this finding may be based on the presence of few neutralizing epitopes in their sequences, most of which are located in variable regions of the proteins, to the phenomena of glycan shielding for epitopes and to the high variability observed between prrsv virus strains. again, a critical gap of knowledge for prrsv is to precisely characterize common epitopes that are present in all prrsv strains. epitopes responsible for generating an efficient immune response eliciting cross-protective immunity remained elusive. taken together, this evidence points to the need for new vaccination approaches that comply with a pathogen free strategy, capable of eliciting effective cellular and antibody responses with mid to long term protection against homologous strains and preferable to heterologous challenge as well. extracellular vesicles(evs) are gaining increased scientific attention as novel vaccines against infectious diseases, including animal diseases of veterinary importance by its capacity of self-antigen presentation, activation of host cell and antibody immune responses and more important, to induce protection in lethal challenge trials ( - ) (box ). in the case of prrsv, artificial micrornas (amirna) were initially synthetized to try suppressing expression of sialoadhesin (sn) or cd by recombinant adenoviral vectors to be contained in exosomes, causing a subexpression of sn and cd at mrna and protein level, and reducing viral titter when porcine macrophages were pre-treated with amirna thus providing new evidence supporting the hypothesis that evs can also serve as an efficient small rna transfer vehicle for pig cells ( ) . more recently, prrsv viral proteins associated to extracellular vesicles (evs) in the size range of exosomes, were reported ( ) . moreover, a targeted-pig trial using evs from sera of infected pigs who had overcome the disease, demonstrated that evs are capable of inducing specific ifn-γ secreting cells after a prime-boost strategy, are safe, free-of-virus and can differentiate infected from vaccinated animals ( ) , moreover, it was demonstrated that those evs contained antigenic viral proteins recognized by pig immune sera and not by the pre-immune one. of interest, however, a recent article indicated that prrsv derived evs are capable of transmitting the virus from one cell to another ( ) . whether these discrepancies are due to in vivo vs. in vitro experimental work and methods applied to isolate evs from serum samples or culture supernatant, remains to be determined. evs have also been explored as novel control strategies in other viral diseases. for example, in respiratory syncytial virus infection, evs are released with a selected modified cargo when compared with uninfected epithelial cells. when analyzed in detail, several viral proteins and diverse species of rna were detected and capable of activating innate immune responses through induction of cytokine and chemokine release ( ) . similar scenarios of viral proteins exported in evs have been observed and extensively reviewed for hiv/hcv/htlv- ( ), ebv ( ) , and other viral diseases. moreover, viral products of various origin and size including ebola virus vp , vp , and np, influenza virus np, crimean-congo haemorrhagic fever np, west nile virus ns , and hepatitis c virus ns , when fused with nef c-terminal domain through dna vectors, were directed to the evs membrane or packaged into them and remained stable after fusion. more importantly, when injected in mice, dna vectors expressing the diverse fusion products elicited a well detectable antigen-specific cd + t cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells, proving its promising results as a cytotoxic t lymphocyte vaccine ( ) . prrsv is a complex disease and several gaps in the knowledge of its economic impact, biology and evolution, genetic polymorphism, mechanism of viral infections, elicitation of protective immune responses and novel control strategies, have been reviewed here (box ). since the late 's, different approaches have permitted to examine more closely this virus allowing the discovery of new features of the complex replication cycle, the identification of proteins and nucleic acids playing a role together with extracellular vesicles and nanotubules in facilitating spreading, and a better understanding of immune evasion (non-neutralizing antibodies, glycan shielding, mutation, recombination events, among others) to further vaccine development. presently available prrsv vaccines have many limitations in terms of heterologous protection, but some efforts have been made by combining new adjuvant formulations with modified live viruses, dna and peptide vaccines, as well as extracellular vesicles a new vaccination approach. advancing in all these gaps in knowledge, will eventually accelerate eliminating and eventually eradicating this devastating veterinary disease of such huge economic importance. sm-t: wrote the first draft of the manuscript. mm, hdp, and lf: wrote sections of the manuscript. all authors contributed to manuscript revision, read, and approved the submitted version. this review received support from the feder project (comrdi - - - ). assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states cost of porcine reproductive and respiratory syndrome virus at individual farm level -an economic disease model isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus ratification vote on taxonomic proposals to the international committee on taxonomy of viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the molecular biology of arteriviruses the structural biology of prrsv a bayesian phylogeographical analysis of type porcine reproductive and respiratory syndrome virus (prrsv) identification of porcine reproductive and respiratory syndrome virus orf a-encoded non-structural proteins in virus-infected cells efficient− frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein nonstructural proteins nsp tf and nsp n of porcine reproductive and respiratory syndrome virus (prrsv) play important roles in suppressing host innate immune responses biogenesis and architecture of arterivirus replication organelles regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein into virions prrsv receptors and their roles in virus infection membrane proteins of arterivirus particles: structure, topology, processing and function influence of n-linked glycosylation of minor proteins of porcine reproductive and respiratory syndrome virus on infectious virus recovery and receptor interaction overview: replication of porcine reproductive and respiratory syndrome virus cd knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus an intact sialoadhesin (sn/siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus role of cd , a tetraspanin, in porcine reproductive and respiratory syndrome virus infection prrsv structure, replication and recombination: origin of phenotype and genotype diversity effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr- ) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread effect of malaria on hiv/aids transmission and progression tunneling nanotubes (tnt) are induced by hiv-infection of macrophages: a potential mechanism for intercellular hiv trafficking tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses innate and adaptive immunity against porcine reproductive and respiratory syndrome virus arterivirus molecular biology and pathogenesis interferon-stimulated genes: a complex web of host defenses production of type i interferons: plasmacytoid dendritic cells and beyond immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc- cells modulation of innate immune signaling by nonstructural protein (nsp ) in the family arteriviridae interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf activation in immortalized porcine alveolar macrophages the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex porcine alveolar macrophage polarization is involved in inhibition of porcine reproductive and respiratory syndrome virus (prrsv) replication porcine reproductive and respiratory syndrome virus nonstructural protein (nsp ) topology and selective isoform integration in artificial membranes the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc- cells nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production the endoribonuclease activity essential for the nonstructural protein of porcine reproductive and respiratory syndrome virus to inhibit nlrp inflammasome-mediated il- β induction porcine reproductive and respiratory syndrome virus (prrsv) inhibits rnamediated gene silencing by targeting ago- novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by prrsv- comparison of host genetic factors influencing pig response to infection with two north american isolates of porcine reproductive and respiratory syndrome virus variation among sows in response to porcine reproductive and respiratory syndrome genetic resistance -an alternative for controlling prrs? porc heal manag genetic analysis of reproductive traits and antibody response in a prrs outbreak herd validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin- production cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus type . lena triggers conventional dendritic cells activation and t helper immune response without infecting dendritic cells tonsil conventional dendritic cells are not infected by porcine reproductive and respiratory syndrome virus response of the cdc and cdc subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus nsp α of porcine reproductive and respiratory syndrome virus strain bb impairs the function of monocyte-derived dendritic cells via the release of soluble cd impact of genotype and of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists sensing of porcine reproductive and respiratory syndrome virus-infected macrophages by plasmacytoid dendritic cells the viral innate immune antagonism and an alternative vaccine design for prrs virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis perforin expression can define cd positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs natural killer cells in host defense against veterinary pathogens suppression of nk cell-mediated cytotoxicity against prrsv-infected porcine alveolar macrophages in vitro evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent role of neutralizing antibodies in prrsv protective immunity passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity the challenge of prrs immunology immunological responses of swine to porcine reproductive and respiratory syndrome virus infection mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes neutralizing antibody responses of pigs infected with natural gp n-glycan mutants of porcine reproductive and respiratory syndrome virus certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein gp expression in marc- cells inhibits porcine reproductive and respiratory syndrome virus infection by inducing beta interferon activity porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses immune response to orf a protein immunization is not protective against porcine reproductive and respiratory syndrome virus infection regulatory t cells and infection: a dangerous necessity european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin- and regulatory t-lymphocytes (treg) regulatory t cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication immune control of prrs: lessons to be learned and possible ways forward immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) antiviral regulation in porcine monocytic cells at different activation states porcine reproductive and respiratory syndrome virus isolates differ in their susceptibility to neutralization effector mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective. front microbiol sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wild-type prrsv strains porcine reproductive and respiratory syndrome (prrs) virus-specific interferon-γ + t-cell responses after prrs virus infection or vaccination with an inactivated prrs vaccine failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate t cell antigens an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination plant-based porcine reproductive and respiratory syndrome virus vlps induce an immune response in mice vectored vaccines to protect against prrsv development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs generation of porcine reproductive and respiratory syndrome (prrs) viruslike-particles (vlps) with different protein composition exosomes as potent cell-free peptide-based vaccine. i dendritic cellderived exosomes transfer functional mhc class i/peptide complexes to dendritic cells induction of protective immunity against experimental eimeria tenella infection using serum exosomes extracellular vesicles in parasitic diseases exosomes from plasmodium yoelii-infected reticulocytes protect mice from lethal infections serumderived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins b lymphocytes secrete antigen-presenting vesicles biological properties of extracellular vesicles and their physiological functions inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirusand/or exosome-delivered the artificial micrornas targeting sialoadhesin and cd receptors targeted-pig trial on safety and immunogenicity of serumderived extracellular vesicles enriched fractions obtained from porcine respiratory and reproductive virus infections exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (prrsv) respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells exosomes and their role in the life cycle and pathogenesis of rna viruses pathogenic role of exosomes in epstein-barr virus (ebv)-associated cancers an exosome-based vaccine platform imparts cytotoxic t lymphocyte immunity against viral antigens we are particularly grateful to christa helwig for english editorial assistance. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sg declared a past supervisory role with one of the authors mm to the handling editor.copyright © montaner-tarbes, del portillo, montoya and fraile. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -g rohhtt authors: bautista, elida m.; faaberg, kay s.; mickelson, dan; mcgruder, edward d. title: functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: g rohhtt abstract porcine reproductive and respiratory syndrome virus (prrsv) is a member of the positive-strand rna virus family arteriviridae. although considerable research has focused on this important pathogen, little is known about the function of most prrsv proteins. to examine characteristics of putative nonstructural proteins (nsp) encoded in orf b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the prset expression system. one region, nsp , encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. prrsv nsp was found to possess a thermolabile and ph-sensitive ntpase activity that was modulated by polynucleotides and to unwind dsrna in a ′ to ′ polarity. these results provide the first evidence of the functional properties of prrsv helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases. porcine reproductive and respiratory syndrome virus (prrsv) emerged in the late s and became a pathogen of scientific interest due to its economic importance. analysis of prrsv structure, genomic organization, and expression and its biological, physicochemical, and pathogenic properties has shown that prrsv is related to equine arteritis virus (eav), lactate dehydrogenaseelevating virus of mice (ldv), and simian hemorrhagic fever virus (shfv) collins et al., ; meulenberg et al., ; plagemann and moennig, ) . these viruses are grouped in a new family designated arteriviridae and placed with the coronaviridae family within the order nidovirales (cavanagh, ) . prrsv has been revealed to consist of two distantly related genotypes, north american (na, . kb) and european (eu, . kb), based on sequence analysis (allende et al., ; meulenberg et al., ; murtaugh et al., murtaugh et al., , nelsen et al., ) . the genome of prrsv encodes at least eight open reading frames (orfs) that are expressed in the infected cell as a nested set of subgenomic mrnas. orf , translated from the full-length rna template, codes for replicase-related nonstructural proteins and is the focus of this study. orfs to encode mostly structural polypeptides. orf contains two long open reading frames, designated orf a and orf b, that are autocatalytically processed by virally encoded proteases (meulenberg et al., ) . orf b is only expressed by ribosomal Ϫ frame shifting due to a slippery sequence and pseudoknot structure in the overlapping orf a/orf b junction (allende et al., ; den boon et al., ; brierly et al., ; meulenberg et al., ; nelsen et al., ; wootton et al., ) . the catalytic domains and cleavage sites for eav orf have been identified. the eav proteases, located within nsp , nsp , and nsp , have been shown to cleave eav orf ab into nonstructural proteins (nsps), in orf a and in orf b (den boon et al., ; snijder et al., snijder et al., , a snijder and meulenberg, ; van dinten et al., . eav nsp autocatalytically cleaves the orf protein immediately downstream between g and g ; nsp is proposed to cleave far downstream between two glycine residues located at eav amino acids and , and nsp , the major protease, cleaves orf between amino acids glutamine and glycine (eg) or glutamine and serine (es) (ziebuhr et al., ) . based on significant homology in the catalytic domains and in predicted cleavage sites, it was surmised that prrsv polymerase may undergo similar proteolytic processing as that of eav (snijder and meulenberg, ; wassenaar et al., ; ziebuhr et al., ) . the four predicted orf b nonstructural polypeptides comprise a putative rna-dependent rna polymerase (pol or nsp ), a protein (nsp ) containing a putative metal-binding domain (mbd) and nucleoside triphos-phate binding or helicase motif (hel), and two proteins of unknown function (nsp and ). nsp contains a domain (corona) that is conserved in all nidoviruses (allende et al., (allende et al., , den boon et al., ; godeny et al., ; gorbalenya et al., ; herold et al., ; meulenberg et al., ; nelsen et al., ; shen et al., ; snijder and meulenberg, ; van dinten et al., wootton et al., ) . despite extensive research on the molecular and biological properties of prrsv, there are important aspects of the disease and biology that remain unclear. the role of the nonstructural genes encoded in orf ab in viral replication, pathogenesis, and immunity have not been demonstrated. this information is necessary to devise effective strategies to prevent and control prrsv infection of swine. the knowledge available for the genes encoded in orf ab is limited to sequence information for a few virus isolates (allende et al., (allende et al., , jiang et al., ; meulenberg et al., ; nelsen et al., ; shen et al., ; wootton et al., ) and predictions on their function based on sequence homology and studies on eav and coronaviruses (gorbalenya et al., ; herold et al., ; snijder and meulenberg, ; . to elucidate the role of the nonstructural genes of arteriviruses in virus replication, previous studies have been done with eav ( . kb) to develop and characterize infectious clones and their derivative mutants (de vries et al., ; van dinten et al., van dinten et al., , van marle, ) . these studies have provided important insights as to the critical role of the genes encoded in orf ab in arterivirus replication. however, prrsv is genetically divergent from eav, possessing only approximately % (orf a) to % (orf b) nucleotide identities to the sequenced eav cell-adapted avirulent strain . in addition, infectious clones of prrsv have been more difficult to prepare and analyze, apparently due to the increased genomic size of prrsv ( . - . kb), the restricted cell tropism of this virus, and the difficulty in transforming these permissive cells with nucleic acids . we have chosen a virulent field isolate of prrsv to explore phenotypic properties of this virus as it exists in nature. to demonstrate and characterize prrsv orf b polypeptides, we have generated recombinant individual nsp proteins that were then analyzed under controlled conditions. this article describes the first functional studies of the putative prrsv helicase. two overlapping orf b gene segments were amplified by rt-pcr and designated cf and ih. these gene segments were cloned and confirmed to be prrsv- specific nucleotides by sequence analysis. the nucleotide sequence of prrsv- orf b has been deposited in genbank (accession no. af ). nucleotide sequence comparison of prrsv- orf b revealed that this isolate is closely related to prrsv-na strains (data not shown). sequence analysis also determined that the predicted cleavage sites for prrsv orf b were maintained in isolate prrsv- (allende et al., ; . these predicted cleavage sites appear to be well conserved among prrsv strains (table ) . genome clones cf and ih both contained an overlapping unique saci site that was used to generate a -nt ch fragment, which comprised all orf b flanked by minimal orf a sequence at the Ј end and by a small amount of orf sequence at the Ј end. the ch fragment was cloned and used as a template to generate pcr-amplified orf b nsp gene fragments (fig. a) . these gene fragments were cloned into prset-b plasmids for protein expression and designated ptprrsv nsp - . western blot analysis with t . ab demonstrated that the produced recombinant proteins corresponded to polypeptides of the predicted molecular size (fig. b) . to confirm the specificity of the individual nsps, antipeptide rabbit sera were generated as described under materials and methods. western blot analyses with nsp antipeptide rabbit sera (␣-hel, fig. b ) or other gene-specific anti-peptide sera (data not shown) demonstrated the specificity of prrsv- -specific individual nsps. experiments were conducted to obtain soluble recombinant prrsv- orf b proteins. optimal conditions for nsp , which is predicted to have rna helicase and atpase functions (meulenberg et al., ) , were obtained with the escherichia coli strain bl (de )plyse, which has been designed to have more stringent expres- note. orf b amino acid alignment of north american and european isolates (genbank accession nos. are indicated in the text) was performed using the pileup program of seqweb version . of the wisconsin package version . . the predicted cleavage sites are underlined and based on sequence homology to the predicted sites described for eav (meulenberg et al., ; nelsen et al., ; van dinten et al., . lowercase indicates amino acid difference at that position compared to the reference na strain. sion of the t polymerase. in addition, culturing of transformed bacteria at °c and the use of reduced-nutrient media further improved expression. under these conditions and after h of iptg induction, approximately - % of expressed recombinant nsp protein was soluble. efforts were directed to obtain purified nsp and to characterize its predicted ntpase function and demonstrate its helicase activity. to obtain purified recombinant prrsv- helicase, proteins in soluble extracts from bacteria transformed with ptprrsv -nsp were adsorbed by affinity to cobalt or nickel resins and eluted as described. samples obtained in the last elution fractions from nickel columns provided a highly purified helicasesoluble protein preparation (Ͼ % pure based on coomassie blue staining, fig. ). protein preparations obtained with cobalt resins also yielded adequate yields but had reduced atpase activity compared to nickel affinity-purified protein (data not shown). therefore, nickel affinity-purified nsp helicase was used for the characterization of its inherent atpase and helicase activities. with similar conditions, soluble nsp and nsp were obtained (data not shown) and used as controls in the subsequent experiments as indicated. as initial proof of helicase-like activity, experiments were performed to test the ability of purified prrsv- nsp protein to hydrolyze radiolabeled ribonucleotides in the presence or absence of polyribonucleotides. from the autoradiographic films of the chromatographs (fig. ) , it was apparent that prrsv nsp helicase hydrolyzed all four nucleotides in the absence of polynucleotides. polynucleotides did not appear to significantly affect the level of nucleotide hydrolysis, except for a slight reduction in the level of hydrolysis of ctp in the presence of poly(u). to further characterize the ntpase function of prrsv nsp helicase, the subsequent studies were performed with atp as substrate. primers used for amplification and the numbers in italics represent the predicted molecular size of the amplified products. the ih-and cf-amplified gene segments were selected for cloning and sequencing. the cloned ih and cf gene fragments were consequently ligated at the saci restriction site to generate the ch fragment (represented by the cross-hatched bar) that contains the complete orf b gene of prrsv- . the recombinant polypeptides from prrsv- orf b that were expressed in the prokaryotic system are presented by lines. the number in italics on the lines represent the predicted molecular size in kilodaltons (kda) of prrsv- orf b polypeptide fragments. the histidine (his)-t . tag derived from the expression vector increased the molecular size in approximately . kda. (b) western blot analysis of the expressed recombinant proteins with the indicated antibodies described in the text. molecular markers (mm) are indicated by the numbers at the left in kda. a positive control protein for the monoclonal antibody t . was included (t ϩc). as negative control, bacterial extracts that were transformed with empty plasmids (prset) were included to test each antibody. the protein bands that were specifically recognized by the antibodies and had the expected molecular size of the predicted polypeptide are indicated by arrows. the level of atpase activity in recombinant prrsv helicase was compared to the atpase activity in control samples. the control samples were obtained following the same procedure used for purification of recombinant helicase and consisted of protein preparations (equalized by protein concentration) obtained from bacteria transformed with one of the following plasmids: ( ) empty prset, ( ) ptprrsvnsp , or ( ) ptprrsvnsp . the results demonstrated that the hydrolysis of atp appeared specific for recombinant nsp (prrsv helicase) and not for possible contaminating bacterial proteins. these results also established that both predicted nsp and predicted nsp products lacked a specific atpase activity (fig. ) . the atpase activity of prrsv helicase was shown to be dependent on recombinant protein concentration (fig. ). prrsv helicase atpase activity was also determined to be dependent on ph (optimal ph range was between . and . ) and influenced by the assay buffer used (fig. a ). with tris buffer, the atpase activity of prrsv helicase was stable over a broader ph range than with the other buffers tested (fig. a ). there was no significant differences seen in the level of atpase activity using tris buffer concentrations in the range of to mm (data not shown). further characterization of the atpase activity was performed using mm tris buffer at ph . . prrsv helicase activity was dependent on the presence of divalent ions, as the presence of edta inhibited the recombinant proteins' atpase activity in a dose-dependent manner (fig. b ). in addition, there was no atpase activity observed in the absence of divalent ions (fig. c ). optimal atpase activity was detected at - mm for all the four divalent ions tested. whereas the level of atpase activity of prrsv helicase consistently remained stable over a broader range of mgcl concentrations, increasing concentrations of mnso inhibited the activity in a dose-dependent manner and to a greater extent than the other divalent ions tested (fig. c) . lastly, the atpase activity of prrsv helicase was very sensitive to temperature. optimal activity was detected between and °c. the atpase activity was significantly reduced at . °c and was only detected at background levels at temperatures at and above °c and completely abolished at °c (fig. d ). kinetic analysis of recombinant helicase atpase activity revealed dependence on time (fig. a ) and substrate concentration (fig. b) . the michaelis-menten constant (k m ) for the atpase activity of prrsv helicase was determined to lie in the range of - mm atp, with a maximum velocity of . - mm atp hydrolyzed per min (fig. c ). to assess whether the level of atpase activity would be affected by the presence of polynucleotides, kinetic analysis was performed in the presence or absence of poly(u). poly(u) significantly enhanced the atpase activity of prrsv helicase at a given atp level (fig. a ). although maximum velocity of enzymatic activity was not reached, comparison of the initial velocities of the relative atpase activities showed that the presence of poly(u) increased the k m and v max at least -and fold, respectively. however, at low atp concentrations, poly(u) did not have an effect or tended to reduce the atpase activity ( table ). the fold increase in the level of atp hydrolyzed induced by poly(u) was in a close linear proportion (r ϭ . ) to the concentration of atp substrate (fig. b) . these data indicate that the effect of polynucleotides on the atpase activity of prrsv is dependent on atp substrate concentration. this result also explains our initial findings (fig. ) , in which no apparent effect of the polynucleotides was detected when the atp concentration was limited and the level of atpase activity present in the assay was nearly % of the substrate. last, the effect of the presence of other mononucleotides on the level of atpase activity was ascertained using the bioluminescence detection method (mcelroy and deluca, ). this assay has been shown to spe- fig. . specificity of the atpase activity of prrsv- helicase. the specificity of the atpase activity was determined by testing the indicated protein concentrations of purified recombinant proteins prepared as described in the text. the atpase assay was performed using . m of atp substrate and the level of atp remaining in the samples was measured after min incubation. the level of atpase activity is expressed as the percentage of atp hydrolyzed in the atpase assay. data represent the mean Ϯ standard deviation of triplicate samples. cifically detect atp (moyer and henderson, ) . in addition, we confirmed that the measurement of atp by the bioluminescence method was not affected by the presence of ctp, gtp, or utp (data not shown). as shown in fig. , ctp, gtp, and utp inhibited the level of atp hydrolysis by prrsv helicase only when they were present at concentrations at least -fold higher than the atp substrate. this result confirms that prrsv nsp helicase protein can interact with all four nucleotides and, furthermore, this helicase protein may have a higher affinity for atp than for the other nucleotides. to demonstrate whether recombinant prrsv helicase possessed functional rna unwinding activity, various substrates were designed and tested in a standard helicase assay. these substrates consisted of two partially complementary rna strands containing single-stranded (ss) regions at the Ј or Ј ends of one or both strands. one set of substrates designed to contain single-strand regions at both Ј and Ј ends did not initially form the expected partial duplex structures under initial hybridiza-tion conditions. one explanation is that these long oligonucleotides are predicted to form secondary structures (energies of Ϫ to Ϫ kcal/mol; data not shown) and therefore might not have initially annealed to the other strand. however, duplexes or other higher ordered structures appear to form in the presence of recombinant helicase (data not shown). one suggestion for this result is that prrsv helicase first promoted unwinding of initial interoligonucleotide structures, then winding to form the expected duplexes, and/or introducing secondary structures in the labeled rna strand. to further analyze the predicted helicase activity of the prrsv nsp protein, experiments were performed using duplex substrates prepared with synthetic oligonucleotides containing single-strand regions at the Ј or Ј ends and short duplex regions ( and bp). results obtained with rna Јduplex and Ј Јduplex indicated that prrsv helicase lacked Ј-to- Ј unwinding activity (fig. a ). in contrast, results with the rna Јduplex revealed that prrsv helicase has unwinding activity in the Ј-to- Ј orientation (fig. b) . the Ј-to- Ј orientation of the unwinding activity of the prrsv helicase was further confirmed in experiments using substrates prepared with in vitro transcribed rna that contained singlestrand regions at the Ј end of both strands and duplex regions of bp ( Ј Јduplex # ) and bp ( Ј Јduplex # ), as shown in fig. b . these results, together with the atpase data described above, demonstrate that the recombinant prrsv nsp helicase is functionally active and shares the properties reported for helicases of other members of the nidovirales (seybert et al., a,b) . the nonstructural polypeptides encoded in nidovirales orf b regions have been predicted to have essential roles in virus replication and gene expression (allende et al., ; gorbalenya et al., ; meulenberg et al., ; nelsen et al., ; van dinten et al., ) . evidence for the significant role of these proteins in arteriviruses has been derived using an infectious clone of eav (van dinten et al., (van dinten et al., , van marle et al., ) . however, as stated previously, eav is genetically distinct from other arteriviruses, possessing only approximately % nucleotide identity and . % amino acid identity in the orf b region when compared to prrsv-na . thus, it is crucial to gather basic information on distantly related arteriviruses to examine variability in orf b nucleotide composition and function and to assess outcomes of potential orf b region mutations. this study was completed to provide molecular tools to facilitate understanding of the biochemical and functional properties of prrsv-na-predicted orf b-processed polypeptides. accordingly, we have cloned and expressed predicted orf b protein products of prrsv- in a prokaryotic system and determined optimal conditions for functional expression of soluble nsp , nsp , and nsp . of these three polypeptides, nsp was chosen for further biochemical characterization, as this predicted protein has been shown by sequence comparison to contain conserved ntpase and helicase domains (allende et al., ; nelsen et al., ; meulenberg et al., ) of the superfamily (sf) of helicases (kadare and haenni, ; seybert et al., a,b) . prrsv-na nsp bears only approximately % amino acid similarity to nsp of eav , illustrating remarkable interfamily divergence and rendering possible differences in precise dynamics for this critical protein. the described studies demonstrated that the predicted nsp product containing the metal binding and ntpase-helicase domains of prrsv orf b has ntpase activity. the initial ntpase analysis, determined by thin-layer chromatography, revealed that prrsv helicase hydrolyzes the four ribonucleotides in the absence of polynucleotides. the interaction of the recombinant prrsv helicase with the nucleotides was confirmed by demonstrating that the presence of excess gtp, ctp, and utp inhibited the level of atp hydrolysis. the ability of prrsv helicase to catalyze hydrolysis of different nucleotides is a characteristic shared by various reported viral helicases (bisaillon et al., ; kadare et al., ; li et al., ; preugschat et al., ; rikkonen et al., ) . we demonstrated that the activity observed for the recombinant his-tagged nsp is specific for the helicase because the recombinant prrsv orf b nsp and nsp polypeptides that were expressed and purified under the same conditions as prrsv helicase did not have specific atpase activity. although atpase activity has been previously reported for the human coronavirus e helicase (heussip et al., ; seybert et al., a) , a member of the order nidovirales, and more recently for eav (seybert et al., b) , this is the first study reporting the characterization of the ntpase activity of the putative helicase of prrsv. the characterization of the atpase activity revealed some features of prrsv helicase that are common among known viral helicases such as the requirement for divalent cations and sensitivity to extreme ph and temperatures. in contrast to other viral helicases (bisaillon et al., ) , prrsv helicase appears to be extremely sensitive to temperatures above physiological levels. this finding suggests that viral or cellular factors may be required to stabilize prrsv helicase function. the atpase activity of prrsv helicase was also dependent . prrsv helicase activities. rna substrates were generated by annealing two partially complementary rna strands for use in a standard helicase (hel) assay. rna substrate predicted duplex structures are indicated by diagrams above each assay result. a sample lacking prrsv helicase (Ϫh) was included to indicate migration of intact radiolabeled duplex, and denatured substrates (Ϫh, °c) were included to indicate migration of unduplexed labeled short strand. migration of substrates after the helicase assay is indicated on each electropherogram as single-stranded (ss) or double-stranded (ds) oligomers. (a) results of the helicase assay with substrates containing Ј single regions at one ( Јduplex) or both ( Ј Јduplex) strands and duplex regions of and bp, respectively. (b) prrsv helicase activity displaced the labeled short strand of Ј tailed rna substrates with duplex lengths of ( Јduplex), ( Ј, Јduplex-# ), and bp ( Ј Јduplex-# ). note. the atpase assay was performed as described under materials and methods at the indicated atp concentrations in the presence (ϩ) or absence (Ϫ) of poly(u) in triplicate. * statistical significance was determined by paired two-sample t-test with alpha at . . on protein concentration. although the presence of polynucleotides was not required for detecting the ntpase activity of prrsv helicase using the standard tlc method, poly(u) had a significant effect on this activity when the activity was measured at various atp substrate concentrations. interestingly, the effect of poly(u) in the atpase activity of prrsv helicase was dependent on the substrate concentration. at atp concentrations lower than the estimated k m , poly(u) had an inhibitory effect, whereas at higher atp concentrations, poly(u) had a stimulatory effect on the atpase activity of prrsv helicase. it was also interesting to note that the level of effect of poly(u) was in a close linear relationship with the atp substrate concentration. the substrate concentration-dependent modulatory effect of polynucleotides on the atpase activity of prrsv helicase found in this study has not been previously described. data reported for other viral helicases are consistent with a stimulatory effect that varies depending on the type of helicase and polynucleotide used (rikkonen et al., ; tamura et al., ) . the level of nucleic acid stimulation reported for the known members of the viral sf and sf helicases has only been in the range of a twofold increase, whereas higher levels of stimulation have been reported for sf helicases (kadare and haenni, ) . the atpase activity of the human coronavirus e, which belongs to the same nidovirales order as prrsv, was found to be highly stimulated with polynucleotides and the major stimulatory effect was found with poly(u), which increased the ntpase activity of the helicase up to -fold (heusipp et al., ; seybert et al., a) . a -fold increase in the atpase activity by polynucleotides was found for the eav helicase (seybert et al., b) . for prrsv helicase we found that whereas the overall effect of poly(u) on the k m and v max reached levels of -and -fold increase, respectively, the effect on the amount of atp hydrolyzed varied from ϳ . -fold (at . m atp substrate) to . fold (at m atp substrate). experimental data analyzing the effect of polynucleotides under the range of atp substrate concentrations used in this study have not been reported for other viral helicases. therefore, it is not clear whether our findings represent a unique feature of prrsv helicase. we suggest that the modulatory effect of poly(u) on the atpase activity of prrsv helicase discovered in this study may have biological relevance regarding virus replication and gene expression. it is tempting to speculate that if prrsv helicase is involved in the processes of its genome replication and transcription, as suggested by the studies with the mutant eav infectious clones (van dinten et al., (van dinten et al., , van marle et al., ) , prrsv helicase needs to be regulated to achieve a balance between nucleotide hydrolysis and viral rna synthesis. analysis of the crystal structure of prrsv helicase will be important to determine whether the regulatory effect of polynucleotides in the atpase activity of prrsv helicase is dependent on conformational changes differentially induced by various ratios of atp substrate to polynucleotide bound to the protein. to further characterize prrsv helicase function in terms of its major predicted role in rna winding, studies analyzing the ability of the protein to interact with various substrates were performed. the recombinant helicase did not exhibit Ј to Ј unwinding activity on synthesized oligomer duplexes of or bp and single-strand regions of - nucleotides. the prrsv helicase did demonstrate Ј-to- Ј unwinding activity on duplexed substrates with single-stranded segments on one or both Ј ends. these results are consistent with the Ј-to- Ј unwinding properties found for eav (seybert et al., b) and hcov (seybert et al., a) , supporting the finding that nidovirales sf helicases are closely related with functional properties different to those of other viral helicase families. this study provides the first evidence on both atpase and helicase-like activities of prrsv helicase and suggests a regulatory role of polynucleotides on its atpase activity and unwinding properties. the molecular tools developed in this study will be useful for further studies directed to understand the role of the predicted intermediate orf b gene protein products in prrsv replication. a prrsv isolate, designated prrsv- , was obtained from the national veterinary services laboratories (nvsl, ames, ia). selection of isolate prrsv- was based on its clinical history and its ability to replicate well in the marc- cell line (kim et al., ) . the virus was isolated from the serum of an -day-old piglet exhibiting interstitial pneumonia. the piglet had come from an alabama farm experiencing a reproductive disease outbreak in march of . prrsv- was propagated in marc- cells to passage level - . culture fluid containing virions was concentrated by ultracentrifugation through a . -m sucrose cushion and further purified by sucrose gradient centrifugation as described (bautista et al., ) . viral rna was extracted from the purified virus with trizol reagent (gibco-brl), concentrated by alcohol precipitation, suspended in rnase-free double distilled water, and stored at Ϫ °c until analyzed. rt-pcr, cloning, and sequencing analysis of prrsv- orf b genomic segments prrsv- gene fragments containing orf b sequences were amplified by rt-pcr. reverse transcription was performed in a -l reaction containing g of purified viral rna, . g random hexamers, and u superscript ii reverse transcriptase (gibco-brl), following the manufacturer's recommendations. polymer-ase chain reactions (pcr) were performed in l reactions using l viral cdna, m dntps, u high-fidelity vent (new england biolabs), and various set of primers (table ) according to the strategy depicted in fig. a . the reactions were performed by an initial denaturation for min at °c, followed by cycles and min final extension at °c. the cycles consisted of denaturation at °c for s, annealing at °c for min, and extension at °c for . min ϩ s/cycle. the extension time for gene products longer than nt was performed for . min ϩ s/cycle. two overlapping fragments, designated cf and ih (fig. a) , were cloned into the pcrblunt vector system (invitrogen). plasmid dna from selected cf and ih clones were purified using a plasmid purification kit (qiagen) according to the manufacturer's instructions and confirmed by enzymatic restriction and sequence analysis. sequence analysis was performed with plasmid-specific primers at the automated sequence facility at eli lilly and co., indianapolis, in. prrsv sequences used for nucleotide and amino acid comparison included published genbank sequences u (vr- ; nelsen et al., ), af (respprrs; yuan et al., ) , af (primepac; yuan et al., ) , af (sp, shen et al., ) , af (pa , wootton et al., ) , and m . (lelystad; meulenberg et al., ) . cloned cf and ih fragments were joined at a unique saci restriction site to generate a -nt ch fragment (fig. a) , which comprised the last nt of orf a, all of orf b, and the first nt of orf . four ch clones (ch , ch , ch , and ch ) were confirmed for proper frame and orientation by restriction digest and sequence analysis. the ch clone was used as a template for further orf b nsp gene subcloning. gene fragments encoding predicted orf b nsps (nsp to ) were amplified by pcr and cloned into the pcrblunt vector using prrsv- -specific primers (table ) designed to contain restriction sites for cloning into the prokaryotic prset-b expression vector (invitrogen). prset-b bacterial expression vector cloning was performed following standard techniques (sambrook, ) . briefly, the gene fragments were inserted into the bamhi and kpni sites of the prset. the recombinant prset plasmids containing orf b nsp genes were confirmed for proper orientation and frame by enzymatic restriction analysis and by sequencing the region at the insertion sites. the recombinant plasmids were designated pt-prrsvnspn, where n corresponded to the number of the cloned orf b nsp. expression analysis of predicted orf b nsp, optimization of protein expression, and protein purification the prset vector system allows the expression of recombinant proteins via transcription by iptg-induced t polymerase. orf b nsps were expressed as recombinant proteins attached at their amino-terminus to a tag containing a polyhistidine peptide, the antigenic peptide t . , and an enterokinase cleavage site. ten to fifty nanograms of recombinant prset-b plasmid dna containing the corresponding orf b fragments were placed in e. coli bl (de )plyss-or bl (de )plyse-competent cells by bacterial transformation following the manufacturer's recommendations (invitrogen). expression of soluble recombinant proteins was optimized by culturing transformed bacterial clones at various temperatures, in different culture media, and by varying iptg-induction times. recombinant protein expression was confirmed by western blot analysis of crude protein extracts with a chemiluminescence detection kit (boehringer mannheim) using selected antibodies, including a monoclonal antibody to the polyhistidine peptide (clontech), a t . tag antibody (novagen), and peptide-specific polyclonal antisera generated to prrsv- nsp , , and (described below). after optimal conditions were determined, soluble recombinant proteins were purified from bacterial extracts by affinity to cobalt (novagen) or ni-nta (qiagen) resins according the manufacturer's recommendations. recombinant proteins were eluted with mm imidazole. eluted fractions were subjected to electrophoresis, followed by coomassie blue staining, and western blot analysis. fractions containing the purified recombinant protein of interest were pooled and dialyzed against mm tris ph . containing % glycerol and mm dtt. purified proteins were stored at Ϫ °c until further analysis. purified recombinant protein concentrations were determined by colorimetric assay (smith et al., ) using the bca protein assay reagent kit (pierce) with bovine serum albumin (bsa) as standard. antigenic regions for development of antipeptide sera were selected by computer analysis of peptide hydrophobicity/antigenicity indices of the predicted amino acid sequences of prrsv- nsp , , and genes. the amino acid sequences of the peptides used to generate the antibodies were (c)hrpstypaknsmagingrfptkd for nsp , (c)eqgltpldpgryqtrrg for nsp , and (c)reylddrere for nsp . peptide synthesis, khl conjugation, polyclonal rabbit antibody production, and affinity purification of antibodies were contracted with zymed laboratories, inc. affinity-purified antibodies were preadsorbed with lysates of porcine alveolar macrophages, marc- , and e. coli cells at °c overnight, clarified by centrifugation, filtered through a . -nm filter, and aliquots stored at Ϫ °c. optimal antibody concentration was determined by testing serial dilutions of antibodies by western blot of crude protein extracts. analysis and characterization of ntpase activity of prrsv- nsp (helicase) the functionality of the recombinant nsp helicase was initially determined by assessing ntpase activity on standard thin layer-chromatography (gwack et al., ) . purified protein samples were incubated with . ci of ␣- p-labeled ribonucleotide in l of a solution containing mm dtt, mm nacl, and g/ml bsa at different conditions. the samples were analyzed under various divalent ion concentrations, temperatures, time periods, protein concentrations, ph conditions, and buffer systems. the divalent ions tested included mgcl , mgso , mncl , and mnso . different ph conditions were tested using buffers containing mm mes, mm pipes, mm hepes, or mm tris. reaction products ( l) were spotted onto polyethylenimine (pei)-cellulose f plates (em science). chromatographs were developed in . m kh po (ph . ), dried, autoradiographed, and analyzed by densitometry or phosphoimaging. the atp bioluminescence assay kit cls ii (boehringer mannheim) was used for quantitative analysis of nsp atpase activity. in this assay, the bioluminescent signal emitted by the oxidation of luciferin is directly proportional to the atp concentration present in the test sample (mcelroy and deluca, ) . this assay was performed in triplicate at conditions described above ( -l reactions) with . to m atp (promega) as substrate. for kinetic analysis, the level of atp hydrolysis produced by the recombinant nsp helicase was measured at different concentrations of atp and at various incubation times. after incubation, the helicase was inactivated at °c for min and samples were diluted with vol of mm tris-hcl ph . buffer containing mm edta. diluted samples ( l) were transferred in triplicate to microfluor b -well flat-bottom plates (dynex technologies inc. this assay was performed using nm protein in l reaction buffer containing mm tris ph . , mm mgcl , g/ml bsa, mm nacl, mm dtt with various concentrations of atp at °c for min. the -min incubation was selected because it was within the range at which the rate of atp hydrolysis was linear (fig. b) . the k m of prrsv- helicase atpase activity was determined by nonlinear regression analysis of the michaelis-menten equation using the prism program version . (graphpad software) according to the method described by motulsky ( ). duplexed rna substrates were prepared using both synthetic rna oligos and in vitro transcribed rna. the synthetic oligonucleotides Ј-gggagtagctccaattcgccc- Ј (ss ), Ј-tttttttttttttttgggcgaattggagctactccc- Ј (ls Ј), Ј-gggcgaattggagctactccctttttttttt-ttttt- Ј (ls Ј), Ј-cgagccattctaggtcaaaccaatt-gccgccgtcgactt- Ј (ss - Ј), and Ј-actagtggtt-tcacctagaatggctgcgtcccttcttttcctc- Ј (ls - Ј) were purchased from annovis (aston, pa). the ss and ss - Ј were labeled at the Ј end with [␥- p]-atp (amersham, ci/mmol) using t polynucleotide kinase and purified using the qiaquick nucleotide removal kit (qia-gen). duplexes were generated by annealing the labeled short strands with -fold excess unlabeled long strands as described (seybert et al., b) . the following substrates were obtained by annealing rna oligos for min at °c: Ј duplex (ss ϩ ls- Ј), Јduplex (ss ϩ ls- Ј), Ј Јduplex (ss - Ј ϩ ls - Ј). in vitro transcribed rna was generated using plasmids pgem-l and pgem-s as templates. dna fragments were generated by rt-pcr amplification of strain vr- Ј-untranslated region from a previously prepared clone (ptvrsgorf ; unpublished data) using adapter primers ( Ј- -apai: Ј-actag-ggcccttaacaaaaaaaaaaaaaaaaaaa; Ј-utr-ndei: Ј-actagcatatggcactagtgattccggaat; Ј- -apai: Ј-actagggcccttaattggcgagaac-catgcggc) to generate Ј-ttaattggcgagaaccat-gcggccgaaattaacaaaaaaaaaaaaaaaaaaagcg-gccgcgaattccggaatcactagtgcca- Ј (l fragment) and Ј-ttaacaaaaaaaaaaaaaaaaaaagcggccgcgaatt-ccggaatcactagtgcca- Ј (s fragment). after apai and ndei digestion, these fragments were cloned into the respective sites of pgem-t to produce two plasmids (pgem-s and pgem-l). these plasmids were subsequently digested with either apai (sp orientation), saci (t orientation), or nsii (t orientation), purified using the qiaquick pcr purification kit (qiagen), and blunt-ended prior to in vitro transcription. in vitro transcription was performed with sp and t polymerases using ribomax large scale rna production system (promega). pgem-s was transcribed in the presence of [␣- p]-utp (amersham, ci/mmol). transcripts were treated with dnase i and purified by two rounds of phenolchloroform-isoamyl alcohol extraction, followed by two rounds of chloroform-isoamyl alcohol extraction. the substrates Ј Јduplex# (l-sp -apai ϩ s-t -saci), Ј Јduplex# (l-t -saci ϩ s-sp -apa ), Ј Јduplex# (l-t -nsii ϩ s-sp -apai), and Ј Јduplex# (l-sp -apai ϩ s-t -nsii) were generated by annealing the short radiolabeled transcript with -fold excess unlabeled long strand as described (seybert et al., b) . the unwinding assay was performed in l reactions containing m protein, m substrate in mm tris buffer (ph . ), or hepes buffer (ph . ) containing mm mgcl , mm dtt, g/ml bsa, % glycerol, mm atp, and mm nacl. reactions were incubated at °c for h and stopped by adding l of ϫ loading buffer ( % sds/ % ficoll/ mm edta/ . % bromphenol blue). controls included in each assay were native (annealed) and denatured (heated at °c) substrate in the absence of helicase. the reaction products were analyzed by native tbe-polyacrylamide gel ( - % gradient gel, bio-rad) electrophoresis and autoradiography. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus structural polypeptides of the american (vr- ) strain of porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambdal protein characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot nidovirales: a new order comprising coronaviridae and arteriviridae isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicative-form rnas complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis rnastimulated atpase and rna helicase activities and rna binding domain of hepatitis g virus nonstructural protein characterization of coronavirus rna polymerase gene products identification of an atpase activity associated with a -kilodalton polypeptide encoded in gene of the human coronavirus e isolation and genome characterization of porcine reproductive and respiratory syndrome virus in p.r atpase, gtpase, and rna binding activities associated with the -kilodalton protein of turnip yellow mosaic virus virus-encoded rna helicases enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line the serine protease and rna-stimulated nucleoside triphosphatase and rna helicase functional domains of dengue virus type ns converge within a region of amino acids firefly and bacterial luminescence: basic science and applications lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav infectious transcripts from cloned genome-length cdna of porcine reproductive and respiratory syndrome virus analyzing data with graphpad prism nucleoside triphosphate specificity of firefly luciferase comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus genetic variation in the prrs virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents analysis of orf in european porcine reproductive and respiratory syndrome virus by long rt-pcr and restriction fragment length polymorphism (rflp) analysis lactate dehydrogenaseelevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses a steady-state and pre-steady-state kinetic analysis of the ntpase activity associated with the hepatitis c virus ns helicase domain atpase and gtpase activities associated with semliki forest virus nonstructural protein nsp molecular cloning. a laboratory manual biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases the human coronavirus e superfamily helicase has rna and dna duplexunwinding activities with Ј-to- Ј polarity determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion measurement of protein using bicinchoninic acid the Ј end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease proteolytic processing of the n-terminal region of the equine arteritis virus replicase the coronaviruslike superfamily proteolytic processing of the replicase orf a protein of equine arteritis virus the arterivirus nsp protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the arterivirus nsp protease is the prototype of a novel group of chymotrypsin-like enzymes, the c-like serine proteases the arterivirus replicase. the road from rna to protein(s), and back again the molecular biology of arteriviruses rna-stimulated ntpase associated with the p protein of the pestivirus bovine viral diarrhea virus processing of the equine arteritis virus replicase orf b protein: identification of cleavage products containing the putative viral polymerase and helicase domains an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate recombination between north american strains of porcine reproductive and respiratory syndrome virus complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains virus-encoded proteinases and proteolytic processing in the nidovirales we thank drs. stephen wessman and randall levings from the national veterinary services laboratories (nvsl, ames ia) for providing prrsv isolates and the marc- cell line. we appreciate dr. thomas w. molitor from the university of minnesota (st. paul, mn) for kindly providing prrsv isolates and anti-prrsv sera useful in the characterization of prrsv isolates. the technical advice of drs. elcira villareal and jesus gutierrez is deeply appreciated. we thank the personnel from the sequencing facility at eli lilly and co. for help in the automated nucleotide sequencing. the technical assistance of anita jackson and the preparation of material transfer agreements by the lilly's legal department are greatly appreciated. financial support for these studies was provided by elanco animal health, a subsidiary of eli lilly and co., greenfield, in. key: cord- - o viv authors: rahe, michael c.; murtaugh, michael p. title: mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o viv the adaptive immune response is necessary for the development of protective immunity against infectious diseases. porcine reproductive and respiratory syndrome virus (prrsv), a genetically heterogeneous and rapidly evolving rna virus, is the most burdensome pathogen of swine health and wellbeing worldwide. viral infection induces antigen-specific immunity that ultimately clears the infection. however, the resulting immune memory, induced by virulent or attenuated vaccine viruses, is inconsistently protective against diverse viral strains. the immunological mechanisms by which primary and memory protection are generated and used are not well understood. here, we summarize current knowledge regarding cellular and humoral components of the adaptive immune response to prrsv infection that mediate primary and memory immune protection against viruses. porcine reproductive and respiratory syndrome virus (prrsv) is the most severe enemy of porcine health and wellbeing. the highly mutable, enveloped, rna virus was discovered nearly years ago but, while extensive research has been carried out and many vaccines have been developed, there is still no reproducible immunological intervention that develops a broadly protective immune response against virulent prrsv. prrs disease was first described on farms in north carolina in the usa at the end of the s. outbreaks were marked by reproductive losses, post-weaning pneumonia, and increased mortality in growing pigs. initial efforts to identify an etiological agent responsible for the new disease syndrome were unsuccessful, leading to the disease being temporarily designated mystery swine disease (msd) in north america. koch's postulates for msd were fulfilled in with a previously unidentified rna virus discovered in europe, named lelystad virus [ , ] . the discovery was quickly followed by isolation of the virus, initially referred to as swine infertility and respiratory syndrome virus or sirs virus, in north america [ ] . the name prrsv was introduced in and encompasses prrsv- (genotypes first isolated in europe) and prrsv- (genotypes first isolated in north america) [ , ] . today, both virus types are globally distributed, with prrsv- viruses predominantly in europe and prrsv- viruses largely in north america, asia and south america [ ] . recent discovery of multiple arteriviral nucleotide sequences in nonhuman primates has led to a reclassification of prrsv as two distinct viruses, prrsv- and prrsv- [ ] . here, we use the generic prrsv to refer broadly to both viruses when evidence indicates that are equivalent, and the specific prrsv- and prrsv- is used when a distinction is desired. the reasoning is based on the many similarities of the two viruses in fine details of genome structure and organization, transcriptional strategy, host preference, clinical signs of disease, and prrsv infects cells of the macrophage/monocyte lineage, including dendritic cells [ ] [ ] [ ] [ ] . permissive cells express cluster of differentiation (cd) , a hemoglobin-haptoglobin scavenger, which is the necessary receptor for prrsv infection and replication [ ] [ ] [ ] . macrophages and dendritic cells are common members of the mononuclear phagocyte system that plays a varied, and important, role in many aspects of tissue remodeling, development, immunity and immunopathology [ ] . classically designated as part of the innate immune system, these leukocytes are critical for the development of a productive adaptive immune response. macrophages and, particularly, dendritic cells take up and present antigen to t cells and b cells, thus initiating an adaptive immune response against the presented antigen [ , ]. if a pathogen is able to infect and destroy, manipulate, or maintain itself within macrophages or dendritic cells, it then has the potential to modulate the immune response into a favorable situation for its own replication and survival. therefore, many pathogens employ strategies for macrophage infection as a way to make the host more amenable to infection. recent research into mycobacterium tuberculosis (mtb) has shown that, after phagocytosis, the bacterium arrests phagosome maturation and intra-phagosome lipolysis resulting in mtb survival and an increased supply of nutrients for growth [ , ] . human immunodeficiency virus (hiv) infects macrophages to establish reservoirs within the host for the chronic stage of the disease when cd + t cells are largely depleted and neutralizing antibodies may be present [ ] [ ] [ ] . leishmania major is a protozoan which infects phagocytes to subvert the immune system. the parasite expresses glycoprotein (gp) , a multifaceted surface-expressed pathogenicity factor that is responsible for preventing antigen presentation and killing by natural killer (nk) cells [ ] [ ] [ ] . indeed, there are many more examples of burdensome pathogens which target phagocytic cells, especially macrophages and dendritic cells, in an attempt to gain a foothold within the immune system and allow for unchecked survival and replication [ ] [ ] [ ] . prrsv is one of these pathogens. the ability of prrsv to subvert the immune system has not been investigated as extensively as more prominent pathogens of humans, such as hiv. prrsv has been shown to inhibit the production, or the downstream effects, of type interferons, particularly interferon (ifn)-α, on intracellular signaling [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, multiple prrsv proteins (nonstructural protein (nsp) , nsp , nsp , nsp , nsp and nucleocapsid) have been reported to possess interferon inhibiting abilities. in addition, a number of in vivo experiments have reproduced earlier in vitro findings showing that interferon-α is inhibited during the early stages of prrsv infection [ , , ] . while the impact of type interferon suppression is likely to create a favorable environment for the virus to replicate and survive in phagocytic cells, it is still unclear what effect, if any, suppression of type interferon activity has on the adaptive immune response to infection [ ] . future investigations could clarify the relative contributions of viral proteins on modulation of interferon production and their impacts on viral growth, survival, and the subsequent development of the adaptive immune response. apart from interfering with interferon expression, prrsv has also displayed the in vitro ability to subvert the immune system by spreading from cell to cell. recent work has uncovered the ability of the virus to spread infectious viral rna, several replicases, and certain structural proteins between cells via intercellular nanotubules [ , ] . while this activity theoretically allows for prrsv to avoid neutralizing antibodies, the presence and significance of this mechanism in prrsv pathogenesis has yet to be fully elucidated. future studies are needed to determine if this process operates in naturally permissive macrophages and dendritic cells, if it can be interrupted, for example by intracellular antibodies, and what effect it might have on viral propagation [ , ] . vaccines depend upon innate immune stimulation to promote effective adaptive immune response to antigen, resulting in production of antibodies and cytotoxic t cell responses. the ability of a pathogen to successfully infect and replicate within innate immune cells makes the development of a protective immune response more difficult. as a result, the generation of effective vaccines against pathogens that target immune cells is fraught with challenges. extensive variation in viral genetics, primary immune responses, and cross-protection indicates that much remains to be learned about cellular pathogenesis in order to arrive at better immunological solutions. immunosuppression refers to suppression of the immune system and its ability to fight infection. hiv and infectious bursal disease virus are examples of viral infections that destroy entire lymphoid cell populations that ablate or disable adaptive immune responses. lymphoproliferative cancers block cellular differentiation and deprive the body of mature, effector lymphocytes, thus causing immunosuppression in a different manner. prrsv does neither; infection does not lead to severe lymphoid depletion or ablation, and it does not interfere profoundly with lymphocyte differentiation or maturation. leukocyte perturbations in lymphoid tissues are associated with prrsv infection, suggesting that adaptive immunity might be weakened, though not destroyed [ ] [ ] [ ] [ ] [ ] [ ] . the immune system also maintains peripheral tolerance to self and commensal bacteria through immunosuppressive mechanisms that include regulatory t cells (tregs), characterized as cd + cd + forkhead box p (foxp ) + t lymphocytes [ ] . treg suppressive properties were discovered when thymectomized or treg-depleted mice succumbed to autoimmune reactions [ , ] . tregs suppress effector and effector memory t cell proliferation by cytokine deprivation leading to polyclonal apoptosis, and by suppression of antigen presenting cells by cytotoxic t lymphocyte-associated antigen- (ctla- ) and other mechanisms [ ] . studies in prrsv infections give an ambiguous picture about the role of tregs. prrsv- strains are reported to induce a strong treg response which included transforming growth factor (tgf)β- secretion in vitro as well as in vivo [ , ] . other studies did not show treg responses to infection with either prrsv- or prrsv- [ , ] . interleukin- (il- ), an immunosuppressive cytokine expressed by various cell types including tregs, was induced by prrsv- vaccination in weaned pigs in one study, but was not induced in weaned or adult pigs in another study [ ] . additional in vitro and in vivo studies reported il- mrna transcription and cytokine production after prrsv infection [ ] [ ] [ ] . however, kinetic analysis in serum of viremic pigs of various ages showed that elevated il- levels were primarily a function of age and were not associated with infection status [ ] . the only exception was in weaned pigs infected with a virulent virus, in which a transient increase was associated with viral pathogenesis [ ] . on balance, the immunological evidence for prrsv inducing a state of immunosuppression does not appear to be compelling. secondary infections following prrs disease outbreak in swine herds, suggesting a reduced ability to fight infection, is an alternative indicator of immunosuppression. an early study showed concurrent pulmonary bacterial infections in % of prrs cases [ ] . however, the study did not determine if bacterial infections were present before the prrs outbreaks. the immunosuppression question also was addressed in more controlled settings using dual infection models with prrsv and various bacterial species. a summary of published literature in showed no predisposition to bacterial disease in of coinfection models, three ambiguous outcomes, and four cases in which severity of disease was increased [ ] . more recent studies found a positive association between prrsv infection and replication of porcine circovirus (pcv ) or swine influenza virus [ , ] . it is possible that bacterial infections in swine herds increase following prrs outbreaks due an increased burden of viral infection on host resilience to pathogen burden. subclinical viral and bacterial infections are common, with pcv , salmonella enterica, haemophilus parasuis, various mycoplasma species, leptospira, and escherichia coli being examples. control of infection is maintained by a combination of immune resistance to microbial replication and tissue tolerance to damage. in a coinfection model of influenza virus and legionella pneumophila, it was clearly demonstrated that l. pneumophila infection was subclinical in healthy mice, but was lethal in the presence of influenza virus [ ] . overwhelming disease was due to loss of tissue resilience, since the bacterial load was unchanged [ ] . this model might account for mortalities observed in experimental swine following prrsv exposure [ ] . given the variable results of prrsv coinfection models in swine and an alternative mechanism for increased disease in prrsv-infected herds, generalized immunosuppression does not appear to be a key feature of prrsv pathogenesis. prrsv, like many viruses, has developed countermeasures to host immune responses that enable it to survive and replicate for extended periods of time before the infection is resolved. prrsv modulation of intracellular antiviral defense mechanisms has been reviewed extensively [ ] . the effects of prrsv infection on adaptive immune response, i.e., antigen-specific t cell, b cell, and antibody responses, are less well characterized. the antiviral response of t cells to prrsv, examined primarily by the ifnγ enzyme-linked immunospot (elispot), appears to develop slowly over a period of weeks, and is not associated with changes in viral loads in blood or in infected lung and lymphoid tissues [ , ] . peripheral blood mononuclear cells (pbmc) from young, weaned pigs show limited ifnγ responses even when stimulated by phytohemagluttinin, which might account for the low anti-prrsv responsiveness after re-stimulation in vitro [ ] . however, pbmc from growing pigs and mature sows, which showed higher levels of ifnγ sensitivity, still showed limited responsiveness [ ] . these findings indicate that prrsv may interfere with specific cell-mediated immunity, but more direct evidence is needed for a fuller understanding. by contrast, the interaction of prrsv with pigs does not appear to retard or attenuate the development of humoral immunity or b cell differentiation. induction of antibody responses to prrsv proteins, both structural and non-structural, occurred in the same time frame as antibody responses to keyhole limpet hemocyanin (klh), an irrelevant protein antigen [ ] . the antibody response to klh was also the same in the presence or absence of prrsv infection [ ] . similarly, prrsv infection did not inhibit cellular or humoral immune protection in response to pseudorabies virus vaccination [ ] . thus, the adaptive b cell response is not delayed or suppressed by prrsv. an extended viremia and prolonged survival in lymphoid tissues is characteristic of prrsv infection. these features show that prrsv has mechanisms of immune avoidance that are not present in viruses such as influenza virus and foot and mouth disease virus, in which sterilizing immunity is achieved within - days. it appears from the findings of field observations and experimental investigations that some type of prrsv-specific t cell interference is present, whereas specific b cell inhibition or a generalized state of immunosuppression are not immunological hallmarks of prrsv infection. the antibody response to prrsv typically dominates discussions of prrsv immunity, as neutralizing antibodies are the crucial component of immune-mediated protection against most viral infections [ , ]. as a result, shortly after the identification of prrsv as the causative agent of mystery swine disease, there was a strong push to identify the presence and dynamic response of neutralizing antibodies against prrsv and then to characterize their specificity for prrsv variants. early work suggested that neutralizing antibodies against homologous prrsv could be found as early as - days after inoculation [ ]. however, this was likely the non-affinity matured immunoglobulin (ig)m response, as anti-swine igm ablated the previously observed neutralizing activity. subsequent research showed that the high affinity neutralizing igg response, detected at around - days post-inoculation, is specific for the inoculating virus with partial neutralizing activity against heterologous viruses [ ] [ ] [ ] [ ] [ ] . following the identification of prrsv neutralizing antibodies, the effectiveness of immunoglobulins in protecting against infection was evaluated with passive transfer studies. these experiments displayed the effectiveness of neutralizing antibodies at preventing clinical infection and disease against homologous challenge [ , ] . however, these studies also showed that immune protection can be quite limited, especially between prrsv- and prrsv- [ ]. within prrsv- or prrsv- , protection against homologous inoculation is consistently solid, whereas protection against heterologous challenge is variable for unclear reasons [ - ]. however, genetic similarity, based primarily on orf sequence comparisons, shows no relationship with degree of protection [ ]. these results appeared to explain the potential field problem, in which vaccinated or live virus inoculated animals become infected with a variant prrsv genetically different enough from the inoculating strain to evade the immune system, propagate, and then cause disease. hence, ever since the mutability, antigenic variability, and resultant immunological elusiveness of prrsv were first appreciated, a broadly neutralizing antibody response to prrsv has been coveted by immunologists and practitioners [ ] . recent research shows that there are animals capable of developing a broadly neutralizing antibody response to genetically disparate viruses [ , ] . however, this immune capability has only been found in a proportion of animals in groups of similar genetics age, sex, and exposure history [ ] . the seemingly random ability of some animals to develop broadly neutralizing antibodies suggests that the inherent variation of the adaptive immune response may play a role in conferring broadly neutralizing capabilities to certain animals. investigations into this ability are needed at the lymphocyte level and while the obvious target is the b cell, t cells cannot be overlooked, as the induction of a humoral immune response requires antigen-specific t cell driven help [ , ] . therefore, animals able to develop a strong neutralizing antibody response would require both b cells and t cells that are capable of recognizing neutralizing epitopes. the conditions needed to achieve cross-neutralizing antibody production are not known, but may involve multiple exposures to the same or different virus isolates. sows with high titered, broadly neutralizing antibodies were found in herds with multiple exposures to virulent field viruses [ ] . in an experimental study, cross-neutralization was reported in animals exposed first to a prrsv vaccine strain followed by homologous or heterologous virus challenge [ ] . however, the majority of data analyzed were below the neutralization assay cutoff. duration of viremia, up to days, was linked with increased breadth of neutralizing antibodies following a single viral infection [ ] . however, since cross-neutralization activity and titer data were not presented, it was not possible to further interpret the results. the animals were not subsequently challenged, so it is not known if the cross-neutralizing activity in serum was predictive of protection. other studies showed that significant neutralizing antibody responses are not commonly observed during viremic infection of young pigs, as well as in adult sows [ , [ ] [ ] [ ] . recently, vaccinology research in hiv has shown that sequential immunizations, tailored for specific stages of the immune response, may be useful for inducing broadly neutralizing antibodies [ ] [ ] [ ] . the approach is based on the finding that early immune responses to hiv resulted in neutralizing antibodies against the circulating virus which quickly led to immune escape of the virus and the ineffectiveness of generated antibodies. the antibody-resistant virus then stimulated a secondary antibody response which again selected for antibody resistant virus. this virus-antibody hide and seek continued, eventually resulting in the selection of several neutralization targets of the virus as well as the generation of broadly neutralizing antibodies [ ] [ ] [ ] . cloning of the antibodies showed that somatic mutations are generally necessary for antibody neutralizing capabilities against hiv- [ , ] . these findings have shown that the b cell response of the host adapts in the germinal center as the virus evolves, suggesting that tailored sequential immunization could lead to the development of a broadly neutralizing antibody response [ ] . the consistent generation of a broadly neutralizing antibody response to prrsv on the herd level has evaded the swine health industry since the emergence of prrsv. there are multiple proposed mechanisms by which prrsv may evade or inhibit the development, or the effectiveness, of a neutralizing antibody response, such as glycan shielding of envelope glycoprotein (gp) or gp [ , ] , the existence of decoy epitopes in gp [ ] , lymphocyte dysregulation [ ] , and inhibition of the innate immune response [ ] . comprehension of defense mechanisms employed by prrsv makes the development of a broadly neutralizing immune response appear to be a daunting task. however, as previously shown, some animals are capable of developing such a response. simply, the key to adapting the immune phenomenon of some animals to a vaccine capable of inducing broadly protective immunity in many animals lies in identifying conserved epitopes on surface proteins which are necessary for infection. while the purported targets of neutralization have been extensively discussed in recent reviews, it is worth noting that several epitopes on the membrane (m) protein, gp , gp , gp , and gp , have been shown, or implicated, to harbor neutralizing activity [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, knocking out only cd in the pig is sufficient to render animals non-susceptible to prrsv infection and replication [ , , ] . it is proposed that following endocytosis, cd associates with the virus within the endosome, resulting in uncoating of the virus and the release of the viral genome into the cellular cytoplasm [ ] . since cd is necessary for viral infection and replication, the logical next step is to identify the conserved regions of viral surface proteins, most likely the minor glycoproteins (gp , gp , and gp ), that interact with cd [ , ] . traditionally, the non-neutralizing antibody response to prrsv has been considered useful only for its ability to identify if an animal had been exposed and seroconverted to virus. indeed, there are many structural and non-structural proteins of prrsv which make this possible through their ability to induce a robust humoral immune response [ , , ] . however, recent research on other pathogens has shown that non-neutralizing antibodies may play a much larger role in immunity than was previously appreciated [ ] [ ] [ ] [ ] . alternative antibody functions, such as antibody dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc), and antibody-dependent complement-mediated virolysis may be important in the clearance of virus and virally infected cells from an animal. to our knowledge, there are only two published papers investigating non-neutralizing antibody functions in the context of prrsv infection [ , ] . both of these in vitro studies utilized a prrsv- virus and failed to find an effect of adcc and cdc on infected cells. however, experiments focused on prrsv- viruses with extended time points beyond h are warranted. a more extensive review of non-neutralizing antibody functions can be found in the cited review [ ] . if antibodies are the most important effectors of the immune system against viral infection, then b cells that make the antibodies are the most important cells. previous research on the interaction between prrsv and the porcine b cell is contradictory. it has recently been suggested that prrsv infection results in lymphocyte apoptosis and immune impairment [ ] . several sources have shown that prrsv largely or exclusively induces a specific humoral response to infection [ , ] . other studies report that prrsv infection results primarily in polyclonal b cell activation leading to hypergammaglobulinemia and the development of immune complexes [ ] [ ] [ ] [ ] . the majority of work describing infection leading to polyclonal activation and hypergammaglobulinemia was performed in germ-free isolator piglets. this model is very effective for comparing b cell and antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [ ] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [ , ] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure ). the second line of defense is memory b cells (figure ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [ ] . antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [ ] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [ , ] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure ). the second line of defense is memory b cells (figure ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [ ] . currently, there is scant research on the memory b cell response to prrsv. strong memory responses have been shown against nsp , nsp , n, and the end of gp [ , ] . the specific memory b cells are abundant in tonsil, lymph nodes draining the lungs and reproductive tract, and spleen. unfortunately, there are many questions about the porcine memory response to prrsv which have yet to be answered, including if memory cell kinetics closely mimic antibody kinetics, the response of prrsv-specific memory pools upon homologous or heterologous viral challenge, and the importance of these cells in conferring protection against challenge. the development of sensitive and specific reagents, such as b cell tetramers, is a first step in being able to answer these critical questions. additionally, it is possible that the key to understanding the broadly neutralizing response to prrsv lies within circulating or lymphoid organ resident memory b cells. the potential to investigate these cells for identification of heavy and light chain antibody sequences is reviewed in rahe and murtaugh [ ] . plasma cells are terminally differentiated b cells responsible for making antibodies. apart from the immature plasmablast, two types of plasma cells have been defined in the mouse and human [ , ] . short-lived plasma cells quickly boost antibody titers while long-lived plasma cells maintain circulating antibody titers in the face of continual antibody degradation. mulupuri et al. identified prrsv-specific plasma cells in several secondary lymphoid organs, such as the spleen, tonsil, sternal lymph node, and inguinal lymph node [ ] . interestingly, no prrsv-specific or klh-specific plasma cells were found in the bone marrow of immune pigs [ ] . this was surprising, as the bone marrow has been long considered as the reservoir for long-lived plasma cells in both mice and humans [ ] [ ] [ ] . it then begs the question, do pigs have long-lived plasma cells and, if so, where do they reside? mulupuri et al. found prrsv and klh specific plasma cells in secondary lymphoid organs days after inoculation [ ] . however, these cells may not be "long lived" as the prolonged viremia of prrsv may result in a somewhat continuous stimulation of memory b cells resulting in the appearance of this plasma cell population in secondary lymphoid organs. it seems unlikely that pigs do not have long lived plasma cells, as the half-life of porcine antibodies in serum is, on average, approximately nine days [ , ] . therefore, without long lived plasma cells, pigs would quickly lose humoral protection as antibody titers waned. the identification of the anatomic location as well as the understanding of mechanisms for inducing a strong long lived plasma cell response may be important for future vaccine design as well as comprehending host-pathogen interactions. interestingly, even though neutralizing antibodies have historically garnered the majority of attention in prrsv immunology, it is well-known that pigs readily control infection in the absence of neutralizing antibodies. furthermore, viremia is reported in the presence of neutralizing antibodies [ , ] . therefore, there must be other facets of the immune system which effectively function to control infection and eliminate prrsv from the host. while some of this activity may be attributed to non-neutralizing functions of antibodies, the t cell response to infection demands further investigation. a recent prrs immunity review summarized previous research on functional t cell subsets, and prrsv epitope targets, as well as gaps in t cell immunity [ ] . here, we provide context for the understanding of novel results that have not been comprehensively reviewed. early research on the t cell response to prrsv identified a large, transient decrease in the cd + /cd + t cell ratio early, usually within the first week, in the course of infection [ ] . the change in this ratio could have been due to a temporary loss of cd + cells through apoptosis or to an increase in cd + cells due to antigen-specific proliferation [ ] . the importance of these findings for clearance of prrsv or protection from infection were not known at the time, and other explanations, such as fluxes in cell populations between spleen, other lymphoid tissues, and blood could not be discounted. experiments to address the helper t cell type /helper t cell type (th /th ) paradigm in the pig showed that prrsv induced a strong th response, as expected, identified in vivo by an increased expression of th -specification factor tbx (t-bet) in cd + cells [ ] . however, the finding is at odds with previously reports indicating that prrsv infection results in the production of il- , a cytokine classically associated with a th phenotype. similarly, monocyte-derived dendritic cells (mo-dcs) infected with prrsv down regulate swine leukocyte antigen (sla)-i, sla-ii, cd and cd as well as promote il- secretion over il- secretion [ ] . delineation of the th /th response to prrsv, elucidation of th /th -specific cytokine markers in swine, as well as identifying associated cytokine responses of dendritic cells within secondary lymphoid organs where t cell proliferation and differentiation is most likely to occur, would help to resolve these outstanding questions [ ] . the th cell has classically been identified, in mouse and human, as playing an important role in extracellular bacterial immunity through the production of the pro-inflammatory cytokines, il- a, il- f, and il- [ , ] . il- producing th cells are known to exist in the pig [ ] . the importance of this t cell subset in the context of prrsv infection has recently been investigated. a strain of chinese highly pathogenic prrsv (hp-prrsv) appeared to suppress th cells in the peripheral blood and lungs of pigs, resulting in an increased susceptibility to secondary bacterial infections [ ] . remarkably, the effect was prrsv strain-specific, as a non-hp prrsv strain failed to elicit the same response. future research into the t cell response to prrsv, especially with t cell tetramers and functional elispots, will be essential for the characterization of both cd + and cd + antigen specific t cells. understanding how antigen-specific t cells interact with both infected and uninfected antigen presenting macrophages and dendritic cells will be helpful for advancing the field of prrsv immunity. the natural killer cell is an innate lymphoid cell which can have a profound impact on adaptive immunity, but is also able to induce an early and rapid innate response against pathogens through a variety of mechanisms. nk cells produce cytokines, such as ifnγ, show cytotoxic activity against infected cells not expressing mhci, can induce dendritic cell maturation, and effect the destruction of infected cells in adcc [ ] . however, nk cells may deploy even more extensive and important functions in porcine immunity than are currently realized. an early clue that nk cells were involved in innate responses to prrsv was a sharp peak in serum ifnγ shortly after infection [ ] . the acute response was attributed to nk cells, as the result was deemed too early for a t cell response, and suggested that decreased viral burdens in the lung prior to humoral or t cell responses could be due to the function of nk cells. however, it is known that porcine macrophages are also capable of producing ifnγ in the presence of prrsv infection [ , ] . furthermore, prrsv appears to suppress the nk cell response without significantly affecting nk cell numbers [ ] [ ] [ ] [ ] . the cause of this suppression has yet to be determined, although viral proteins, rather than soluble factors from cells, may be responsible [ ] . potential roles of additional nk cell functions, such as adcc, in prrsv immunity are poorly understood [ ] . prrsv has tormented the health and wellbeing of swine worldwide since its discovery in the late s. unfortunately, after almost years of research into the porcine immune response to prrsv, there is still no effective means for inducing a broadly protective immune response at the herd level. the reasons for this failure are not completely known, but presumably include mechanisms by which the virus subverts the immune system. the ability of the virus to rapidly mutate while not losing fitness challenges the host immune system to keep pace. at the same time, infection of macrophages, a key player in immunoregulation, challenges both innate and adaptive immune cell mobilization as well as induction of a coordinated response that is needed for effective control and elimination of the virus. fortunately, foundational advances in the understanding of viral pathogenesis and immunity are enabling more informative investigations. the identification of cd as the necessary and sufficient receptor for infection supports the implications of broadly neutralizing antibodies that a conserved target is present on all prrsv. understanding how prrsv surface glycoproteins interact with cd should lead to the identification of conserved epitopes which are necessary for infection. if, as appears to be the case, there is only one conserved way into the cell, then there must be a conserved viral sequence, or structure, which enables viral entry. furthermore, the knowledge that pigs eventually develop sterilizing immunity, if given enough time, supports the concept that conserved epitopes exist on the virus. therefore, the study of mature animals, which have cleared the virus, may provide the key to understanding how the immune system eventually gets the upper hand on the virus and cures infection. even with seminal advances in several aspects of the study of prrsv, there remains much to be understood and clarified. currently, the published literature presents conflicting views on many aspects of prrsv adaptive immunity, especially related to t and b cell responses and the production, or inhibition, of cytokines in the face of infection. the continued development of antigen-specific reagents, of high sensitivity and specificity, is needed for understanding how the host responds to prrsv infection. furthermore, it is important that future prrsv studies focus on the relevant host animal, the conventional pig. while the study of this outbred animal species is perhaps challenging at times, it affords the ability to study the host-pathogen interaction in the only species in which the virus naturally interacts. additionally, knowledge gained about the immunology of conventional pigs will accelerate immunological elucidation of other pig-pathogen interactions. in conclusion, prrsv continues to be the most burdensome pathogen of pigs worldwide, due to its propensity for immune evasion and manipulation. however, the continued study of the porcine immune response to infection, with improved reagents and methods, will illuminate those aspects of the host-pathogen interaction that are now hidden. it is through these discoveries that the complex question that is prrsv will finally be answered. mystery swine disease in the netherlands: the isolation of lelystad virus experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions porcine reproductive and respiratory syndrome virus (porcine arterivirus) reorganization and expansion of the nidoviral family arteriviridae prrsv, the virus broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus the ever-expanding diversity of porcine reproductive and respiratory syndrome virus innate and adaptive immunity against porcine reproductive and respiratory syndrome virus chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses localization and fine mapping of antigenic sites on the nucleocapsid protein n of porcine reproductive and respiratory syndrome virus with monoclonal antibodies evolutionary diversification of type porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with mac- staining at days post-infection virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus genetic engineering alveolar macrophages for host resistance to prrsv interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity antigen-specific b-cell responses to porcine reproductive and respiratory syndrome virus infection porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread porcine reproductive and respiratory syndrome virus (prrsv) infection spreads by cell-to-cell transfer in cultured marc- cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection schwartz-cornil, i. rotavirus anti-vp secretory immunoglobulin a contributes to protection via intracellular neutralization but not via immune exclusion intracellular neutralization of viral infection in polarized epithelial cells by neonatal fc receptor (fcrn)-mediated igg transport the chinese highly pathogenic porcine reproductive and respiratory syndrome virus infection suppresses th cells response in vivo thymic depletion of lymphocytes is associated with the virulence of prrsv- strains identification of apoptotic cells in the thymus of piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus suppression of nk cell-mediated cytotoxicity against prrsv-infected porcine alveolar macrophages in vitro the comparative profile of lymphoid cells and the t and b cell spectratype of germ-free piglets infected with viruses siv, prrsv or pcv type porcine reproductive and respiratory syndrome virus infection mediated apoptosis in b-and t-cell areas in lymphoid organs of experimentally infected pigs the molecular mechanisms of regulatory t cell immunosuppression. front. immunol. , , study on cellular events in post-thymectomy autoimmune oophoritis in mice. ii. requirement of lyt- cells in normal female mice for the prevention of oophoritis t cell-mediated maintenance of natural self-tolerance: its breakdown as a possible cause of various autoimmune diseases induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus induction of inducible cd +cd +foxp + regulatory t lymphocytes by porcine reproductive and respiratory syndrome virus (prrsv) prrsv-infected monocyte-derived dendritic cells express high levels of sla-dr and cd / but do not stimulate prrsv-naïve regulatory t cells to proliferate european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine upregulation of interleukin- gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus cytokines transcript levels in lung and lymphoid organs during genotype porcine reproductive and respiratory syndrome virus (prrsv) infection changes in lymphocyte subsets and cytokines during european porcine reproductive and respiratory syndrome: increased expression of il- and il- and proliferation of cd -cd high concurrent respiratory infections in cases of prrs virus pneumonia. j. swine health prod prrs plus"-prrsv infection in combination with other agents; national pork board porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type (pcv ) subtypes pcv a and pcv b by prolonging pcv viremia and shedding in vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses role of tissue protection in lethal respiratory viral-bacterial coinfection pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (prrsv) are related to viral load in acute infection porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic antibody response to porcine reproductive and respiratory syndrome virus (prrsv) nonstructural proteins and implications for diagnostic detection and differentiation of prrsv types i and ii the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load mechanisms of t cell-b cell interaction quantitative analysis of porcine reproductive and respiratory syndrome (prrs) viremia profiles from experimental infection: a statistical modelling approach immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection the challenge of prrs immunology serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus immunization for hiv- broadly neutralizing antibodies in human ig knockin mice hiv- vaccines. hiv- neutralizing antibodies induced by native-like envelope trimers hiv- broadly neutralizing antibody precursor b cells revealed by germline-targeting immunogen analysis of a clonal lineage of hiv- envelope v /v conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors maturation pathway from germline to broad hiv- neutralizer of a cd -mimic antibody early antibody lineage diversification and independent limb maturation lead to broad hiv- neutralization targeting the env high-mannose patch broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals rational hiv immunogen design to target specific germline b cell receptors progress toward active or passive hiv- vaccination immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain interaction between innate immunity and porcine reproductive and respiratory syndrome virus the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies gp of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function porcine reproductive and respiratory syndrome virus entry into the porcine macrophage arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture comparison of antibody-dependent cell-mediated cytotoxicity and virus neutralization by hiv- env-specific monoclonal antibodies opportunities to exploit non-neutralizing hiv-specific antibody activity non-neutralizing epitopes induce robust hcv-specific antibody-dependent cd + nk cell responses in chronic hcv-infected patients the immune correlates of protection for an avian influenza h n vaccine in the ferret model using oil-in-water adjuvants porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis effector mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus polyclonal activation of b cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (prrsv)-infected pigs lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs porcine reproductive and respiratory syndrome virus subverts repertoire development by proliferation of germline-encoded b cells of all isotypes bearing hydrophobic heavy chain cdr polyclonal hypergammaglobulinemia and formation of hydrophobic immune complexes in porcine reproductive and respiratory syndrome virus-infected and uninfected pigs antibody repertoire development in fetal and neonatal piglets. xv. porcine circovirus type infection differentially affects serum igg levels and antibodies to orf in piglets free from other environmental factors antibody repertoire development in fetal and neonatal piglets. iv. switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification normalizing the environment recapitulates adult human immune traits in laboratory mice dirty mice might make better models immunological memory and protective immunity: understanding their relation heterogeneity in the differentiation and function of memory b cells interleukin- drives proliferation and differentiation of porcine memory b cells into antibody secreting cells humoral immunity due to long-lived plasma cells competence and competition: the challenge of becoming a long-lived plasma cell immunoglobulin synthesis by human lymphoid tissues: normal bone marrow as a major site of igg production antibody formation in mouse bone marrow. i. evidence for the development of plaque-forming cells in situ bone marrow is a major site of long-term antibody production after acute viral infection half-lives of immunoglobulins igg, iga and igm in the serum of new-born pigs half-life of porcine antibodies absorbed from a colostrum supplement containing porcine immunoglobulins an interferon inducing porcine reproductive and respiratory syndrome virus vaccine candidate elicits protection against challenge with the heterologous virulent type strain vr- in pigs virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (prrs) virus a novel lineage transcription factor based analysis reveals differences in t helper cell subpopulation development in infected and intrauterine growth restricted (iugr) piglets the role of porcine reproductive and respiratory syndrome virus infection in immune phenotype and th /th balance of dendritic cells species specialization in cytokine biology: is interleukin- central to the t h -t h paradigm in swine? interleukin (il)- and il- are coexpressed by th cells and cooperatively enhance expression of antimicrobial peptides influenza a inhibits th -mediated host defense against bacterial pneumonia in mice cd engagement strongly induces cd expression on porcine dendritic cells and polarizes the t cell immune response toward th natural killer cells in host defense against veterinary pathogens infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs expression of interferon-gamma and tumour necrosis factor-alpha in pigs experimentally infected with porcine reproductive and respiratory syndrome virus immunohistochemical staining of ifn-γ positive cells in porcine reproductive and respiratory syndrome virus-infected lungs porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs the authors declare no conflicts of interest. key: cord- -rp ugk authors: jing, huiyuan; song, tao; cao, sufang; sun, yanting; wang, jinhe; dong, wang; zhang, yan; ding, zhen; wang, ting; xing, zhao; bao, wenqi title: nucleotide-binding oligomerization domain-like receptor x restricts porcine reproductive and respiratory syndrome virus- replication by interacting with viral nsp date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: rp ugk porcine reproductive and respiratory syndrome virus (prrsv) causes one of the most economically important diseases of swine worldwide. current antiviral strategies provide only limited protection. nucleotide-binding oligomerization domain-like receptor (nlr) x is unique among nlr proteins in its functions as a pro-viral or antiviral factor to different viral infections. to date, the impact of nlrx on prrsv infection remains unclear. in this study, we found that prrsv infection promoted the expression of nlrx gene. in turn, ectopic expression of nlrx inhibited prrsv replication in marc- cells, whereas knockdown of nlrx enhanced prrsv propagation in porcine alveolar macrophages (pams). mechanistically, nlrx was revealed to impair intracellular viral subgenomic rnas accumulation. finally, mutagenic analyses indicated that the lrr (leucine-rich repeats) domain of nlrx interacted with prrsv nonstructural protein (nsp ) rdrp (rna-dependent rna polymerase) domain and was necessary for antiviral activity. thus, our study establishes the role of nlrx as a new host restriction factor in prrsv infection. porcine reproductive and respiratory syndrome (prrs) is one of the most economically important viral diseases affecting the swine industry worldwide since it was first reported in (harding et al., ; snijder et al., ) . the etiological agent of this devastating disease, prrs virus (prrsv), is a positive-sense single-stranded rna virus that belongs to the family arteriviridae, order nidovirales (loving et al., ) . the virus engages with the host cellular protein interaction network during infection, facilitating virus hijacking of the host molecular machinery to fulfill the viral life cycle (ke and yoo, ; lunney et al., ; rahe and murtaugh, ; shi et al., ; sun et al., ) . based on their genetic and antigenic differences, prrsv strains are classified into distinct genotypes, prrsv- (european type) and prrsv- (north american type) (han and yoo, ; murtaugh et al., ) . the ∼ kb viral genome contains at least open reading frames (orfs), which encode at least nonstructural proteins (nsp) and structural proteins (han and yoo, ) . among the nonstructural proteins, nsp contains an rna-dependent rna polymerase (rdrp) domain in its c-terminal portion, which is critical for viral rna synthesis, and replication efficiency (fang and snijder, ; yang et al., ; zhou et al., ) . in this regard, recent advances show that amino acids at positions , , and in nsp contribute to enhanced pathogenicity and determine the fatal virulence of the virus zhao et al., ) . in addition, two highly conserved t-cell epitopes have been identified in nsp , which may provide broad cross-protection against diverse prrsv strains (parida et al., ; rascon-castelo et al., ) . noteworthy, multiple novel antiviral treatments, including nsp specific camel single-domain antibodies, and sirna targeting nsp , as well as vaccine strategy of de-optimization of codon pair bias in nsp , significantly decreased prrsv replication in vitro and in vivo liu et al., ; xie et al., ; zheng et al., ; zhu et al., ) . finally, nsp was found to associate with at least prrsv encoded proteins, including nsp α, nsp β, nsp , nsp α, nsp β, nsp , nsp , nsp , and nucleocapsid (n) protein, validating the notion of this protein as a core component of the viral replication/transcription complex (rtc) liu et al., ; nan et al., ) . further exploring the interaction of nsp with host cellular proteins and analyzing the biological significance are necessary for fully understanding the replication mechanisms and pathogenesis of prrsv . nucleotide-binding oligomerization domain (nod)-like receptor (nlr) x (also known as clr . and nod ), a member of the nlr family proteins, is initially identified as key mediators of immune defense and inflammation (arnoult et al., ; coutermarsh-ott et al., ; eitas et al., ; imbeault et al., ; kang et al., ; kanneganti, ; koblansky et al., ; lupfer and kanneganti, ; moore et al., ; philipson et al., ; singh et al., ; soares et al., ; tattoli et al., ; ting et al., ; wang et al., ) . to date, accumulated evidence indicated that nlrx inhibits nf-κb (nuclear factor-kappa b) signaling, inflammasome activation, double-stranded rna (dsrna) activated kinase pkr and type i interferon (ifn) production but potentiates reactive oxygen species (ros) production and autophagy (abdul-sater et al., ; allen et al., ; feng et al., ; guo et al., ; hung et al., ; kim et al., ; lei et al., ; moore et al., ; o'neill, ; qin et al., ; stokman et al., ; tattoli et al., ; theus et al., ; xia et al., ; yin et al., ; zeng et al., ) . recently, nlrx was also identified to facilitate human immunodeficiency virus (hiv- ) (guo et al., ) , herpes simplex virus (hsv- ) (guo et al., ) , hepatitis c virus (hcv) (qin et al., ) and kaposi's sarcoma-associated herpesvirus (kshv) infection, whereas restricts influenza a virus (iav) (jaworska et al., ) , and hepatitis a virus (hav) (feng et al., ) replication. these conflicting reports about whether nlrx functions as a pro-viral or antiviral factor imply that the role of nlrx in viral infection is more complex than previously thought and remains to be clarified. until now, the specific role of nlrx in prrsv infection was scarcely known. thus, the aim of the present study is to seek evidence for a potential role of nlrx in prrsv infection. hek t and pams cells (jing et al., b) were cultured and maintained in rpmi- supplemented with % heat-inactivated fetal bovine serum (fbs). hela and marc- cells were cultured and maintained in dmem supplemented with % heat-inactivated fbs. prrsv strain hn (genbank: ay . ) is a highly pathogenic prrsv- (north american) strain, which was isolated from the lung of pigs suffering from "high fever" syndrome in henan province, china (cao et al., ) . the virus was amplified and titered in marc- cells. uv-inactivated prrsv was generated by irradiating the virus with short-wave uv light ( nm) for h. the loss of infectivity was confirmed by the inability of the uv-exposed virus to produce a cytopathic effect on monolayer of marc- cells. mouse monoclonal anti-ha, anti-flag, and anti-β-actin antibodies were purchased from abclonal biotechnology (china). anti-flag polyclonal antibody (macgene, china), anti-nlrx polyclonal antibody (proteintech, china), were purchased and used according to the manufacturers' recommendations. hrp-conjugated anti-mouse and antirabbit igg light (or heavy) chain specific antibodies (abbkine science, usa) were purchased and used according to the manufacturers' recommendations. a monoclonal antibody directed against prrsv n protein was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant n protein (jing et al., b) . the flag or ha epitope tag was amplified by pcr and cloned into the pcaggs-mcs vector to generate the pcaggs-flag or pcaggs-ha plasmid, encoding an n-terminal flag or ha tag (jing et al., b) . expression plasmids for flag-tagged nsp (full-length and truncated) were constructed by pcr amplification of the cdna from prrsv-infected marc- cells. plasmids encoding full-length and truncated nlrx (nm_ ) were constructed by pcr amplification using the specific primers listed in table . all constructs were confirmed by dna sequencing. the cells were transfected with expression plasmids using lipofectamine (invitrogen) according to the manufacturer's instructions. where necessary, empty control plasmid was added to ensure that each transfection receives the same amount of total dna. for sirna knockdowns, cells plated in -well plates and transfected with nm the indicated sirnas twice over a -h period by using lipofectamine . sirna sequences used are as follows: si-nlrx - # , ′-uugucaaucugcugcgcaa- ′; si-nlrx - # , ′-gugcugggc uugcggaaga- ′; si-nlrx - # , ′-gcaugucuuccgccgggau- ′; negative control sirna, ′-uucuccgaacgugucacgutt- ′ (table ) . prrsv titers were expressed as the tissue culture infectious dose (tcid ) per milliliter using the reed-muench method as previously described (jing et al., a) . briefly, marc- cells were seeded in -well plates, following, infected with serial -fold dilutions of prrsv samples in eight replicates. plates were incubated for - h before virus titers were calculated. total rna was isolated at the indicated time points using trizol reagent. qpcr was performed using sybr green real time pcr master mix (toyobo biologics, osaka, japan) in a lightcycler (roche table the sequences of primers used for construction of nlrx , and nsp protein mutants. sequence the restriction enzyme sites used for cloning are underlined in italics. the sequences of primers used for real-time pcr. primer names genebank sequence ( '- ') jing, et al. virus research ( ) - molecular biochemicals). individual transcripts in each sample were assayed three times. the pcr conditions were as follows: initial denaturation for min at °c, followed by cycles of s at °c, s at °c and s at °c. the fold change in gene expression relative to normal was calculated using the delta delta cycles to threshold (ΔΔct) method. gene expression in marc- cells and pams was normalized to that of gapdh and β-actin, respectively. primers were designed using the primer express software (version . ; applied biosystems, carlsbad, ca). absolute quantitative mrna levels were calculated using standard curves as previously described . cells cultured in -mm dishes were prepared by adding μl of × lysis buffer a ( mm tris−hcl [ph . ], % sodium dodecyl sulfate (sds), % dl-dithiothreitol, and % glycerol). the cell extracts were boiled for min, and then resolved with %- % sds-page. the separated proteins were electroblotted onto a polyvinylidenedifluoride (pvdf) membrane (millipore, billerica, ma). run for - h at v on ice. the western blotting was probed with specific antibodies. the expression of β-actin was detected with a mouse monoclonal antibody to demonstrate equal protein sample loading. densitometry quantification of protein bands of interest was performed using imagej software (table ) . hela cells seeded on microscope coverslips and placed in -well dishes were transfected with flag-tagged nsp expression plasmid when the cells reached approximately - % confluence. at h after transfection, cells were fixed with % paraformaldehyde for min, subsequently permeated with . %triton x- for min at room temperature. after three washes with pbs, cells were sealed with pbs containing % bovine serum albumin for h, followed by incubation with rabbit polyclonal antibody against nlrx and mouse monoclonal antibody against flag tag for h at room temperature separately. successively, cells were treated with fitc-labeled goat anti-mouse and cy -labeled goat anti-rabbit (invitrogen) antibodies for h, with dapi for min at room temperature. after washing with pbs, fluorescent images were acquired using a confocal laser scanning microscope (olympus fluoview ver. . , japan). to investigate the interactions between proteins, hek t cells were lysed in immunoprecipitation lysis buffer (ripa). after the lysates were incubated for h at °c with rnase and dnase, the lysate proteins were incubated overnight at °c with the indicated antibodies. protein a + g agarose beads ( μl; beyotime) were then added to each immunoprecipitation reaction for another h. the agarose beads were then washed three times and the captured proteins were resolved on %- % sds-page, transferred to pvdf membranes, and analyzed by immunoblotting. the results represent the means and standard deviations from three independent experiments. graphpad prism software (graphpad software, san diego, ca, usa) was used for data analysis using a twotailed unpaired t-test *p < . ; **p < . . our work has previously shown that prrsv infection up-regulates the expression of nlrx in marc- cells, a highly permissive cell line derived from epithelial cells of a monkey kidney (jing et al., ) . to ascertain this result in the main target cells of acute prrsv infection, porcine alveolar macrophages (pams) were infected with prrsv at an moi of . . real-time pcr was performed at h, h, and h post infection (hpi) to determine the expression levels of nlrx . as shown in fig. a , the data showed that nlrx expression was significantly upregulated at h, and increased at a steady-state level in prrsv-infected pams when compared to non-infected cells (> -folds). moreover, uv-inactived prrsv failed to induce nlrx mrna expression, indicating that the up-regulation of nlrx dependents on viral replication (fig. a) . next, pams were infected at different moi with prrsv for h before analysis for the mrna expression of nlrx . result indicated that prrsv up-regulated nlrx in a dose dependent manner (fig. b) . aiming to clarify whether prrsv induces nlrx expression in protein level, pams were infected at an moi of . with prrsv or uvinactived prrsv for the designated time. nlrx protein levels measured by western blotting reflected the changes in mrna observed, suggesting that endogenous nlrx expression was upregulated by prrsv infection (fig. c) . in order to investigate whether the upregulation of nlrx might have a correlation with prrsv infection, we transfected marc- cells with nlrx expression plasmid, followed by infection with prrsv. nlrx activity on viral infection was monitored by measuring prrsv titers in the culture supernatants collected at , and hpi. as shown in fig. a , overexpression of nlrx consistently reduced viral titers of prrsv at - hpi. next, marc- cells transfected with nlrx were infected with different moi of prrsv, a tcid assay was used to determine the effect of nlrx on the production of infectious prrsv particles. as expected, tcid data suggested that overexpression of nlrx inhibited prrsv proliferation, especially at low infection doses (fig. b) . since marc- cells are monkey derived though they are prrsv permissible, we wondered whether endogenous nlrx had the same effect on prrsv replication in primary pam cells. to this end, three pairs of sirna duplexes against nlrx were individually transfected into pams. the knockdown efficiency of these sirna was evaluated by real-time pcr. result indicated that si-nlrx - # achieved the highest efficiency (fig. a) . western blot analysis further showed that si-nlrx - # reduced the level of nlrx protein by ∼ % compared to the controls cells transfected with nc sirna (fig. b) . to investigate the function of endogenous nlrx in prrsv replication, pams were treated with nc sirna or si-nlrx - # followed table the sequences of primers used for quantification of total viral rna and sgrnas. sequence ( '- ') jing, et al. virus research ( ) - by prrsv infection. then viral titers were measured by tcid at - hpi. we observed that reduced nlrx protein expression was correlated with increased prrsv in pams (fig. c ). in aggregate, these data suggested that nlrx can potentially inhibit replication of prrsv in pams. nlrx contains a carboxy-terminal lrr domain, a central nod domain and a unique n-terminal card-related x domain (moore et al., ) (fig. a ). to better understand how nlrx impaired prrsv replication, we mapped the domains of nlrx required for this restriction. to this end, we transfected marc- cells with plasmids encoding either the full-length nlrx or its mutants prior to prrsv infection. progeny virus in culture supernatants was determined by tcid assay. the results showed that constructs lacking the x domain (Δx) impaired prrsv replication. whereas constructs lacking the lrr domain (Δl) did not. as shown in fig. b , construct encoding lrr alone or lrr in combination with nod domain also impaired prrsv replication, suggesting that lrr is the key domain of nlrx that impairs prrsv replication. the c-terminal lrr domain of nlrx has been shown to bind rna, and participate in regulation of steady-state levels of a subset of mitochondrial rna (feng et al., ; hong et al., ; singh et al., ) . the observations in fig. b led to our working hypothesis that nlrx modulated viral rna synthesis. to test this, increasing dose of nlrx was transfected into marc- cells followed by prrsv infection, qpcr was then performed to test the total viral rna levels. result in fig. c showed that nlrx inhibited the synthesis of viral rnas in a dose dependent manner. furthermore, knockdown of endogenous nlrx in pams increased the levels of viral total rna (fig. d) . the prrsv genome is a single-strand positive-sense rna flanked by the ′ and ′ un-translated regions (utrs). a set of ′-coterminal subgenomic rnas (sgrnas) are produced during infection (han and yoo, ) . thus, we evaluated the role of nlrx in viral sgrnas synthesize. these synthesized viral sgrnas were then quantified by qpcr after the infection of the marc- cells with prrsv. as shown in fig. e , the relative levels of viral grna and sgrnas, including grna, fig. . prrsv infection up-regulates nlrx expression in pams. (a) pams were infected at an moi of . with prrsv or uv-inactived prrsv for the designated time. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx . gene expression was normalized to that of β-actin. significant differences from the uninfected cells are denoted by asterisks (**) for p < . . (b) pams were infected at the indicated moi with prrsv for h. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx . (c) pams were infected at an moi of . with prrsv or uv inactived prrsv for the designated time. cell lysates were blotted with the indicated antibodies. sgrna to , apparently declined in nlrx -overexpressing cells compared to the control, but the relative levels of viral sgrna - had no significant differences. this result suggested the inhibitory role of nlrx in the process of synthesizing long sgrnas and genomic rna. nucleocapsid (n)-nsp interaction has been shown to be involved in the production of prrsv sgrnas . to explore the mechanism by which nlrx suppresses viral sgrnas synthesis, we investigated whether nlrx interacts with prrsv nsp /n protein. to this end, hek t cells were transfected with plasmids expressing flag-nsp /n and ha-nlrx and co-immunoprecipitated with flag antibody or ha antibody. even though no co-immunoprecipitation of n protein was detectable with overexpressed nlrx in hek t cells (data not shown), ha-nlrx was co-precipitated with flag-tagged nsp and vice versa (fig. a) . next, we investigated whether nlrx could colocalize with nsp . hela cells were transfected with plasmids expressing flag-nsp . confocal data revealed ectopically expressed flag-nsp and endogenous nlrx partially co-localized with each other in the cytoplasm (fig. b) . to identify the binding region within nlrx involved in the nsp -nlrx interaction, hek t cells were co-transfected with various combinations of ha-tagged full-length or deleted versions of nlrx and flag-tagged nsp . mapping studies by co-ip revealed that nsp interacted with the c-terminal lrr domain of nlrx , but not with its central nod domain or n-terminal x domains (fig. c ). prrsv nsp contains n-terminal rdrp domain and a c-terminal domain of unknown function zhao et al., ) . to further investigate which domain within nsp is required for binding with nlrx , plasmids expressing nsp mutants were constructed and their ability to interact with nlrx was assessed by co-ip. result showed that wt nsp and nsp c, but not the nsp n, bound to nlrx (fig. e) . the results showed that the rdrp domain of nsp is responsible for interaction with nlrx . nlrx was the first nlr shown to reduce type-i ifn production and facilitate viruses infection, by binding to mavs (mitochondrial antiviral signaling protein) on mitochondria and sting (stimulator of interferon genes) on endoplasmic reticulum (er), as demonstrated for sindbisvirus, hiv- , hsv- and kshv (guo et al., ; ma et al., ; moore et al., ; qin et al., ) . however, all of these functions reported for nlrx are not without controversy. for example, nlrx prevented iav-induced macrophage apoptosis. its deficiency led to increased pulmonary viral titer, inflammation, and reduced pulmonary function during iav infection (jaworska et al., ) . analogously, nlxr was required for immediate irf (interferon regulatory factor )-directed antiviral responses. replication of both hepatitis a virus (hav) and hepatitis c virus (hcv) was enhanced in nlrx -deficient hepatocytes (feng et al., ) . adding to these similarities, we show here that nlrx acts to restrict prrsv replication. based on these observations, it is possible that the effect of nlrx during viral infection is a doubleedged sword, potentially weakening irf mediated responses as well as presenting a hurdle to be overcome by the virus. nlrx and iav pb -f interaction is critical for the control of iav replication and inflammation, indicating that interaction between nlrx and viral component might be a key factor in determining the outcome of viral infections (jaworska et al., ) . our study shows that nlrx inhibits prrsv replication by suppression its rna synthesis. this is achieved by the association of prrsv nsp with lrr domain of nlrx . strikingly, c-terminal lrr domain of nlrx has been shown to possess a rna binding site (hong et al., ) . in this regard, nlrx interacts with rhinovirus (rv) rna in polarized airway fig. . enhancement of prrsv replication by nlrx knockdown in pams. (a) pams were transfected with a scrambled control sirna (nc) or three different sirna duplexes against nlrx for h. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx . β-action level was monitored as an internal control. (b) pams were transfected with nc sirna or sirna # against nlrx . after h, cell lysates were blotted with anti-nlrx or anti-β-actin antibody. (c) pams were transfected with the control sirna or the nlrx sirna for h and then infected at an moi of . with prrsv. virus titers were determined by tcid assay at , , and h post infection. epithelial cells (unger et al., ) . likewise, nlrx also binds hav genomic rna to suppress pkr activation (feng et al., ) . collectively, these findings support a model in which nlrx interacts with various viral factors to achieve an optimal immune state against diverse viruses. future studies will be necessary to assess whether the nsp of prrsv- interacts with nlrx . nsp , the key enzyme encoded by orf b for rna-templated rna synthesis (rdrp) (fang and snijder, ) , is closely related to the replication efficiency and pathogenicity for piglets (li et al., b; music and gagnon, ; xu et al., ; zhao et al., ) . as a consequence, several host proteins have been reported to interact with prrsv nsp . on the one hand, interaction between nsp and cellular annexin a , retinoblastoma protein (prb), ddx positively regulates the replication of prrsv in vitro, demonstrating that prrsv heavily relies on host cellular proteins to complete life cycle (dong et al., ; li et al., a; zhao et al., ) . on the other hand, recent literature indicated that the interaction of nsp with sumo e conjugating enzyme ubc and cellular protein interleukin- enhancer binding factor (ilf ) through its rdrp domain resulted in a significantly decrease of virus titers, indicating that cells utilize host antiviral factors as defense mechanisms to limit prrsv infection wen et al., ) . altogether, these results provide insights into the complex interactions of prrsv rdrp with the host proteome. noteworthy, the nsp c-terminal fragment consisting of amino acid residues - was also identified as the n protein binding region , strengthening the concept that the c-terminal rdrp domain is a common protein interacting platform. thus, the c-terminal rdrp domain of nsp might be promising drug targets for interfering with prrsv replication. even though our knowledge on the unique discontinuous transcription of arterivirus and coronavirus has rapidly grown over the last years, the molecular mechanisms controlling template switch still remain unclear (di et al., ; sola et al., ) . in coronavirus, rna helicase ddx was reported to benefit the synthesis of longer sgrnas by unwinding the hairpin loop structure of viral grna, implying that rna secondary structure may hinder the balance between shorter and longer sgrnas synthesis (wu et al., ) . in line with this, a recent study suggested that prrsv sgrna - , but not sgrna - , were impaired in helicase dhx -knockdown cells . in this study, we observed that ectopic expression of nlrx had no effects on fig. . nlrx suppresses the synthesis of viral subgenomic rnas. (a) schematic representation of full-length nlrx and corresponding deletion mutants is shown. protein motifs are indicated. (b) marc- cells were transfected with ha-tagged nlrx , or its deletion mutants. after h, cells were infected at an moi of . with prrsv for h. virus titers were determined by tcid assay. (c) marc- cells were transfected with an increasing amount of nlrx expression plasmid or control plasmid and then infected with prrsv (moi = . ) for h. prrsv total rna were measured by qpcr. (d) pams were transfected with control sirna or the nlrx sirna for h and then infected at an moi of . with prrsv. viral total rnas were measured by qpcr at h post infection. (e) marc- cells were transfected with the empty vector or nlrx expression plasmid for h and then infected at an moi of . with prrsv. the levels of viral rnas were monitored at hpi by qpcr. the viral rna levels in control cells were normalized to a value of . the abundance of shorter sgrna - . we therefore hypothesized that longer sgrnas transcript could stop upon reaching nlrx -nsp -rna complex, thereby switching to produce shorter sgrnas. further investigations will be required to dissect the nlrx -dependent mechanism for the effective control of continuous and discontinuous rna synthesis. in summary, these above findings indicate that nlrx inhibits the replication of prrsv by interacting with viral nsp in vitro, providing more knowledge for understanding the mechanisms associated with the replication regulation of prrsv. significant effort is still required to further uncover the potential interaction of nsp with host cellular proteins and translate the current understanding of rdrp biology into effective antiviral drug development. none. ha-tagged nlrx was cotransfected with flag-tagged nsp into hek t cells. cell lysates were immunoprecipitated (ip) with anti-ha antibody or anti-flag antibody and then blotted as indicated. (b) hela cells were transfected with flag-tagged nsp for h. cells were then fixed and incubated with anti-nlrx and anti-flag antibodies. dapi, ′ diamidino- -phenylindole. (c) flag-tagged nsp was cotransfected with the indicated ha-tagged nlrx mutants into hek t cells. cell lysates were immunoprecipitated with anti-ha antibody and blotted with the indicated antibodies. (d) the schematics of nsp and corresponding truncation constructs. numbers indicate the residues where deletions begin or end. (e) ha-tagged nlrx was cotransfected with the indicated flag-tagged nsp mutants into hek t cells. cell lysates were immunoprecipitated with anti-flag antibody and blotted with the indicated antibodies. h. jing, et al. virus research ( ) - enhancement of reactive oxygen species production and chlamydial infection by the mitochondrial nod-like family member nlrx nlrx protein attenuates inflammatory responses to infection by interfering with the rig-i-mavs and traf -nf-kappab signaling pathways an nterminal addressing sequence targets nlrx to the mitochondrial matrix inhibition of highly pathogenic porcine reproductive and respiratory syndrome virus replication by recombinant pseudorabies virusmediated rna interference in piglets structural analysis of porcine reproductive and respiratory syndrome virus non-structural protein alpha (nsp alpha) and identification of its interaction with nsp nlrx suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of nf-kappab signaling new insights about the regulation of nidovirus subgenomic mrna synthesis the interaction of nonstructural protein with retinoblastoma protein benefits the replication of genotype porcine reproductive and respiratory syndrome virus in vitro the nucleotide-binding leucine-rich repeat (nlr) family member nlrx mediates protection against experimental autoimmune encephalomyelitis and represses macrophage/microglia-induced inflammation the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins nlrx promotes immediate irf -directed antiviral responses by limiting dsrna-activated translational inhibition mediated by pkr hp-prrsv is attenuated by de-optimization of codon pair bias in its rnadependent rna polymerase nsp gene nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses engineering the prrs virus genome: updates and perspectives novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by prrsv- structure and functional characterization of the rna-binding element of the nlrx innate immune modulator nlrx modulates differentially nlrp inflammasome activation and nf-kappab signaling during fusobacterium nucleatum infection nlrx regulates neuronal cell death nlrx prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus pb -f protein porcine reproductive and respiratory syndrome virus nsp alpha inhibits nf-kappab activation by targeting the linear ubiquitin chain assembly complex porcine reproductive and respiratory syndrome virus infection activates nod -rip signal pathway in marc- cells dexd/ h-box helicase signaling via myeloid differentiation primary response gene contributes to nf-kappab activation to type porcine reproductive and respiratory syndrome virus infection suppression of nlrx in chronic obstructive pulmonary disease central roles of nlrs and inflammasomes in viral infection the viral innate immune antagonism and an alternative vaccine design for prrs virus cholesterol -hydroxylase inhibits porcine reproductive and respiratory syndrome virus replication through enzyme activity-dependent and -independent mechanisms fas-associated factor- positively regulates type i interferon response to rna virus infection by targeting nlrx the innate immune receptor nlrx functions as a tumor suppressor by reducing colon tumorigenesis and key tumor-promoting signals the mitochondrial proteins nlrx and tufm form a complex that regulates type i interferon and autophagy nlrx attenuates apoptosis and inflammatory responses in myocardial ischemia by inhibiting mavs-dependent nlrp inflammasome activation the interaction between host annexin a and viral nsp is beneficial for replication of porcine reproductive and respiratory syndrome virus nsp and nsp contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china an intracellularly expressed nsp -specific nanobody in marc- cells inhibits porcine reproductive and respiratory syndrome virus replication porcine reproductive and respiratory syndrome virus nucleocapsid protein interacts with nsp and cellular dhx to regulate viral rna synthesis innate and adaptive immunity against porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the expanding role of nlrs in antiviral immunity nlrx negatively modulates type i ifn to facilitate kshv reactivation from latency nlrx is a regulator of mitochondrial antiviral immunity the everexpanding diversity of porcine reproductive and respiratory syndrome virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis the network of interactions among porcine reproductive and respiratory syndrome virus nonstructural proteins innate immunity: squelching anti-viral signalling with nlrx location of t-cell epitopes in nonstructural proteins and of type-ii porcine reproductive and respiratory syndrome virus modeling the regulatory mechanisms by which nlrx modulates innate immune responses to helicobacter pylori infection nlrx mediates mavs degradation to attenuate hepatitis c virus-induced innate immune response through pcbp mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus immunological features of the non-structural proteins of porcine reproductive and respiratory syndrome virus prrsv receptors and their roles in virus infection nlrx acts as tumor suppressor by regulating tnf-alpha induced apoptosis and metabolism in cancer cells nlrx resides in mitochondrial rna granules and regulates mitochondrial rna processing and bioenergetic adaptation arterivirus molecular biology and pathogenesis the mitochondrial protein nlrx controls the balance between extrinsic and intrinsic apoptosis continuous and discontinuous rna synthesis in coronaviruses nlrx dampens oxidative stress and apoptosis in tissue injury via control of mitochondrial activity interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus nlrx is a mitochondrial nod-like receptor that amplifies nf-kappab and jnk pathways by inducing reactive oxygen species production nlrx acts as an epithelial-intrinsic tumor suppressor through the modulation of tnf-mediated proliferation loss of nlrx exacerbates neural tissue damage and nf-kappab signaling following brain injury the nlr gene family: a standard nomenclature nod-like receptor x- is required for rhinovirus-induced barrier dysfunction in airway epithelial cells interaction of porcine reproductive and respiratory syndrome virus proteins with sumo-conjugating enzyme reveals the sumoylation of nucleocapsid protein the involvement of nlrx and nlrp in the development of nonalcoholic steatohepatitis in mice interleukin- enhancer binding factor interacts with the nsp or nsp of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription nlrx negatively regulates tlr-induced nf-kappab signaling by targeting traf and ikk inhibition of porcine reproductive and respiratory syndrome virus by specific sirna targeting nsp gene nonstructural protein residues and are critical sites in determining the replication efficiency and fatal virulence of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus lipid rafts both in cellular membrane and viral envelope are critical for prrsv efficient infection nlrx accelerates cisplatin-induced ototoxity in hei-oc cells via promoting generation of ros and activation of jnk signaling pathway interactions of traf and nlrx gene polymorphisms with environmental factors on the susceptibility of type diabetes mellitus vascular complications in a southern han chinese population two residues in nsp contribute to the enhanced replication and pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus the deadbox rna helicase positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp in vitro inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using dnabased short antisense oligonucleotides mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase in vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification key: cord- -sed xyte authors: zheng, hao; liu, changlong; zhuang, jinshan; yuan, shishan title: baculovirus expression of cloned porcine arterivirus generates infectious particles in both insect and mammalian cells date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: sed xyte studies on several viral pathogens have been hampered by the lack of appropriate in vitro systems for their propagation and amplification. porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus containing a single-stranded positive-sense rna genome (∼ kb), was served as a model virus and its genomic cdna was recombinated into baculovirus. we investigated whether infectious virus particles could be generated by expression of the full-length cloned genome from the modified baculovirus vector. the recombinant baculovirus, acaprrs, was used to infect sf cells. immunofluorescence assay demonstrated the presence of prrsv nonstructural protein (nsp) and nucleocapsid (n) protein and electron microscopy revealed prrsv particles in the culture supernatant. infectious prrsv particles were also produced in susceptible marc- cells inoculated with acaprrs, and the growth characteristics of the prrsv generated were similar to those of the parental prrsv strain. infectious prrsv particles were also generated following acaprrs transduction of bhk- cells and vero cells that are not sensitive to prrsv. titers of prrsv obtained from bhk- and vero cells were up to ( . ) tcid( )/ml. these findings open a new route to the propagation of the virus in vitro and will be of utility in vaccine development. many viral pathogens including norwalk virus and hepatitis c virus (hcv), with the exception of strain jfh- , have as yet no in vitro cell culture system (asanaka et al., ; duverlie and wychowski, ) . the absence of an appropriate in vitro system for their propagation and amplification has constrained studies on such viruses and has hindered vaccine development. attempts have been made to employ fowlpox virus, adenovirus and baculovirus as delivery vectors for expression of hepatitis b virus (hbv) genomic dna or hcv genomic cdna in hepatocytes, and infectious virus particles were reported to be produced in the transduced cells (lucifora et al., ; ren and nassal, ; yao et al., ; yap et al., ) . the insect baculovirus is a versatile tool that has been applied to the production of high levels of recombinant proteins, the display of foreign peptides on virus particles, and the gene delivery in mammalian cells and in vivo (kost et al., ; tani et al., ) . in addition, recombinant baculovirus has been used to amplify an adeno-associated virus vector in insect cells and recently rhopalosiphum padi virus (rhpv), an insect virus that infects aphids, was successfully generated in insect cells from recombinant baculovirus (aslanidi et al., ; pal et al., ) . however, it remains an open question whether vertebrate viruses can be generated in insect cells by use of appropriate baculovirus recombinants. in this paper, we have used porcine reproductive and respiratory syndrome virus (prrsv) as a model virus to address whether infectious mammalian virus particles can be generated in insect cells from recombinant baculovirus. prrsv is a small enveloped virus belonging to the family arteriviridae, order nidovirales. the prrsv genome, a singlestranded positive-sense rna approximately kb in length with a cap and a polya-tail, contains at least nine open reading frames (orfs). orfs a and b encode a polyprotein that is processed co-and post-translationally to generate the mature nonstructural protein (nsp) required for virus replication. the other seven orfs encode seven structural proteins designed glycoprotein (gp) , envelope (e) protein, gp , gp , gp , membrane (m) protein and nucleocapsid (n) protein, respectively (snijder and meulenberg, ) . expression of prrsv structural proteins, as reported for coronavirus structural proteins, requires subgenomic (sg) mrna synthesis by a mechanism of discontinuous transcription (pasternak et al., ) . the only known natural host for prrsv is the pig, and the virus has been shown to infect primary porcine alveolar macrophages (pam) as well as a monkey kidney epithelial cell line, ma- , and its derivatives including marc- cells. in mainland china, prrs is the most economically important disease of swine herds. a new prrs outbreak was first described in mid- , and 'porcine high fever disease' (phfd) subsequently spread throughout the chinese swine industry, resulting in the culling of an estimated million pigs. a highly virulent prrsv variant has been confirmed to cause phfd (lv et al., ; tian et al., ) . several different types of inactivated or modified live vaccines have been trialed against phfd but have only met with limited success, and a major research focus has been to develop a safe and effective vaccine against the new variant. in the present study, we explored the use of a recombinant baculovirus vector to direct the expression of the full-length prrsv genome under the dual control of cmv and polyhedrin promoter sequences. the recombinant virus was used to infect insect or transduce mammalian cells. we report that infectious prrsv particles can be generated after either infection of sf insect cells or transduction of mammalian cells. these findings suggested a new strategy for virus amplification in vitro and vaccine development. spodoptera frugiperda sf cells were cultured at • c in sf- serum-free medium (invitrogen). marc- cells were cultured at • c under % co in eagle's minimal essential medium (jrh) supplemented with % fetal bovine serum (fbs). bhk- cells and vero cells were grown at • c under % co in dulbecco's modified eagle medium (dmem) supplemented with % fbs. the prrsv strain aprrs (genbank accession no. gq ) and its infectious clone papprs were prepared and maintained as described previously (yuan and wei, ) . recombinant baculovirus aclacz was generated from sf cells transfected with the bacmid isolated from escherichia coli dh bac tm and was used as a control virus. the cmv promoter segment was amplified from pcmv-script (stratagene) with the primer pair pcmvf and pcmvr (table ) and pfuultra ® hotstart dna polymerase (stratagene). the amplified segment was cleaved with noti and ligated into noti-and ecori-cleaved pbluescript sk(+) to generate recombinant plasmid pbcmv. a -segment of the prrsv genome was amplified from paprrs, using pfuultra ® hotstart dna polymerase and primers cmv-sf /sr (table ) , and digested with sphi before cloning. plasmid pbcmv was digested with sali, filled in using t polymerase, and cleaved with sphi before insertion of the -end segment by ligase treatment, generating plasmid pc-ars. plasmids pc-ars and paprrs were cleaved with noti plus aflii and religated to substitute the t promoter sequences in paprrs by cmv promoter sequences, generating plasmid pcaprrs (fig. ). hepatitis delta virus (hdv) antigenomic ribozyme sequences were pcr amplified with primers sf and phdvr , phdvr , phdvr , and xbai and xhoi cleavage and religation was used to insert the segment containing hdvr into pcaprrs. a recombinant baculovirus containing the genomic cdna of aprrs, designated acaprrs, was generated using the bac-to-bac baculovirus expression system (invitrogen). first, the full-length cdna in clone pcaprrs was excised with noti plus xhoi, and ligated into pfastbac digested with the same enzymes. the recombinant donor plasmid, pbacaps, was identified by smai digestion and transformed into e. coli dh bac tm . recombinant bacmids were selected following manufacture's instruction, and identified by pcr using of the primer pairs sf /sr and sf /sr ( table ). the recombinant baculovirus was generated following manufacture's instruction. briefly, sf cells were grown to % confluence in mm culture dishes and transfected with the recombinant bacmid using cellfectin (invitrogen). days after transfection, cell supernatants were harvested. recombinant baculovirus was confirmed by pcr with two pairs of primers, sf /sr and sf /sr (table ) . this preparation was used to infect sf cells, generating a virus stock. this stock was stored as aliquots at − • c. sf cells ( × ) in t- flasks were inoculated with acaprrs at a multiplicity of infection (moi) of . . after incubation for h at • c, ml of fresh medium was added to the flasks and incubation continued for a further days at • c. supernatants were clarified by centrifugation in two steps, first at × g for min and then at × g for min (sigma, h- ). to remove acaprrs, supernatants were centrifuged twice at , × g for h at • c (beckman, sw ti). prrsv particles were then precipitated by centrifugation through a % sucrose cushion at , × g for h (beckman, sw ti). the pellet was resuspended in ste buffer. a drop of the final suspension or acaprrs suspension was placed on a formvar-carbon-coated copper grid, negatively stained with % phosphotungstic acid for min, then observed under the electron microscope (jem ). to detect prrsv genomic rna, the final suspension as described above were used to isolate prrsv rna with rnaprep pure cell/bacteria kit (tiangen) according to the manufacturer's instructions. dnasei was used to eliminate contaminating dna. reverse transcription (rt) employed the primer qst and sr , and was followed by pcr using the primer pairs sf /sr and sf /sr . aliquots of sf cells ( × /dish) in mm plates were inoculated with acaprrs or aclacz at an moi of . , or with prrsv aprrs at an moi of , respectively. after incubation for h at • c, cells were washed once with sf- serum-free medium, ml fresh medium was added to each dish, and incubation was continued for days at • c. cell supernatants were discarded and the cell layer was washed once with phosphate-buffered saline (pbs) before ifa. marc- cells ( × /well) in -well plates were inoculated with acaprrs or aclacz at an moi of , or with prrsv aprrs at an moi of . , respectively. after h incubation at • c, cells were washed twice with pbs before adding fresh medium (mem plus % fbs). cells were incubated at • c for a further days and washed once with pbs before ifa. bhk- cells and vero cells ( × /well) in -well plates were inoculated with acaprrs, aclacz, or prrsv aprrs at an moi of and incubated for h at • c. cells were washed twice with pbs before adding fresh medium (dmem plus % fbs). after days, cells were washed once with pbs before ifa. for ifa, cell layers were fixed by adding ice-cold anhydrous methanol and incubating for min at − • c. cells were then permeabilized with . % tween- in pbs at room temperature for min after blocking with % bsa in pbs for min at • c, the cells incubated for . h at • c with anti-prrsv n protein monoclonal antibody (mab), anti-prrsv nsp mab (kindly provided by dr. ying fang at south dakota state university), or anti-acmnpv gp protein mab (acv , santa cruz) at : dilution in pbs plus % bsa. after washing, bound antibody was detected with a goat anti-mouse igg conjugated to alexa fluor (invitrogen). prrsv titers were determined as tcid /ml on marc- cells. serial -fold dilutions of virus were prepared in mem and transferred to -well plates ( l/well). l aliquots of marc- cells ( × cells/ml) were added to each well and the plates were incubated for days at • c under % co . titers in tcid /ml were calculated from cytopathic effects (cpe) according to the reed-muench formula. the virus vasm, in vitro generated from marc- cells inoculated with acaprrs, or prrsv aprrs was serially -fold diluted. . ml viruses were inoculated onto marc- monolayers in well plates. after h at • c, cell monolayers were washed once with pbs and overlaid with ml mem containing % (w/v) low melting-point agarose and % fbs. after incubation for a further days at • c, plaques were stained using % (w/v) crystal violet. to determine the sequences of -and -ends of vasm and its passage on marc- cells, the -full race kit and -full race core set (takara) were used according to the manufacturer's protocols. extraction of total rna employed the qiaamp viral rna mini kit. for -race, primer sr was used for the rt step and the primers sr and sr (table ) were used for nested pcr in combination with the universal primers provided with the kit. for -race, primer sf and the primer provided with the kit were employed. pcr products were purified using a gel extraction kit (watson inc.) and cloned into vector pgem-t for sequencing. genomic clone paprrs was modified to produce a virus capable of generating prrsv on transfection of mammalian cells. first, the t promoter sequence was replaced by the major cytomegalovirus (cmv) promoter. second, the self-cleaving hepatitis delta virus ribozyme (hdvr) sequence was inserted at the -end of the prrsv genomic cdna (fig. a) . the donor plasmid, pbacaps (fig. b) , was constructed to allow transfer to the baculovirus genome. then, a recombinant baculovirus, acaprrs, was generated using the bac-to-bac system. recombinant baculovirus acaprrs contained a complete prrsv genomic cdna bp downstream of the polyhedrin promoter and immediately downstream of the cmv promoter. at the the genomic cdna was followed by the selfcleaving hdvr sequence and a sv polyadenylation site. to determine whether the prrsv genome contained within acaprrs was expressed in insect cells, sf cells were infected with acaprrs, and immunofluorescence was performed days postinfection using mab specific for prrsv proteins n and nsp . sf cells infected with acaprrs displayed positive immunofluorescence for both n and nsp , whereas the sf cells inoculated with prrsv aprrs and aclacz were negative (fig. ) . these results demonstrated that native prrsv was unable to infect sf cells, but the prrsv genome of acaprrs was transcribed and translated to generate prrsv-specific proteins. the production of prrsv structural proteins requires expression of subgenomic mrna (snijder and meulenberg, ) . this also demonstrated that the prrsv genomic cdna carried by acaprrs was subject to genomic rna transcription, and then subgenomic mrna was discontinuously transcribed. to investigate whether prrsv particles were generated following infection of insect cells with the recombinant baculovirus, sf cells were infected with acaprrs and supernatants were harvested days later. the parental acaprrs virus was first removed by centrifugation at , × g. virus particles still present in the supernatant were collected by high-speed centrifugation ( , × g) and examined by electron microscopy. as shown in fig. a , the final supernatants contained virus particles - nm in diameter that were morphologically similar to prrsv virions. however, the particles of acaprrs were morphologically identic to baculovirus virions (fig. b) . to address whether these particles in final supernatants contained prrsv genomic rna, the preparations were digested with dnasei and then subjected to rt-pcr using prrsv-specific primers. as shown in fig. c and d, omission of the rt step failed to generate any pcr product (lane ), whereas rt-pcr generated a pcr product of the expected size (lane ). because pcr amplification of prrsv sequences was crucially dependent on rt, we concluded that virions containing prrsv rna were generated in sf cells. prrsv particles observed by electron microscopy. residual acaprrs particles were removed by centrifugation ( , × g) and prrsv virions ( - nm; arrowed) were collected by high-speed centrifugation ( , × g) and visualized after negative staining. (b) acaprrs particles observed by electron microscopy. the particles of acaprrs were precipitated by centrifugation ( , × g) and resuspended in ste. acaprrs particles (arrowed) were negatively stained and observed under the electron microscope. (c) pcr amplification of prrsv sequences with primer pairs sf /sr . clarified supernatants were examined following ultracentrifugation ( , × g) and dnasei treatment. pcr with primer pairs sf /sr was then performed either with or without a rt step with the primer qst as indicated. the signal in lane was insensitive to prior dnasei treatment, but was abolished when the rt step was omitted (lane ). lane m, dna marker mixture dl . (d) pcr amplification of prrsv sequences with primer pairs sf /sr . after the preparations digested with dnasei, pcr with primer pairs sf /sr was then performed either with or without a rt step with the primer sr . the signal in lane was insensitive to prior dnasei treatment, but was abolished when the rt step was omitted (lane ). lane m, dna marker mixture dl . cultured marc- cells were inoculated with acaprrs; control cultures were inoculated with either aclacz or prrsv aprrs. cells were examined by immunofluorescence at days postinoculation. as shown in fig. a , cells inoculated with either acaprrsv or prrsv aprrs showed positive staining with mab directed against prrsv n protein; cells inoculated with aclacz were negative. the subcellular localization of n protein from acaprrs was indistinguishable from that produced from prrsv aprrs. these results indicated that marc- cells inoculated with acaprrs might generate infectious prrsv virions. to address this possibility, we examined cpe on the marc- cells after inoculation of the acaprrs and control viruses. this revealed pronounced cpe of marc- cells inoculated with acaprrs (moi = . ) at days post-infection, whereas no cpe were detectable in cells inoculated in parallel with aclacz at the same moi. the supernatant of the marc- cells inoculated with acaprrs, designated vasm, was diluted : and reinoculated onto fresh marc- cells. as a control, the undiluted supernatant of the marc- cells inoculated with aclacz was also inoculated onto fresh cells. inspection of the plates revealed strong cpe at days post-infection in the cells inoculated with the vasm preparation, whereas control cells and cells infected with the aclacz supernatants showed no cpe even at days post-inoculation. the presence of prrsv sequences in the reinoculated cells was investigated using rt-pcr. this revealed that both the first and second passages of the marc- supernatants (vasm) were positive for prrsv sequences, whereas parallel cultures of aclacz were systematically negative (fig. b) . these results argued that infectious prrsv particles were produced following marc- cells inoculated with acaprrs. to confirm that virions produced in insect culture were indeed infectious to mammalian cells, sf cells were infected with acaprrs (moi = . ) and supernatant samples were taken at different timepoints, indicated in fig. c . these were titrated by serial dilution, inoculation onto marc- cells, and examination for prrsv production. as shown in fig. c , sf culture supernatant harvested at h post-infection contained prrsv at . tcid /ml, and the peak titer ( . tcid /ml) was achieved by the -day post-inoculation samples (fig. c) . we then compared the growth properties of newly generated prrsv with those of the parental prrsv aprrs strain. marc- cells were infected with either the vasm preparation or with aprrs; culture supernatants were titered for infectious particles at different timepoints post-infection. as shown in fig. a , the growth kinetics of vasm were similar to those of aprrs. although vasm grew slightly slower than aprrs, the peak titers of the two preparations were similar. in addition, the plaque sizes of vasm and aprrs on marc- cells were not significantly different (fig. b) . these findings argued that the growth characteristics of the baculovirus-derived vasm were highly similar to those of the parental prrsv aprrs virus. the results of -and -race showed that the terminal sequences of both the first and second passages of vasm were identical to those of prrsv aprrs (data not presented). marc- cells have previously been shown to be susceptible to infection by prrsv, and we therefore studied whether other mammalian cell lines can express prrsv antigens and/or generate virus particles. first, bhk- and vero cells were inoculated with acaprrs; immunofluorescence analysis was performed using mab specific for prrsv n protein. as shown in fig. a , both cell lines were positive for prrsv fluorescence, whereas controls cultures infected with either prrsv aprrs or aclacz were negative. these results demonstrated that both bhk- and vero cell lines were refractory to infection with prrsv, but moreover that the recombinant baculovirus carrying prrsv genomic cdna can transduce these cells and express the prrsv genome. we then examined whether these cells can also generate infectious prrsv particles. when day post-inoculation supernatants were inoculated onto marc- cells, cpe were detected as early as days post-inoculation, whereas no cpe were detected following inoculation from control aclacz-infected cultures even after days of incubation. we used rt-pcr to confirm that the supernatants of the marc- cells were positive for prrsv sequences whereas aclacz supernatants were negative (fig. b) . these results indicated that the transduction of either bhk- or vero cells with acaprrs led to the generation of infectious prrsv particles. to address the kinetics of prrsv virion production in the bhk- and vero lines, cells were inoculated with acaprrs (moi = . ) and cell supernatants were titered at different timepoints. as shown in fig. c , infectious prrsv particles were generated somewhat faster on bhk- cells than on vero cells, but the peak prrsv titer on vero cells ( . tcid /ml) was slightly higher than that obtained with bhk- cell ( . tcid /ml). the finding that prrsv titers rose markedly following inoculation further argues that prrsv virions were actively generated from bhk- cells and vero cells transduced with acaprrs. the baculovirus autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is a large double-stranded dna virus with a circular genome of about kb in length (ayres et al., ) . in view of their large genome size and flexibility, baculoviruses can accommodate insertion of large segments of foreign dna (hu, ) . we reported the construction of a recombinant baculovirus, acaprrs, containing a fragment of ∼ kb comprising the fulllength genomic cdna of prrsv under the control of the major cmv promoter. baculovirus vectors have previously been exploited to amplify viruses that propagate on hepatocytes, including hbv and hcv, but the baculovirus approach has not yet been applied to the amplification of viruses that lack appropriate cell lines for in vitro propagation (lucifora et al., ; yao et al., ) . we report that infection of insect sf cells with the recombinant baculovirus acaprrs led to the synthesis of prrsv proteins and the culture supernatant contained particles that were morphologically indistinguishable from prrsv virions. these findings demonstrated that the prrsv genomic cdna contained within acaprrs was transcribed for protein synthesis and prrsv rna was actively packaged into virus particles. the recombinant baculovirus approach could therefore provide a general method for propagating viruses, notably rna viruses, for which no appropriate in vitro culture system is as yet available. in the present study, the titers of prrsv particles produced from baculovirus-infected insect cells were relatively modest (∼ . tcid /ml). this may be due to the fact that the genomic cdna is inserted downstream of two promoters, the polyhedrin promoter and the cmv promoter. transcription initiation at the cmv promoter was thought to generate intact genomic prrsv rna, but the cmv promoter is only poorly active in insect cells (he et al., ) . further experiments will be required to address whether direct linkage of prrsv genomic sequences to a strong promoter sequence active in insect cells would significantly increase virus titers. in our study, when the recombinant baculovirus acaprrs was inoculated onto bhk- or vero cells, prrsv titers of . tcid /ml or . tcid /ml were obtained, respectively. baculovirus is generally considered to be a safe vector. transduction of mammalian cells appears to take place efficiently but there are no manifest adverse effects and the baculovirus genome is not transcribed and is unable to replicate (kost and condreay, ; tjia et al., ) . baculovirus can transduce diverse mammalian cell types and has been exploited as a vector for gene therapy and for dna vaccine delivery (kost and condreay, ; tani et al., ) . it was previously reported that baculovirus expressing the e glycoprotein of hcv under the control of the cmv immediate-early promoter-enhancer elicited specific humoral and cell-mediated immune responses following intramuscular inoculation of mice (facciabene et al., ) . in this study, we have demonstrated that a recombinant baculovirus carrying prrsv genomic cdna was able to generate infectious prrsv following transduction of mammalian cells that are themselves refractory to infection with prrsv in vitro. this approach may therefore provide an alternative route for vaccine development, particularly if combined with the expression of genomic cdna derived from an attenuated virus strain. it is possible that direct inoculation of such a recombinant baculovirus into animals or humans could generate infectious vaccine virus in vivo and thereby induce effective anti-viral immunity. because baculovirus can be cultured on insect cells in the absence of serum, this avoids the potential risks of contamination associated with the use of animal products. in conclusion, we have constructed a recombinant baculovirus carrying prrsv genomic cdna downstream of two promoters: the polyhedrin promoter active in insect cells and a cmv promoter active in mammalian cells. when the recombinant baculovirus was inoculated onto insect sf cells or mammalian vero and bhk- cells, infectious prrsv virions were generated. the approach described here provides a new baculovirus-based strategy for vaccine development and the production of viruses for which no appropriate in vitro culture system is as yet available. replication and packaging of norwalk virus rna in cultured mammalian cells an inducible system for highly efficient production of recombinant adeno-associated virus (raav) vectors in insect sf cells the complete dna sequence of autographa californica nuclear polyhedrosis virus cell culture systems for the hepatitis c virus baculovirus vectors elicit antigenspecific immune responses in mice wssv ie promoter is more efficient than cmv promoter to express h hemagglutinin from influenza virus in baculovirus as a chicken vaccine baculovirus as a highly efficient expression vector in insect and mammalian cells recombinant baculoviruses as mammalian cell genedelivery vectors baculovirus as versatile vectors for protein expression in insect and mammalian cells initiation of hepatitis b virus genome replication and production of infectious virus following delivery in hepg cells by novel recombinant baculovirus vector an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome a baculovirusexpressed dicistrovirus that is infectious to aphids nidovirus transcription: how to make sense? hepatitis b virus (hbv) virion and covalently closed circular dna formation in primary tupaia hepatocytes and human hepatoma cell lines upon hbv genome transduction with replication-defective adenovirus vectors the molecular biology of arteriviruses in vitro and in vivo gene delivery by recombinant baculoviruses emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark autographa californica nuclear polyhedrosis virus (acnpv) dna does not persist in mass cultures of mammalian cells baculovirus mediated production of infectious hepatitis c virus in human hepatoma cells stably expressing t rna polymerase expression of target genes by coinfection with replication-deficient viral vectors construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins this study was co-supported by eu fp and shanghai governmental sci-agriculture fund ( - - ) to sy. key: cord- -tnwfp je authors: revilla-fernández, sandra; wallner, barbara; truschner, klaus; benczak, alexandra; brem, gottfried; schmoll, friedrich; mueller, mathias; steinborn, ralf title: the use of endogenous and exogenous reference rnas for qualitative and quantitative detection of prrsv in porcine semen date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: tnwfp je semen is known to be a route of porcine reproductive and respiratory syndrome virus (prrsv) transmission. a method was developed for qualitative and quantitative detection of the seminal cell-associated prrsv rna in relation to endogenous and exogenous reference rnas. as endogenous control for one-step real-time reverse transcription (rt)-pcr ube d mrna was selected. particularly for the analysis of persistent infections associated with low copy numbers of prrsv rna, ube d mrna is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = ). however, the amount of ube d mrna in porcine semen varied (up to -fold), thus its use is limited to qualitative detection of prrsv rna. for quantitation, a synthetic, non-metazoan rna was added to the rna isolation reaction at an exact copy number. the photosynthesis gene ribulose- , -bisphosphate carboxylase/oxygenase large subunit (rbcl) from arabidopsis thaliana was used as an exogenous spike. unexpectedly, prrsv rna was detected in a herd of specific pathogen-free (spf) boars which were tested elisa-negative for anti-prrsv antibodies. therefore, rt-pcr for seminal cell-associated prrsv is a powerful tool for managing the spf status during quarantine programs and for routine outbreak investigations. the porcine reproductive and respiratory syndrome virus (prrsv) belonging to the arteriviridae family is an enveloped single-stranded rna virus with a plus-sense genome which replicates via a -coterminal nested set of subgenomic mrnas (dea et al., ) . the prrsv- and prrsv- strains (formerly european-and north american-type prrsv, respectively; (drew, ) ) and the monophyletic lithuanian prrsv strains (plagemann, ; stadejek et al., ) can be differentiated. in semen, prrsv may be shed from the bulbourethral gland (christopher-hennings et al., ) and can be located in immature sperm cells (sur et al., ) or macrophages (christopher-hennings et al., ) . prrsv can be detected in semen as early as - days postinoculation (p.i.) (christopher-hennings et al., ; legeay et al., ) and can be transmitted by insemination (refs. in sur et al., ) . prrsv-infected boars show no significant clinical signs, and seroconversion and/or viremia is not correlated with shedding of virus in semen (wills et al., ) . while another arterivirus, equine arteri-tis virus, is shed in semen for at least - years, a prolonged carrier state (>day p.i.) in prrsv-infected boars has not been reported (christopher-hennings et al., ) . reference rnas (internal positive controls, internal control rnas) can be classified into exogenous (spike-in rna, rna extraction control, exogenous reference transcript) and endogenous controls. exogenous controls spiked at a defined copy number to the sample before rna isolation and endogenous controls can be applied for relative quantitation of viral rnas using real-time rt-pcr. therefore, reference rna expression should be comparable with that of the target and independent from the experimental conditions, e.g. the presence of a virus. both reference types serve as controls for sample transport and storage conditions, isolation performance, normalise for differences in total rna input or contribute to the detection of false negative results caused by sample-specific inhibitors. they were applied for monitoring the course of infection or intermittent shedding of viruses and for differentiation between a viremic or persistent stage of infection (moody et al., ; pasquier et al., ) . exogenous controls are used for testing of cell-free body fluids. their copy number can be controlled precisely and adjusted easily to the copy number of the gene of interest. endogenous controls are a cost efficient alternative for the detection of viral rnas in veterinary diagnostic work. artificial insemination or conventional sexual reproduction-mediated prrs virus transmission is of great importance for prrsv epidemic. for detection of prrsv in boar semen candidate endogenous reference genes were selected among common housekeeping genes and other genes found previously to be expressed uniformely in human and mouse tissues (hamalainen et al., ; warrington et al., ) . they were chosen from different functional categories based on the panther classification system (thomas et al., ) which reduces significantly the chance that genes might be co-regulated (vandesompele et al., ) . for the development of one-step real-time rt-pcr for four endogenous reference rnas (hprt, ube d , ppia, and hmbs) appropriate target regions were selected and the assay conditions were optimised for amplification efficiency. next, their expression levels and the amplification from genomic dna contamination were examined. the ube d mrna was found to be a suitable endogenous control for qualitative molecular diagnostics of prrsv rna in the cell-associated part of porcine semen. however, due to the variable expression of ube d mrna, an exogenous reference rna derived from a plant gene was developed for quantitative detection of the viral rna. the prrsv- reference sample used for assay validation was generated as follows. a -week-old piglet of a cross-bred (landrace × large white) tested negative for antibodies against prrsv was infected artificially with a . tcid of the spanish isolate olot/ (prrsv- , genbank accession number x ) and slaughtered at day p.i. lung tissue was shipped on dry ice and kept at − • c until rna isolation. lung tissues ( prrs viral rna-negative and prrsv- -or/and prrsv- -positive) for studying ube d mrna expression originated from the breeds large white, landrace, piétrain, large white × piétrain and (large white × landrace) × piétrain. ejaculates were derived from two boar herds (set a and b boars). the set a boars (n = ; piétrain, landrace, and large white breed) were - . years old and lived in the herd since - months. the boars of set b (n = ; piétrain) which joined the herd at the age of months were between and months old. both sets of boars were housed under specific pathogen-free conditions. this was guaranteed by the quarantine and testing program for incoming boars involving two serological tests with an interval of weeks against classical swine fever virus (csfv), pseudorabies virus (prv), swine influenza virus (siv), transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), chlamydophila, brucella, and leptospira species. after quarantine, the boars are tested for these pathogens once a year. according to the management program for the set a boars the absence of prrsv antibodies was required. this was achieved by testing routinely with the idexx (herd-chek) elisa (idexx laboratories, woerrstadt, germany) for which a sample-to-positive (s/p) ratio of ≥ . is considered positive. at the time of sampling the set a boars were elisa negative (− . to . ). in addition an immunoperoxidase monolayer assay (ipma (wensvoort et al., ) ) confirmed the prrs-negative serology. the set b boars showed a high positive serostatus when semen samples were taken (idexx elisa values: . - . for n = , and . - . for n = ) indicating a recent contact with prrsv. total rna from tissue samples and viral rna contained in the porcilis ® prrs vaccine (intervet, unterschleissheim, germany) were isolated with the trizol ® reagent and the trizol ® ls reagent (invitrogen, lofer, austria) according to the instructions of the manufacturer and finally dissolved in l diethyl pyrocarbonate (depc)-treated water. the qiaamp viral rna mini kit (qiagen, hilden, germany) was used to extract total rna from l serum samples and from the modified live prrsv vaccine (ingelvac ® prrs mlv; boehringer ingelheim pharma, ingelheim am rhein, germany) applying l elution buffer. the vaccine is a cell culture-adapted derivative of the pathogenic prrsv- isolate deposited at the american type culture collection under the number atcc-vr (genbank: u ). for seminal rna isolation ejaculated boar semen was collected into a sterile container with an integrated filter (us bag tm , minitube, tiefenbach bei landshut, germany) and was immediately stored at • c to guarantee that the non-sperm cells remained intact. the interval until further processing did not exceed eight hours. rna was isolated using the genelute tm mammalian total rna kit (sigma-aldrich, vienna, austria) with modifications. from a l-semen aliquot the cellular fraction was pelleted by centrifugation at × g for min. the pellet was lysed by vortexing for min in l lysis buffer containing mm -mercaptoethanol. the rna was eluted in a volume of l and stored at − • c. the plasmid p bsk (m.p. murtaugh, university of minnesota, usa) containing a kb fragment of the prrsv- reference strain vr and an arabidopsis thaliana cdna prepared as reported previously (karsai et al., ) were used as pcr templates. amplification was carried out with the primers clus-orf -f and clus-orf -r , and rbcl f and rbcl r (rbcl gene; genbank: atu ), respectively. pcr products were gel purified and subcloned into the pgem ® -t easy vector system i (promega, mannheim, germany). plasmids were purified by a modified alkaline lysis method, verified by dna sequencing, linearised by sali digestion and recovered by phenolchloroform extraction followed by ethanol precipitation. in vitro transcription reactions were performed in l containing g linearised plasmid, × transcription buffer with mm dtt, . mm of each rntp and u t rna polymerase (mbi fermentas, st. leon-rot, germany) for h at • c. subsequently, the reaction was incubated with u rnase-free dnase i (ambion, austin, usa) at • c for min. in vitro transcripts were recovered by phenolchloroform extraction and ethanol precipitation and quantitated by spectrophotometry. the housekeeping genes hprt (genbank: af ), ube d (ubch b), ppia (cypa; genbank: ay ), and hmbs (pbgd) were selected as candidate endogenous reference mrnas considering previous reports (foss et al., ; hamalainen et al., ; steele et al., ) and the fact that in contrast to hprt, ube d and ppia there is no pseudogene for hmbs in the human genome ( (zhang et al., ) ; www.pseudogene.org). human pcr primer sequences targeting conserved regions were selected in a human/mouse-nucleotide alignment (genbank: u and m ) to determine partial porcine ube d and hmbs cdna sequences and subsequently to select primers and probes for real-time rt-pcr of ube d and hmbs. in addition to pcr product sequencing the partial porcine hmbs cdna was determined by sequence anal-ysis of subclones derived from tissue cdnas (data not shown). the amplification of cdna for pcr product sequencing was carried out in a l-reaction volume containing l of the reverse transcription reaction, × buffer, mm magnesium, . mm of each dntp, nm of each primer and u taq dna polymerase. following denaturation at • c for min, targets were amplified by cycles at • c for s, • c for s and • c for min. for sequencing the abi prism bigdye tm terminator cycle sequencing chemistry and the abi prism sequencer (applied biosystems, vienna, austria) were used. one-step quantitative real-time rt-pcr was carried out with a two-enzyme system. separate reverse transcription and dna polymerisation reactions allow testing for dna contamination by including a "minus rt" control, thus assaying for amplification from processed and non-processed pseudogenes. the two-enzyme system used (taqman ® one-step rt-pcr master mix reagents kit, applied biosystems) contains the moloney murine leukemia virus reverse transcriptase and amplitaq gold ® dna polymerase. a volume of l rna per l reaction and primer/probe concentrations of nm/ nm and nm/ nm were applied for the viral and the reference rna assays, respectively. total rna was reverse transcribed for min at • c. the hot start dna polymerase was activated by incubation at • c for min followed by amplification for cycles at • c for s and • c for min. fluorescence "real-time" measurements recorded by either the abi prism ® or ht sequence detection systems (applied biosystems) were transformed into c t values using the sds software versions . . and . , respectively. dilution series of standard rnas were made to characterise the linearity, precision, specificity, and sensitivity of the quantitation assays. standard dilutions were prepared in depc-treated water containing ng/l yeast trna carrier (invitrogen) and were amplified at least in duplicates. the c t values were plotted either against the log of the target copy number or against the log of the input rna mass to generate a regression curve with the formula y = sx + b (s, slope; b, intercept) and to determine the reaction efficiency e = − /s − . as internal quality parameter for a standard curve a regression coefficient (r ) of > . was used. optimal efficiency was concluded if e ≥ . (s < − . ). to identify conserved sites for primer and probe binding in the orf gene coding for the most conserved structural protein (m proteins of prrsv- and - show amino acid sequence identity of - % (dea et al., ) ) the following sequences were aligned using the sequence navigator software (applied biosystems): (i) seven prrsv- sequences: m , aj , x , l , af , af , af , and (ii) prrsv- sequences: u , af , af , aj , l , u , af , af , u -u , z , af , l -l , u , u -u , ab , d , af , af , af , af , af , af , and aj -aj . primers (invitrogen), taqman probes (mwg biotech, ebersberg, germany) and taqman minor groove binder (mgb) probes (applied biosystems, weiterstadt, germany) were designed using the primer express tm . software (applied biosystems, vienna, austria) and are given below as - sequence. standard taqman probes were labeled with the fluorescent quencher dye -carboxytetramethylrhodamine (tamra) and with the reporter dyes -carboxyfluorescein ( fam) or -carboxy- , , , -tetra-chlorofluorescein (tet). in the taqman mgb probe format a non-fluorescent quencher dye (nfq) and a minor groove binder (kutyavin et al., ) were applied. the complete orf and the partial orf sequences were determined by pcr product sequencing using the primer pairs eu - f (cctcgaaggggttaaagctca) and eu - r (cacgaggctccgaagtcct), and eu - f (gtagaaagtgctgcaggtctcca) and eu - r (gcactgtatgagcaaccgg), respectively. in addition, the primers cleu - f (gctcaacccttgacgag-gact), cleu - r (gctctggtttttaccggcc), clus-orf -f (ataaccagagtttcagcggaaca), clus-orf -r (tctcttctgctgcttgccgt), rbcl f (cttgaaggagacagggagtcaact) and rbcl r (catgcttccagagctactcgg) were applied. to determine the partial porcine sequences for the hmbs housekeeping version and for ube d , the primers hshmbs-f (atgtctggtaacggc, exon ), hshmbs-r (gggtac-gaggctttc, exon ) and hube d -seq f (tgaaga-gaatccacaaggaattga, exon ) and hube d -seq r (ccgagcaatctcaggcactaaa, exon ) which were deduced from the homologous human genes (genbank: nm and u ) were used. primers and probes for the endogenous control assays for ppia and hprt developed on the basis of known porcine sequences (genbank: ay and af ) were pcyc- f (gccgcgtctccttcgag), pcyc- r (gcaggaac-ctttataaccaaatcct) and pcyc-vic/mgb (vic-cagaaaacttccgtgctc-nqf-mgb), and phprt- f (tcatggactaattatggacaggactg), phprt- r (tttatatcgcccgttgactggt) and phprt-tet/tamra (tet-tgggaggccatcacatcgtagcc-tamra). oligonucleotides for quantitation of porcine hmbs and ube d mrnas based on sequences determined in this study were phmbs- f (cgcaacg-gcggaagaaa), phmbs- r (ttcagcgttgccac-cacac) and phmbs- fam/tamra ( fam-cca-gctggcccgcatacaaacg-tamra); pube d - f (aggtcctgttggagatgatatgtt), pube d - r (ttgggaaatgaattgtcaagaaa) and pube- d - fam/tamra ( fam-ccaaatgacagtccc-tatcagggtgga-tamra). real-time rt-pcr for prrsv- targeted the orf gene with the primers eu - f and eu - r (see above) and the taqman mgb probe eu -mgb ( fam-ctgtgagaaagcccggac-nfq-mgb). the orf sequence of prrsv- was quantified with the primers us - f (tccagatgccgtttgtgctt) us - r (gacgccggacgacaaatg) and the probe us -mgb ( fam-ccctgcccaccacgt-nfq-mgb). both probe binding sites were completely homologous among the respective prrsv sequences listed above. the primers rbcl f (see above) and rbcl r (taccgcggcttcgatctt) were applied in combination with the taqman probe rbcl tet (tet-tcgcgcagtaaatcaacaaagccca-tamra) to quantitate the exogenous rna standard. if possible primer and probe sequences for real-time pcr were selected to target regions with no obvious secondary structure using the rna and dna folding server (www.bioinfo.rpi.edu/∼zukerm/). a significant amplification from non-processed and processed pseudogenes of endogenous reference genes during real-time rt-pcr was prevented by primer annealing across an exon boundary (ube d and hprt assays) and by primers flanking a large intron (hmbs and ppia assays). the porcine exon-intron structure was concluded from the homologous human and mouse genes (batzoglou et al., ) . we also considered that the human and the mouse genomes contain a processed pseudogene for ube d , but not for hmbs (www.pseudogene.org). the qualitative detection of the endogenous reference ube d mrna in prrsv-infected and non-infected pigs was evaluated by the student's t-test and by the nonparametric wilcoxon rank-sum (mann-whitney) test for two independent samples included in the statistical analysis software spss for windows . . (spss inc., chicago, usa). the spearman's rank correlation test was used to analyse the relationship between ube d expression in semen rnas and the sperm cell concentration. the (porcine) ube d real-time rt-pcr assay was submitted to the real-time pcr primer and probe database (rtprimerdb id: ; (pattyn et al., ) ) and can be used for detection of human ube d mrna due to complete primer/probe homology. sequences determined within this study were submitted to the genbank under the following accession numbers: af (olot/ -g a), af (porcilis prrs vaccine), ay (hmbs), ay (ube d ), af (hprt variant) and af (ppia-like pseudogene). the sequence of the austrian prrsv- isolate ags- (af ) which mismatched in the center of the orf probe binding site targeted by a previous taqman rt-pcr assay (egli et al., ) was also submitted. endogenous control genes were designated according to the homologous human genes using locuslink (www.ncbi.nlm.nih.gov). the dynamic ranges and the amplication efficiencies of the real-time rt-pcr assays for hprt, ube d , ppia, and hmbs were analysed by generating standard curves using lung and lymphoid tissue rnas (fig. a , and data not shown). the equations for the regression lines illustrate that the reaction efficiencies for the endogenous control assays (e > . , i.e. optimal) were comparable to those of the viral assays ( e < %). a dnase i treatment step to exclude cross-amplification from genomic dna was made redundant by assay design/selection (see section ). using porcine tissue rnas we demonstrated that amplification from any contaminating dna was not significant ( c t for mrna and the corresponding minus rt control > ). for rna quantitation in semen specimens ube d and hmbs were analysed in more detail since in semen the differences between the c t value for ppia or hprt and the corresponding minus rt sample controling for genomic dna contamination were small (data not shown). the ube d and hmbs expression was analysed in native semen samples derived from two sets of boars (a, b) with negative and high positive prrsv serostatus. independently of the prrsv serostatus or presence of low prrsv rna copy numbers (see below), the c t values obtained for hmbs mrna were at or near the detection limit (table ; semen rnas for set b boars were not analysed), whereas the ube d mrna was detected in all ejaculates of the individuals analysed (table and data not shown). no correlation between ube d expression and the sperm cell num-ber was found (spearman correlation coefficient r = − . , p = . ; n = in table ). the ube d expression varied from . -fold (set b boars, n = , c t > for prrsv rna, positive prrsv serostatus), over -fold (set a boars, n = , seronegative for prrsv) to -fold (set b boars, n = , only one semen rna with detectable prrsv- orf copy numbers, c t > for prrsv- rna, high positive prrsv serostatus). to prove that ube d would be invariant in the primer and probe binding sites among different individuals of common pig breeds random porcine cdnas (n = ) were sequenced. complete homology in the binding sites was found. moreover, human and pig ube d nucleotide sequences were completely homologous in the primer/probe binding sites. since in semen the variation of ube d expression exceeded the high stringency criteria applied in gene expression studies (variation < five-fold (dheda et al., ) ), we developed a non-metazoan exogenous control rna spike. this reference rna is based on the chloroplast-encoded gene for the large subunit of ribulose- , -bisphosphate carboxylase/oxygenase (rbcl) from a. thaliana (clegg, ) . the standard curve obtained for the in vitro transcript documents optimal amplification efficiency (fig. b) . spiking the cellular fraction of a semen rna sample with , copies of the rbcl in vitro transcript, c t values of . and . were obtained. a silica membrane-based kit was selected to standardise isolation for quantitation of prrsv rna in semen specimens. different semen volumes and different sperm cell numbers were tested with a constant amount of absorbent material ( mm silica column) to determine the lowest c t value for a target mrna (ube d mrna) by one-step realtime rt-pcr. cell numbers of the twelve experimental ejaculates ranged from . to . × cells/ml. duplicate c t measurements for the , , and l semen aliquots ranged between . ± . and c t > (no amplification). the lowest c t values were obtained for the l and/or the l semen aliquots (c t s: . ± . to . ± . , and . ± . to c t > ). no correlation was observed between c t value and the number of spermatozoa. the successful removal of inhibitors for real-time rt-pcr was obtained since one-step real-time rt-pcr yielded optimal amplification efficiency (e > . for ube d mrna, prrsv- and - rnas; fig. a ). one-step real-time rt-pcr assays for prrsv- and - rna allowed quantitation with optimal efficiency (fig. a ; standard curves for two additional viremic pigs infected with prrsv- (data not shown)) as achieved for endogenous and fig. . one-step real-time rt-pcr yields optimal amplification efficiency for quantitation of prrsv rnas, endogenous (a) and exogenous reference rnas (b). c t > for minus rt controls of ube d and hmbs mrnas. prrsv- infection was mimicked by spiking virus-free semen with the ingelvac ® prrs mlv vaccine. exogenous reference rna assays ( e < %, fig. and see above) . since a variation in the detection limits for the different prrsv strains due to snps within the amplicon (barnard et al., ) or in the primer binding sites cannot be excluded, the sensitivity of the assay for a single prrsv reference strain (atcc-vr , prrsv- ) was determined. for this purpose, prrsv rna-free seminal cell fraction-derived rna (see below) was spiked with known copy numbers of the prrsv- orf in vitro transcript (serial four-fold dilutions). the resulting detection limit for the analysis of prrsv- rna in the cellular fraction of semen was orf rna copies. the specificity of the real-time rt-pcr assays for prrsv- and - was examined by using viral rna from the opposite prrsv type as a non-amplification control. viral rna contained in the porcilis prrs vaccine (prrsv- ) and the ingelvac prrs mlv vaccine (prrsv- ) was purified and used at a concentration of . × orf copies/reaction as target and non-target copy numbers (and vice versa) in the quantitation assays. these copy numbers exceeded several-fold the copy numbers found in clinical samples of serum and sperm (data not shown). the real-time rt-pcr quantitation assays (e > . ) did not detect any non-target amplification demonstrating absence of cross-amplification between the two prrsv types (data not shown). the absence of any cross-amplification with samples tested positive for classical swine fever virus (csfv), transmissible gastroenteritis virus (tgev), porcine parvovirus (ppv), pseudorabies virus (prv) and porcine circovirus type ii (pcv ) was also confirmed (data not shown). using the amplicon sequences of the prrsv real-time rt-pcr assays as query in a nucleotide-nucleotide blast (blastn) the specificity of the prrsv primer and probe sequences was demonstrated. the prrsv- and - quantitation assays were used for examination of semen samples derived from two sets of boars with negative (a) and high positive (b) prrsv serostatus (see section ). unexpectedly, semen samples with prrsv- rna copy numbers near the detection limit were detected in both sets (table and data not shown). four of the twelve sera of the set a boars were positive for prrsv- rna but negative for prrsv- rna (table ; serum rnas for set b boars were not analysed). a method was developed for qualitative and quantitative detection of the seminal cell-associated prrsv rna in relation to endogenous (ube d mrna) and exogenous (rbcl rna) reference rnas. neither rrna expression or total rna amount (reviewed by bustin, ) are applicable for normalisation of relative quantitation of prrs viral rna in the seminal cell fraction due to the large difference in expression between viral and rrna genes during persistent infections and the limited practicability of spectrophotometric analysis for routine diagnostic application. ube d mrna is an appropriate control for routine qualitative detection of prrsv rna due to its reliable and low expression in all semen samples tested. preliminary data indicate similar expression of ube d mrna in animals with prrs viral rna-positive or negative status (c t s for tissue samples: . ± . and . ± . , respectively). however, we cannot exclude that for example a selection of the individuals in the two groups based on age, breed, stage of infection or a study design testing in the same animal under the same conditions with and without prrsv would reveal differential expression of ube d depending from the status of prrsv infection. the c t values determined for ube d mrna in semen were comparable to those determined for samples with low viral rna copy numbers which is especially important for reliable virus quantitation during persistent infections. the reasons for the varying c t values in the ube d assay ( c t = . , table ) were not addressed and might involve differences in the rna quality among the samples, in the number and/or the expression level of ube d mrna expressing cells. summarising, the validation of an endogenous reference which would be a cost-efficient alternative for quantitative detection of prrsv still remains a challenge due to the heterogeneous cell population of semen and its varying cell number. the real-time rt-pcr assays for hprt, ppia, hmbs and ube d developed in this study will be useful for future gene expression studies in the pig. due to the occurrence of processed pseudogenes (or intron-less paralogs), housekeeping gene mrnas used in a previous study as endogenous references for human semen samples required the application of dnase i (juusola and ballantyne, ) . this pretreatment step is assumed to impair rna quality and increases assay-related costs and time. here we show that it is possible by a careful primer and probe design to prevent significant amplification from genomic dna without a dnase i digest. quantitation of viral rna relative to a reference rna requires knowledge of the exact copy number contained in this control. due to the considerable variation (or complete absence in the case of hmbs mrna in two samples) in expression in porcine semen rna among the candidate reference genes studied we replaced the endogenous control by an exogenous control rna. it was selected to avoid crosshybridisation with rnas from the organism studied. in the present study the photosynthesis gene rbcl from a. thaliana (clegg, ) was used. artificial sequences or sequences from other organisms are also possible as exogenous controls (baker and o'shaughnessy, ; smith et al., ) . in contrast to a homologous exogenous rna (mimic) which has the same primer but different probe binding sites as the target rna (westcott et al., ) , the use of a heterologous exogenous reference can be applied easily for detection of different target rnas (for review see freeman et al., ) . real-time rt-pcr assays for detection of viral rna in the plasma fraction of semen have been reported (balasuriya et al., ; westcott et al., ) . prrsv appears most consistently in the cellular fraction of semen and not in whole semen or seminal plasma (christopher-hennings et al., . we therefore developed a quantitation assay for seminal-cell associated prrsv rna removing the seminal plasma which has been noted to contain pcr inhibitors (refs. in bourlet et al., ) . the real-time rt-pcr assays for the endogenous and exogenous references as well as for prrsv- and - yielded optimal efficiency (e = . to . ). this allows the relative quantitation of viral rna by the c t -method (ref. in pfaffl, ) avoiding the elaborate amplification of standards in parallel. the detection limit of the prrsv real-time rt-pcr was determined for prrsv- only. future assay validation should involve (i) sequence analysis of the assay target region for new isolates, (ii) the use of primers and/or probes which are degenerated at defined nucleotide positions in order to maximise the detection of all known strains and isolates, (iii) determination of efficiency and sensitivity for isolates varying in the primer and/or probe target sites, and (iv) spiking of quantitation standards which represent these polymorphic isolates into an adequate number of semen samples before rna extraction. the occurrence of viral rna in serum and semen (set a boars in table ) of seronegative boars housed under spf conditions was unexpectedly demonstrated. the high c t values reflect low copy numbers, thus virus isolation would not be expected to be successful (wills et al., ) . the occurrence of seroconversion is highly unlikely due to these low c t s, considering the routine prrs antibody testing carried out in the spf boar stud and the fact that eight of these boars were elisa-and ipma-negative days after showing low c t s for prrs viral rna in semen (superscript b in table ). the sporadic detection of viral rna in serum after a period of negative testing was reported, and pigs which have returned to seronegative status based on elisa may still harbor infectious prrsv (batista et al., ; martelli et al., ; wills et al., ) . the serological status is also not an adequate indicator of prrsv and prrsv rna shedding in the semen (christopher-hennings et al., ) . this underlines the need to complement immunoassays with more sensitive nucleic acid-based methods for a comprehensive approach especially for the characterisation of persistent prrsv infections (batista et al., ) , e.g. during the quarantine and testing program for incoming boars to reduce later conflicts in the interpretation of the prrsv status. it is possible that our results obtained for the boars of set a, retrospectively, document only a contact between the animals and the virus before joining the herd, since the incoming boars were selected on the basis of their serological, but not of their viral rna status, and contact of the boars with prrsv before entering the herd was possible in principle. note during the processing of this paper, quantitative realtime rt-pcr tests for the detection of prrsv in whole boar semen and in the cell-associated part of the ejaculate were reported, respectively (van rijn, p.a., wellenberg, g.j., hakze-van der honing, r., jacobs, l. expression of prostaglandin d synthetase during development in the mouse testis detection of equine arteritis virus by real-time taqman reverse transcription-pcr assay pcr bias toward the wild-type k-ras and p sequences: implications for pcr detection of mutations and cancer diagnosis detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative elisa s/p ratios human and mouse gene structure: comparative analysis and application to exon prediction multicenter quality control for the detection of hepatitis c virus rna in seminal plasma specimens quantification of mrna using real-time reverse transcription pcr (rt-pcr): trends and problems persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars identification of porcine reproductive and respiratory syndrome virus in semen and tissues from vasectomized and nonvasectomized boars chloroplast gene sequences and the study of plant evolution current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates validation of housekeeping genes for normalizing rna expression in real-time pcr a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus quantitative taqman rt-pcr for the detection and differentiation of european and north american strains of porcine reproductive and respiratory syndrome virus regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde- -phosphate dehydrogenase and beta-actin mrna expression in porcine immune cells and tissues quantitative rt-pcr: pitfalls and potential identification and validation of endogenous reference genes for expression profiling of t helper cell differentiation by quantitative real-time rt-pcr messenger rna profiling: a prototype method to supplant conventional methods for body fluid identification evaluation of a homemade sybr green i reaction mixture for real-time pcr quantification of gene expression -minor groove binder-dna probes increase sequence specificity at pcr extension temperatures development of a rt-pcr test coupled with a microplate colorimetric assay for the detection of a swine arterivirus (prrsv) in boar semen intermittent shedding of prrsv in semen of seronegative boars measuring infectious bursal disease virus rna in blood by multiplex real-time quantitative rt-pcr intermittent detection of hepatitis c virus (hcv) in semen from men with human immunodeficiency virus type (hiv- ) and hcv rt-primerdb: the real-time pcr primer and probe database a new mathematical model for relative quantification in real-time rt-pcr porcine reproductive and respiratory syndrome virus: origin hypothesis exogenous reference rna for normalization of real-time quantitative pcr identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses variable expression of some "housekeeping" genes during human keratinocyte differentiation porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis panther: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes comparison of human adult and fetal expression and identification of housekeeping/maintenance genes mystery swine disease in the netherlands: the isolation of lelystad virus use of an internal standard in a closed one-tube rt-pcr for the detection of equine arteritis virus rna with fluorescent probes duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus comparative analysis of processed pseudogenes in the mouse and human genomes we thank juan plana-duran for the infected lung, michael p. murtaugh for the gift of a plasmid containing vr prrsv cdna, marie-theres hauser for providing the a. thaliana cdna, irene sommerfeld-stur for help with statistical data analysis, birgit strobl for valuable discussion, and hanno schreiber, elisabeth hofmann, arnulf hertweck, georg walcher, kurt höller, and wolfgang sipos for assistance. we acknowledge the veterinary diagnostic laboratory labovet and the austrian agency for health and food safety for performing the elisa and the ipma tests. this work was supported by a grant of the university of veterinary medicine vienna to r.s. while s.r.-f. and g.b. declare their commercial interest in prrsv molecular diagnostics, the remaining authors have no competing financial interests. key: cord- -xq iq ai authors: frossard, jean-pierre; grierson, sylvia; cheney, tanya; steinbach, falko; choudhury, bhudipa; williamson, susanna title: uk pigs at the time of slaughter: investigation into the correlation of infection with prrsv and hev date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xq iq ai hepatitis e virus (hev) and porcine reproductive and respiratory syndrome virus (prrsv) and are both globally prevalent in the pig population. while hev does not cause clinical disease in pigs, its zoonotic potential has raised concerns in the food safety sector. prrs has become endemic in the united kingdom (uk) since its introduction in , and continues to cause considerable economic losses to the swine industry. a better understanding of the current prevalence and diversity of prrsv and hev in the uk, and their potential association, is needed to assess risks and target control measures appropriately. this study used plasma, tonsil, and cecal content samples previously collected from pigs in abattoirs in england and northern ireland to study the prevalence of several pathogens including prrsv and hev. the diversity of prrsv strains detected in these samples was analyzed by sequencing open reading frame (orf ), revealing no substantial difference in prrsv strains from these clinically unaffected pigs relative to those from clinical cases of disease in the uk. despite the potential immuno-modulatory effect of prrsv infection, previously demonstrated to affect salmonella and hev shedding profiles, no significant association was found between positive prrsv status and positive hev status. hepatitis e virus (hev) is the cause of hepatitis e in humans, typically a self-limiting hepatitis but more serious in those with pre-existing liver conditions and in the immunocompromised [ ] . in pigs, hev infection alone does not cause clinical disease. hev genotypes hev- and hev- are the cause of sporadic cases of hepatitis e in developed countries, and are ubiquitous in the pig population worldwide [ ] . hepatitis e is a foodborne zoonosis, for which pork or pork products from infected pigs is one of the risks identified in europe [ , ] and consumption of processed pork products in the united kingdom (uk) has been shown to be associated with an increased risk of acquiring hev [ ] . hence, there is a need to better understand factors influencing hev entering the food chain. porcine reproductive and respiratory syndrome virus (prrsv) was first confirmed in the uk in and is now considered endemic [ ] . the economic and welfare impacts of the disease are considerable, as both the breeder and grower segments of the pig industry are affected [ ] . all prrsv infections in the uk characterized to date have been identified as being caused by genotype virus, but the genetic diversity of the virus is continually increasing [ ] . the phylogenetic analyses of uk prrsv sequences have previously been based on data from samples submitted for diagnostic purposes, originating from clinical cases of prrs, thereby possibly introducing a bias in our coverage of circulating strains. it is therefore possible that prrsv strains circulating in apparently healthy pigs in the uk may represent a different subset from those causing disease. prrsv infection has been suggested to modulate pig immune responses, thereby rendering pigs more susceptible to other infections [ ] [ ] [ ] . for example, previous studies have shown significant associations between prrsv presence and salmonella shedding [ ] . salines et al. [ ] reported that experimental co-infection of hev and prrsv affected the dynamics of hev infection. however, a direct immune-regulation in infected pigs could not be confirmed for genotype prrsv [ ] , rather suggesting a role for co-infection viruses influencing each other more directly. moreover, less pathogenic strains of genotype prrsv seem to cause a more persistent infection than highly pathogenic ones which are better resolved by the immune response [ ] . notably, viruses closely related to the modified-live vaccine used in the uk have previously been found circulating on farms with clinical prrs [ ] . in , an abattoir-based study was undertaken to estimate the prevalence of various pathogens including hev and prrsv in uk-reared pigs at slaughter and seroprevalence for prrsv was . % ( / ) [ ] , while hev seroprevalence was . % ( / ). approximately . % of pigs were hev viremic ( / ) and around one in five pigs had evidence of an active hev infection ( / ), with hev rna detected in serum or cecal contents [ ] . to follow up these studies, we report here on ( ) the investigation of prrsv active infection (rna in tonsil) using the same abattoir survey sample-set and ( ) an analysis of the correlation of prrsv and hev infection in these pigs, which could be of significance for the control of both diseases and in informing farming practices for reducing hev in the food chain. the overall study design for the abattoir survey has been described previously [ ] . briefly, qualifying pigs were sampled at abattoirs in england and northern ireland, between january and may . these pigs originated from farms, with between and pigs from each. the majority of pigs were from farms in england ( . %), followed by northern ireland ( . %), scotland ( . %), and wales ( . %), which is representative of the uk pig population [ ] . of the samples collected from each selected carcass along the processing line, those relevant for the investigation of hev and prrsv were a blood sample (whole blood with ethylenediaminetetraacetic acid (edta)), tonsil, and the whole cecum. antibody to prrsv had been detected in of plasma samples, with additional samples being inconclusive [ ] . only of these plasma samples were also analyzed for the presence of antibodies to hev, with of these being sero-positive for prrsv. tonsil samples from all seropositive or inconclusive pigs were then tested by a real-time reverse transcription polymerase chain reaction (rt-pcr) to detect prrsv nucleic acid. the tonsils of four other pigs for which plasma samples-and hence enzyme-linked immunosorbent assay (elisa) results-were missing were also tested by pcr, while for five seropositive animals the tonsil samples were not available, so a total of tonsil samples were analyzed; only of these animals also had matching hev pcr data. rna was extracted from the tonsil samples using the magna pure lc dna isolation kit ii (tissue) following the manufacturer's instructions (roche diagnostics, burgess hill, uk), setting the sample volume to µl and the elution volume to µl. a real-time rt-pcr assay targeting the nucleocapsid (orf ) gene of prrsv and differentiating genotypes and was used [ ] with a stratagene mx p qpcr system (agilent genomics, wokingham, uk). sequencing of the prrsv open reading frame (orf ) gene was subsequently performed on nucleic acids from tonsil samples testing positive in the diagnostic prrsv pcr to characterize the viruses present [ ] . the pcr amplicons were purified using the beckman ampure solid phase reversible immobilisation (spri) technique (beckman coulter ltd., high wycombe, uk). cycle sequencing was performed using forward and reverse primers and abi bigdye chemistry (applied biosystems ltd., warrington, uk) at the end of which the dye terminators were removed using beckman cleanseq spri (beckman coulter ltd., high wycombe, uk). samples were sequenced on an abi capillary electrophoresis dna analyser (applied biosystems ltd., warrington, uk) and the raw data analysed by abi seqscape software (applied biosystems ltd., warrington, uk). the resulting sequences were aligned with reference sequences from genbank, and with other uk prrsv orf sequences from samples submitted to the animal and plant health agency (apha) for prrs diagnostic testing between and , using the clustalw algorithm [ ] in mega [ ] . the phylogenetic tree was generated in mega using the neighbour-joining method [ ] , with the evolutionary distances being computed using the maximum composite likelihood method [ ] . the sequences obtained were deposited in genbank with accession numbers mf to mf . existing data for prrsv seropositivity [ ] and hev seropositivity and active infection [ ] were collated with data obtained in this study for prrsv active infection. associations between the two viruses, or antibodies to them, in the same carcasses were investigated using χ tests, with stata v. (statacorp, college station, tx, usa). collated data for prrsv seropositivity and hev seropositivity and active infection (rna in plasma and/ or cecal contents) were available for pigs. collated data for prrsv active infection (rna in tonsil) and seropositivity and active infection for hev were available for pigs. prrsv rna was detected in of tonsil samples. this corresponds to . % of the prrsv seropositive finisher pigs showing active prrsv infection at slaughter. importantly, all pcr-positive samples were of genotype (european), which is endemic in the uk, and no genotype virus, which is exotic to the uk, was detected. while the elisa-positive pigs tested by pcr originated from farms in counties, pcr-positive pigs were only identified from farms in eight of those counties. almost two-thirds of the pcr-positive pigs were from farms in east anglia or east riding and north lincolnshire. while seropositivity had been found to vary significantly between age groups (p = . ) with the highest level found in pigs aged less than six months ( . %) and lowest in those aged > months ( . %) [ ] , the prevalence of prrsv rna-positive tonsils was similar across the age groups (table ) . sequencing of the orf prrsv gene was undertaken on of the tonsil samples from which prrsv rna was detected. two pcr-positive tonsils were not suitable for sequencing as there was insufficient viral nucleic acid in the samples. six samples did not yield useable sequence data. all of the sequences confirm that the viruses belong to prrsv genotype . only three of the sequences may be considered to possibly originate from the currently licensed attenuated vaccine, with greater than % similarity between the sample and vaccine strain orf sequences ( . %, . %, and %). the phylogenetic trees (figure ) illustrate the genetic diversity of the orf genes from the samples in this study in comparison to the vaccine virus licensed in the uk at the time and published reference sequences representing the different genotypes and subtypes ( figure a ) and in more detail, in the context of previously sequenced viruses specifically from uk pigs between and (unpublished data) ( figure b ). in the within-uk analysis, there is no clear association between geographic origin and the clade in which the prrsv strains belong. all of the sequences are found in clades where other uk strains were already identified, and no distinct clustering is observed. analyses were performed to identify potential associations between prrsv serology or pcr status (prrsv rna detected in tonsil sample) and hev serology or pcr status (hev rna detected in serum or cecal contents). these are summarized in tables and . of the six animals that were pcr positive for both hev and prrsv, one was less than six months old, three were six months of age, and two were greater than six months of age. there was no evidence from this study that prrsv infection, as detected by serology or pcr, increased the likelihood of hev infection in pigs at the time of slaughter. the only associations identified suggested that for the pigs in this study, prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma or cecum (table ) ; prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma (table ) ; prrsv pcr positive animals were less likely ( . % vs. . %, p = . ) to be hev seropositive (table ) . it has been suggested that infection with prrsv renders pigs more susceptible to secondary bacterial [ , , , ] or viral [ , ] infections. in this study, we further investigated the active prrsv infection at slaughter age and the association between prrsv infection and an active hev infection in pigs entering the food chain in the uk. since the majority of uk pig farms that vaccinate against prrsv use a live vaccine [ ] it was considered that prrsv in both the vaccinated and naturally infected pigs may modulate hev infection. the abattoir survey had found a prevalence of antibodies to prrsv in slaughter-age pigs of . % [ ] . antibody to vaccine and field prrsv cannot be distinguished but vaccination of rearing pigs is less common than that of breeding pigs in the uk and is generally performed when there is an expectation of field prrsv challenge during the rearing period. therefore, seropositivity in finishers is considered a reasonable indicator of the presence of prrsv infection on the respective rearing units. from the prrsv seropositive pigs in the study, the prevalence of prrs viral rna in tonsils, where prrsv may persist up to days post infection [ ] , was . %. as tonsils from most seronegative pigs were not tested by pcr, it is possible that detection of a few prrsv-positive pigs in the early stages of infection were missed, although prrsv rna was not detected in any of the tonsils from seronegative pigs in a pilot study (data not shown). as the pigs were not showing obvious clinical signs, this is a significant finding, highlighting that a proportion of healthy pigs from prrsv-infected units may be infectious at slaughter, and if still shedding virus, may be able to transmit the virus on to other farms, for example through contaminated vehicles [ ] , underlining the need for good biosecurity during and after transport to slaughter. these findings triggered the genetic characterization of the viruses in order to further evaluate the potential risks associated with their presence. overall, the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to be representative of the overall diversity of prrsv strains circulating in the uk. none of them was from an eastern european subtype of genotype prrsv. of the successfully-sequenced viruses, three showed greater than . % similarity to the modified-live vaccine used in the uk at the time. these 'vaccine-like' viruses may derive from vaccine virus, and have been identified in previous years, although at a lower rate [ ] , possibly because most samples previously sequenced were from disease outbreaks, whereas these were detected in clinically normal pigs. the other viruses showed between . % and . % similarity to the vaccine sequence. although the degree of genetic difference of a field prrs virus from the vaccine strain cannot alone predict the degree of protection that would be afforded by the vaccine to infection by the field virus, nor allow for a determination of the strain's pathogenicity, these results further illustrate the diverse nature of field prrsv in the uk. interestingly, some of the viruses from this study are sufficiently similar to one another to be potentially linked epidemiologically, even when they originate from pigs from different geographic regions. conversely, for two pigs from the same farm, the viruses detected in them did not show a great degree of similarity. there was no relation between four prrsv sequences from animals co-infected with hev (no sequence data was available for the other two), as they each grouped into different clusters in the phylogenetic analysis, one being homologous with the porcilis prrs vaccine strain sequence. the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to fit within the overall diversity of prrsv strains previously found to be circulating in the uk. the existence of infected slaughter-age pigs with the potential, if shedding, to transmit the virus to susceptible pigs is thereby confirmed. we found no evidence in this study that exposure to, or infection with prrsv enhanced hev infection rates in pigs entering the food chain. indeed, pigs exposed to prrsv were less likely to be viremic (p = . ). the age-range of the six co-infected pigs identified reflected that of the overall population sampled. the calculated virus load in plasma for hev showed no association with either prrsv serology or pcr status (data not shown). several other studies have specifically investigated prrsv and hev co-infection [ , , ] , some by experimental infection. one report of an association of co-infection with disease was restricted to investigation of a single pig [ ] . martelli et al. [ ] found no association between these pathogens in an investigation of diagnostic submissions. the abattoir survey had investigated the presence of a number of other pathogens in clinically healthy pigs, but no evidence was found that the prrsv-seropositive pigs were more likely to carry salmonella or yersinia or have antibodies to toxoplasma [ ] . interestingly, salines et al. [ ] reported that experimental prrsv and hev co-infection did affect the hev infection dynamics in five-week old pigs with no maternal antibody for these two endemic viruses. the simultaneous co-infection resulted in delays to both the latent period ( . vs. . days with hev alone) and the humoral response ( . vs. . days) to hev, as well as increasing the infectious period ( . vs. . days), in association with an increased hev viral load. in contrast, several other studies have failed to demonstrate any association between prrsv infection and the dynamics of other viral infections [ , , ] . while the present study failed to show any positive association overall between prrsv and hev infections, this may be an age-dependent effect, and variation in the timing of the respective infections may also affect their outcomes. future investigations of natural prrsv and hev co-infections should perhaps be directed towards younger pigs, since these were under-represented in this study, and may provide different outcomes. a field study showed that the majority of pigs were infected by weeks of age [ ] . experimental infections to further characterize prrsv and hev co-infections must consider non-simultaneous infections, as they may be more relevant to the situation in the field. other viral co-infections such as porcine circovirus type (pcv- ) and prrsv or hev also remain to be investigated, with at least one report of fatal disease associated with hev and pcv- [ ] . an active hev infection in pigs entering the food chain is a potential risk to public health and there is a need to better understand factors that may influence this. the uk abattoir survey had found that one in five pigs had an active hev infection as they entered the food chain [ ] . this same finding of active hev infections in slaughter-age pigs is found worldwide [ ] [ ] [ ] [ ] . there was no evidence from the current study that prrsv infection adversely affected the proportion of hev infected pigs entering the food chain. further studies are needed to investigate factors influencing the dynamics of hev infection in the pig and within farms and that may then be used to inform means of reducing infection in slaughter-age pigs. no association was found between prrsv and hev infections in the slaughter age pigs sampled. in addition, there was no difference in strain diversity of prrsv sampled from clinically unaffected pigs in this study relative to those identified from clinical cases of disease in the uk. hepatitis e: an emerging infection in developed countries zoonotic origin of hepatitis e hepatitis e virus in england and wales: indigenous infection is associated with the consumption of processed pork products the cost of endemic disease in pig production porcine reproductive and respiratory syndrome virus: genetic diversity of recent british isolates in utero infection with prrs virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility in streptococcus suis infection infection of porcine reproductive and respiratory syndrome virus suppresses the antibody response to classical swine fever virus vaccination risk factors for salmonella enterica subsp enterica shedding by market-age pigs in french farrow-to-finish herds hepatitis e virus chronic infection of swine co-infected with porcine reproductive and respiratory syndrome virus host-pathogen interactions during porcine reproductive and respiratory syndrome virus infection of piglets increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance a prevalence study of salmonella spp., yersinia spp., toxoplasma gondii and porcine reproductive and respiratory syndrome virus in uk pigs at slaughter prevalence of hepatitis e virus infection in pigs at the time of slaughter porcine reproductive and respiratory syndrome virus: antigenic and molecular diversity of british isolates and implications for diagnosis improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice molecular evolutionary genetics analysis version . the neighbour-joining method: a new method for reconstructing phylogenetic trees molecular evolutionary genetics analysis (mega) software version . in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections risk factors for porcine reproductive and respiratory syndrome virus infection and resulting challenges for effective disease surveillance duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus an experimental model to evaluate the role of transport vehicles as a source of transmission of porcine reproductive and respiratory syndrome virus to susceptible pigs one case of swine hepatitis e virus and porcine reproductive and respiratory syndrome virus co-infection in weaned pigs detection of hepatitis e virus (hev) in italian pigs displaying different pathological lesions effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus hepatitis e virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm fatal disease associated with swine hepatitis e virus and porcine circovirus co-infection in four weaned pigs in china hepatitis e virus load in swine organs and tissues at slaughterhouse determined by real-time rt-pcr hepatitis e virus in swine and effluent samples from slaughterhouses in brazil genetic characterization and serological prevalence of swine hepatitis e virus in shandong province the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -w ynbewh authors: lee, sang-myeong; kleiboeker, steven b. title: porcine arterivirus activates the nf-κb pathway through iκb degradation date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: w ynbewh nuclear factor-kappab (nf-κb) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. the present study demonstrated for the first time that a virus belonging to the arteriviridae family activates nf-κb in marc- cells and alveolar macrophages. in porcine reproductive and respiratory syndrome virus (prrsv)-infected cells, nf-κb activation was characterized by translocation of nf-κb from the cytoplasm to the nucleus, increased dna binding activity, and nf-κb-regulated gene expression. nf-κb activation was increased as prrsv infection progressed and in a viral dose-dependent manner. uv-inactivation of prrsv significantly reduced the level of nf-κb activation. degradation of iκb protein was detected late in prrsv infection, and overexpression of the dominant negative form of iκbα (iκbαdn) significantly suppressed nf-κb activation induced by prrsv. however, iκbαdn did not affect viral replication and viral cytopathic effect. prrsv infection induced oxidative stress in cells by generating reactive oxygen species (ros), and antioxidants inhibited nf-κb dna binding activity in prrsv-infected cells, suggesting ros as a mechanism by which nf-κb was activated by prrsv infection. moreover, nf-κb-dependent expression of matrix metalloproteinase (mmp)- and mmp- was observed in prrsv-infected cells, an observation which implies that nf-κb activation is a biologically significant aspect of prrsv pathogenesis. the results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by prrsv. prrsv is an enveloped, positive-stranded rna virus that is a member of the order nidovirales, family arteriviridae, along with lactate dehydrogenase-elevating virus of mice, equine arteritis virus, and simian hemorrhagic fever virus. prrsv causes one of the most economically important diseases of swine which is characterized by severe and sometimes fatal respiratory disease and reproductive failure. infection with prrsv also predisposes pigs to infection by bacterial and viral pathogens such as steptococcus suis, haemophilus parasuis, mycoplasma hyopneumoniae, acti-nobaccillus pleuropneumoniae, salmonella spp., and swine influenza virus (benfield et al., ; done and paton, ; galina et al., ; groschup et al., ; kawashima et al., ; zeman et al., ) . the most consistent pathological lesions caused by prrsv during acute infection are interstitial pneumonia and mild lymphocytic encephalitis (halbur et al., ; plagemann, ; rossow et al., rossow et al., , . tissue macrophages and monocytes are the major target cells during both acute and persistent infection (molitor et al., ) , although pneumocytes and epithelial germ cells of the testis have also been shown to be infected (sur et al., (sur et al., , . viruses are known to control cellular signal transduction pathways, and the nf-nb pathway is a common target of many viruses. nf-nb is an inducible transcription factor that plays a key role in inflammation, innate immune responses, the regulation of cell proliferation, and cell survival (caamano and hunter, ; li and verma, ) . activation of nf-nb by viral infection is a key trigger to inducing type i interferon (ifn) transcription and other immune responses, including pro-inflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases (mmps), cyclooxygenase (cox ), and inducible nitric oxide synthase (inos) (caamano and hunter, ; santoro et al., ) . these molecules are involved in initiating adaptive immune responses by recruiting immune cells to the site of infection. furthermore, it was recently demonstrated that temporally activated nf-nb confers an essential innate antiviral response against cytoplasmic rna viruses (human parainfluenza virus type and respiratory syncytial virus) in an ifn-independent manner, showing the importance of nf-nb in the innate antiviral response (bose et al., ) . nf-nb exists as a homodimer or heterodimer comprised of one or two of five subunits, rela (p ), p , relb, c-rel, and p . the predominant form is a heterodimer composed of p and rela subunits . when inactive, nf-nb is sequestered in the cytoplasm by associating with inhibitory proteins of the inb family, including inba, inbh, and inbe, which mask the nuclear localization signal. in response to a wide range of stress signals (e.g., lipopolysaccharide (lps), tumor necrosis factor (tnf), interleukin (il)- , and virus infection), the inactive nf-nb-inb complex is dissociated via serine phosphorylation by inb kinase (ikk) and degradation of inb in proteasomes. these events lead to the unmasking of the nuclear localization sequence of nf-nb, which then allows nf-nb to enter the nucleus and activate transcription of target genes . while the immune response against prrsv is not fully characterized, experimental work has demonstrated that the adaptive immune response of prrsv-infected pigs is generally ineffective (horter et al., ; murtaugh et al., ; wills et al., wills et al., , . specific evidence of an ineffective adaptive immune response includes a slow neutralizing antibody response, which is typically not detected until weeks p.i. (albina et al., b) and does not reach maximum levels until - weeks p.i. (nelson et al., ; yoon et al., ) . while the importance of a cellmediated response for protection against prrsv is well accepted, the effectiveness of this response during the early phases of disease also appears to be suboptimal (murtaugh et al., ) . for example, the t-cell response to prrsv is weak and transient and cannot be re-stimulated for more than weeks post-challenge (molitor et al., ; xiao et al., ) . additionally, ifn-g responses of prrsvinfected pigs were relatively weak and increased slowly in comparison to pseudorabies-virus-infected pigs (meier et al., ) . although the precise mechanisms for the ineffective nature of the adaptive immune response to prrsv are not known, prrsv evasion of the innate immune responses, such as the type i ifn response, may set the stage for subsequent subversion of the adaptive immune response. previous studies demonstrated that prrsv appears to elicit weak innate interferon and cytokine responses compared to other viruses such as swine influenza virus (siv), porcine respiratory coronavirus (prcv), transmissible gastroenteritis (tge) virus, and pseudorabies virus (albina et al., a; meier et al., ; van reeth and nauwynck, ; van reeth et al., . because nf-nb is important for regulation of type i ifn and after h, cells were fixed and permeabilized followed by ifa. nuclear translocation of nf-nb was detected by confocal laser microscopy with fitc staining following incubation with a specific antibody recognizing the nf-nb p subunit. (b) nuclear translocation of nf-nb detected by western blot of nuclear extracts from marc- cells and pam cultures. both cell types were infected with prrsv at moi = . . at , , , , and (only in pam) h post-infection, nuclear extracts were prepared and subjected to western blot analysis, as described in materials and methods. cytokines (lenardo et al., ; mogensen and paludan, ) , we hypothesized that prrsv infection may inhibit nf-nb activation to prevent antiviral responses. the experiments presented herein were designed to determine if prrsv infection modulates nf-nb activation in host cells. after observing nf-nb activation following prrsv infection, the mechanisms by which prrsv mediates nf-nb activation, as well as the role of nf-nb activation in prrsv replication, were studied using a dominant negative form of inba. the present study provides a basis for understanding the molecular pathways of pathology and immune evasion associated with disease caused by prrsv. nuclear translocation, which is one of the key steps during activation of nf-nb, was detected by indirect fluorescent antibody (ifa) staining following infection with prrsv (fig. a) . in uninfected marc- cells, p staining was predominantly cytoplasmic, whereas prrsv infection (multiplicity of infection (moi) = . ) for h resulted in nuclear localization of nf-nb p staining and increased cytoplasmic staining of nf-nb p . in fig. b , western blot analysis shows that infection with prrsv led to accumulation of nf-nb protein in the nucleus. compared to uninfected control cells, the amount of nf-nb increased as prrsv infection progressed in marc- cells and pam cultures. increased nf-nb concentrations in the nucleus were apparent at h p.i. in marc- cells and at h p.i. in pam cultures. in these (and subsequent) experiments, cell viability was assessed by trypan blue staining, and cultures were consistently found to be approximately % viable in mock-infected cells and % in prrsv-infected cells at h p.i. to determine if nuclear translocated nf-nb was capable of binding nb binding motifs following prrsv infection, an nf-nb p transcription factor assay was performed using nuclear extracts of marc- cells and pam cultures infected with prrsv. marc- cells and pam cultures were either mock-infected or infected with prrsv at moi = . , and nuclear extracts were prepared at the indicated times after virus infection. following infection with prrsv, nf-nb p dna binding activity increased as prrsv infection progressed. although a slight, though statistically significant decrease was consistently observed at h p.i. in pam cultures (but not marc- cells), the predominant effect of prrsv infection in both cell types was an increase in nf-nb dna binding activity. as shown in fig. , significant increases in nf-nb dna binding activity in prrsv-infected cells were measured, especially at and h p.i, compared with that in mock-infected cells. at h p.i., nf-nb p dna binding activities increased . -fold in marc- cells and . -fold in pam cultures. the nf-nb dna binding activity observed in these assays was ablated by an excess of unlabeled competitor, but not by an excess of unlabeled noncompetitor (data not shown). taken together, these results demonstrated that prrsv induces nuclear translocation of nf-nb followed by increased dna binding activity of nf-nb both in marc- cells and pam cultures. prrsv enhances nf-jb-regulated gene expression, and nf-jb activation by prrsv is dependent on viral dose and active viral replication translocation into the nucleus allows nf-nb to stimulate expression of target genes. thus, an nf-nb reporter assay was used to determine if prrsv infection enhanced nf-nb-regulated gene expression. marc- cells were transiently transfected with an nf-nb luciferase reporter plasmid (nf-nb-luc), which contains nb binding motifs under the control of a cmv promoter. therefore, luciferase expression is under control of nf-nb activation. after transfection, cells were infected with prrsv for h or left uninfected. nf-nb-regulated luciferase expression was significantly enhanced during prrsv infection at and h p.i. which correlated with an increased level of nf-nb dna binding activity (fig. a) . nf-nb activity was . -fold and . -fold higher in prrsv-infected cells at and h p.i. compared to uninfected control cells. these findings show that prrsv infection stimulated nf-nbregulated gene expression late in infection, which means that nf-nb activated by prrsv is transcriptionally active and functional. to determine if there was a relationship between prrsv replication and nf-nb activation, marc- cells were infected at various mois and nf-nb activation was monitored by measuring nf-nb-regulated luciferase expression. as shown in fig. b , a higher moi resulted in higher levels of nf-nb activation, suggesting that nf-nb activation by prrsv is viral dose-dependent. in some viral infections, binding of the viral particle to a cellular surface receptor is sufficient to trigger signaling cascades that activate nf-nb. to test this possibility, uv-inactivated prrsv was used to determine if prrsv binding to its receptor mediates nf-nb activation. as shown in fig. b , uv-inactivation of prrsv decreased nf-nb activation compared to infection with noninactivated (fully infectious) prrsv. nf-nb-regulated gene expression was reduced from . -fold to . -fold at moi = , from . -fold to . at moi = . , and from . -fold to . -fold at moi = . . these data demonstrated that nf-nb levels are elevated primarily as a result of prrsv replication and that uv-inactivation of prrsv significantly decreased this effect. a key step that leads to nf-nb activation in response to many extracellular stimuli is degradation of inb proteins. therefore, it was determined if prrsv-mediated changes in the inb proteins correlated with increased nf-nb activity. protein levels of inba, inbh, and inbe were monitored by western blot analysis following a time course of infection in marc- cells and pam cultures. results in fig. show that inba, inbh, and inbe were found in uninfected marc- cells and pams and the protein levels were mostly unchanged throughout the time course. in marc- cells (fig. a ), prrsv infection resulted in a lower concentration of inba at h p.i. compared to uninfected control cells, suggesting proteosomal degradation of inba. this correlated with the highest nf-nb activity at h p.i. among the time points tested. however, inba was still weakly detectable in prrsv-infected cells at late times p.i. inbh and inbe remained relatively constant at all time points in prrsv-infected marc- cells. in pam cultures, degradation of inba and inbe was detected after prrsv infection at h and h p.i. as shown in fig. b . the degradation of inbh protein was detected at h p.i. the onset of the degradation of inb proteins correlated with the nf-nb activation later in prrsv infection as observed above. for both marc- cells and pam cultures, the same blot was also reacted with an actin-specific antibody to confirm that comparable amounts of protein were loaded in each lane. #p < . compared to mock-infected control, *p < . compared to mock-infected control. results are representative of at least three independent experiments. (b) marc- cells transfected with nf-nb-luc and phrg-tk plasmid were infected with prrsv or uv-inactivated prrsv at moi = , . , and . for h. after washing with pbs, fresh medium was added. at h post-infection, cells were lysed. firefly and renillar luciferase activities were measured by using a dual luciferase reporter assay kit. the luciferase assays were performed in triplicate. firefly luciferase activity was normalized by renillar luciferase activity. results are shown as the relative fold change compared to that of mock-infected cells. the symbol c indicates p < . for results from infectious virus compared to mock-infected controls. the symbols #, *, and k indicate p values < . , < . , and < . , respectively, for results from uvinactivated virus compared to infectious virus. these results are representative of at least three independent experiments. each bar represents the averaged data from one representative experiment. values are shown as the mean t sd from triplicate wells. to determine if nf-nb activation by prrsv was dependent on inba degradation in marc- cells, the nf-nb pathway was blocked by using an adenovirus vector expressing a dominant negative form of inba (ad-inbadn) which lacks both constitutive (barroga et al., ) and inducible (brown et al., ) phosphorylation sites. thus, inbadn is a potent nf-nb inhibitor. the same adenovirus vector expressing gfp (ad-egfp) instead of inbadn was used as a control. marc- cells were infected with ad-egfp or ad-inbadn at various mois ( , , or ) for h and then transfected with pnf-nb luc plasmid and phrg-tk plasmid followed by superinfection with prrsv at moi = . . after h, cells were lysed and analyzed for luciferase activity. overexpression of inbadn significantly suppressed constitutive nf-nb activity compared to that in the ad-egfp, and such inbadn expressing cells failed to activate nf-nb in response to prrsv infection (fig. ) . it was then determined if nf-nb was required for efficient prrsv replication. cell culture medium was collected at , , , , and h p.i. production of infectious progeny virus was determined by serial -fold dilutions of viral stocks with % tissue culture infectious dose (tcid ) titers calculated by the method of reed and muench ( ) on marc- cells. the results representing three independent experiments are shown in fig. . the kinetics of prrsv replication were compared to that in control cells. the results showed that neither ad-egfp (fig. a ) nor ad-inbadn (fig. b ) affected prrsv replication in marc- cells. in addition, a typical prrsv-induced cpe was observed in both ad-egfp-and ad-inbadn-infected cells. therefore, this result showed that blocking the nf-nb pathway by overexpression of inbadn does not alter production of prrsv progeny viruses. intracellular ros production was detected by staining with the hydrogen-peroxide-sensitive fluorescent dye dcfh-da which is cleaved intracellularly by nonspecific . degradation of inba is required in nf-nb activation induced by prrsv. marc- cells were infected with ad-egfp or ad-inbadn at different moi for h and then transfected with pnf-nb luc plasmid and phrg-tk plasmid. cells were then mock-infected or infected with prrsv at moi = . . at h p.i., cells were lysed, and lysates were analyzed for firefly and renillar luciferase activities using a dual luciferase reporter assay kit. firefly luciferase activity was normalized by renillar luciferase activity. results are shown as the relative fold change compared to that of mockinfected cells. for all assays, analysis was performed in triplicate, and values are shown as mean t sd. these results are representative of at least three independent experiments. *p < . . esterases to form dcfh. ros in the cells then oxidizes dcfh to form the fluorescent product dcf (sawada et al., ) . as shown in fig. a , dcf fluorescence was enhanced in cells infected with prrsv at h p.i, suggesting that prrsv induced ros production in marc- cells. to determine if ros induction by prrsv contributed to activation of nf-nb, cells were treated with antioxidants and nf-nb binding activity was measured at h p.i. the dna binding activity of nf-nb p was markedly reduced in prrsv-infected cells when treated with the antioxidant pdtc or nac as shown in fig. b . the highest concentration of pdtc or nac used in this experiment reduced nf-nb activity to less than % in prrsv-infected cells. to rule out that the observed effect is simply due to inhibition of viral replication by antioxidants, virus titer was determined at and h p.i. (fig. c) . prrsv replication was not significantly affected by either pdtc or nac in all concentration tested. to investigate a possible biological role of nf-nb activation in prrsv pathogenesis, mrna expression of mmp- and mmp- which are regulated by nf-nb were determined at h p.i. as shown in fig. , mmp- and mmp- gene expression were significantly enhanced by prrsv infection which increased expression by approximately -and -fold, respectively. overexpression of inbadn completely blocked mmp- and mmp- gene expression. therefore, these data indicate that the activation of the nf-nb pathway by prrsv was necessary for enhanced mrna expressions of mmp- and mmp- . virus -host interactions lead to both activation and inhibition of complex cellular pathways, resulting in antiviral responses as well as enhanced viral replication and virulence. despite years of research, little is known about intracellular signaling pathways that play key roles after prrsv infection and the role of these pathways in prrsv pathogenesis. the present study demonstrated for the first time that a virus belonging to the arteriviridae family activates nf-nb in host cells and that potential mechanisms of prrsv-mediated nf-nb activation are derived from the inb protein degradation and ros induction. the major target cells of prrsv in vivo are tissue macrophages such as pams. marc- cells are the only continuous cell line that is highly permissive for prrsv infection (kim et al., ) , and they are typically used for in vitro experiments of prrsv as well as virus maintenance and attenuation in the laboratory. viruses could have different effects on nf-nb pathways depending on the cell type infected as demonstrated in epstein-barr virus and measles virus infection (devergne et al., ; dhib-jalbut et al., ; dreyfus et al., ; fang et al., ; helin et al., ) . therefore, in the present study, both marc- cells and pam cultures were used to determine if prrsv activates the nf-nb pathway. this study showed that prrsv infection resulted in increased nuclear translocation of nf-nb and increased dna binding activity both in marc- cells and pam cultures. in addition, nf-nbdependent luciferase expression was significantly increased in marc- cells by prrsv infection. although the nf-nb reporter assay was not successfully performed in pam cultures (due to extremely low transfection efficiencies of pam cultures), results presented here clearly demonstrated that prrsv activates the nf-nb pathway in both its natural target cells, pam, as well as in a continuous cell line, marc- cells. viruses have developed various strategies which lead to either activation or inhibition of nf-nb-dependent gene transcription for their benefits (santoro et al., ) . the nf-nb pathway can be activated as a protective response of the host to viruses. therefore, some viruses, such as vaccinia virus, african swine fever virus, influenza a virus, and mengovirus, have evolved strategies to block nf-nb activation in order to evade the innate immune response (powell et al., ; shisler and jin, ; wang et al., ; zoll et al., ) . the nf-nb pathway can also be activated directly by viruses. viruses including hiv, herpesviruses, hepatitis c virus, encephalomyocarditis virus, reovirus, dengue virus, west nile virus, and herpes simplex virus have evolved strategies to activate nf-nb to exploit nf-nb for optimized replication, or to control host cell proliferation and survival to maximize viral progeny production (connolly et al., ; goodkin et al., ; jan et al., ; santoro et al., ; schwarz et al., ; waris et al., ) . despite the importance of the nf-nb pathway in immune response, it has not been determined if prrsv or other arteriviruses modulate this pathway. previous studies demonstrated that prrsv induced weak type i ifn responses (albina et al., a; lee et al., ; miller et al., ; van reeth et al., ) . therefore, it has been postulated that prrsv inhibited the nf-nb pathway to evade antiviral responses of host cells. however, the present study provides evidence that prrsv actually activates the nf-nb pathway in pams, which are primary target cells in vivo. the synthesis of type i ifn is regulated trascriptionally and post-transcriptionally, and various transcription factors such as the interferon regulatory factor (irf) family as well as nf-nb may be involved (hiscott et al., ; taniguchi and takaoka, ; wathelet et al., ) . therefore, it is possible that prrsv blocks ifn gene expression at transcriptional and/or post-transcription levels but does not inhibit the nf-nb pathway. fig. . prrsv increases mmp- and mmp- gene expression through an nf-nb-dependent pathway. marc- cells were infected with prrsv for h at moi = . , and total rna was extracted and treated with dnase i. quantitative real-time rt-pcr was performed for mmp- or mmp- specific primers. results are expressed as relative fold changes of mmp- or mmp- mrna using cyclophilin as an internal control. values are shown as the means t sd from triplicate wells and represent two independent experiments. *p < . compared to ad-egfp/prrsvinfected cells. a number of studies have suggested that oxidative stress induced by increased generation of ros is involved in the activation of nf-nb (ghosh and karin, ; janssen-heininger et al., ) . virus infections such as human immunodeficiency virus (hiv) (israel and gougerot-pocidalo, ) , cytomegalovirus (cmv) (speir, ) , influenza virus (flory et al., ) , hepatitis b virus (hbv) , hepatitis c virus (hcv) (gong et al., ) , japanese encephalitis virus (lin et al., ) , and herpes simplex virus (mogensen et al., ) activate the nf-nb pathway through ros production. our study demonstrated that prrsv generated ros, and the involvement of ros in nf-nb activation by prrsv was demonstrated by reduced nf-nb binding activity in the presence of pdtc and nac. it has been shown that oxidative stress induced by ros is associated with viral pathogenesis in case of influenza virus and hiv (peterhans, ; schwarz, ) . however, the role of ros in prrsv pathogenesis remains to be elucidated. viruses modulate nf-nb activation through various mechanisms. activation of nf-nb is usually mediated by degradation of inba in a proteasome-dependent mechanism after phosphorylation by ikk (hayden and ghosh, ) . inba is generally thought to be the major inhibitor of nf-nb activation. nf-nb activation by influenza virus is mediated by oxidative radicals and activation of ikk as a result of overexpression of viral proteins in endoplasmic reticulum (flory et al., ) . the tax transactivator oncoprotein of human t-lymphotropic virus- activates nf-nb by interacting directly with ikk (o'mahony et al., ) . hsv- induces persistent translocation of nf-nb by inba degradation (patel et al., ) . in this study, western blot analysis of inb protein levels revealed that inba protein was degraded in prrsv-infected cells and the expression of inba-dn eliminated nf-nb activation by prrsv. this finding demonstrates that nf-nb activation by prrsv is mediated at least in part by inba degradation in marc- cells and the degradation of inba, inbh, and inbe in pam cultures. this result indicates the possibility that different molecules are involved in nf-nb activation in pam culture infected with prrsv compared to marc- cells. however, the precise mechanism through which prrsv influences the inb degradation in both cells is presently unknown. some viruses activate the nf-nb pathway through viral protein-cellular receptor interaction. for instance, hiv gp and ebv gp activate nf-nb signaling pathway through binding to cd and cd (bossis et al., ; d'addario et al., ; sugano et al., ) . however, it is unlikely that nf-nb activation by prrsv is triggered solely by viral binding to its cognate cellular receptor because the level of nf-nb activation by prrsv increased as prrsv replication progression was significantly reduced by uv-inactivation of virus. therefore, it is possible that prrsv replication or viral protein expression is a prerequisite for activation of the nf-nb pathway. alternatively, a soluble factor induced by prrsv could be responsible for the delayed nf-nb activation. previous studies have demonstrated a requirement for nf-nb activation in viral replication. influenza virus infection is dependent on an active nf-nb signaling pathway (nimmerjahn et al., ) . inhibition of nf-nb activation blocked influenza virus infection of susceptible cells, and cells with low nf-nb activity were poorly susceptible to influenza virus infection (nimmerjahn et al., ) . efficient replication of hsv- is promoted by nf-nb activation through the inb kinase-inb-p pathway (gregory et al., ) . however, blocking the nf-nb pathway by overexpression of dominant negative forms of inba did not interfere with prrsv replication, suggesting that activation of the nf-nb pathway is non-essential for efficient viral replication. similarly, other studies with japanese encephalitis virus or cytomegalovirus demonstrated that nf-nb activation is not required for efficient viral replication (benedict et al., ; liao et al., ) . although nf-nb activation does not play an essential role in prrsv replication in vitro, it does not mean that nf-nb has no contribution to prrsv pathogenesis in vivo. macrophages represent an important source for a variety of soluble immune mediators, including cytokines, chemokines, mmps, and adhesion molecules which often contain nf-nb binding sites in their promoters (caamano and hunter, ; kim and koh, ; li and verma, ; mogensen and paludan, ) . the mmps are a group of zinc-and calcium-dependent endopeptidases that degrade an extracellular matrix implicated in tissue remodeling and chronic inflammation. mmps, especially mmp- and mmp- , play a role in immune responses by promoting infiltration of inflammatory cells (kumagai et al., ) . a previous study demonstrated that prrsv infection significantly increased mmp- and mmp- which correlated with the appearance of severe histological lung lesions characterized by massive lymphomononuclear cell infiltration and possible local immunosuppression in prrsv-infected pigs. therefore, it is possible that mmps produced in prrsv-infected cells by nf-nb activation could mediate the influx of new cells of the monocyte/macrophage lineage. it is well established that nf-nb may play a pivotal role in apoptosis of virus-infected cells. the nf-nb activation by viruses could be either an anti-apoptotic response to maximize viral replication by prolonging host cell survival or pro-apoptotic response as a mechanism to increase virus spread (bowie et al., ; mi et al., ) . in cells infected with viruses such as sindbis virus, reovirus, or dengue virus, apoptosis is facilitated by the activation of nf-nb which was triggered by viral infection (connolly et al., ; jan et al., ; lin et al., lin et al., , . in others, activated nf-nb prevents apoptosis and prolongs cell survival (bowie et al., ; goodkin et al., ; grimm et al., ; su et al., ; thomas et al., ) . prrsv has been known to induce apoptosis mostly in bystander cells in vivo (sirinarumitr et al., ) . it is not known yet whether the activation of nf-nb in prrsv infection plays an anti-apoptotic role or pro-apoptotic role. in summary, the present study demonstrated that prrsv activates nf-nb via inb degradation in marc- cells and pam cultures and that nf-nb activation is not required for efficient prrsv replication in vitro. ros induction likely contributes to activation of the nf-nb pathway in prrsvinfected cells. however, the detailed molecular mechanisms and viral components underlying nf-nb activation remain to be elucidated. this study also suggested a possible role of nf-nb activation in prrsv pathogenesis by showing that prrsv increased mmp- and mmp- mrna expression through the nf-nb pathway. future studies will confirm if prrsv activates the nf-nb pathway in vivo. understanding the role of nf-nb activation following prrsv infection will contribute important information about the molecular pathogenesis of prrsv infection. the marc- cell line, which is a clone of the african green monkey kidney cell line ma- , and the hek- cell line, which is derived from human embryonic kidney cells were cultured and maintained in dulbecco's modified eagle medium (dmem) supplemented with % fbs, . ag/ml fungizone, u/ml penicillin, ag/ml streptomycin sulfate, and ag/ml gentamicin (biowhittaker inc., walkersville, md) and then held at -c in a humidified % co incubator. the pam cultures were obtained by bronchoalveolar lavage of -to -week-old domestic piglets from a prrsv negative herd. the lungs were removed from the piglets immediately after euthanizing by electrocution, and rpmi- medium (life technologies, grand island, ny) was introduced through the main stem bronchi. bronchoalveolar lavage fluid was centrifuged at  g for min. after centrifugation, cell pellets were resuspended in rpmi- medium supplemented with % fetal bovine serum (fbs), mm l-glutamine, . ag/ml fungizone, u/ml penicillin, ag/ml streptomycin sulfate, and ag/ml gentamicin (biowhittaker, walkersville, md) and plated at a density of -  cells/well in a -well primaria plate (becton dickinson and company, franklin lakes, nj) . the pam cultures were confirmed to be prrsv-negative by rt-pcr before used in subsequent experiments. pam cultures were incubated for h at -c in a humidified % co incubator and washed once with complete rpmi- media before use. all cells were maintained at -c in a humidified % co incubator. prrsv isolate was obtained from clinical cases submitted to the university of missouri's veterinary medicine diagnostic laboratory. virus stocks of prrsv were prepared in marc- cells. a low multiplicity of infection (moi < . ) was used to prepare viral stocks. for virus infection, cells were initially adsorbed with virus at the indicated moi for h at -c. after h of adsorption, cells were gently washed with medium. at indicated time points post-infection (p.i.), culture media were harvested for the virus titration, and cells were lysed to prepare cellular extracts. adenovirus vectors expressing a dominant negative inba or green fluorescent protein (gfp) were kind gifts from dr. steven l. bachenheimer (university of north carolina, usa). adenovirus stocks were prepared in hek- cells. subconfluent monolayers of marc- cells in a well plate were transfected with ng of prl-tk (promega, madison, wi) and ng of pnf-nbluc (stratagene, la jolla, ca) using the lipofectamine transfection reagent (invitrogen carlsbad, ca). the prl-tk plasmid contains the renilla reniformis (sea pansy) luciferase gene under the transcriptional control of the herpesvirus thymidine kinase promoter and constitutively expresses low levels of renillar luciferase. the pnf-nbluc plasmid contains the firefly luciferase gene under the transcriptional control of a synthetic promoter containing five direct repeats of the nf-nb binding element. after transfection, cells were mock-infected or infected with viruses at the indicated moi. at various time points post-infection, cell monolayers were lysed in al of passive lysis buffer (promega, madison, wi) followed by cell lysate analysis for both luciferase activities by using the dual luciferase reporter assay (promega, madison, wi). luciferase activity was measured as relative light units (rlus) using a luminometer (turner biosystems, inc. sunnyvale, ca). for all assays, experiments were performed in triplicate. for each experimental point, the average of the firefly luciferase activity was divided by the average of sea pansy luciferase activity to correct for differences in transfection efficiencies. the resulting ratios were used to compare the expression of the firefly luciferase gene in virus-infected cells to that present in uninfected (mock) cells. cytoplasmic and nuclear protein extracts from marc- cells and pam cultures were prepared with the nuclear extraction kit (active motif, inc., carlsbad, ca) according to the manufacturer's protocol. protein concentration was determined by the bio-rad protein assay (bio-rad, hercules, ca) with bovine serum albumin as a standard. nf-jb p transcription factor assay nf-nb binding to nb sites was assessed using the trans-am nf-nb p transcription factor assay kit (active motif, inc., carlsbad, ca). in this assay, an oligonucleotide containing the nf-nb consensus site is attached to a -well plate. the active form of nf-nb contained in cell extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies using enzyme-linked immunosorbent assay technology with absorbance reading. ten microgram of nuclear proteins was analyzed for p binding to nb oligonucleotide according to the manufacturer's instructions. the specificity of the assay was monitored by competition with free wild-type nb consensus oligonucleotide or mutated nb consensus oligonucleotide. marc- cells were washed three times with pbs, fixed and permeabilized in cold methanol for min at -c, and washed three times with pbs. cells were incubated for h at room temperature (rt) with a : dilution of primary mouse anti-nf-nb p antibody (santa cruz biotechnology, santa cruz, ca). the cells were then washed five times in pbs followed by incubation for h with the anti-mouse igg secondary antibody conjugated with fitc (sigma, st. louis, mo). the cells were washed again with pbs and then examined with an olympus x microscope fitted with a bio-rad mrc- confocal laser (bio-rad, hercules, ca). western blotting was performed by utilizing a standard protocol (davis et al., ) . briefly, cytoplasmic or nuclear extracts were diluted ( : ) in  sample buffer and boiled for min. twenty micrograms of each extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to a nitrocellulose membrane (amersham biosciences, piscataway, nj). the membrane was washed with phosphate-buffered saline-tween (tpbs), blocked in a solution of tpbs containing % nonfat dry milk, and then washed three times. the membrane was then incubated with primary antibody overnight at -c or h at rt, washed three times with tpbs, and incubated with the secondary antibody horseradish peroxidase (hrp) conjugate solution for h at rt. samples were washed three times with tpbs, and then the signal was detected with the chemiluminescent protein detection system (amersham biosciences, piscataway, nj). antibodies used for western blot are anti-nf-nb p (santa cruz biotechnology, santa cruz, ca), anti-inba (cell signaling, beverly, ma), anti-inbh (santa cruz biotechnology, santa cruz, ca), anti-inbq (santa cruz biotechnology, santa cruz, ca), anti-rabbit igg-hrp (amersham biosciences, piscataway, nj), and anti-actin (sigma, st. louis, mo). cells were treated with v, v-dichlorofluorescein diacetate (dcfh-da, calbiochem, la jolla, ca) for min and washed twice with pbs. the cells were observed under a fluorescence microscope. the antioxidants used in this study were n-acetyl-lcysteine (nac; calbiochem, la jolla, ca) in pbs and pyrollidine dithiocarbamate (pdtc; sigma, st. louis, mo) in pbs. concentrations which did not show cytotoxicity were used in this study. none of the solvents alone affected nf-nb activation in the concentrations used in this study (data not shown). extraction of rna was performed using trizol (invitrogen, carlsbad, ca), and the nucleospin rna ii kit (bd biosciences inc., palo alto, ca) with dnase i digestion performed directly on the spin column according to the manufacturer's instructions. heterologous competitor rna for quantification of mmp- , mmp- , or cyclophilin was synthesized using the respective real-time rt-pcr primer sequences in a methodology previously described (kleiboeker, ) . the concentration of purified competitor rna was estimated by measuring the absorbance at nm, and the purity was assessed by determining the ratio of absorbance at nm to the absorbance at nm. samples were considered to be relatively pure and suitable for use as quantification standards if the ratio was ! . . following purification, the rna was serially diluted in rnase-free dh o and stored as aliquots at À -c. the number of molecules of competitor rna/al was estimated based on the rna concentration and the molecular weight of the transcript. amplification of al rna was performed using the qiagen quantitect probe rt-pcr kit (qiagen inc., valencia, ca) with thermocycling, and detection was performed in a stratagene mx (stratagene inc., la jolla, ca). samples were analyzed in triplicate. thermocycling conditions were: -c ( min), -c ( min), followed by cycles of denaturation ( -c, s) and annealing/ extension ( -c, s). the primers and probe used for exonuclease (taqman) amplification of mmp- were v-ccaccacaacatcacctattgg- v (forward), v-gaa-ggcgcgggcaaa- v (reverse), and -fam-tccaaaa-ctactcggaagacttgccgc-bhq - v (probe). the primers and probe used for v exonuclease (taqman) amplification of mmp- were v-ccgtcgcccatcat-caa- v (forward), v-caggtattgcactgccaactct- v (reverse), and -fam-cgatgtcgcccccaaaacgga-bhq - (probe). amplification of cyclophilin was performed as previously described (miller et al., ) and was used for normalization of mmp- and mmp- transcript quantities. all oligonucleotide primers were used at a final concentration of . am, and the dual-labeled probes were used at a final concentration of . am. all oligonucleotide primers and probes were synthesized by integrated dna technologies, inc. (coralville, ia). the relative copy numbers of mmp- and mmp- compared to cyclophilin were determined as previously described (pfaffl, ) . the student's t test was used for the statistical analyses. p values of less than . were considered statistically significant. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) constitutive phosphorylation of i kappa b alpha by casein kinase ii neutrality of the canonical nf-kappabdependent pathway for human and murine cytomegalovirus transcription and replication in vitro characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) temporal activation of nf-kappab regulates an interferon-independent innate antiviral response against cytoplasmic rna viruses nf-kappab activation upon interaction of hiv- envelope glycoproteins with cell surface cd involves ikappab kinases viral appropriation of apoptotic and nf-kappab signaling pathways control of i kappa b-alpha proteolysis by site-specific, signal-induced phosphorylation nf-kappab family of transcription factors: central regulators of innate and adaptive immune functions reovirus-induced apoptosis requires activation of transcription factor nf-kappab epstein -barr virus envelope glycoprotein gp induces nf-kappab activation and il- beta synthesis in human monocytes -macrophages involving pkc and pi -k basic methods in molecular biology association of traf , traf , and traf with an epstein -barr virus lmp domain important for blymphocyte transformation: role in nf-kappab activation failure of measles virus to activate nuclear factor-kappa b in neuronal cells: implications on the immune response to viral infections in the central nervous system porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression inactivation of nf-kappab by ebv bzlf- -encoded zebra protein in human t cells mechanism of measles virus failure to activate nf-kappab in neuronal cells influenza virus-induced nf-kappab-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase interaction between streptococcus suis serotype and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets missing pieces in the nf-kappab puzzle nf-kappa b and rel proteins: evolutionarily conserved mediators of immune responses human hepatitis c virus ns a protein alters intracellular calcium levels, induces oxidative stress, and activates stat- and nf-kappa b nf-kappab is required for apoptosis prevention during herpes simplex virus type infection efficient replication by herpes simplex virus type involves activation of the ikappab kinase-ikappab-p pathway ebv latent membrane protein- protects b-cells from apoptosis by inhibition of bax serological studies on the potential synergism of porcine reproductive and respiratory syndrome virus and influenza-, corona-and paramyxoviruses in the induction of respiratory symptoms in swine comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus signaling to nf-kappab measles virus activates nf-kappa b and stat transcription factors and production of ifn-alpha/beta and il- in the human lung epithelial cell line a convergence of the nf-kappab and interferon signaling pathways in the regulation of antiviral defense and apoptosis characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection oxidative stress in human immunodeficiency virus infection potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and nf-kappab are sequentially involved recent advances towards understanding redox mechanisms in the activation of nuclear factor kappab detection of porcine reproductive and respiratory syndrome virus and mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress lipopolysaccharide activates matrix metalloproteinase- in endothelial cells through an nf-kappab-dependent pathway enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line signaling pathways to the assembly of an interferon-beta enhanceosome. chemical genetic studies with a small molecule applications of competitor rna in diagnostic reverse transcription-pcr inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes the involvement of nf-kappa b in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction nf-kappab regulation in the immune system salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa b activation thiol agents and bcl- identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor nf-kappa b suppression of steady-state, but not stimulus-induced nf-kappab activity inhibits alphavirus-induced apoptosis replication-incompetent virions of japanese encephalitis virus trigger neuronal cell death by oxidative stress in a culture system signal transduction through nf-kappa b gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination induced apoptosis supports spread of adenovirus vectors in tumors. hum interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc- cells molecular pathways in virus-induced cytokine production. microbiol activation of nf-kappa b in virus-infected macrophages is dependent on mitochondrial oxidative stress and intracellular calcium: downstream involvement of the kinases tgf-beta-activated kinase , mitogenactivated kinase/extracellular signal-regulated kinase kinase , and i kappa b kinase immunity to prrsv: double-edged sword immunological responses of swine to porcine reproductive and respiratory syndrome virus infection serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus active nf-kappab signalling is a prerequisite for influenza virus infection human t-cell lymphotropic virus type tax induction of biologically active nf-kappab requires ikappab kinase- -mediated phosphorylation of rela/p herpes simplex type induction of persistent nf-kappa b nuclear translocation increases the efficiency of virus replication oxidants and antioxidants in viral diseases: disease mechanisms and metabolic regulation a new mathematical model for relative quantification in real-time rt-pcr lactate dehydrogenase-elevating virus and related viruses an ikappab homolog encoded by african swine fever virus provides a novel mechanism for downregulation of proinflammatory cytokine responses in host macrophages method of determining fifty percent endpoints pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs fetal microscopic lesions in porcine reproductive and respiratory syndrome virus-induced abortion nf-kappab and virus infection: who controls whom analytical and numerical techniques for the evaluation of free radical damage in cultured cells using scanning laser microscopy oxidative stress during viral infection: a review nf-kappab-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in p knockout mice the vaccinia virus k l gene product inhibits host nf-kappab activation by preventing ikappabalpha degradation a pneumo-virulent united states isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo cytomegalovirus gene regulation by reactive oxygen species. agents in atherosclerosis role of nf-kappab and myc proteins in apoptosis induced by hepatitis b virus hbx protein epstein -barr virus binding to cd activates the initial viral promoter via nf-kappab induction in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis the interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the irf family of transcription factors respiratory syncytial virus inhibits apoptosis and induces nf-kappa b activity through a phosphatidylinositol -kinase-dependent pathway proinflammatory cytokines and viral respiratory disease in pigs differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding influenza a virus ns protein prevents activation of nf-kappab and induction of alpha/beta interferon mitochondrially associated hepatitis b virus x protein constitutively activates transcription factors stat- and nf-kappa b via oxidative stress hepatitis c virus ns a and subgenomic replicon activate nf-kappab via tyrosine phosphorylation of ikappabalpha and its degradation by calpain protease virus infection induces the assembly of coordinately activated transcription factors on the ifn-beta enhancer in vivo porcine reproductive and respiratory syndrome virus: a persistent infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection laboratory investigation of prrs virus infection in three swine herds the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord- -jhm u ka authors: wang, dang; chen, jiyao; yu, chaoliang; zhu, xinyu; xu, shangen; fang, liurong; xiao, shaobo title: porcine reproductive and respiratory syndrome virus nsp antagonizes type i interferon signaling by targeting irf date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: jhm u ka porcine reproductive and respiratory syndrome virus (prrsv) is an arterivirus from the nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. the prrsv-encoded nonstructural protein (nsp ) is a nidovirus-specific endoribonuclease (nendou) that is conserved throughout the arteriviridae and coronaviridae families. previously, our research and that of others demonstrated that prrsv nsp inhibits type i interferon (ifn) production through nendou activity-dependent mechanisms. here, we found that prrsv nsp also inhibited ifn-stimulated response element (isre) promoter activity and subsequent transcription of ifn-stimulated genes (isgs). detailed analysis showed that nsp targeted interferon regulatory factor (irf ), but not transducer and activator of transcription (stat ) or stat , key molecules in the type i ifn signaling pathway. furthermore, the nsp -irf interaction impaired the formation and nuclear translocation of the transcription factor complex ifn-stimulated gene factor (isgf ) in both nsp -overexpressed and prrsv-infected cells. importantly, nsp mutations (h a, h a, and k a) that ablate nendou activity or its cell cytotoxicity also interacted with irf and retained the ability to block ifn signaling, indicating that the nsp -irf interaction is independent of nendou activity or cell cytotoxicity of nsp . taking the results together, our study demonstrated that prrsv nsp antagonizes type i ifn signaling by targeting irf via a nendou activity-independent mechanism, and this report describes a novel strategy evolved by prrsv to counteract host innate antiviral responses, revealing a potential new function for prrsv nsp in type i ifn signaling. importance the nidovirus-specific endoribonuclease (nendou) encoded by prrsv nonstructural protein (nsp ) is a unique nendou of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. previous studies have revealed that the nendou of nidoviruses, including porcine reproductive and respiratory syndrome virus (prrsv) and human coronavirus e (hcov- e), acts as a type i interferon (ifn) antagonist. here, for the first time, we demonstrated that overexpression of prrsv nsp also inhibits ifn signaling by targeting the c-terminal interferon regulatory factor (irf) association domain of irf . this interaction impaired the ability of irf to form the transcription factor complex ifn-stimulated gene factor (isgf ) and to act as a signaling protein of ifn signaling. collectively, our data identify irf as a natural target of prrsv nendou and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling. agent, prrs virus (prrsv) (family arteriviridae, order nidovirales), has a positive-polarity single-stranded rna genome, approximately kb in length, encoding two nonstructural polyproteins (orf ab and orf b) and eight structural proteins (gp , e, gp , gp , gp , orf a, m, and n) ( , ) . after being cleaved by four virus-encoded proteases, nonstructural protein ␣ (nsp ␣), nsp ␤, nsp , and nsp , the two polyproteins are further cleaved into individual nonstructural proteins that perform different functions during the viral life cycle ( ) . to reproduce rapidly and establish a persistent infection in pigs, prrsv has developed the ability to resist host interferon (ifn) signaling, which is the key signaling pathway for innate immune responses against viral invasion or replication ( , , ) . innate immune responses are activated by host pattern recognition receptors (prrs), which recognize molecular structures called pathogen-associated molecular patterns (pamps) that are structurally conserved within a large number of pathogenic organisms ( ) . upon recognition of pamps, prrs initiate signaling pathways that ultimately trigger the production of type i ifns. subsequently, the janus kinase/signal transduction and transcription activator (jak/stat) pathway is activated by type i ifns ( , ) . briefly, type i ifns bind to their surface receptors (ifnar and ifnar ) and then activate and phosphorylate the two intracellular tyrosine kinases janus kinase (jak ) and tyrosine kinase (tyk ). these activated tyrosine kinases mainly phosphorylate two transcription factors, namely, signal transducer and activator of transcription (stat ) and stat , which interact with ifn regulatory factor (irf ) to form ifnstimulated gene factor (isgf ) ( ) . the transcription factor complex isgf (stat / stat /irf ) is transported to the nucleus, where it recognizes ifn-stimulated response elements (isres), leading to the induction of hundreds of ifn-stimulating genes (isgs) ( ) . many isgs function as potent antiviral effectors, directly preventing viral infections by targeting viral processes such as viral entry, viral genome replication, viral protein synthesis, or viral body release ( ) . during coevolution with their hosts, many viruses have developed elaborate strategies to circumvent the jak/stat pathway in ifn signaling ( ) . among several known viral evasion strategies, targeting the transcription factor complex isgf is a particularly powerful means of inactivating ifn signaling, because this strategy effectively inhibits common downstream isg expression. for example, our recent study showed that porcine deltacoronavirus blocks the jak/stat pathway by c-like protease-mediated cleavage of stat ( ) . the nonstructural protein (nsp ) of rotavirus mediates the degradation of irf by targeting the c-proximal irf-binding domain and inhibits ifn-mediated phosphorylation of stat ( , ) . sendai virus c protein binds stat to block the formation of stat /stat heterodimers or stat /stat homodimers, which inhibit ifn signaling ( ) . previous studies have shown that prrsv nsp inhibits type i ifn production through nidovirus-specific endoribonuclease (nendou) activitydependent mechanisms ( ) ( ) ( ) . however, the nendou activity of overexpressed nsp unexpectedly exhibited extensive substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells, indicating that the inhibition of ifn production by wild-type (wt) prrsv nsp may be due to its cytotoxicity ( ) . here, we found that prrsv nsp also inhibits isre promoter activity and the transcription of isgs, thereby interfering with the type i ifn signaling pathway. importantly, mutations that eliminate nendou activity and its cytotoxicity in nsp retain the ability to block ifn signaling. detailed analysis showed that nsp inhibited type i ifn signaling by targeting irf , a key molecule in the isgf complex, revealing a potential novel function of prrsv nsp in type i ifn signaling. identification of prrsv nsp as an antagonist of type i ifn signaling. type i ifn signaling induces a potent antiviral response in cells by inducing the expression of hundreds of isgs, which is vital for the control of viral infections ( ) . to assess the potential role of prrsv nsp in type i ifn signaling, the mrna levels of ifn-stimulated gene (isg ), isg , isg , and '- '-oligoadenylate synthetase (oas ) were analyzed in human embryonic kidney cells (hek- t) overexpressing hemagglutinin (ha)-tagged prrsv nsp . as shown in fig. a , prrsv nsp significantly inhibited the transcription of isgs induced by ifn-␣ compared with the control group results. because of the presence of isre in the isg promoter regions, various concentrations of prrsv nsp expression plasmid and isre-luciferase reporter plasmid were cotransfected into hek- t cells or porcine kidney cells (pk- ). the results showed that nsp strongly inhibited ifn-␣-induced isre promoter activity in a dose-dependent manner in hek- t cells (fig. b) and pk- cells (fig. c) . these results confirm the antagonistic nature of prrsv nsp in type i ifn signaling. prrsv nsp inhibits type i ifn signaling in an endoribonuclease activityindependent manner. in arterivirus, the nsp endoribonuclease is important for viral replication ( , , ( ) ( ) ( ) ( ) . several previous studies have shown that prrsv nsp inhibits type i ifn production in a nendou activity-dependent manner ( ) ( ) ( ) . we considered such a possibility for prrsv nsp -mediated inhibition of type i ifn signaling to be associated also with its nendou activity. on the basis of their chemical properties and known residue requirements, the three catalytic residues of endoribonucleases (his , his , and lys [numbering based on prrsv nsp ]) were shown to be directly involved in catalysis ( , , ) . thus, three endoribonuclease catalytic residue mutations, his ala (h a), his ala (h a), and lys ala (k a), were introduced into prrsv nsp and the corresponding eukaryotic expression plasmids expressing the n-terminally ha-tagged nsp endoribonuclease inactive mutants were constructed. to test whether the ha-tagged nsp mutants lose endoribonuclease activity, ha-tagged wt nsp and three mutants were expressed in escherichia coli. the recombinant proteins were purified ( fig. a) , and fluorescence resonance energy transfer (fret) assays were performed to detect the endoribonuclease activity. as shown in fig. b , the levels of endoribonuclease activity of the three ha-tagged nsp mutants were significantly decreased compared with the wt nsp (fig. b) . however, none of the endoribonuclease inactive mutants (h a, h a, or k a) showed a loss of the ability of nsp to inhibit isre promoter activity in cells overexpressing ha-tagged nsp mutants (fig. c) . we also used prrsv nsp as a negative control and porcine epidemic diarrhea virus (pedv) nsp as a positive control. in agreement with a previous study ( ) , pedv wt nsp suppressed ifn-␣-induced isre promoter activity whereas such an inhibitory effect was not observed in cells expressing a pedv nsp endoribonuclease inactive mutant (h a) or prrsv nsp (fig. c) . these data suggest that although the nendou activity of pedv nsp is important for inhibiting ifn signaling, prrsv nsp does not depend on the nendou activity. we further tested the ability of nsp mutants to inhibit ifn-␣-induced isg expression. as shown in fig. d , each endoribonuclease inactive mutant (h a, h a, or k a) of nsp also inhibited the ifn-␣-induced transcription of isgs. since these three mutants are also devoid of cell cytotoxicity ( ), our results indicated that the inhibition of ifn signaling observed in nsp -expressing cells is independent of its endoribonuclease activity and cell cytotoxicity. prrsv nsp disrupts isgf -mediated activation of the isre promoter. isgs are antiviral effectors induced by ifns through the formation of a tripartite transcription factor, isgf , which is composed of stat , stat , and irf ( ) . given the pivotal role of the transcription factor complex isgf in type i ifn signaling, we further investigated whether overexpression of nsp inhibits isgf -mediated signaling. as shown in fig. , coexpression of the components of transcription factor complex isgf (stat , stat , and irf ) significantly activated the isre promoter compared with the results seen with the empty plasmid control. however, activation of the isre promoter by isgf was significantly inhibited in the presence of prrsv nsp (fig. ) . similarly to the results seen with wt nsp , each endoribonuclease inactive mutant (h a, h a, or k a) was also able to suppress the isgf -mediated ires promoter (fig. ) , suggesting that the endoribonuclease activity of nsp does not govern the ability of nsp to block the activity of the isgf -induced ires promoter. consequently, we speculated that prrsv nsp might target the isgf complex to inhibit type i ifn signaling. prrsv nsp does not degrade or block the phosphorylation of stat and stat . since downregulation of molecules responsible for ifn-activated signal transduction is a mechanism commonly employed by viruses ( , ( ) ( ) ( ) , we investigated whether nsp impairs the endogenous protein levels of stat , stat , and irf . as shown in fig. , no reduction was observed in the protein levels of stat , stat , and irf as a consequence of the presence of wt nsp or expression of its endoribonuclease inactive mutants (h a, h a, and k a [compare lanes and to lane ]), suggesting that nsp does not induce downregulation of the isgf complex. the type i ifn-activated isgf transcription complex containing tyrosine-phosphorylated stat and stat associated with irf is rapidly translocated to the nucleus fig prrsv nsp inhibits isgf -induced isre promoter activity. hek- t cells were cotransfected with prrsv nsp or its endoribonuclease activity-defective mutants ( . g/well), along with porcine isgf complex (stat /stat /irf ; . g/well), isre-luc plasmid ( . g/well), and prl-tk plasmid ( . g/well). after h, cells were harvested for luciferase assays. *, p Ͻ . . after ifn treatment, and phosphorylation-dependent activation of stat and stat is critical to mediate ifn-inducible antiviral responses ( , ) . to investigate whether prrsv nsp alters the stat and stat phosphorylation status after ifn-␣ stimulation, lysates from nsp -expressing cells were subjected to western blotting using phospho-stat (stat -y ) and phospho-stat (stat -y ) antibodies (abs), respectively. the levels of phosphorylated stat and stat were greatly increased after ifn-␣ treatment, and no difference in the levels was found between nsp -expressing cells and nsp -nonexpressing cells (fig. , lanes and ) . similarly, nsp mutants lacking endoribonuclease activity (h a, h a, and k a) had no effect on the phosphorylation of stat and stat (fig. [compare lane to lanes to ]). these results indicated that prrsv nsp and its endoribonuclease inactive mutants do not target the isgf complex for degradation or prevent type i ifn-induced stat proteinactivating tyrosine phosphorylation. prrsv nsp interacts with the irf-association domain (iad) of irf . previous studies have shown that many viral proteins can interact with components of the isgf complex to inhibit type i ifn signaling ( , , ) . thus, we next investigated whether nsp functions by interacting with the isgf component. to this end, hek- t cells were transfected with expression constructs encoding ha-tagged prrsv nsp protein and flag-tagged porcine stat , stat , and irf . coimmunoprecipitation (co-ip) and immunoblotting analyses showed that nsp interacted with porcine irf , but not stat or stat ( fig. a to c). to further confirm the interaction of nsp with endogenous irf , coprecipitated endogenous irf and prrsv nsp were analyzed by immunoblotting with an anti-irf antibody and an anti-ha antibody, respectively. as shown in fig. d , reciprocal pulldown of both nsp and endogenous irf further confirmed the interaction between nsp and irf . previous studies demonstrated that irf contains an n-terminal dna-binding domain (dbd; amino acids [aa] to ) and a c-terminal iad (aa to ) connected by a flexible linker (fl) ( ) ( ) ( ) . to investigate which domain of porcine irf is involved in nsp protein binding, four mutants with deletions of different domains of irf , including irf (aa to ; dbd), irf (aa to ; dbd-fl), irf (aa to ; iad), and irf (aa to ; fl-iad), were constructed by mutagenesis (fig. e ). hek- t cells were cotransfected with various combinations of flag-tagged full-length or deleted versions of irf and the ha-tagged prrsv nsp protein. as shown in fig. f , prrsv nsp -irf interaction impairs the ifn-induced formation and nuclear accumulation of isgf . since the function of isgf relies on the selective interaction between phosphorylated stat and the irf-association domain of irf ( , ) , the observed interaction between nsp and irf -iad led us to speculate that this interaction may impair the recruitment of phosphorylated stat by irf and the subsequent nuclear accumulation of isgf . to test this hypothesis, hek- t cells were transfected with the indicated nsp expression plasmids and then treated with ifn-␣. the prrsv nsp protein and phosphorylated stat /stat (stat -y and stat -y ) were immunoprecipitated with an anti-irf antibody. as shown in fig. a , ifn-␣ treatment significantly induced the phosphorylation of stat and stat , and irf efficiently pulled down phosphorylated stat /stat after ifn-␣ treatment (compare lanes and ). however, the amount of phosphorylated stat /stat that bound to irf remarkably decreased in the presence of prrsv nsp and its mutants (h a, h a, and k a) (fig. a, lanes and to ) . to further test whether prrsv nsp prevents the ifn-induced nuclear accumulation of isgf , we performed nuclear and cytoplasmic fractionation of the cells following ifn-␣ treatment. antibodies against stat -y and stat -y were used to detect the presence of the phosphorylated proteins in the two fractions. as expected, higher levels of stat -y and stat -y were found in the nuclear fraction than in the cytoplasmic fraction after ifn-␣ treatment of mock-transfected cells (fig. b , lanes and ). in contrast, in nsp -transfected cells after ifn-␣ stimulation, more isgf complex components (stat -y , stat -y , and irf ) were detected in the cytoplasmic fraction than in the nuclear fraction (fig. b, lanes and ) . taken together, these findings indicate that prrsv nsp impairs the formation and nuclear translocation of isgf . because the endoribonuclease inactive mutants of nsp retained the ability to block type i ifn signaling, these mutants in particular were still capable of interacting with irf . next, we investigated whether these mutants, lacking endoribonuclease activity, still impaired the formation and nuclear accumulation of isgf . indeed, our data revealed that the prrsv nsp mutants (h a, h a, and k a) still significantly inhibited the ifn-induced formation and nuclear accumulation of isgf and did so to similar extents (fig. b [compare lanes to and lanes to ] ). this provided further support for the notion that the ability of prrsv nsp to block type i ifn signaling is independent of its endoribonuclease activity and cell cytotoxicity, because these three mutants had similar effects with respect to inhibiting the formation and nuclear translocation of isgf compared with wt nsp . prrsv infection inhibits the formation of isgf through the interaction of nsp with irf . to exclude the possibility that the observed prrsv nsp -irf interaction was an artifact of plasmid overexpression in cell culture, we analyzed this interaction in the context of prrsv infection. african green monkey kidney cells (marc- ) were infected with prrsv for h and then collected at h after ifn-␣ treatment for immunoprecipitation with anti-nsp or anti-irf antibodies to detect the interaction between nsp and endogenous irf . as shown in fig. a , nsp was coimmunoprecipitated with endogenous irf in prrsv-infected cells. furthermore, phosphorylated stat /stat was also coimmunoprecipitated with endogenous irf upon ifn-␣ stimulation. however, the formation of stat /stat /irf heterotrimers was significantly inhibited following prrsv infection (fig. a [compare lanes and ] ). hek- t cells were transfected with expression vectors encoding ha-nsp and its mutants. endogenous irf was precipitated and immunoblotting analysis was performed with an anti-pstat antibody (the top panel), anti-pstat antibody (the second panel), or anti-ha antibody (the fourth panel) to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in nuclear and cytoplasmic fractions. subcellular fractionation of hek- t cells, h after ifn-␣ treatment, was performed for nuclear and cytoplasmic fractions, followed by western blotting using stat -y antibody, stat -y antibody, anti-hsp antibody as a cytoplasmic marker, and anti-parp antibody as a nuclear protein marker, as indicated. ha antibody was used to detect the expression of prrsv nsp and its mutants (ha-nsp ). to detect the interaction with endogenous irf . (b) phosphorylated stat and stat in the nuclear and cytoplasmic fractions. an experiment parallel to that outlined for panel a was performed. subcellular fractionation of marc- cells was performed to obtain nuclear and cytoplasmic fractions, followed by western blotting using anti-stat -y , anti-stat -y , anti-irf , and anti-nsp antibodies, along with anti-hsp antibody as a cytoplasmic marker and anti-parp antibody as a nuclear protein marker, as indicated. (c to e) marc- cells were infected with prrsv (moi ϭ . ) or uninfected. at h postinfection, the cells were mock-treated or treated with ifn-␣ ( iu/ml) for h. after the cells were fixed and permeabilized, p-stat (c), p-stat (d), and irf (e) was visualized by immunofluorescence staining with rabbit anti-pstat , rabbit anti-pstat , or rabbit anti-irf antibody. the nsp protein was detected by the use of a nsp -specific mab. to further address whether the ifn-induced nuclear accumulation of isgf was also affected by prrsv infection, nuclear and cytoplasmic fractionation was performed. compared with uninfected cells, the levels of stat -y , stat -y , and irf proteins were reduced in the nuclear fraction of prrsv-infected cells after ifn-␣ stimulation (fig. b [compare lanes to and lanes to ]) . immunofluorescence experiments showed that prrsv infection inhibited ifn-induced nuclear accumulation of stat -y (fig. c) , stat -y (fig. d) , and irf proteins (fig. e ) compared to mock-infected cells. in prrsv-infected cells with ifn stimulation, nsp and irf were primarily colocated in the cytoplasm (fig. e) . together, these data indicated that prrsv infection inhibits the formation and nuclear accumulation of isgf through the nsp -irf interaction, which was consistent with the results of gene transfection experiments in cells expressing prrsv nsp . during coevolution with their hosts, many viruses have acquired mechanisms to circumvent host innate immune responses. of note, prrsv infection has been found to produce abnormally low levels of type i ifns and to inhibit the ability of type i ifns to induce antiviral responses ( , ) . the endoribonuclease encoded within the prrsv nsp sequence, which has the uridylate-preferred cleavage site for rna that is necessary for virus replication, has been found to be a multifunctional protein ( , ) . previously, it has been demonstrated that prrsv nsp is involved in the inhibition of ifn production through multiple distinct mechanisms, as follows. (i) nsp represses the transcription of type i ifns by inhibiting the activation of transcription factors irf and nuclear factor kappa b (nf-b), which directly activate the promoters of type i ifns ( , ) . (ii) nsp reduces the levels of transcripts and proteins of mitochondrial antiviral signaling protein (mavs) and retinoic acid-inducible gene i (rig-i), two critical factors in the ifn induction pathway ( ) . (iii) nsp removes the ubiquitin chains from ib␣ (inhibitor of nf-b alpha), thereby preventing the proteasomal degradation of ib␣ and subsequent liberation of nf-b ( ). (iv) nsp recruits the otu deubiquitinase with linear linkage specificity (otulin) to enhance its ability to remove the cellular protein ubiquitin associated with innate immunity, resulting in the additive effect of suppressing type i ifn production ( ) . however, whether nsp also regulates ifn signaling remains unclear. in this report, we present evidence that prrsv nsp suppressed type i ifn signaling by targeting irf , a key molecule in the jak/stat pathway, revealing a potential new function for the nidovirus endoribonuclease in type i ifn signaling. prrsv nsp , a conserved nendou within the arteriviridae and coronaviridae families, belongs to the xenopus laevis poly(u)-specific endoribonuclease (xendou) superfamily and plays an important role in nidovirus replication and pathogenesis ( ) . the structures of the arterivirus nsp , coronavirus (cov) nsp , and xendou catalytic domains, essential for endoribonuclease activity, and particularly the active site residues (his , his , and lys ; numbering based on prrsv nsp ), were found to be highly conserved. besides prrsv nsp , several other studies have reported that cov nsp inhibits ifn production in ectopic expression experiments. in support of this, infection with nendou activity-deficient covs, such as pedv, murine hepatitis virus, and human cov e (hcov- e), produced a remarkably high level of type i ifn in primary cells compared with wt infection, which effectively demonstrated the ifnantagonistic properties of nsp of covs ( , , ) . these data appear to indicate that endoribonuclease activity is sufficient for nendou-mediated ifn transcriptional repression; however, the nendou activity of overexpressed nsp /nsp may unexpectedly mediate nonspecific cleavage, thereby inducing cytotoxicity ( , ) . unfortunately, it was impossible to determine whether the cell cytotoxicity also contributed to the inhibition of ifn induction by nendou, as mutations disrupting the ability of nendou to block ifn production abrogated not only endoribonuclease activity but also its cell cytotoxicity ( ) . thus, the observation of ifn transcriptional suppression in previous studies could have been the result of cytotoxicity as a consequence of ectopic over-expression of nendou. previous studies have shown that prrsv wt nsp is toxic to e. coli and that the expression levels of wt nsp are extremely low and that one of the nendou mutants (nsp k a) can be expressed at high levels under identical conditions ( , ) . similar phenomena were observed in our experiments designed to express and purify nsp . indeed, in our previous study ( ) , we found that prrsv wt nsp exhibited cytotoxicity to eukaryotic cells whereas no cytotoxicity associated with the nendou mutants of nsp was detected. similarly, recombinant nsp of equine arteritis virus (eav) has been reported to be extremely toxic to a variety of hosts ( ) . thus, the lower level of expression of prrsv wt nsp may have been due to its cytotoxicity (fig. ) . interestingly, our results clearly showed that the prrsv nsp mutants (h a, h a, and k a) devoid of both nendou activity and cell cytotoxicity retained the ability to block type i ifn signaling by targeting irf , as did wt nsp , indicating that nsp has evolved a new mechanism for the impairment of ifn signaling that operates in an endoribonuclease activity-independent and cell cytotoxicity-independent manner, distinct from the inhibition of ifn induction. as a potent antiviral response, type i ifn signaling can control viral infections by activating the transcription factor complex isgf (stat /stat /irf ), resulting in increased transcription of hundreds of isgs and contributing to the development of an antiviral state ( , ) . it is becoming increasingly apparent that irf is a central factor not only for mediation of but also for regulation and direction of type i ifn responses ( ) . while many ifn effects, in particular, those associated with type i ifns, require all three canonical signaling molecules, studies of irf deficiency revealed unique roles for irf that were distinct from those of stat and stat ( , ) . recently, evidence has emerged that irf is the main viral target of the host's innate immune response. for example, our previous study ( ) revealed that porcine bocavirus nonstructural protein (ns ) inhibits the dna-binding activity of isgf by interacting with irf . another study showed that the e oncoprotein of papillomavirus binds to irf to block the formation of the isgf complex ( ) . moreover, many virus-encoded proteins, such as varicella-zoster virus orf , adenovirus early region a protein (e a), and rotavirus nsp , mediate irf degradation ( , , ) . human cytomegalovirus also reduces the protein levels of irf in human embryonic lung fibroblasts ( ) . to survive in the host, prrsv has also evolved strategies to block ifn signaling. previous studies have revealed that prrsv nsp ␤, a papain-like proteinase, blocks the nuclear translocation of isgf by inducing degradation of karyopherin-␣ ( , ) . here, we showed that nsp is another prrsvencoded antagonist of ifn signaling. prrsv nsp adopts a mechanism distinct from that of nsp ␤, i.e., interaction with irf , an essential component in the formation of isgf . our data not only highlight the multifaceted control of type i ifn signaling by prrsv but also uncover a novel mechanism by which prrsv antagonizes innate immune signaling. it is well established that irf exhibits several functionally conserved regions among the members of the irf family, such as an n-terminal dna-binding domain that recognizes and binds to isre motifs in the promoter region of most isgs and a c-terminal irf-association domain that is responsible for selective interaction with the coiled-coil domain (ccd) of stat ( , ) . recently, rengachari and colleagues reported the crystal structures of irf -iad alone and in a complex with stat -ccd ( ) . the structure of the irf -iad/stat -ccd complex revealed that surface features had deviated from their respective paralogs to enable a specific interaction between irf -iad and stat -ccd required for isgf function in cells ( ) . in this study, we found that prrsv nsp specifically interacted with irf -iad. thus, it may be the case that the targeting of irf -iad by nsp sequesters the interaction between irf and stat . this may help to explain why the formation and nuclear translocation of isgf were severely impaired in prrsv nsp -transfected cells and prrsv-infected cells. interestingly, prrsv nsp mutants (h a, h a, and k a) also impaired the formation and nuclear translocation of isgf by interacting with irf . furthermore, the three-dimensional ( d) structures of nendou activity-defective arterivirus nsp (k a in prrsv nsp ; h a and k a in eav nsp ) are remarkably similar to those of wt prrsv nsp ( , ) , with an overall root mean square deviation (rmsd) range of . to . Å (data not shown), suggesting that the ala substitution in his , his , and lys of prrsv nsp in this study might not prevent the core conformations of nsp protein from folding correctly. on the basis of data presented here and those reported by others mentioned above, we speculate that the ability to interact with irf correlated with the correct folding of nsp but not with nendou activity or cell cytotoxicity. future investigations will be required to identify the structural basis of the nsp -irf interaction. although our data clearly demonstrated that prrsv nsp could be coimmunoprecipitated with endogenous irf in prrsv-infected cells, thereby inhibiting the formation and nuclear translocation of isgf (fig. ) , whether prrsv nsp also functions as an antagonist of ifn signaling during prrsv infection has not been fully understood. one of the obstacles to studying arterivirus with nendou activitydeficient nsp mutants in cell culture is that catalytic residue mutations have been reported to nearly completely abolish viral replication even in ifn-deficient cells ( , ) . future investigations designed to identify other nonactive site residues of nsp that are involved in the nsp -irf interaction but that do not affect prrsv replication will be required to fully elucidate the function of nsp in ifn signaling. in summary, our data identify prrsv nsp as a newly recognized antagonist in ifn signaling and show that the nsp -irf interaction is involved in inhibition of the formation and nuclear translocation of isgf . this novel function of prrsv nsp offers new insight into the interaction between a viral endou and the ifn signaling pathway, potentially aiding the development of novel therapeutic targets and more-effective vaccines against prrsv. cells and viruses. hek- t, marc- , and pk- cells, obtained from the china center for type culture collection, were cultured at °c and % co in dulbecco's modified eagle's medium (invitrogen, usa) supplemented with % fetal bovine serum. prrsv strain wuh (genbank accession number hm . ), isolated from the brain of a pig suffering from "high fever" syndrome in china at the end of , was amplified and titrated as described previously ( ) . plasmids. the luciferase reporter plasmid isre-luc and the cdna expression constructs encoding porcine stat , stat , and irf with an n-terminal flag tag have been described previously ( ) . the luciferase reporter plasmid isre-luc contained ifn-stimulated response elements in the promoter of most of the isgs cloned upstream of the firefly luciferase reporter gene. prl-tk plasmid (promega) was used as an internal control for normalization of the transfection efficiency. the nsp gene of prrsv strain wuh was amplified and cloned into pcaggs-ha with an n-terminal ha tag. prrsv nsp mutants (h a, h a, and k a) were constructed by overlap extension pcr using specific mutagenic primers (available upon request) in a pcaggs-ha background. prrsv nsp and its mutants with an n-terminal ha tag were cloned into pgex- p- with a glutathione s-transferase (gst) tag in the n terminus. irf deletion mutants were also amplified by pcr and constructed in a pcaggs-flag background. pedv nsp was amplified from pedv strain aj ( ) and cloned into vector pcaggs-ha with a ha tag. pedv nsp mutants (h a) were constructed using pedv nsp as the template. all constructed plasmids were confirmed by sequencing. luciferase reporter gene assay. hek- t or pk- cells grown in -well plates were cotransfected with reporter plasmid isre-luc ( . g/well), prl-tk ( . g/well), and the indicated expression plasmids or an empty control plasmid. where indicated, cells were further treated with ifn-␣ (pbl assay science) ( , u/ml) h after the initial cotransfection. cells were lysed h later, and firefly luciferase and renilla luciferase activities were measured using a dual-luciferase reporter assay system (promega) according to the manufacturer's protocol. the relative levels of firefly luciferase activities were standardized as prl-tk activities. data are presented as means and standard deviations (sd). p values of Ͻ . were considered statistically significant, and p values of Ͻ . were considered highly statistically significant. rna extraction and quantitative real-time pcr. in brief, total rna was extracted from cells using trizol reagent (invitrogen), and rna ( g) was reverse transcribed into cdna using avian myeloblastosis virus reverse transcriptase (takara, japan). the cdna ( l of l) was then used in a sybr green real-time pcr assay (applied biosystems). the abundance of individual mrna transcripts in each sample was determined three times and normalized to that of glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna. all quantitative pcr (qpcr) primers used in this study have been described previously ( ) . protein expression and purification. for protein expression, the recombinant prokaryotic expression plasmids encoding the n-terminally gst-ha-tagged wt nsp and mutant (h a, h a, k a) proteins were transformed into e. coli strain trans bl (de ) and cultured at °c in lb medium containing g/ml ampicillin. when the culture optical density at nm (od ) reached . to . , the cells were induced by the use of . mm iptg (isopropyl-d- -thiogalactopyranoside) for h at °c. for protein purification, the cultured cells were collected by centrifugation at , rpm for min and resuspended in phosphate-buffered saline (pbs) followed by disruption with ats ah- . the supernatant was filtered by the use of a filter with a . -mm-pore-size membrane and was then loaded onto a glutathione-sepharose b column (ge healthcare). to gain gst-free proteins, the harvested fusion proteins with an n-terminally gst tag were incubated with gst- c rhinovirus protease at a ratio of : for h at °c, and the cleavage products in the cleavage buffer were further separated by reloading onto glutathione-sepharose b column. finally, the target proteins were eluted by the use of ml cleavage buffer. the purified proteins were stored at Ϫ °c for detection of enzyme activity. enzyme activity assay. the rna substrate ( =- -carboxyfluorescein-da-ru-da-da- -carboxy-n,n,n,n-tetramethylrhodamine- =) was purchased from nanjing genscript company. the n-terminally ha-tagged prrsv nsp /h a/h a/k a proteins and the rna substrate were placed in a reaction buffer containing mm hepes (ph . ), mm kcl, and mm dithiothreitol (dtt) diluted with . % diethyl pyrocarbonate-treated water. the concentration of proteins used in the assay was m, and the substrate concentration was m. the endoribonuclease activity was monitored every min for min in a fluorescence plate reader, with excitation at a wavelength of nm and emission at a wavelength of nm. the results were analyzed by the use of graphpad prism software . . western blot analyses and subcellular fractionation. cells were grown in -mm-diameter dishes and harvested with lysis buffer (beyotime, china) plus nm phenylmethylsulfonyl fluoride (pmsf) and phosstop phosphatase inhibitor (sigma, usa). samples were then separated by sds-page and transferred to polyvinylidene difluoride membranes (millipore, usa) to determine the protein expression levels. overexpression of stat , stat , irf , and irf deletion mutants was evaluated using mouse monoclonal anti-flag antibody (macgene, china). the expression of prrsv nsp and its mutants was analyzed using mouse monoclonal anti-ha antibody (mbl, japan). rabbit polyclonal antibodies against irf (santa cruz, usa), stat (santa cruz, usa), stat (abclonal, china), phospho-stat (stat -y , cell signaling technology, usa), and phospho-stat (stat -y , abclonal, china) were used to detect the protein levels and phosphorylated forms of each of the respective endogenous proteins. monoclonal antibody (mab) against prrsv nsp was produced from hybridoma cells derived from sp / myeloma cells and spleen cells of balb/c mice immunized with recombinant nsp protein from prrsv strain wuh . the isotype of nsp -specific mab is mouse igg . the specificity of mab against prrsv nsp was confirmed by specific reactions performed with pcaggs-ha-nsp -transfected cells and prrsv-infected cells. to analyze the nuclear translocation of isgf , nuclear fractions were extracted from cells using an ne-per nuclear and cytoplasmic extraction reagents kit (thermo fisher scientific, usa) according to the manufacturer's protocol. the cytoplasmic and nuclear fractions were subjected to western blotting. successful isolation was assessed using rabbit polyclonal antibodies against heat shock protein (hsp ; proteintech, china) and parp (proteintech, china) with cytoplasmic and nuclear protein markers, respectively. coimmunoprecipitation and immunoblotting analyses. to test the interactions between proteins, hek- t cells or marc- cells from each -mm-diameter dish were lysed in radioimmunoprecipitation assay (ripa) lysis buffer containing mm tris-hcl (ph . ), mm nacl, % triton x- , % sodium deoxycholate, . % sds, nm pmsf, and phosstop phosphatase inhibitor (sigma, usa), and the protein concentration was measured and adjusted. for each immunoprecipitation, g of cell lysate protein was incubated with . g of the indicated antibody and l of protein aϩg agarose (beyotime, china) overnight at °c. the agarose beads were then washed three times with ml of lysis buffer. the precipitates were subjected to % sds-page and subsequent immunoblot analysis using the indicated antibodies. in some immunoblotting analyses, the horseradish peroxidase-conjugated goat anti-rabbit igg light chain (ipkine, usa) were used to eliminate the interference of the rabbit igg heavy chain. indirect immunofluorescence assay. marc- cells cultured on coverslips in -well plates were infected with prrsv at a multiplicity of infection (moi) of . . at h postinfection, the infected cells were mock treated or treated with recombinant human ifn-␣ (pbl assay science) ( , u/ml) for h. the cells were then fixed with % paraformaldehyde for min and immediately permeabilized using methanol (precooled at Ϫ °c) for min at room temperature (rt). after three washes with pbs, cells were incubated with % bovine serum albumin (bsa)-pbs overnight at °c and incubated with the primary antibody for h. the antibodies used were as follows: anti-phospho-stat (cell signaling technology, usa), anti-phospho-stat (cell signaling technology, usa), anti-irf (abclonal, china), and anti-nsp mab. anti-mouse igg (hϩl) antibody conjugated to alexa fluor or anti-rabbit igg (hϩl) antibody conjugated to alexa fluor was diluted to : for use as the secondary antibody, after which the cells were stained with ', -diamidino- -phenylindole (dapi; beyotime, nantong, china)-pbs for min. fluorescent images were acquired with a confocal laser scanning microscope (olympus fluoview ver. . , japan). porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the ever-expanding diversity of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins prrsv structure, replication and recombination: origin of phenotype and genotype diversity the structural biology of prrsv antiviral strategies against prrsv infection porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins the janus protein tyrosine kinase family and its role in cytokine signaling tyrosinephosphorylated stat and stat plus a -kda protein all contact dna in forming interferon-stimulated-gene factor how cells respond to interferons interferon-stimulated genes and their antiviral effector functions interplay between janus kinase/signal transducer and activator of transcription signaling activated by type i interferons and viral antagonism porcine deltacoronavirus nsp antagonizes type i interferon signaling by cleaving stat rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp protein inhibits interferon-mediated stat activation structural basis of the inhibition of stat activity by sendai virus c protein nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp interferon signalling network in innate defence structural biology of the arterivirus nsp endoribonucleases nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc- cells biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat ns of dengue virus mediates stat binding and degradation zika virus targets human stat to inhibit type i interferon signaling porcine bocavirus np negatively regulates interferon signaling pathway by targeting the dna-binding domain of irf the ebola virus interferon antagonist vp directly binds stat and has a novel, pyramidal fold the irf family transcription factors in immunity and oncogenesis stat nuclear trafficking mapping of nine porcine interferon regulatory factor genes distinct stat structure promotes interaction of stat with the p subunit of the interferon-alpha-stimulated transcription factor isgf two domains of isgf gamma that mediate protein-dna and protein-protein interactions during transcription factor assembly contribute to dna-binding specificity the viral innate immune antagonism and an alternative vaccine design for prrs virus the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-kappab signaling by means of its deubiquitinating activity the superimposed deubiquitination effect of otulin and prrsv nsp promoted the multiplication of prrsv an "old" protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication the emerging role of interferon regulatory factor in the antiviral host response and beyond the type i interferon-alpha mediates a more severe neurological disease in the absence of the canonical signaling molecule interferon regulatory factor mice deficient in stat but not stat or irf develop a lethal cd ϩ t-cellmediated disease following infection with lymphocytic choriomeningitis virus the human papillomavirus e oncoprotein abrogates signaling mediated by interferon-alpha varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms effects of adenovirus e a protein on interferonsignaling human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction porcine reproductive and respiratory syndrome virus nsp beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha degradation porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation structural basis of stat recognition by irf reveals molecular insights into isgf function mir- b reduces porcine reproductive and respiratory syndrome virus replication by negatively regulating the nf-kappab pathway complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china this work was supported by the major project of the national natural science foundation of china (grant ), the national basic research program ( ) of china (grant cb ), the national natural science foundation of china (grants key: cord- - v pjqt authors: zhao, jun; zhu, ling; xu, lei; huang, jianbo; sun, xiangang; xu, zhiwen title: porcine interferon lambda (ifn-λ ) shows potent anti-prrsv activity in primary porcine alveolar macrophages (pams) date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: v pjqt background: porcine reproductive and respiratory syndrome virus (prrsv) is a serious viral disease of swine. at present, there are vaccines for the control of prrsv infection, but the effect is not satisfactory. the recombination of attenuated vaccines causes significant difficulties with the prevention and control of prrsv. type iii interferons (ifns), also called ifn-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. results: therefore, primary porcine alveolar macrophages (pams) were used for this investigation. to this end, we found that the replication of prrsv in pams was significantly reduced after pre-treatment with ifn-λ , and such inhibition was dose- and time-dependent. the plaque formation of prrsv abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary pams were treated with ifn-λ at ng/ml. in addition, ifn-λ in our study was able to induce the expression of interferon-stimulated genes (isg ), ′- ′-oligoadenylate synthase (oas ), ifn-inducible transmembrane (ifitm ), and myxoma resistance protein (mx ) in primary pams. conclusions: ifn-λ had antiviral activity against prrsv and can stimulate the expression of pivotal interferon-stimulated genes (isgs), i.e., isg , mx , oas , and ifitm . so, ifn-λ may serve as a useful antiviral agent. supplementary information: the online version contains supplementary material available at . /s - - - . type i interferons (ifn-α/β) and type iii ifns (ifn-λs), as the first line of defence in innate immunity, play a crucial role in the body's resistance to exogenous pathogens [ ] . type iii ifns, also called ifn-λs, were first described in [ , ] , consist of ifn-λ , ifn-λ , ifn-λ and ifn-λ in humans [ , ] , ifn-λ and ifn-λ in mice [ , ] , ifn-λ and ifn-λ in swine [ ] [ ] [ ] . both ifn-α/β and ifn-λs bind to unique receptors and induce the previous signalling pathway and expression of the ifn-stimulated genes (isgs) to mediate antiviral activity. type i ifn interacts with a receptor formed by interferon alpha/beta receptor (ifnar ) and interferon alpha/beta receptor (ifnar ). type iii ifns bind to the specific receptor chain ifn-λr and il- r [ ] . type i ifn receptor is ubiquitously expressed in various types of cells and organs; however, ifn-λr is widely expressed on epithelial cells, dendritic cells, human peripheral blood monocytes or macrophages [ ] , which means that ifn-λs may provide a focused antiviral response against mucosal and immune organ infections. interferon bind to its receptors on the cell surface and induce the production of a large number of isgs, including isg , myxoma resistance protein (mx) family, ′- ′-oligoadenylate synthase (oas) family and the ifninducible transmembrane (ifitm) family, through jak-stat signal transcription [ ] . isg is one of the most highly induced isgs, isg can inhibit viral translation, replication, or egress [ ] . mx is a broadly inhibitor and inhibits a wide range of viruses by blocking the endocytic traffic of incoming virus particles and the uncoating of ribonucleocapsids [ ] . oas can recognise the dsrna produced by the virus in the infected cells and play an antiviral role by activating the ribonuclease l (rnase l) to degrade the diseased mrna [ ] . the ifn-inducible transmembrane (ifitm) family has a role in blocking virus entry [ ] . ifitm has high potency against influenza a virus and severe acute respiratory syndrome (sars) coronavirus [ ] . prrsv is a member of the family arteriviridae in the order nidovirales, which causes severe reproductive failure in sows and respiratory distress in piglets and growing pigs [ ] . also, prrsv is an immunosuppressive virus that can infect the lymphatic system of the whole body and produce viraemia after infection [ ] . prrsv mainly infects and destroys porcine alveolar macrophages and leads to severe immunosuppression, which promotes the infection of mycoplasma pneumoniae, streptococcus, a. pleuropneumoniae, and other pathogens [ , ] . the primary pams derived from piglet alveoli is an appropriate model for studying the interaction of prrsv immune responses and host-pathogen in vitro. an in vivo antiviral test of type iii interferons from pigs has not been reported. in this study, the antiviral activity of porcine ifn-λ against prrsv in primary pams was evaluated, and the expressions of isgs genes induced by ifn-λ was also investigated in primary pams. previous study has confirmed that porcine ifn-λ possess the high specific activity against porcine epidemic diarrhoea virus (pedv), classical swine fever virus (csfv), hepatitis e virus (hev) and so on [ , , ] . in this study, we verified the antiviral effect of ifn-λ against prrsv in vitro on pams. as shown in fig. , treatment of primary pams with ifn-λ could reduce the multiplication of prrsv. the degree of cytopathic effect (cpe) decreased with the increase in ifn-λ concentration ( fig. a-e) . the number and size of viral plaques also decreased with the increase in ifn-λ concentration ( fig. f-j) . the virus titre was significantly reduced with the increase of ifn-λ treatment dose ( , fig. the cpe of primary pams treated with porcine ifn-λ and infected with prrsv. the primary pams were untreated or pre-treated with ifn-λ ( , , ng/ml). b the primary pams not treated. b the primary pams treated with ng/ml ifn-λ . c the primary pams treated with ng/ml ifn-λ . d the primary pams treated with ng/ml ifn-λ . e control primary pams. k the primary pams were treated or untreated with ng/ml of ifn-λ for h and then were infected with prrsv nj strain at . moi. infected cells were cultured for , , or h after infection. f, g, h, i, j corresponds to a, b, c, d, e with the same treatment. magnifications, × , ng/ml), and the maximum treatment dose could reduce the virus titre by four orders of magnitude compared with the control group ( fig. k, the raw data are shown in supplementary table s ). these results indicate that the ifn-λ could significantly inhibit the replication of prrsv in a dose-dependent manner in primary pams. to investigate the time-dependent manner of the ifn-λ inhibits the replication of prrsv, we used ifn-λ of ng/ml concentration to treat pams cells and infected with the prrsv. cell cultures were collected at specific time and determine the virus titre. as shown in fig. (the raw data are shown in supplementary file , table s ), the inhibition of ifn-λ on prrsv decreased with time in primary pams, but the inhibition still existes. prrsv proliferation slowed down within h to h in primary pams that were treated with ifn-λ . the above results showed that ifn-λ could maintain a potent anti-prrsv activity in the later stage and significantly inhibit the replication of prrsv in a timedependent manner in primary pams. isg , mx , oas and ifitm have well-known antiviral properties and may affect prrsv replication. therefore, we accessed the quantification of isgs induce by ifn-λ . as seen in fig. a to d, a dose-dependent induction of isg , mx , oas and ifitm has been observed in primary pams treated with ifn-λ . the expression of mrna for isg , mx , oas , and ifit m was up-regulated by , , , and times respectively at the concentration of ng/ml in primary pams. as shown in fig. e and f (the full-length blots are presented in supplementary file ), a dosedependent induction of the antiviral proteins isg , mx and oas has been observed in primary pams treated with ifn-λ . the expression concentration of three antiviral proteins increased with the increasing ifn-λ concentration. both isg and mx showed low expression in the untreated condition, and ifn-λ induced a large amount of expression. the expression of isg , mx and oas tended to be stable when the concentration of ifn-λ was higher than ng/ml (fig. f ). the results in our research confirm that the porcine ifn-λ shows potent anti-prrsv activity in primary pams. pams are the first line of defence against pathogenic microbe infections in the lung. prrsv replicates in monocytic lineage cell types, particularly in pams, and causes immunosuppression in swine [ ] . therefore, we selected the primary pams to carry out the ifn-λ in vitro anti-prrsv study. alveolar macrophages are resident phagocytes of the alveolar space [ ] . the expression of the ifn-λ receptor in alveolar macrophages has been confirmed and reported [ ] . macrophages express il- rβ and il- rα at both the mrna and protein levels [ ] . ifn-λ has the strongest antiviral function in ifn-λs [ ] . in our study, the prrsv proliferation reduced when primary pams were treated with ifn-λ ( ng/ml) (fig. i) . ifn-λ treatment could significantly reduce the virus titre of prrsv proliferation on pams, and the virus titre of the ng/ml treatment group was four orders of magnitude lower than that of the control group (fig. b) . consistent with these results, treatment with , or [ ] . the study of two other kinds of viruses targeted porcine intestinal epithelial, pedv and csfv, confirming that ifn-λ inhibits their infection in vitro [ , ] . all of these imply that porcine ifn-λ can inhibit the proliferation of porcine viruses such as csfv, pedv and prrsv. the antiviral activities of ifn-λ are due to isg induction and ifn-λ can induce the expression of isg. ifn-λ exerts its anti-hiv function by activating jak-stat pathway-mediated innate immunity in macrophages [ ] . ifn-λ can bind to cell surface receptors and induce the high expression of interferon-stimulating genes of the mx, oas and ifitm families [ , , ] . the gene transcription profile induced by ifn-λ , particularly the gene transcription profile induced by ifn-λ in primary pams has not been reported. in our study, we assessed whether the antiviral efficacy of ifn-λ was caused by the levels of isg expression induced by ifn-λ . consistent with the expecting result, the expression of isg , oas , mx , and ifitm was provoked in primary pam cells. the mrna transcription and protein translation of the isg , oas and mx showed dose-dependence. however, the rangeability of mrna and protein expression levels of isg , oas and mx were different. the expression levels of protein reached its peak when treated with ng/ml ifn-λ while the expression of mrna continuous increased (fig. ) . in summary, our data demonstrated that ifn-λ could inhibit the replication of prrsv in primary pams, and such inhibition is dose-and time-dependent. alveolar macrophages are one of the earliest immune defence cells in the lungs that contact pathogenic microorganisms. they are essential components of the innate and specific immunity of the host [ ] . pams are an essential host cell for prrsv natural infection. ifn-λ can stimulate the expression of pivotal isgs, i.e. isg , mx , oas , and ifitm . this study indicated that porcine ifn-λ might serve as a promising therapeutic agent against prrsv and other viruses in swine in the future. supplementary file (a to d) . the grey value of protein bands was measured by image j (f). data were presented as mean ± sem (n = ). *p < . ; **p < . ; ***p < . ; ****p < . by unpaired t-test to determine the anti-prrsv activity of ifn-λ in the primary pams, the e. coli-derived ifn-λ was prepared in our laboratory. to explore the dose-dependent of ifn-λ antiviral, primary pams were untreated or pretreated with ifn-λ ( , , ng/ml) for h. then, the cells were infected with prrsv nj strain at . moi for - h, washed and replenished with fresh medium containing the indicated ifn-λ . infected cells were cultured for h after infection. to explore the time-dependent effect of ifn-λ antiviral, primary pams were pre-treated with ng/ml ifn-λ for h. then, the cells were infected with prrsv nj strain at . moi for - h, washed and replenished with fresh medium containing the indicated ifn-λ . infected cells were cultured for , , , h after infection. all of the cells were submitted to two freeze-thaw cycles and titrated by % tissue culture infective dose (tcid ) in marc- cells. the cytopathic effect (cpe) units in culture plates were counted, and the viral titre analysis made use of the reed-muench method. to examine the level of isg expression in primary pams following ifn-λ stimulation, the cells were stimulated with the indicated concentrations ( , , ng/ml) of ifn-λ in -well plates for h. cells were then lysed, total rna was extracted for subsequent qpcr analysis and total protein was extracted for western blot analysis. every treatment group in this study had three duplicate samples (n = ). total rna was extracted from the cellular supernatant or cell lysates using the ez- spin column total rna isolation kit (sangon biotech (shanghai) co., ltd., china) according to the manufacturer's instructions and the rna concentration was measured using a nucleic acid concentration analyzer (scandrop , analytik jena, germany). reverse transcription was performed using the prime script™ ii st strand cdna synthesis kit (takara), and qpcr was performed in a light cycler (roche, switzerland) with tb green® premix ex taq™ ii (tli rnaseh plus) (takara). the thermal cycling conditions were °c for s, followed by cycles of °c for s, and °c for s. all acquired data were obtained using light cycler real-time pcr machines (roche) and analysed with light cycler software . based on the cycle threshold (ΔΔct) method. primers were designed using oligo . software and are shown in table . total protein was extracted from the cell lysates using the western and ip cell lysis buffer (sangon biotech (shanghai) co., ltd., china) according to the manufacturer's instructions and protein concentration was determined using the bca protein assay kit (sangon biotech (shanghai) co., ltd., china). after gel electrophoresis, the proteins were transferred to nitrocellulose membranes (bio-rad, usa), and blocked in % skim milk at °c overnight. after washing with pbst ( . % tween- in pbs), the membrane was incubated with primary antibodies for h at °c. after washing, the membrane was incubated with horseradish peroxidase (hrp)-conjugated igg antibody (abcam, no: ab ) for h at °c. the protein bands were detected using supersignal™ west pico plus chemiluminescent substrate (thermo scientific, usa) and chemiluminescence imaging system (bio-rad, chemidoc mp, california, usa). the primary antibodies of isg (no: ab ), oas (no: ab ), mx (no: ab ) and β-actin (no: ab ) was purchased from abcam. statistical analysis was performed and histogram were drawn using graphpad prism™ . (graphpad software, usa), paired student t-test, and one-way anova was used to test differences between different groups. p values< . were considered significant. the gray intensity of protein bolts was analyzed by image j (national institutes of health, usa). the layouts and cropping of the pictures were completed by adobe illustrator cs (adobe systems incorporated, california, usa). the online version contains supplementary material available at https://doi. org/ . /s - - - . additional file : table s . the viral titer at h after the pams stimulated with different dose of ifn-λ . table s . the viral titer at , , or h after the pams stimulated with ifn-λ ( ng/ml). abbreviations ifn-λ : interferon lambda ; pams: porcine alveolar macrophages prrsv: porcine reproductive and respiratory syndrome virus; ifns: interferons isg : interferon-stimulated genes ; oas : ′- ′-oligoadenylate synthase ifitm : ifn-inducible transmembrane ; mx : myxoma resistance protein isgs: interferon-stimulated genes; ifn-α: interferon alpha; ifn-β: interferon beta; ifn-λs: interferon lambdas; ifn-λ : interferon lambda ; ifn-λ : interferon lambda ; ifn-λ : interferon lambda ; ifnar : interferon alpha/beta receptor ; ifnar : interferon alpha/beta receptor ; ifn-λr : interferon lambda receptor ; il- r : interleukin receptor ; mrna: messenger rna pedv: porcine epidemic diarrhoea virus; csfv: classical swine fever virus hev: hepatitis e virus; cpe: cytopathic effect; il- rβ: interleukin receptor beta; il- rα: interleukin receptor alpha; hiv: human immunodeficiency virus african green monkey embryonic kidney epithelial cells dmem: dulbecco's modified eagle's medium; fbs: foetal bovine serum pcv : porcine circovirus type ; prv: pseudorabies virus; moi: multiplicity of infection; tcid : % tissue culture infective dose; qpcr: real-time polymerase chain reaction; pbst: . % tween- in pbs; hrp: horseradish peroxidase interferon-inducible antiviral effectors il- , il- and their class ii cytokine receptor il- r ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex ifn-λ : the paradoxical new member of the interferon lambda family interferon-lambda: a new addition to an old family murine interferon lambdas (type iii interferons) exhibit potent antiviral activity in vivo in a poxvirus infection model characterisation of the mouse ifn-lambda ligand-receptor system: ifnlambdas exhibit antitumor activity against b melanoma molecular cloning, expression and antiviral activity of porcine interleukin- (poil- ) antiviral activity of porcine ifn-λ against porcine epidemic diarrhea virus in vitro molecular characterisation and antiviral analyses of porcine type iii interferons contribution of type iii interferons to antiviral immunity: location, location, location interferon-stimulated genes: a complex web of host defences interferon-induced isg pathway: an ongoing virus-host battle structural basis of oligomerisation in the stalk region of dynamin-like mxa viral encounters with ′, ′-oligoadenylate synthetase and rnase l during the interferon antiviral response a diverse range of gene products are effectors of the type i interferon antiviral response distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus porcine reproductive and respiratory syndrome emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china effects of porcine reproductive and respiratory syndrome virus (isolate tw ) on porcine alveolar macrophages in vitro alveolar macrophages: plasticity in a tissue-specific context emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark ifn-lambda mediates antiviral protection against porcine epidemic diarrhoea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and haemophilus parasuis in vitro lambda interferon inhibits human immunodeficiency virus type infection of macrophages comparison of antiviral activity of lambda-interferons against hiv replication in macrophages lambda interferon inhibits hepatitis b and c virus replication short communication: antiviral activity of porcine ifn-k against porcine epidemic diarrhoea virus in vitro ifn-λ inhibits hiv infection of macrophages through the jak-stat pathway antiviral activity of type i and type iii interferons against porcine reproductive and respiratory syndrome virus (prrsv) ifnlambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha alveolar macrophages: plasticity in a tissue-specific context quantitative nitric oxide production by rat, bovine and porcine macrophages publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank prof. zhu for her insightful comments on the design of the study. all data generated during this study are included in supplementary file (table s , s ). however, the raw data is available from the corresponding author upon reasonable request. the sichuan provincial laboratory animal management committee (licence no: syxk (chuan) - ) approval has been received. the "guidelines for experimental animals" of the ministry of science and technology (beijing, china) were followed. not applicable. the authors declare that they have no competing interest.received: november accepted: october key: cord- -o t srim authors: chaudhari, jayeshbhai; liew, chia-sin; workman, aspen m.; riethoven, jean-jack m.; steffen, david; sillman, sarah; vu, hiep l. x. title: host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: o t srim both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (prrsv) strains can establish persistent infection in lymphoid tissues of pigs. to investigate the mechanisms of prrsv persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated prrsv strain at days post infection. a total of differentially expressed genes (degs) were detected of which degs were upregulated and degs were downregulated. specifically, genes involved in innate immune responses and chemokines and receptors associated with t-cell homing to lymphoid tissues were down regulated. as a result, homing of virus-specific t-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific t-cell in lymphoid tissue than in peripheral blood. genes associated with t-cell exhaustion were upregulated. likewise, genes involved in the anti-apoptotic pathway were upregulated. collectively, the data suggested that the live-attenuated prrsv strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, t-cell homing, and preventing cell apoptosis. porcine reproductive and respiratory syndrome virus (prrsv) is a positive sense, single stranded rna virus that belongs to the family arteriviridae, under the order nidovirales [ ] . based on phylogenetic analysis, prrsv was originally classified into two types: type or prrsv- , which originated in europe, and type or prrsv- , which originated in north america. the international committee on taxonomy of viruses (ictv) recently updated the arterivirus taxonomic structure in which prrsv- and prrsv- are now respectively classified as two species: betaarterivirus suid and betaarterivirus suid [ ] . prrsv infects pigs of all ages; however, clinical manifestations are more severe when the virus infects pregnant sows and young pigs, causing reproductive failure and respiratory distress, respectively (reviewed in [ ] ). prrsv is endemic in most swine producing countries worldwide, causing significant economic losses to swine producers [ ] . genes involved in innate immune response and genes encoding chemokines and receptors important for t-cell homing and trafficking were downregulated. on the other hand, genes involved in the anti-apoptotic pathway and t-cell exhaustion were upregulated. functional studies revealed that the frequencies of virus-specific ifn-γ-secreting cells are lower in lymphoid tissue than in peripheral blood mononucleated cells (pbmcs). collectively, the results shed important insight into the mechanisms of prrsv persistence in the host. the attenuated prrsv strain designated con used in this study was recovered from an infectious cdna clone constructed using viral rna extracted from the attenuated con-p [ ] . marc- cells, a monkey kidney cell line [ ] , were cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and × penicillin-streptomycin ( units/ml of penicillin, and µg/ml of streptomycin (life technologies, grand island, ny, usa). pbmcs and lymph node-derived cells were cultured in complete rpmi- medium (crpmi) supplemented with % fbs, × of glutamax- (life technologies, grand island, ny, usa), units/ml of penicillin, and µg/ml of streptomycin (sigma-aldrich, st. louis, mo, usa). mouse monoclonal antibody specific to prrsv n protein sdow was purchased from the national veterinary services laboratories (ames, ia, usa). anti-porcine cd ε (clone bb - e - c ; fitc-conjugated), anti-porcine cd (clone - - ; pecy -conjugated), anti-porcine cd (clone - - ; alexa flour -conjugated), and anti-porcine ifn-γ (clone p g ; pe-conjugated), were purchased from bd biosciences (san diego, ca, usa). alexa flour -conjugated goat anti-mouse igg was purchased from invitrogen (eugen, or, usa). the pig experiment conducted in this study was approved by the university of nebraska-lincoln (unl) institutional animal care and use committee under the protocol number . ten -week-old prrsv, porcine circovirus type (pcv ), and siv negative pigs were purchased from the midwest research swine (glencoe, mn, usa). the pigs were randomly assigned to two groups of five pigs, which were accommodated in two separate rooms in the animal biosecurity level (absl- ) research facilities at unl. after week of acclimation, pigs in group were injected with dmem to serve as negative controls, whereas pigs in groups were inoculated intramuscularly with . tcid of live-attenuated prrsv strain con . whole blood samples with anticoagulant edta were collected to obtain plasma and pbmcs. plasma samples were stored at − • c for measurement of viremia and antibody response. a portion of freshly isolated pbmcs were used for measurement of t cell responses while the rest of pbmcs were cryopreserved for future analysis. at days post-infection (dpi), sample of inguinal lymph node (iln) was aseptically collected from the pigs under anesthesia. one half of the inl was stored in crpmi for lymphocyte isolation while the other half of the inl was minced and stored in trizol reagent (life technologies, carlsbad, ca, usa) for rna extraction. viral loads in plasma and tissues were measured by a commercial rt-qpcr kit (tetracore inc., rockville, md, usa). viral loads in plasma was reported as log copies per ml, whereas viral loads in tissues were reported as log copy per µg of total rna used in the rt-qpcr reaction. for statistical purposes, samples that had no detectable levels of viral rna were assigned a value of log copies. pbmcs were isolated from edta-whole blood as previously described [ , ] . single cell suspension was isolated from iln immediately after collection as follows. the tissue was processed to viruses , , of remove connecting tissue and cut into small pieces which were placed in a -µm nylon cell strainer (corning, durham, nc, usa) in the presence of crpmi. the tissue pieces were pressed against the nylon mesh by using the plunger of a ml syringe. the resulting cell suspension was collected into a ml conical tube which was passed through a -µm cell strainer one more time to remove large tissue debris. cells were pelleted by centrifugation at × g for min at room temperature and treated with a ml red blood cell (rbc) lysis buffer (life technologies, carlsbad, ca, usa). rbc lysis reaction was ceased by adding ice cold pbs containing % fbs, followed by centrifugation at × g for min at room temperature. cell pellet was resuspended in crpmi. to determine cell concentration and viability, samples of pbmc and iln-derived cells were stained with acridine orange and propidium iodide (viastaintm aopi staining solution, nexcelom, lawrence, ma, usa) and counted using an automatic cell counter (cellometer auto , nexcelom, lawrence, ma, usa). freshly isolated cells were used for elispot and flow cytometric analysis. the remaining cells were cryopreserved in % dmso, % fbs, and % rpmi- and stored in liquid nitrogen. prrsv antibody levels in plasma were measured at the veterinary diagnostic center of the university of nebraska by using the commercial elisa idexx prrs x ab test (idexx laboratories, westbrook, me, usa) following the manufacturer's instructions. the serum-virus neutralization (svn) assay was performed as previously described [ ] using plasma rather than serum. results were expressed as the log of the reciprocal of the highest dilution that showed a ≥ % reduction in the number of fluorescent foci presenting in the control wells. the frequencies of ifn-γ-secreting cells (ifn-γ scs) in pbmcs and iln-derived cells were measured by using an ifn-γ elispot assay as previously described [ , ] . briefly two replicates of , pbmcs or iln cells freshly collected from each pig were plated into two wells of a -well plate with pvdf membrane that were coated with anti-porcine ifn-γ antibody. the cells were stimulated with µl of crpmi containing . × tcid of con . for positive control, cells were plated at cells per well, followed by stimulation with a µl crpmi containing ng/ml of phorbol myristate -acetate (pma) and µg/ml of ionomycin. for negative control, cells were simply cultured in crpmi. at h post stimulation, the plate was washed with pbs-containing . % tween (pbs-t) followed by incubation with biotin-labeled antibody against porcine ifn-γ (clone p c , bd biosciences pharmingen, san diego, ca, usa). spots were developed by using alkaline phosphatase-conjugated streptavidin (southern biotech, birmingham, al, usa) in conjunction with alkaline phosphatase substrate (vector laboratories, burlingame, ca, usa). spots were counted and analyzed using an aid elispot reader version . (aid gmbh, strassberg, germany). freshly isolated pbmcs and iln cells were ex vivo stimulated with × tcid of con as described above. a cocktail solution consisting of ng/ml of pma and µg/ml of ionomycin was included as a positive control while crpmi served as a negative control. at hrs post-stimulation, µl of crpmi containing µg/ml of golgi-plug brefeldin a (bd bioscience, san jose, ca, usa) was added and further incubated for hrs. samples were centrifuged at × g for min at room temperature. cells were resuspended in flow cytometry staining buffer (facs buffer) (pbs with % fbs and . % sodium azide), and stained with anti-porcine cd ε, cd , and cd monoclonal antibodies and incubated on ice for min in the dark. cells were washed thrice using facs buffer. the cells were fixed and permeabilized with % paraformaldehyde and . % triton x- , respectively, followed by intracellular staining with an ifn-γ antibody. cells were analyzed by using a cytek dxp cytometer (cytek biosciences, fremont, ca, usa) and acquired data were analyzed using the flowjo software (bd biosciences, san jose, ca, usa) with gating based upon fluorescence minus one (fmo) control. for each sample, , events were acquired. relevant cell population was gated on cd + prior to analyzing the cd + and cd + cells ( figure s a ). ifn-γ positive cells were counted from individual cd + , cd + , and cd + cd + double positive (dp) cells ( figure s b ). total rna was isolated from iln collected from the infected and non-infected using the trizol reagent according to the manufacturer's protocol. rna was re-purified using rneasy mini kit (qiagen, hilden, nrw, germany) according to the manufacturer instructions, followed by a dnase treatment using turbo dna-free tm kit (life technologies, v.a. graiciuno , vilnius, lithuania) to remove dna contamination. purified rna was submitted to novogene bioinformatics technology co.ltd for rna sequencing. sequencing libraries were generated using nebnext ® ultratm rna library prep kit for illumina ® (neb, ipswich, ma, usa) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. briefly, mrna was purified from mg of total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in nebnext first strand synthesis reaction buffer ( x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of ends of dna fragments, nebnext adaptor with hairpin loop structure were ligated to prepare for hybridization. in order to select cdna fragments of preferentially ~ bp in length, the library fragments were purified with ampure xp system (beckman coulter, beverly, ma, usa). then µl user enzyme (neb, ipswich, ma, usa) was used with size-selected, adaptor-ligated cdna at • c for min followed by min at • c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers and index (x) primer. finally, pcr products were purified using ampure xp system and library quality was assessed on the agilent bioanalyzer system. the libraries were sequenced on an illumina platform hiseq and approximately million raw bp/ bp paired-end) reads were generated. rna-seq reads were first trimmed with trim galore version . . [ , ] , which include filtering reads shorter than bp and low-quality ends from reads (phred score: < ) and the option of removing reads with ns. the quality of reads before and after trimming was checked with fastqc version . . [ ] . the filtered reads were aligned to the sus scrofa genome (sscrofa . ; gcf_ . ) using tophat version . . [ ] [ ] [ ] with a read mismatch of and a maximum multi hits of and further default parameters. alignment files generated by tophat were then used to generate gene counts using htseq version . . (htseq-count), with a minimum alignment quality of [ ] . a data matrix of gene counts for all samples was created using a custom python script and the data matrix was used to run the differential gene expression analysis in deseq version . . [ ] . genes with an adjusted p value smaller than . and a log fold change larger than were considered as differentially expressed. the differential expression result table generated by deseq was annotated with gene information obtained from ensembl biomart for sus scrofa, supplemented by annotation from the gtf file. to visualize the level of prrsv rna genome, reads mapped to the prrsv con genome were counted base by base, normalized to counts per million and subsequently used to generate a coverage track using deeptools version . . [ ] . gene ontology (go) in the database (http://www.geneontology.org/) is an international standardized classification system for gene function, and it supplies a set of controlled vocabulary to comprehensively describe the properties of genes and gene products. go enrichment analysis of degs was implemented by the goseq r package [ ] , in which gene length bias was corrected. go terms with viruses , , of corrected p-value less than . were considered significantly enriched by differentially expressed genes. kyoto encyclopedia of genes and genomes (kegg) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). kobas version . [ ] was used to test the statistical enrichment of differential expression genes in kegg pathway. virus-specific t-cell data were analyzed by unpaired t-test analysis using graphpad prism software version . . (graphpad software, llc, san diego, ca, usa). a p < . was considered significant. sequencing data from rna-seq were deposited in the ncbi geo and are available under accession number geo: gse all pigs inoculated with con became viremic starting from dpi. the viremia level increased and peaked at dpi ( figure a ). pigs inoculated with con did not displayed any clinical signs throughout the course of this study. at dpi, iln was collected and rna was extracted. rt-pcr analysis revealed that viral rna genome was detected in all five con -infected pigs but none of the sham-inoculated pigs ( figure b) . subsequently, the rna samples were subjected to rna-seq. an average of million paired-end reads were obtained for each rna sample (table s ). to confirm the presence of viral rna during persistent infection in pigs, the reads were mapped to the con genome. viral rna reads were only detected from iln of con -infected pigs which mapped throughout the viral genome ( figure c ). the results demonstrate that all five pigs in this study were persistently infected with the attenuated prrsv strain con . to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles ( figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). to examine the host responses to con persistent infection, rna reads were mapped to the reference pig genome (sscrofa . ; gcf_ . ). principal component analysis (pca) indicated that control and con -infected pigs formed separated clusters indicating that they had distinct transcriptional profiles (figure a ). the infected pigs showed more variability than the control pigs. in addition, one pig in the con -infected group (con_ ) showed a unique transcriptional profile, as it associated closer to the control pigs. this association is likely due to the different genetic makeup of this pig than the other four animals within this group. direct comparison of rna transcripts between control and con -infected animals revealed a profound change in the transcriptional profiles in the iln tissue of con -infected pigs. out of genes in the annotated porcine genome, there were degs (fdr < %, |log | fold change ≥ ), of which degs were upregulated and degs were downregulated ( figure b and table s ). the control animals are indicated by red dots labeled "dmem_ animal number," while the con -infected animals are indicated by cyan dots labeled "con_animal number"; (b) the volcano plot of differentially expressed genes between con -infected and control pigs. red dots denote significantly down-regulated genes (p < . , log fold change ≤− ), green dots indicated significantly up-regulated genes (p < . , log fold change ≥ ), and grey dots represent genes that were not differentially expressed. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. to understand their biological functions, degs were subjected to go enrichment analysis. go terms having a corrected value of p < . were considered statistically significant (table s ) . significantly enriched go terms were grouped under three categories: biological processes, molecular function, and cellular components. thirty highly enriched go terms in con -infected pigs are shown (figure a) . a large number of degs are enriched into the category of biological processes (cytoskeleton organization, multicellular organism development, regulation of intracellular signal transduction, cell communication, cell surface receptor signaling, regulation of programmed cell death etc.), followed by molecular function (cellular macromolecular catabolic processes, kinase activity, phosphotransferase activity, dna binding, metal ion binding) and cellular components (ribonucleoprotein complexes, bounding membrane of organelles). kegg pathway analysis revealed multiple enriched pathways including apoptosis, chemokine signaling, cellular, senescence, mitophagy, lysosome, endocytosis, and mapk ( figure b and table s ). detailed analysis of selected enriched pathways is presented in the respective sections below. the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an anti-inflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the innate immune system is the first line of defense against invading pathogens, with the major influence on the development of strong adaptive immune responses. con is capable of inducing type i ifns both in vitro and in vivo [ ] . in the current study, we did not detect differential expression of type i ifn or interferon stimulated genes (isg) rna transcripts, suggesting that the type-i ifn was not induced in iln of con-infected pigs at dpi. we observed an upregulation of rna-sensing molecules including tlr (dsrna), tlr , and tlr (ssrna) (figure a ). on the other hand, no differential expression of signaling molecules downstream of tlr pathways including interferon regulatory factors (irf) irf and irf , was observed (table s ). in addition, we observed an upregulation of tlr mrna in con -infected pigs. while the ligands and signaling pathway involving tlr remain poorly understood, it has been demonstrated that tlr can act as an antiinflammatory receptor which can also suppress tlr -induced ifn-β production [ ] . interestingly, we observed an increased expression of cd (ox ) and its receptor cd r (figure a ). cd r is an inhibitory immune receptor that is expressed on myeloid cells and b-and t-lymphocytes [ ] , while cd is widely expressed on multiple cell types including endothelial cells, neurons, and lymphocytes [ ] . cd /cd r interaction suppresses cytokine production, and inflammatory responses [ ] . the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and upregulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that the complement system is another crucial component of the innate immunity. it acts as an important link between innate and adaptive immune system. thus, viruses have developed a mechanism to modulate complement responses by down-regulating activation proteins and up-regulation of regulatory proteins [ ] . in the current study, increased expression of regulatory proteins (cfh, cfi, and cd ) was observed from con -infected animals (figure b) . conversely, c q, a classical complement component involved in increasing virus neutralizing ability of antibodies [ ] was down-regulated ( figure b) . collectively, the data suggest that the con virus suppresses both complement activation and inflammatory responses at the site of persistent infection. apoptosis is a powerful innate immunity mechanism to curtail viral spread through eliminating virally infected cells. apoptosis can be triggered by both extrinsic and intrinsic stimuli [ ] . it is well documented that prrsv induces apoptosis in tissues of infected pigs, especially during an acute phase of the infection [ , , ] . in this study, expression of tnfrsf a (tnfα receptor ) gene that contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . contains a death domain [ ] , was downregulated in the iln of infected pigs ( figure ) . similarly, expression of pro-apoptotic genes including aifm , chac , and osr , was downregulated ( figure ). on the other hand, expression of birc and bfl- /a (bcl a ), two apoptosis inhibitors that interfere with caspase activation [ ] , was upregulated. furthermore, expression of several negative regulators of apoptotic genes including bcl- -associated killer (bak ), damage induced apoptosis suppressor (ddias), x-linked inhibitor of apoptosis protein (xiap), mcl , api, bnip , and faim was upregulated. collectively, these results suggest that the pro-apoptotic signaling pathway was suppressed in iln tissue of pigs persistently infected with con . lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . celladhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of tcell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of lymphocyte activation occurs in the secondary lymphoid organs. activated t-cells then migrate to the sites of infection where they exert their effector functions to eliminate infected cells [ ] . cell-adhesion molecules, chemokines, and receptors play a central role in regulating t-cell migration [ ] . prrsv mainly persists in lymphoid tissue of infected pigs [ , ] . in this study, expression of several important chemokine ligands (ccl , ccl , ccl , ccl , cx cl , and ccl ) and chemokine receptors (ccr and ccr ) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (apcs) to the lymphoid tissues was down regulated in iln of con -infected pigs (figure a ). on the other hand, expression of cd (pd- ), a marker of t-cell exhaustion, was upregulated ( figure b) . likewise, expression of inhibitory receptors havcr (also known as tim ) and tgit, which transmit the inhibitory signals for t-cell differentiation and effector activities [ ] , was upregulated. we also found increased expression of other co-inhibitory molecules (btla, faslg, fas, and ido ) that are associated with the regulation of t-cell exhaustion during chronic viral infection (figure b) . together, the results suggest that t-cell migration to iln, one of the sites of prrsv persistence, might be affected because of the down regulation of important chemokines and receptors, and that the t-cells in iln might be exhausted. interestingly, markers of regulatory t-cells (t reg ) were downregulated in iln of infected pigs (figure c) , suggesting that t reg might not be present in iln at dpi. regulatory t-cells (treg) were downregulated in iln of infected pigs (figure c ), suggesting that treg might not be present in iln at dpi. since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsvspecific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). since the expression of chemokines and receptors important for t-cell migration was downregulated in iln, we sought to compare the frequencies of virus-specific t-cells in pbmcs and in iln using the ifn-γ sc elispot assay. the number of spots was similar when pbmcs and iln cells were stimulated with non-specific t-cell activator pha/ionomycin. however, the number of spots was significantly lower in iln than in pbmcs when the cells were stimulated with whole prrsv antigen (figure a ). the results clearly indicate that the frequencies of prrsv-specific ifn-γ scs were significantly lower in iln than in pbmcs. to further elucidate the phenotypes of prrsv-specific t-cells, we used a multi-color flow cytometric assay to identify subsets of t-cells that secrete ifn-γ in response to prrsv activation ex vivo. cd + cd + dp cells were the major t-cell population secreting ifn-γ, both in pbmcs and iln. swine have a significant population of extrathymic cd + cd + dp t cells that represent memory t-cells [ ] . similar to the elispot assay, the flow cytometric assay also indicated that the percentage of t-cell secreting ifn-γ was comparatively lower in iln than in pbmcs (figure b ). cells collected from dmem-inoculated pigs did not show any significant t-cell reactivities after ex vivo stimulation with con , both in elispot and in flow cytometry assays (data not shown). viruses , , x for peer review of a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virusneutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). a number of genes associated with th , or humoral response, were upregulated in the iln of con -infected pigs (figure a ). il- , rgs , and nuggc are involved in the development of germinal center (gc) and activation of b-cell follicles [ ] . tnfsf b and tnfsf are potent activators of b-cell lineage and ig class switching, respectively [ ] . b-cell surface antigens ms a (cd ), activation-induced cytidine deaminase (aicda) [ ] , and rafting family member (rftn ) are associated with b-cell receptor signaling. the upregulated expression of these genes in iln of infected animals suggested that the humoral immune response to con -infection was not affected. to corroborate transcriptome data, we measured both virus-specific antibody levels at various time points post-infection. high levels of non-neutralizing antibodies specific to viral n protein (measured by a commercial elisa) were detected in all pigs (figure b ). however, only minimal levels of virus-neutralizing antibodies (titer : ) were detected in the serum of infected pigs at dpi (figure c ). together, both transcriptomic and serological data indicate that b-cell development and antibody production are not affected by con -infection. however, low levels of virus-neutralizing antibodies are likely due to the virus ability to escape antibody neutralization (see below). viruses , , x for peer review of prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current prrsv persists in lymphoid tissue of infected pigs for several months [ , ] . the mechanism of prrsv persistence is not fully understood. we performed genome-wide transcriptome analysis of lymphoid tissue collected from pigs persistently infected with an attenuated prrsv strain using rna-seq technology that detects both host and viral rna. viral rna reads were detected in iln of all five infected pigs. it was reported previously that prrsv genome mainly exists in dsrna forms in lymphoid tissues during persistent infection [ ] . since only mrna (purified by using poly-t oligo-attached magnetic beads) was used for library construction and rna sequencing, the viral rna reads detected in this study must be derived from either viral genomic rna or sub-genomic mrna, not from dsrna. the viral rna reads map throughout the viral genome. however, we are not able to discern whether these reads are derived from genomic or sub-genomic mrna because we used a short-read rna sequencing platform. it would be interesting to use long-read rna sequencing to study the viral transcriptome at different states of infection in pigs [ ] . it was reported previously that no significant degs were observed in lymphoid tissue of persistently infected animals [ ] . in the current study, we identified a large number of degs in persistently infected animals (figure b ). this might be due to the difference in the experimental setup. in the previous study, pigs were infected with a wild-type prrsv- whereas in this current study, pigs were infected with an attenuated synthetic prrsv strain [ , ] . besides, the previous study looked at a small set of selected genes while in this study we look at the genome-wide rna transcriptome. go terms and kegg analysis revealed that genes involved in the innate immune (complement and tlr) pathways, apoptosis, cytokine-chemokine signaling, t-cell exhaustion, and humoral responses are highly differentially expressed in prrsv-infected animals compared to control. the complement system is a constituent of innate immunity that serves by neutralizing cell-free viruses, lysing virus-infected cells, and boosting virus specific responses [ ] . it also links the innate and adaptive immune responses, enhances humoral immunity, regulates antibody effector mechanisms, and modulates t-cell function [ ] . many viruses have developed a strategy to evade the complement pathway by recruiting or enhancing the production of host transcription regulatory components. during acute infection ( dpi), prrsv significantly represses the expression of complement regulatory components (cd and c bpb) in lung tissue [ ] . early complement activation during acute infection facilitates the release of newly formed virions from infected cells, yet, chronic viral infection is reported to suppress the activity of complement activation proteins and increases the activity of regulatory proteins (reviewed [ ] ). in the current study, we found overexpression of cfh and cfi along with another regulatory factor cd (decay-accelerating factor). overexpression of cfh, a major soluble regulator of the alternative pathway, results in inhibition of c and c convertase enzymes. cfi suppresses the complement active proteins c b (opsonin) and c b via mediating cleavage to their inactive form [ , ] . regulatory factor cd is an inhibitor of c convertase which prevents c b deposition on the cell surface [ ] . apart from that, we also see the downregulation of c q, which increases neutralizing and hemagglutination inhibition activity of anti-influenza antibodies [ ] . available data from previous studies and the current study suggest that activation of complement pathway during acute infection helps to disseminate virus, while suppression of complement components like c q, c r, and c , and upregulation in regulatory components during persistent infection facilitates the virus to escape from the complement system to maintain persistence in lymphoid tissues. most naturally occurring prrsv strains suppress type i ifn production by inhibiting the activation and nuclear translocation of irf /irf and nf-kb [ , ] . deficiency of irf / results in severe mortality to infection with west nile virus (wnv), chikungunya virus (chikv), and ross river virus infection, and promotes viral persistence in the infected hosts [ ] [ ] [ ] . it has been reported that prrsv nsp β inhibits irf phosphorylation and nuclear translocation; thus, inhibiting ifn production [ , , ] . interestingly, the synthetic prrsv-con and its attenuated form con were able to induce type i ifns [ , ] . contrary to its nature to induce type i ifn, in the current study no upregulation of canonical type i ifn signaling pathway-associated genes was observed. however, expression of rna sensing molecules including tlr , tlr , and tlr was upregulated in con -infected animals. on the basis of available data, we hypothesize that reduced levels of viral replication and sequestration of viral rna in infected cells during persistent infection might limit further activation of type i ifn signaling pathway by preventing interaction with cytoplasmic pattern recognition receptors (prrs). apoptosis or programmed cell death is a potential host immune mechanism against virally infected cells to curtail the spread of newly formed viral progenies. apoptosis is induced by two distinct, yet inter-connected signaling pathways, the extrinsic and intrinsic pathways [ ] . in order to successfully establish persistent infection, viruses have developed mechanisms to inhibit apoptosis. for instance, adenovirus (e b- k), human cytomegalovirus (ul ), poxviruses (f l), and myxoma virus (m l) have an inhibitory effect on proapoptotic proteins bak/bax [ ] . it appears that prrsv can also modulate apoptosis. studies of pulmonary alveolar macrophages (pam) infected with prrsv in vitro reveal that the virus stimulates anti-apoptotic pathways early in infection while it induces apoptosis late in infection [ ] . on the other hand, the virus induces apoptosis in the tissues of infected animals during acute infection, but the frequencies of apoptotic cells reduced to normal levels observed in control, non-infected pigs from day post-infection [ ] . in this study, expression of multiple anti-apoptotic genes including xiap, bfl- /a (bcl a ), and birc was upregulated while expression of pro-apoptotic genes (aifm , chac , and osr ) was downregulated. xiap is an endogenous caspase and inhibitor [ ] . bfl- /a (bcl a ) is a transcriptional target of nuclear factor-kb (nf-kb) that suppresses caspase activation and apoptosis in response to death-inducing stimuli like tnfα [ ] . birc is an interaction partner to tnfrsf b and inhibits apoptosis by interfering with the caspase activation [ ] . the pro-apoptotic bcl- family member like bcl- -associated killer (bak ), which allows the release of cytochrome c via formation of homo-oligomers and stable insertion into outer mitochondrial membrane was significantly suppressed. thus, the data suggest that apoptosis was suppressed in the iln of con -infected animals during persistent infection. chemokine molecules ccl and ccl play a crucial role in migration, activation, expansion, and survival of antiviral t-cells. suppression of the ccr and ccl /ccl axis results in dysfunction of t-cells during viral infection [ , ] . ccl -ccr axis plays role in protection against multiple viruses, such as hiv [ ] , herpes simplex virus (hsv- ) [ ] , hsv- , hepatitis c virus [ ] , and pseudorabies [ ] . acute prrsv infection increases the expression of chemokines, leading to the infiltration of immune cells toward the sites of infection [ , ] . in this study, expression of both ccl and ccl was significantly downregulated in con -infected lymph node (figure a ). additionally, expression of different chemoattractant molecules (fractalkine/cx cl , ccl , ccl , ccl , ccl , and ccl ) and receptors (ccr and ccr ) was also downregulated. it is possible that downregulation of these chemokines and receptors might impair t-cells trafficking to inguinal lymph node, the site of prrsv persistence. our transcriptional data are supported by the functional data which demonstrate that the frequencies of ifn-γ sc in iln were significantly lower than in pbmcs (figure ) . chronic or persistent viral stimulation results in hierarchical loss of effector functions of t-lymphocytes, including proliferation, cytokine production (e.g., ifn-γ and il- ), and cytolytic responses [ , , ] . although the molecular signatures involved in t-cell exhaustion are not completely understood, the overexpression of cell surface inhibitory receptors (e.g., pd- , ctla- , and others) primarily mediates cd + t-cell dysfunction [ ] [ ] [ ] . t-cell exhaustion seems to be a common phenomenon caused by arteriviruses, as t-cell exhaustion was also reported in horses persistently infected with eav [ ] . recently, it was shown that prrsv infection alone or co-infection with porcine circovirus type (pcv ) can significantly upregulate surface expression of pd-l (cd ) on porcine monocytes-derived dendritic cells (modcs) [ ] . increased expression of pd-l on the surface of antigen presenting cells (apcs) possibly contributes to the ineffective t-cell responses during the prrsv infection. in the present study, expression of several markers for t-cells exhaustion such as pd- (cd ) and its ligand, cd (pd-l ) was upregulated in con -infected animals, suggesting that the t-cells in lymph nodes of persistently infected animals might be exhausted (figure b) . however, additional studies (e.g., t-cell cytotoxicity and cytokine production) are required to fully assess the functionality of t-cells during prrsv persistence. germinal centers (gcs) are the specialized locations in the secondary lymphoid tissues where b-cell maturation, differentiation, somatic hypermutation, and class switching of isotypes take place [ ] . in the current study, the gc development-associated genes, which includes il- produced by follicular helper t-cells [ ] , the regulator of g protein signaling (rgs ) [ ] , and gc-associated nuclear gtpase (nuggc) [ ] , were upregulated in the lymph node of con -infected animals. thus, prrsv infection does not seem to impair gc formation and b-cell activation. this is supported by the fact that all five con -infected pigs developed a robust antibody response, as measured by the idexx elisa ( figure b) . however, only minimal titers of virus-neutralizing antibodies were detected at dpi. perhaps, prrsv does not suppress the humoral immune response. instead, the virus evades from antibody-mediated neutralization through different mechanisms such as glycan shielding and decoy epitopes [ ] [ ] [ ] . in summary, we identified a robust host transcriptional response in the inguinal lymphoid tissue of pigs persistently infected with the attenuated prrsv strain con . genes involved in the innate immune responses are downregulated. similarly, chemokines and receptors associated with t-cell homing to the lymphoid tissue are downregulated. this might lead to the lower frequencies of virus-specific t-cells in lymphoid tissue than in peripheral blood. additionally, the genes involved in the anti-apoptotic pathways are upregulated. collectively, the data suggest that prrsv can create a pro-survival microenvironment at the lymphoid tissue which allows the virus to persist for an extended period. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . representative flow cytometry gating strategy used to identify ifn-γ secreting cd α + , cd α + , and cd α + cd α + (dp) t cells from pbmcs and iln. table s . summary of sequencing quality control and mapping data of samples. arterivirus molecular biology and pathogenesis ictv-international committee on taxonomy of viruses. virus taxonomy: release. ec porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus type porcine reproductive and respiratory syndrome virus infection increases apoptosis at the maternal-fetal interface in late gestation pregnant gilts open reading frame of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis characterization of the porcine reproductive and respiratory syndrome virus glycoprotein (gp ) in stably expressing cells induction of apoptosis by the nonstructural protein and of porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach expression of toll-like receptor mrna and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines simian hemorrhagic fever virus: recent advances lactate dehydrogenase-elevating virus equine viral arteritis genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro cd + t cell susceptibility/resistance to equine arteritis virus infection equine arteritis virus long-term persistence is orchestrated by cd + t lymphocyte transcription factors, inhibitory receptors, and the cxcl /cxcr axis identification of factors associated with virus level in tonsils of pigs experimentally infected with porcine reproductive and respiratory syndrome virus double-stranded viral rna persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination location of t-cell epitopes in nonstructural proteins and of type-ii porcine reproductive and respiratory syndrome virus a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b use of interleukin to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection a wrapper script to automate quality and adapter trimming cutadapt removes adapter sequences from high-throughput sequencing reads a quality control tool for high throughput sequence data ultrafast and memory-efficient alignment of short dna sequences to the human genome differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions htseq-a python framework to work with high-throughput sequencing data moderated estimation of fold change and dispersion for rna-seq data with deseq manke, t. deeptools : a next generation web server for deep-sequencing data analysis gene ontology analysis for rna-seq: accounting for selection bias kobas . : a web server for annotation and identification of enriched pathways and diseases tlr is a negative regulator of both myd -dependent and -independent tlr signaling the inhibitory cd r is differentially expressed on human and mouse t and b lymphocytes cd and membrane protein interactions in the control of myeloid cells cd /cd r paired potent inhibitory molecules regulating immune and inflammatory responses; part ii: cd /cd r potential clinical applications complement evasion strategies of viruses: an overview. front influenza a virus m blocks the classical complement pathway through interacting with c qa apoptosis: a review of programmed cell death apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines nf-kappab antiapoptosis: induction of traf and traf and c-iap and c-iap to suppress caspase- activation bfl- /a functions, similar to mcl- , as a selective tbid and bak antagonist activation, differentiation, and migration of naive virus-specific cd + t cells during pulmonary influenza virus infection regulation of t cell migration during viral infection: role of adhesion molecules and chemokines molecular and cellular insights into t cell exhaustion functional and phenotypic analysis of porcine peripheral blood cd /cd double-positive t cells speckled-like pattern in the germinal center (slip-gc), a nuclear gtpase expressed in activation-induced deaminase-expressing lymphomas and germinal center b cells member of the tumor necrosis factor family and b lymphocyte stimulator il- and il- collaborate to shape t-dependent antibody responses the architecture of sars-cov- transcriptome a synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection viral mimicry of the complement system the complement system in regulation of adaptive immunity control of the amplification convertase of complement by the plasma protein beta h human complement c b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta h for cleavage of c b and c b in solution decay accelerating factor (cd ): a target for cancer vaccines? porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions regulation of interferon regulatory factor- by the hepatitis c virus serine protease infectious chikungunya virus in the saliva of mice, monkeys and humans induction of ifn-beta and the innate antiviral response in myeloid cells occurs through an ips- -dependent signal that does not require irf- and irf- interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus the cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein antagonizes interferon regulatory factor activation identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain bax insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and sh-redox regulation porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages structural biology. controlling the caspases ccr and its ligands: balancing immunity and tolerance ccr coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs ccl induces mucosal homing of hiv- -specific iga-secreting plasma cells in mice immunized with hiv- virus-like particles influence of ccr ligand dna preexposure on the magnitude and duration of immunity enhancement of immune responses by co-delivery of ccl /mip- beta chemokine plasmid with hcv core dna/protein immunization genetic co-transfer of ccr ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular signature of cd + t cell exhaustion during chronic viral infection t cell exhaustion t cells and viral persistence: lessons from diverse infections pd-l expression is increased in monocyte derived dendritic cells in response to porcine circovirus type and porcine reproductive and respiratory syndrome virus infections role of nf-kappa b in cell survival and transcription of latent membrane protein -expressing or epstein-barr virus latency iii-infected cells influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank dirk anderson at flow cytometry core facility, nebraska center for biotechnology for assistance on flow cytometric analysis and the staff members of unl life sciences annex and veterinary diagnostic center for the care of animals. the use of product and company names is necessary to accurately report the methods and results; however, the united states department of agriculture (usda) neither guarantees nor warrants the standard of the products. the use of names by the usda implies no approval of the product to the exclusion of others that may also be suitable. the usda is an equal opportunity provider and employer. key: cord- - uaj hmx authors: desmonts de lamache, d.; moges, r.; siddiq, a.; allain, t.; feener, t. d.; muench, g. p.; mckenna, n.; yates, r. m.; buret, a. g. title: immuno-modulating properties of tulathromycin in porcine monocyte-derived macrophages infected with porcine reproductive and respiratory syndrome virus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: uaj hmx porcine reproductive and respiratory syndrome virus (prrsv) is a positive-stranded rna virus that grows in macrophages and causes acute pneumonia in pigs. prrsv causes devastating losses to the porcine industry. however, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control prrsv infection. the common occurrence of prrsv infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. the macrolide antibiotic tulathromycin (tul) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. the aim of this study was to characterize the anti-viral and immunomodulating properties of tul in prrsv-infected porcine macrophages. our findings indicate that blood monocyte-derived macrophages are readily infected by prrsv and can be used as an effective cellular model to study prrsv pathogenesis. tul did not change intracellular or extracellular viral titers, not did it alter viral receptors (cd and cd ) expression on porcine macrophages. in contrast, tul exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against prrsv. tul had an additive effect with prrsv on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. tul significantly attenuated prrsv-induced macrophage pro-inflammatory signaling (cxcl- and mitochondrial ros production) and prevented prrsv inhibition of non-opsonized and opsonized phagocytic function. together, these data demonstrate that tul inhibits prrsv-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury. a a a a a responsible for estimated losses exceeding us$ million/year in the usa alone, porcine reproductive and respiratory syndrome (prrs) is a devastating disease in the swine industry [ ] . first identified in europe and north america in the late s [ ] , this syndrome is currently prevalent in most swine-producing countries [ ] . its causative agent, the porcine reproductive and respiratory syndrome virus (prrsv), is a small enveloped positive-sense singlestranded rna virus, member of the arterivirus genus [ ] . sequence comparison between viral isolates demonstrated that prrsv exists in at least two distinct genotypes, the european genotype (eu type or type i) commonly referred to as prrsv- , and the north american genotype (na type or type ii) known as prrsv- [ ] . prrsv has a very narrow cell tropism, and may induce persistent asymptomatic infections [ , ] . in its natural host, the virus targets alveolar macrophages (am) [ , ] , and is able to infect most cells of the monocyte-macrophage lineage such as intravascular and lymph node macrophages [ , , ] . these cells play a crucial role in immune surveillance, pathogen killing and adaptive immune response stimulation [ ] . prrsv impairs macrophage phagocytic and bactericidal functions, induces host cell death often resulting in an inflammatory response, and perhaps most importantly predisposes the pig to secondary infections [ ] [ ] [ ] [ ] [ ] . indeed, opportunistic pathogens, whether viral-swine influenza virus, pseudorabies virus-or bacterial-streptococcus suis, bordetella brochiseptica-potentiate prrsv-induced pneumonia [ ] . these synergistic effects promote a self-sustaining inflammatory response increasing the severity and the duration of the disease [ ] [ ] [ ] [ ] [ ] . the common occurrence of prrsv infection with bacterial infections combined with the lack of efficient vaccines begs the question of the value of antibiotics for the treatment of prrs. traditionally, antibiotic efficacy is evaluated solely based on their antimicrobial properties. however, some macrolides have been found to modulate ros and pro-inflammatory cytokines such as cxcl- and il- , and to alter the production of lipid mediators that regulate inflammation [ ] [ ] [ ] [ ] [ ] [ ] . these antibiotics accumulate within leukocytes at concentrations that may reach times the systemic levels, which in turn allows them to be transported directly to the site of infection and confers them superior pharmacodynamics [ ] . there is little evidence supporting a direct anti-viral property for macrolides, but their effects on leukocytes support the hypothesis that such macrolides may be beneficial in the context of viral infections such as prrsv [ , ] . in an attempt to uncover new mechanisms whereby macrolides may protect against the detrimental effects of prrsv, the present study investigated the effects of tulathromycin in porcine monocyte-derived macrophages. tulathromycin is a triamilide in which its lactone-ring is comprised of polar amine groups. it is used for the treatment and prevention of swine respiratory diseases associated with actinobacillus pleuropneumoniae a gram-negative bacteria often found in prrsv-infected pigs [ ] . a. pleuropneumoniae exerts cytotoxic effects in macrophage and neutrophils and increases the production of pro-inflammatory il- , cxcl- -also known as interleukin- -and leukotriene b , which ultimately leads to severe pulmonary tissue damage and death [ ] [ ] [ ] [ ] [ ] . recent studies have demonstrated that in addition to its antimicrobial effects, tulathromycin inhibits cxcl- and ltb production in stimulated neutrophils and macrophages [ , , ] . in addition, tulathromycin promotes the apoptotic death of neutrophils and their phagocytic clearance by macrophages -a phenomenon known as efferocytosis-both crucial processes in the resolution of inflammation [ , , [ ] [ ] [ ] . we hypothesized that tulathromycin may generate immunomodulatory benefits in prrsv-infected monocyte-derived macrophages. the findings indicate that tulathromycin, in the absence of a direct anti-viral effect, is able to restore the phagocytic function and to attenuate the pro-inflammatory phenotype of prrsv-infected monocyte-derived porcine macrophages. the african green monkey kidney cell line marc- (crl- ) which is highly permissive to prrsv, was used for viral passage and plaque titration assay, as validated previously [ , , ] marc- cells were cultivated in dulbecco's modified eagle's medium (dmem; thermo fisher scientific, waltham, ma, usa) supplemented with % fbs (invitrogen, carlsbad, ca, usa) and iu/ml penicillin-streptomycin (thermo fisher scientific, waltham, ma, usa). the cells were maintained at ˚c, % co and passaged twice weekly. prrsv- isolate nvsl - (genbank accession no. ay . ) was used in all experiments as previously described [ ] . viral titration was performed via plaque assay. briefly, marc- cells were seeded in well plates (costar; sigma aldrich, saint-louis, mo, usa) and grown until confluency. once at confluency, cells were infected with prrsv, for hour in serum-free dmem to allow attachment of viral particles. following attachment, marc- were overlaid with a solution of x mem diluted : with . % agarose. infection was carried for h and plaques were revealed with neutral red (sigma-aldrich, saint-louis, mo, usa). dr. r. m. yates from university of calgary generously provided both marc- cell line and prrsv- isolate nvsl - . all animal experimental practices and care were conducted according to the standards of the canadian council of animal care guidelines and approved by the university of calgary life and environmental science animal care committee. blood was collected from healthy large white and landrace cross -to weeks old ( -to kg) female and castrated male piglets. the animals were housed at the veterinary science research station (university of calgary) at ˚c ± ˚c with % humidity, light cycles consisted of hours continuous light exposure followed by hours of darkness. piglets were fed twice with the antibiotic-free feed % hog grower (hi-pro feeds, okotoks, ab, canada), water was provided ad libitum. after weeks, animals were euthanized and tissues made available for secondary teaching and research use. in accordance with the standards of the canadian council on animal care, pigs were euthanized by intracardiac injection with sodium pentobarbital. monocytes were obtained and differentiated into macrophages as described previously [ ] . briefly, blood was pooled and centrifuged for minutes at x g, ˚c in a heraeus megafuge r (thermo fisher scientific, waltham, ma, usa). the plasma was removed, and the buffy coat layer was collected into and diluted : in filter-sterilized . % nacl. sterile polysucrose and sodium diatrizoate gradient solution (histopaque; sigma-aldrich, saint-louis, mo, usa) was added into each tube before centrifugation for minutes at x g, ˚c. pbmcs located at the opaque interphase were then collected, washed with sterile-filtered x hank's balanced salt solution (hbss; thermo fisher scientific, waltham, ma, usa) and centrifuged for minutes at x g, ˚c. contaminating erythrocytes were removed by three hypotonic lysis cycles with sterile ice-cold double-distilled water for seconds followed by the addition of x hbss to restore tonicity. pbmcs were then resuspended in serum-free iscove's modified dubelcco's medium (imdm; thermo ficher scientific, waltham, ma, usa) supplemented with iu/ml penicillin-streptomycin. cells were counted using a hemocytometer and viability was assessed by . % trypan blue exclusion (flow laboratories). pbmcs purity was determined by diff-quick staining on cytospin slides (cytospin cytocentrifuge, thermo fisher scientific, waltham, ma, usa). the cells were then plated in tissue-culture treated , , and well plates (costar; sigma aldrich, saint-louis, mo, usa) or in labtek chamber slides (thermo fisher scientific, waltham, ma, usa) at a concentration of . x cells/ ml for two hours to allow attachment. following adhesion, non-adherent mononuclear cells were washed with warm hbss ( ˚c). subsequent adherent monocytes were incubated for days at ˚c, % co in imdm supplemented with % heat inactivated(hi)-pig serum (ge healthcare, chicago, il, usa), iu/ml penicillin-streptomycin and % l supernatant to allow for differentiation into monocyte-derived macrophages (mdms). l -conditionned medium is commonly used to potentiate monocytes to differentiate into homogenous populations of mature macrophages [ , ] . culture media was changed every days. flow cytometry was used to quantify the number of cells expressing cd (a known cluster of differentiation of monocytes and macrophages). more than % of the isolated cells expressed cd (s fig) . on day , as described previously [ ] , macrophage differentiation was monitored by microscopic morphological changes using diff-quick, and esterase staining, a well known feature allowing to distinguish between monocytes and mature macrophages [ ] . at day , more than % of the cell preparations were differentiated macrophages (data not shown). seven days-old differentiated macrophages were incubated with tulathromycin (draxxin; zoetis, parsippany-troy hills, nj, usa) diluted in imdm + % hi-pig serum at a concentration of . mg/ml or mg/ml or with vehicle control (imdm + % pig serum), as established recently [ , ] . at these concentrations and time points, the drug exhibits immunomodulating properties in bovine macrophages without inducing apoptosis [ ] . antibiotics like tulathromycin accumulate within leukocytes at concentrations that may reach > times the systemic levels, which in turn allows them to be transported directly to the site of infection, and hence confers them with superior pharmacodynamics [ , ] . this phenomen is critical to the mode of action of tulathromycin. the drug concentrations used in these present experiments are consistent with this knowledge, and with previous studies that showed that tulathromycin has immunomodulating effects in bovine and porcine neutrophils and macrophages [ , , ] . these recent studies have reproduced the same immunomodulating effects seen in vitro at these drug concentrations than when using live infected cattle and pigs given tulathromycin at the recommended therapeutic dosage [ , , ] . hence the concentrations used here reflect the physiological conditions in which the drug accumulates at high concentrations within these leukocytes. indeed, comparison of intracellular drug concentrations in treated versus untreated animals have been published previously [ ] . using lc/ ms ms, in animals given the recommended dose of . mg/kg body weight, studies have measured the rapid and prolonged distribution of the drug into lung homogenates, pulmonary epithelial ling fluid (pelf), as well as in pelf cells. macrophages are the major constituents of pelf cells in such preparations. drug levels measured in pelf cells reached concentrations times greater than those in plasma [ ] . it is believed that this great affinity for cellular uptake may be related, in part, to the tri-basic chemical structure of the drug and the trapping of ionized drug within acidic phagolysosomes. macrophages were infected with prrsv minutes after tul or vehicle treatment, or not infected (uninfected controls) and incubated for h at ˚c, % co to allow virus attachment and entry (time ; t = ). prrsv was diluted in serum-free dmem to reach a multiplicity of infection (m.o.i) ranging from . to depending on the experiment. culture media was replaced by pre-warmed imdm supplemented with % pig serum for all experimental groups. prrsv infection was performed for another to hours depending on the experiment. all functional assays contained the following experimental groups: untreated and uninfected control (control); tulathromycin-treated (tul); untreated and prrsv-infected (virus); tulathromycin-treated and prrsv-infected (tul+virus); lps-activated; pro-apoptotic positive control (staurosporine; μm) (sts); or pro-necrotic positive control ( . % triton-x) (trit-x) where appropriate. to avoid l cytokine-induced polarization of macrophages, all macrophages activation experiments were performed on monocytes that were grown in l supernatant free. the effects of tulathromycin and prrsv on macrophage differentiation and activation was determined via microscopic observations and cytokine quantification. mdms were treated with tulathromycin ( . or mg/ml) for hour and infected with prrsv (m.o.i. of . ) for , , or h at ˚c, % co . supernantants were collected and frozen at - ˚c until processed and macrophages were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) and observed with a nikon eclipse t microscope to assess morphological changes. images were taken with a retiga x camera (q imaging, surrey, bc, canada) on a leica dmr fluorescent microscope (leica, wetzlar, germany) and analyzed using imagej software. individual macrophage morphology was assessed and classified as "resting" or "fibroblast-like" morphology. at least macrophages per group in independent experiments were assessed. supernatants were processed to measure interleukin- (cxcl- ) and interleukin- (il- ) concentrations using the porcine cxcl- quantikine enzyme-linked immunosorbent assay (elisa; p , r&d systems, minneapolis, mn, usa) and the il- quantikine elisa (p , r&d systems, minneapolis, mn, usa) respectively. samples were processed as per manufacturer's instructions. ros production by mdms following tulathromycin treatment and/or prrsv infection was monitored with the oxiselect intracellular ros assay kit (cell biolabs, san diego, ca, usa). experiments assessed ros production in resting cells, as well as in a group of cells induced by lipopolysaccharide (lps) to determine the effects of the various stimuli under basal conditions in these cells, as well as when they were activated. mdms were infected for , , , or h (m. o.i of . ) or uninfected (uninfected control). the same treatments were performed on macrophage stimulated with lipopolysaccharide ( μg/ml lps from e. coli o :b (sigma-aldrich, saint-louis, mo, usa). mdms were exposed to ', '-dichlorodihydrofluorescin diacetate (dcfh-da) a cell-permeable fluorogenic probe oxidized to highly fluorescent ', '-dichlorodihydrofluorescein (dcf) by ros. fluorescence intensity, proportional to ros levels within the cytosol was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) reading at nm (excitation) and nm (emission). phagocytic capacity of mdms was assessed using non-opsonized zymosan particles and opsonized latex beads. non-opsonized phagocytosis was monitored using fluorescently labelled saccharomyces cerevisiae zymosan a particles (texas red; sigma-aldrich, saint-louis, mo, usa). mdms seeded on labtek chamber slides or on coverslips at x cells/ml were infected for or hours (m.o.i of . ) or not infected (uninfected control). following infection, experimental groups were incubated with zymosan a particles diluted in control media to a final ratio of : (zymosan:cells) for hour. after exposure, extracellular zymosan a particles were washed away with warm pbs and the cells were fixed in ice-cold % acetone solution. actin was stained with the alexa fluor phalloidin antibody (thermo fisher scientific, waltham, ma, usa) and the nucleus was revealed with dapi (thermo fisher scientific, waltham, ma, usa). enumeration of intracellular zymosan was performed using a leica dmr fluorescent microscope. fc-mediated phagocytic index was measured using carboxylate-modified μm diameter latex or silica beads (kisker biotech, steinfurt, germany) covalently coated with bsa and human igg (sigma-aldrich, saint-louis, mo, usa). the beads were subsequently incubated with macrophages for minutes at a : (beads:cells) ratio. following phagocytosis, extracellular beads were washed away with warm pbs and the cells were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) before microscopic observations. macrophages containing one or more zymosan particles or latex beads were considered as 'positive cells', the phagocytic index was calculated as the ratio of positive macrophages versus total macrophages. a minimum of cells per experimental group were counted from randomly selected fields. all pictures were taken using leica dmr fluorescent microscope with a retiga x (q imaging, surrey, bc, canada) and analyzed using imagej software. in order to prevent any counting bias slides labellings were covered with tape prior to microscopic observations. the pro-apoptotic effects of tulathromycin and prrsv were assessed using a cell death detection elisa kit (roche) according to the manufacturer's instructions as previously described [ , ] . absorbance was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) set at nm. mdms were incubated with tulathromycin ( . or mg/ml) for minutes and infected with prrsv (m.o.i. of . ) for , or h at ˚c, % co . similar experimental treatments were conducted with cells stimulated with lps ( μg/ml from e. coli o :b ; sigma-aldrich, saint-louis, mo, usa) to assess apoptosis in activated macrophages. for all experiments, cells incubated with imdm containing % hi-pig serum or staurosporine ( μm) were used as negative and positive controls respectively. annexin v staining (roche) was performed on the same experimental groups to further assess apoptotic cell death. staining was performed as per manufacturer's instructions and fluorescence was observed using a leica dmr fluorescent microscope equipped with a hcx pl fluotar x objective (aperture = . ). images were taken at , and hours post infection (p. i) with a retiga x (q imaging, surrey, bc, canada). importantly, experiments measuring cytokines and ros followed those in which we measured apoptosis. this allowed to adjust concentrations of tulathromycin and virus to levels at which apoptosis was not detected in the cells, and hence cell death would not be a factor in the cellular cytokine and ros responses. necrosis was assessed through the determination of lactate dehydrogenase (ldh) levels using a cytotoxicity detection kit (roche). mdms were treated with vehicle medium alone (control) or with tulathromycin ( mg/ml) for minutes. cells were then infected with prrsv for , , or h (m.o.i. . ) or supplemented with control medium, and % triton x in media was used as positive control. supernatants were collected and processed following manufacturer's instructions. a spectramax m e microplate reader (molecular devices, san jose, ca, usa) was used to measure ldh concentrations in each sample at nm. necrosis was expressed as the absorbance ratios of the experimental cell lysates versus absorbances from controls arbitrarily set at . ( %). all experimental groups were assessed in duplicates. to assess the potential anti-viral effects of tulathromycin, extracellular and intracellular viral particles counts were monitored with plaque titration assays. for extracellular counts, supernatants were harvested at , , and hours p.i and incubated with confluent marc- cells for h as described above. for intracellular counts, macrophages were washed twice with warm ( ˚c) phosphate buffer saline (pbs; sigma-aldrich, saint-louis, mo, usa) and lysed with double distilled water exposure and thorough mixing. cellular debris were spun down at , x g for minutes and supernatants were harvested and incubated with confluent marc- cells as described previously. prrsv staining was performed to further characterize potential antiviral effects. marc- cells were seeded in labtek chamber slides, grown to % confluence and infected with prrsv (m.o.i. . ) for h at ˚c, % co . prrsv foci numbers and size were revealed using the sr- f antibody (rti, llc, brooking, sd, usa). fluorescence ratio was calculated using imagej. five fields of view per well were counted per sample. expression levels of prrsv receptors in mdms in the presence and absence of tulathromycin were assessed by immunofluorescence. seven days old mdms cultivated with or without l conditioned medium were treated with hbss (control) or tulathromycin ( mg/ml for hours). the murine l fibroblast cell line, known to secrete macrophage-colony stimulating factor (m-csf) is widely used to induce macrophage differentiation from monocytes and prevent differentiation into monocyte-derived dendritic cells [ ] . prior to staining, l -grown cells were washed times in ice-cold pbs to remove all l media and then fixed in % paraformaldehyde in pbs for minutes. fixed cells were then washed times in cold pbs and stained for hour with a r-phycoerythrin (rpe) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) and a fluorescein isothiocyanate (fitc) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) at a dilution of to and to respectively. following staining cells were washed times in cold pbs and observed under leica dmr fluorescent microscopy. fluorescence ratios from randomly selected fields were calculated using the software imagej. images were taken with a retiga x camera. to prevent any bias, slide labelling was covered with tape prior to microscopic observations. all statistical analyses were made using prism software and data were expressed as means + standard error from mean (sem). all data sets were tested for normality. data with parametric distribution were compared using student's t-test, or one-way anova with tukey's multiple comparision anlaysis where appropriate. non-parametric data were compared with a kruskal-wallis test. for every assay, a minimum of separate, independent experiments were conducted with all experimental groups assayed in duplicates or triplicates. statistical significance was established at p < . . in order to determine whether blood monocytes and mdms are susceptible to prrsv we isolated blood monocytes from healthy pigs and cultured them for a period of days in medium supplemented with pig serum to mimic biological conditions. after plating, adherent monocytes exhibited a round shape morphology and were approximatively μm in diameter ( fig a) . by day , the cells displayed a larger, macrophage-like, morphology with characterisitic cytoplasmic vacuoles (fig c) . monocyte differentiation was also measured using non-specific esterase (nse) staining. by day more than % cells were esterase-positive cells. oneday-old monocytes were significantly less susceptible to prrsv compared to days old differentiated mdms (fig ) . prrsv viral particle numbers increased by a . log ( -fold increase) in mdms, and by a . log ( -fold increase) in blood monocytes, between and hours p.i. in both cell types, prrsv infection reached a plateau at h p.i. (fig ) . to optimize the macrophage differentiation protocol, mdms were also cultured in a l -conditioned medium. monocytes cultivated in l -conditioned medium showed the same morphology as those cultivated in medium devoid of l -factors. consistent with previous data, microscopic observation and nse staining showed that by day , more than % cells were macrophages. viral titers in monocytes incubated with l were significantly higher at hours p.i. compared to viral titers in monocytes cultivated without l supernatant (fig ) . numbers of prrsv infectious particles were significantly elevated in mdms cultivated with l -supernatants at all time points of the infection (except from the hours p.i. time point) versus mdms cultivated in medium supplemented with hi-pig serum alone (fig ) . in l -cultivated mdms, prrsv infection peaked at h p.i. and declined afterwards, whereas it continued to increase at hours in l -cultivated monocytes (fig ) . based on these observations, we chose to use l -cultivated mdms for functional experiments, unless stated otherwise. considering that l supernatants may contain cytokines other than m-csf (such as il- and il- ) that could influence macrophage polarization and confound our studies, we decided to grow our cells in medium containing only pig serum when assessing macrophage pro-inflammatory signaling. moreover, to limit the impact of tulathromycin and prrsv-induced apoptosis on macrophage numbers and functions, we treated our cells with tulathromycin at a concentration of . mg/ml and decreased prrsv m.o.i from . to . . at these concentrations, neither the virus nor the drugs significantly induced mdm apoptosis at the experimental time points (data not shown). if the cells were treated at a concentration of mg/ml, functional analysis were performed before hours of incubation. prrsv infection induced a sharp fibroblast-like morphological alteration and pseudopod projections in mdms (fig a) . in uninfected cells (control and tul), less than % of cells exhibited this change in morphology, while nearly % of the cells exhibited this phenotype upon prrsv infection (fig b) . tulathromycin pre-treatment significantly inhibited this morphological change in mdms (fig b) . since macrophage shape and function are correlated [ ] , we hypothesized that prrsv-induced morphological changes were associated with a change in macrophage activation. infection of mdms with prrsv caused a -fold increase of cxcl- secretion after hours (fig ) . prrsv-induced cxcl- secretion was significantly inhibited when mdms were pretreated with tulathromycin (fig ) . the positive control lps, also significantly increased the production of cxcl- (fig ) . we then measured the production of mitochondrial ros, a hallmark of pathogenic oxidative damage in inflamed tissues [ , ] . prrsv infection significantly increased intracellular ros (fig ) . tulathromycin treatment abolished prrsv and lps-induced intracellular ros production, however, it did notrestore ros levels to control values in cells exposed to both lps and prrsv (fig ) . interestingly, intracellular ros levels were significantly lower in mdms incubated with lps and prrsv compared to mdms stimulated only with lps (fig ) . as our results indicated that tul inhibited macrophage pro-inflammatory signaling, another set of experiment assessed the effects of the drug on il- , a cytokine with potent anti-inflammatory properties [ ] . resting mdms produced approximately pg/ml il- throughout the course of the experiments (fig ) . prrsv infected cells secreted significantly less il- compared to control cells at and hours p.i. (fig ) . il- levels did not significantly change versus controls when uninfected cells were treated with tulathromycin alone. however, immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages prrsv-induced il- inhibition was abolished when the cells were pre-treated with tulathromycin at and hours post infection (fig ) . tulathromycin ( mg/ml), as well as prrsv alone (m.o.i = . ), or the positive control staurosporine induced mdms apoptosis h post-infection (fig ) . combined pre-treatment with tulathromycin ( mg/ml; h) and prrsv (m.o.i = . ) for hours showed an additive effect to induce further mdms apoptosis versus single treatments (fig a) . to confirm these data, cells were stained with annexin v, a phospholipid-binding protein with high affinity for the early apotptic marker phosphatidylserine (ps) [ ] . at hours, both tulathromycin alone or prrsv alone induced significant levels of apoptosis compared to controls ( -fold increase vs. control) (fig b and c) . when cells were exposed to the combination of tulathromycin immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages and prrsv, levels of apotosis were almost double those measured in cells exposed to single treatments (fig b and c ). prrsv infection (m.o.i = . ) significantly increased the levels of ldh produced during necrosis and hours p.i. (fig ) . treatment with tulathromycin ( mg/ml) significantly reduced prrsv cell necrosis at hours (fig ) . this effect of tulathromycin could no longer be detected at hours. tulathromycin alone did not alter levels of necrosis (fig ) . triton-x (trit-x), used as a pro-necrotic positive control, induced necrosis in mdms (fig ) . another set of experiments assessed the effects of prrsv, and of tulathromycin, on the nonopsonized and opsonized phagocytic functions of mdms. prrsv infection significantly immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages inhibited both phagocytic functions of the cells (figs and ). prrsv-induced phagocytic inhibition was inhibited by tulathromycin (figs b and b ). tulathromycin treatment alone did not alter mdms phagocytosis versus controls (figs and ). during phagocytosis, macrophages can engulf multiple antigens at the same time [ , ] . additional experiments assessed mdms that engulfed less than particles (ie with basal phagocytic indices) versus cells that ingested more than particles (i.e. with elevated phagocytic indices). prrsv infection significantly reduced the number of cells with high phagocytic indeces, an effect that was abolished by tulathromycin (figs c and c) . tulathromycin inhibited the prrsv-induced reduction of basal and high phagocytic indices (figs and ). tulathromycin alone did not change either of the mdms phagocytic indices versus controls. the same results were obtained when the cells were infected for hours (fig ) . another set of experiments assessed whether the effects of tulathromycin described above were associated with direct antiviral properties of the antibiotic, in porcine mdms (fig a) or marc- cells (fig b) . upon incubation with prrsv, extracellular and intracellular viral particles were enumerated via plaque assay. tulathromycin did not change intracellular or extracellular viral titers in either of the cell models (fig a and b ). to verify these results, marc- cells were stained with fitc-conjugated anti-prrsv nucleocapsid antibody sr f antibody. size and numbers of viral foci were calculated in presence or absence of tulathromycin ( fig c) . again, tulathromycin pre-treatment did not alter viral titers compared to exposure to prrsv alone (fig c and d ). to further examine the effects of tulathromycin on prrsv infectivity, experiments measured viral receptor expression in mdms. to date, two major prrsv receptors have been extensively studied (cd and cd ) and it is not entirely clear which one of these two receptors is essential for prrsv infection [ ] [ ] [ ] . since l -conditioned medium increases viral titers, we hypothesized that it might be due to an increase in cell permissivity resulting from an increase in prrsv receptor expression. to test this hypothesis, we cultivated monocytes in medium containing pig serum alone or in l -conditionned medium for days and then treated them with tulathromycin. mdms differentiated in medium devoid of l -supernatant expressed both receptors. approximatively % of cells expressed cd and % of cells expressed cd . tulathromycin treatment did not significantly change the percentage of cd and cd positive cells (respectively % and % of positive cells) (fig a; upper panels; fig b) . mdms incubation in l -supernatant supplemented medium was sufficient to significantly increase the number of cd positive cells (more than % of mdms were cd positive versus less than % in pig serum supplemented medium alone). in addition, following l -supernantant exposure we were not able to detect any cd positive cells (fig a; lower panels; fig b) . tulathromcyin treatment following l -incubation did not have any significant effect on viral receptor expression in these experiments (fig a; lower panels; fig b) . prrs is one of the most devastating diseases of the porcine industry [ , ] . treatment options to control prrs outbreaks are limited and the efficacy of vaccines is thwarted by the antigenic variability of prrsv [ ] . disease severity is closely related to the ability of the virus to dysregulate macrophages functions and induce inflammation. therefore, we hypothesize that targeting either of these components may represent a critical element of novel therapeutic approaches. anti-inflammatory and immunomodulatory properties of macrolides have been well established [ - - ] . whether these effects may be beneficial in the context of viral diseases such as prrs remains obscure. the present study assessed the anti-viral and immunomodulating properties of tulathromycin (tul) in prrsv-infected porcine macrophages. the findings indicate that tul inhibits prrsv-induced inflammatory responses in porcine monocyte-derived macrophages and protects against the phagocytic impairment caused by the virus, in the absence of any direct anti-viral effects. the two most common cellular models are pams and marc- cells [ , ] . both have significant limitations. the isolation of pams requires bronchoalveolar lavages, and the function of these cells depends on the age and environment of the animal [ ] . moreover, shortly after the initiation of a respiratory infection, alveolar macrophages are replaced by monocytederived macrophages, which therefore represent a key cell population in host-prrsv immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages interactions. monkey marc- epithelial cells do not originate from pigs. therefore, the present experiments developed and used a simple porcine monocyte-derived macrophage model system to characterize the impact of tulathromycin on prrsv infection. previous in vitro studies have shown that the virus could infect blood monocyte-derived macrophages (mdms) [ ] . consistent with previous findings, monocytes were less susceptible to prrsv than differentiated monocyte-derived macrophages [ ] . the addition of l supernatant during macrophage differentiation significantly increased their susceptibility to the virus compared to macrophages cultivated in medium supplemented with pig serum alone. it has been well established that l supernatant is a source of m-csf and is used to induce macrophage differentiation [ ] . immunostaining of prrsv receptors showed that the addition of l immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages strongly upregulated cd ( % positive cells to % positive cells) but abolished cd expression. these results indicate that l supernatant modulate the expression of prrsv receptors, and that cd alone is sufficient for prrsv infection. this is consistent with recent observations showing that cd , but not cd , enabled non-permissive cells to become susceptible, and that increased cd correlates with increased susceptibility to prrsv [ , , ] . l supernatant is known to contain m-csf, but very little is known about other cytokines and chemokines present in this supernatant [ ] . considering that cd and cd expression can be induced by il- and ifn-γ respectively, and that il- treatment increases prrsv infectivity while ifn-γ decreases it [ , , ] , the role of these cytokine in the modulation of macrophage susceptibility to infection requires further investigation. the present findings demonstrate that mdms can readily be infected by prrsv, and hence represent a useful cellular model to study prrsv pathogenesis, as suggested recently [ ] . a hallmark of prrsv pathogenesis resides in its ability to alter macrophages survival and function, hence predisposing the host to secondary infections [ , ] . there is correlation between macrophage morphology and function, hence providing an easy way to monitor immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages changes in macrophage polarization [ ] . in this study we found that prrsv infection dramatically altered monocyte-derived macrophage morphology, inducing an elongated phenotype with numerous cytoplasmic pseudopods. recent findings indicate that these pseudopods promote intercellular junctions allowing prrsv to evade host immunity through direct intercellular spread [ ] . tulathromycin pre-treatment was sufficient to prevent the prrsvinduced pseudopod formation and morphological alterations in macrophages. whether tul may prevent intercellular junctions and thus hinder prrsv immune evasion requires more research. to test the hypothesis that change in macrophage morphology was associated with altered function, we measured the production of pro-and anti-inflammatory cytokines (cxcl- and il- respectively) as well as the production of mitochondrial ros. cxcl- is a potent neutrophil chemoattractant secreted by macrophages and other cell types, and is a critical mediator of neutrophil infiltration in inflamed tissues [ ] . the present findings demonstrate that prrsv is a potent inducer of cxcl- in monocyte-derived macrophages. virally induced cxcl- secretion was inhibited by tul. studies in live animals are warranted to assess whether these observations suggest that tul might attenuate prrsv-induced inflammation through cxcl- inhibition. mitochondrial ros production is a hallmark of cell stress and inflammation, and contributes to prrsv-induced tissue damage [ , ] . other reports showed that mitochondrial ros production was implicated in prrsv-induced apoptotic death of marc- cells [ ] . here we demonstrate that prrsv indeed induces ros production in porcine monocyte-derived macrophages, and that this production is inhibited when the cells are pre-treated with the antibiotic. tulathromycin was also able to restore ros levels to control in lps-stimulated cells but not in lps and prrsv exposed to both lps and prrsv. interestingly, in these conditions mdms showed a decrease in ros production compared to cells exposed only to lps. this suggest that prrsv may inhibit intracellular ros production of mdms during bacterial infections.another set of studies sought to determine whether tul inhibition of the viral-induced pro-inflammatory cxcl- coincided with an increase in antiinflammatory signaling. we found that the virus alone was able to inhibit il- secretion, and that tul blocked this effect. these data are in contrast with others from the scientific literature. indeed, it is generally accepted that prrsv induce il- production to increase its infectivity [ ] . in fact, il- activated cells are more permissive to prrsv than unstimulated m -polarized macrophages [ ] . more research is necessary to explain the mechanisms whereby prrsv regulates the production of il- . tulathromycin alone did not induce il- secretion suggesting that cxcl- and mitochondrial ros inhibition by tul was not dependent on il- production. taken together the present findings strongly support the hypothesis that tulathromycin may attenuate prrsv-induced inflammation by inhibiting production of pro-inflammatory cxcl- , and by preventing the suppression of anti-inflammatory il- . consistent with previous studies, we found that prrsv and tul induced macrophage apoptosis [ , , , ] . the present findings also illustrate that tul and prrsv haver additive pro-apoptotic effects. morevoer, the data indicate that prrsv leads to cell necrosis, an effect that was inhibited by tul. necrosis is known to exacerbate local inflammation, to induce the release of cytotoxic molecules, and to lead to extensive tissue damage, while cell apoptosis contributes to the resolution of inflammation [ , ] . more research in live prrsv-infected animals will help determine whether tul is able to promote the resolution of prrsv-induced pulmonary inflammation at least in part via such a mechanism, as well by shifting local cytokine release from pro-inflammatory to anti-inflammatory mediators. it is well established that prrsv infected pigs are often infected by secondary pathogens [ , ] . at present, the mechanisms resulting in the increase of secondary infections during prrsv infections remain incompletely understood. studies have shown that prrsv is directly able to impair macrophage phagocytosis, which in turn may represent a key element of the development of secondary infection [ , , ] . macrophage phagocytosis is triggered when phagocytic receptors including opsonic receptors (fcr) or pattern recognition receptors such as the mannose receptor, are activated [ , ] . using non-opsonized zymosan particles or igg-coated latex beads, the present findings demonstrate that prrsv significantly inhibits both phagocytic pathways. these results are consistent with previous reports showing decreased phagocytosis of latex beads, or live bacteria (streptococcus suis) upon prrsv infection [ , ] . recent findings suggest that prrsv- inhibits phagocytosis through its interaction with sialoadhesin (also referred to as cd ). however, in our model system, l cultivated mdms were negative for cd suggesting either that mechanisms for inhibition of phagocytosis are strain and/or genotype dependent, or that prrsv may inhibit phagocytosis through multiple pathways [ ] . another report recently demonstrated that the same nsvl- - strain as used here may impair phagosomal maturation and nadph oxidasemediated respiratory burst, both implicated in the antimicrobial properties of macrophages [ ] . tul blocked the prrsv-induced inhibition of non-opsonized and igg-mediated macrophage phagocytosis. these observations pave the way towards studies in vivo to assess whether this antibiotic might help control secondary infections during prrsv infections through this mechanisms in addition to its direct anti-microbial properties. the mechanisms whereby tul protects against prrsv-induced inhibition of phagocytosis require further elucidation. some macrolides such as tilmicosin and tylvalosin have been recently demonstrated to possess direct anti-viral effects against prrsv [ , ] , while others like erythromycin do not [ ] . in the experiments described herein, tul did not exhibit any direct anti-viral properties, nor did it significantly alter the expression of the two receptors used by the virus for entry, cd and cd . together, the data indicate that in porcine mdms, tul is able to block prrsvinduced pseudopod formation, necrosis, pro-inflammatory cxcl- and mitochondrial ros production, and inhibition of macrophage phagocytosis. in addition tul also synergized with prrsv to induce pro-resolution cell apoptosis and the production of anti-inflammaotry il- . the results also show that the protective modulation of macrophage structure, function, and behavior by tul occurs in the absence of a direct anti-viral effect. the present observations pave the way towards further studies with a prrsv- strain to determine whether the effects we observed in this study are conserved with the other prrsv genotype. studies in vivo will help determine whether and how these effects may translate into clinical benefits. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers reproductive failure of unknown etiology porcine reproductive and respiratory syndrome virus changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the molecular biology of arteriviruses arterivirus molecular biology and pathogenesis effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus (prrsv): kinetics of infection in lymphatic organs and lung the role of pulmonary intravascular macrophages in porcine reproductive and respiratory syndrome virus infection the spatiotemporal cellular dynamics of lung immunity effect of porcine reproductive and respiratory syndrome virus (prrsv) on alveolar lung macrophage survival and function a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus polymicrobial respiratory disease in pigs immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs interaction of the european genotype porcine reproductive and respiratory syndrome virus (prrsv) with sialoadhesin (cd /siglec- ) inhibits alveolar macrophage phagocytosis dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection effects of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasteurella multocida in pigs concurrent highly pathogenic porcine reproductive and respiratory syndrome virus infection accelerates haemophilus parasuis infection in conventional pigs anti-inflammatory benefits of tilmicosin in calves with pasteurella haemolytica-infected lungs anti-inflammatory effects of macrolides in lung disease immuno-modulation and anti-inflammatory benefits of antibiotics: the example of tilmicosin mechanisms of action and clinical application of macrolides as immunomodulatory medications direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages: inhibition of cxcl- secretion, induction of apoptosis, and promotion of efferocytosis anti-inflammatory benefits of antibiotics: tylvalosin induces apoptosis of porcine neutrophils and macrophages, promotes efferocytosis, and inhibits pro-inflammatory cxcl- , il α, and ltb production, while inducing the release of pro-resolving lipoxin a and resolvin d dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract virulence factors of actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host immunomodulatory effects of tulathromycin on apoptosis, efferocytosis, and proinflammatory leukotriene b production in leukocytes from actinobacillus pleuropneumoniae-or zymosan-challenged pigs il- as a keystone cytokine in health and disease host-pathogen interplay at primary infection sites in pigs challenged with actinobacillus pleuropneumoniae anti-inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase- -dependent neutrophil programmed cell death and inhibits nf-κb signaling and cxcl transcription. antimicrob. agents chemother macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving tgf-β, pge , and paf resolution of inflammation: a new therapeutic frontier evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents infection of porcine bone marrow-derived macrophages by porcine respiratory and reproductive syndrome virus impairs phagosomal maturation granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine l cells identification of markers that distinguish monocytederived fibrocytes from monocytes, macrophages, and fibroblasts rapid and prolonged distribution of tulathromycin into lung homogenate and pulmonary epithelial lining fluid of hostein calves following a single subcutaneous administration of . mg/kg body weight modulation of macrophage phenotype by cell shape porcine arterivirus activates the nf-κb pathway through iκb degradation. virology porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway biology of interleukin- quantitation of apoptosis and necrosis by annexin v binding, propidium iodide uptake, and flow cytometry the cell biology of phagocytosis phagocytosis: receptors, signal integration, and the cytoskeleton sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages an intact sialoadhesin (sn/ siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line variability of neutrophil and pulmonary alveolar macrophage function in swine establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with prrsv- cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses human monocytes express cd , which is upregulated by il- and identical to p interferon-inducible cd / siglec attenuates anti-hiv- effects of ifn-α immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread adenoviral-expressed gp of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions apoptosis and porcine reproductive and respiratory syndrome virus corpse clearance defines the meaning of cell death differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases effects of porcine reproductive and respiratory syndrome virus (isolate tw ) on porcine alveolar macrophages in vitro in utero infection with prrs virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets mechanisms of phagocytosis in macrophages porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability transcriptional analysis of prrsv-infected porcine dendritic cell response to streptococcus suis infection reveals up-regulation of inflammatory-related genes expression antiviral activity of tilmicosin for type and type porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of nf-κb activation antibiotic-mediated inhibition of porcine reproductive and respiratory syndrome virus (prrsv) infection: a novel quinolone function which potentiates the antiviral cytokine response in marc- cells and pig macrophages the authors thank troy feener, barbara smith, and the staff at the veterinary sciences research station at the university of calgary for their help with animal handling. we also thank dr. constance finney and dr. edina szabo for their help with flow cytometry. buret. key: cord- -wm eyaam authors: becares, martina; sanchez, carlos m.; sola, isabel; enjuanes, luis; zuñiga, sonia title: antigenic structures stably expressed by recombinant tgev-derived vectors date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: wm eyaam coronaviruses (covs) are positive-stranded rna viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. transmissible gastroenteritis virus (tgev) was engineered to express porcine reproductive and respiratory syndrome virus (prrsv) small protein domains, as a strategy to improve heterologous gene stability. after serial passage in tissue cultures, stable expression of small prrsv protein antigenic domains was achieved. therefore, size reduction of the heterologous genes inserted in cov-derived vectors led to the stable expression of antigenic domains. immunization of piglets with these tgev vectors led to partial protection against a challenge with a virulent prrsv strain, as immunized animals showed reduced clinical signs and lung damage. further improvement of tgev-derived vectors will require the engineering of vectors with decreased recombination rate. the order nidovirales comprises enveloped single-stranded, positive-sense rna viruses. the nidovirales order includes the coronaviridae family that contains viruses with the largest known rna genome, of around kb (enjuanes et al., ) . coronavirus (covs) infect a wide range of mammalian and avian species. the development of efficient cov reverse genetics systems (almazan et al., (almazan et al., , (almazan et al., , (almazan et al., , casais et al., ; thiel et al., ; yount et al., yount et al., , makes them promising expression vectors, with several advantages over other viral expression systems. covs replicate in the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely (lai and cavanagh, ) . in addition, these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes (enjuanes et al., ; masters, ) . as covs in general infect both respiratory and enteric mucosal surfaces, they may be used to target the antigen to these areas, stimulating the mucosal immune system to induce a pleiotropic secretory immune response, including lactogenic immunity . in fact, it has been described that a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues (saif, ) . moreover, the tropism of covs may be engineered by modifying the spike (s) gene (casais et al., ; sanchez et al., ) , and non-pathogenic cov strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available and therefore are suitable to develop safe virus vectors (cavanagh et al., ; ortego et al., ) . in fact, several studies have reported the construction of covderived viral vectors expressing high levels of heterologous proteins, including reporter and viral proteins (bentley et al., ; ribes et al., ; shen et al., shen et al., , . foreign gene expression levels can be regulated by the use of different transcription-regulating sequences (trss) ranging from intermediate to high gene expression levels (alonso et al., a) . in addition, our group has recently identified an optimized transcription-regulating motif, enhancing by -fold the mrna levels of a viral gene, which can be used in expression vectors based in cov genomes (mateos-gomez et al., ) . additionally, a combination of these trss could be used to drive the expression of two or three heterologous genes from just one infectious cdna (i.e., dicistronic or tricistronic vectors). genetic stability of a heterologous gene within the viral vector is essential for its development as a live immunization vector. in general, the stability of heterologous genes is high for dna viruses and negative rna viruses, in which the low level of recombination contributes to the maintenance of the inserted foreign genes (bukreyev et al., ) in contrast, positive rna viruses are highly prone to recombination, both homologous and non-homologous (alejska et al., ; figlerowicz et al., ) leading to the loss of the inserted genes and avoiding their expression over a long time period. covs are positive rna genomes with high recombination frequency (denison et al., ; lai, ; sanchez et al., ) . genetic instability leading to the loss of heterologous genes has been frequently reported in cov-derived vectors, both in vitro (bentley et al., ; cruz et al., ; sola et al., ) and in vivo (bentley et al., ) . in view of the frequent instability of cov-based vectors expressing proteins of large size, we explored whether the reduction of heterologous gene size was a useful strategy to increase insert stability, by reducing the probability of the presence of toxic domains in the inserted gene or protein. in fact, the expression of small protein domains is a common strategy used to reduce toxicity when toxic proteins are expressed in bacteria (edwards et al., ; samuelson, ) . tgev infects the enteric and respiratory tissues of newborn piglets resulting in a mortality of nearly % (saif and wesley, ) . interestingly, some non-enteric tgev variants with alterations in the s protein have a tropism restricted to the respiratory tract, and show attenuated phenotype (sanchez et al., ) . tgev-derived vectors have been successfully engineered for the expression of green fluorescent protein (gfp). the gfp gene was expressed by replacing the non-essential genes a and b, leading to very stable ( passages in tissue culture) high expression levels of the heterologous protein ( μg/ cells) . recombinant tgev (rtgev) vectors have been engineered for dicistronic expression of heterologous genes, such as porcine reproductive and respiratory syndrome virus (prrsv) gp and m proteins (cruz et al., ) , or rotavirus vp and vp , in which formation of rotavirus virus like particles (vlps) in the cytoplasm of rtgev infected cells was observed (enjuanes et al., ) . tgev has been previously used as an immunization vector to confer partial protection against prrsv infection (cruz et al., ) . in addition, an engineered rtgev in which the tropism was modified replacing the s protein by the homologous one from mouse hepatitis virus (mhv) was used to confer protection against rotavirus infections (ribes et al., ) . the engineered rtgev expressing rotavirus vp protein was then evaluated in the mouse model. the recombinant virus triggered a humoral response via systemic (serum igg and iga) and mucosal (intestinal iga) antibodies. in addition, partial protection against rotavirus-induced diarrhea was observed in % of the challenged animals. porcine reproductive and respiratory syndrome (prrs) is the most important infectious disease affecting swineherds worldwide. it is characterized by reproductive failure in sows, as well as severe pneumonia in piglets (lunney et al., ) . the causative agent of prrs is prrs virus (prrsv) that is included in arteriviridae family, in the order nidovirales. prrsv is an enveloped, singlestranded positive sense rna virus of approximately kb in length that contains open reading frames (orfs). orf a and orf b encode the replicase non-structural proteins, while orfs to encode structural proteins: the small envelope protein (e), the membrane protein (m), nucleocapsid protein (n) and the glycoproteins gp a, gp , gp , gp , and the recently identified protein a (dokland, ; firth et al., ; johnson et al., ) . currently, prrs causes huge economic losses in the swine industry, but commercially available vaccines are only partially effective (charerntantanakul, ) . prrsv infection induces a weak innate immune response, probably contributing to the reduced and delayed subsequent humoral and cellular immune responses, and also to virus persistence (kimman et al., ). this is probably due to the limited interferon alpha (ifn-α) elicited by prrsv (albina et al., ; calzada-nova et al., ) . the knowledge on prrsv correlates of protection is limited. neutralizing antibodies against prrsv are mainly directed to gp protein (kim and yoon, ; ostrowski et al., ) , although neutralizing antibodies recognizing gp and gp have also been described following prrsv infection (costers et al., ; oleksiewicz et al., ; vanhee et al., ) . prrsv m protein is a potent inducer of t-cell proliferation in piglets infected with prrsv, and may also play a role in protection (bautista et al., ; jeong et al., ) . current vaccines against prrsv have a limited efficacy. best results have been obtained using modified live vaccines, although they have several problems such as incomplete protection, virus shedding and possible reversion to virulence (charerntantanakul, ) . vector-based vaccines could represent an advantage to stimulate both humoral and cell immune responses against prrsv (cruz et al., ) . given the potential of cov-derived vectors, and the requirement of more efficient vaccines against prrsv, the work presented here is focused on the use of tgev as a vector for the expression of prrsv antigenic combinations. the expression of prrsv small domains containing the epitopes relevant for protection would lead to a significant increase in vector stability. previous work from our laboratory has shown that rtgevs coexpressing full-length prrsv gp (wild type or modified) and m proteins induced partial protection against prrsv (cruz et al., ) . the modest results obtained may be due to the instability of gp protein in the rtgev system, resulting in a significant loss of gp expression in - passages in tissue culture. expression of full-length prrsv gp or gp proteins was also toxic for rtgev leading to the loss of the heterologous gene sequence (m. becares, s. zuñiga and l. enjuanes, unpublished results) . in this work the stability of the expression of small domains of prrsv gp , gp and gp , previously described as potentially relevant in the induction of protection against prrsv has been studied, in comparison with the expression of full-length proteins, using rtgev vectors. our results showed that reduction of the heterologous genes size inserted in the cov-derived vector is a promising strategy to achieve stable expression. additionally, as prrsv m protein was stable in rtgev, several antigenic structures were engineered using this protein as scaffold for the expression of small antigenic domains, resulting in high stability. furthermore, immunization of piglets with these live attenuated rtgev vectors partially protected against prrsv, with reduction of clinical signs and lung damage as well as a faster viremia decrease. prrsv m protein is a long non-glycosilated membrane protein of around amino acids, which is the most highly conserved structural protein of prrsv (meng et al., ) and has been involved in the induction of t-cell response against prrsv (bautista et al., ) . a rtgev vector expressing prrsv m protein was generated encoding prrsv m gene in the location previously occupied by non-essential genes a and b. prrsv m gene expression was driven by the transcription-regulating sequence of gene a (trs a ) (fig. a) . a rtgev-s . -trs a -m was recovered, with a titer of pfu/ml, as expected for rtgev viruses. in order to test the stability of prrsv m protein expressed by this vector, cloned viruses were serially passaged in tissue culture and the maintenance of the heterologous gene was evaluated at different passages by the analysis of plaque-purified viral clones. the presence of the heterologous sequence in the viral genome was evaluated by rt-pcr, using specific primers flanking the insertion region. after and serial passages of the rtgev-s . -trs a -m, all the isolated viral clones still contained m gene sequence (fig. b) , with the expected size and sequence as revealed by pcr product sequencing. in addition, all the isolated clones expressed m protein mrna (fig. b) , confirming m gene stability in rtgev system. moreover, m protein expression was analyzed by immunofluorescence in st cells infected at moi . . m protein was expressed in % of the infected cells (fig. c) , with expression levels remaining constant through the passages (data not shown). altogether, these results indicated that expression of prrsv m protein in rtgev was fully stable. expression of small domains of prrsv gp protein using rtgev netralizing antibodies recognizing gp are considered the most relevant for protection, with the epitope responsible for the neutralization located in the ectodomain of gp protein (kim and yoon, ; ostrowski et al., ) . previous studies from our group indicated that the partial protection observed after immunization with rtgevs expressing full length gp protein, both wild type or glycosylation mutants, was probably due to the toxicity of this protein in rtgev system, leading to heterologous gene loss with passages (cruz et al., ) . as a consequence, we decided to evaluate the expression of small domains containing epitopes potentially relevant in protection, as the reduction of potential toxic domains could increase vector stability. in a first approach, a rtgev co-expressing gp ectodomain (gp ecto) and m protein was engineered. gp ecto transcription was driven by the trs a , and that of m protein by an optimized trs partially derived from gene n trs (trs n ) (alonso et al., a) . m protein was included in the rtgev construct because it was fully stable and we previously observed that it increased gp stability, probably by forming the heterodimer observed in the native virus (cruz et al., ) . in fact, we postulate that the expressed gp domains and m protein could form a heterodimer similar to the one observed in the virus, what may be important for its immunogenicity. gp ecto consisted in the most n-terminal amino acids of the olot gp protein, which according to bioinformatics predictions cover the ectodomain of the protein. this domain included the protein motifs relevant in protection, such as the immunodominant epitope and the epitope genomic rna (grna) and subgenomic mrna (mrna) encoding prrsv m protein were detected. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. (c) immunofluorescence analysis of st cells infected with the passage rtgev-s . -trs a -m at hpi. a polyclonal antibody specific for tgev and a secondary antibody staining red were used to identify virus-infected cells. expression of prrsv m protein was detected with a monoclonal antibody and a secondary antibody staining green. critical in neutralization as well as the glycosylation sites ( fig. a) . to allow gp ecto protein detection, a hemaglutinin (ha) tag was fused to gp protein domain ( fig. a ). this tag is small ( amino acids) and was previously used for cov protein tagging, without showing any toxicity (alvarez et al., ) . rtgev-s . -trs a -gp ecto-trs n -m was recovered with titers similar to those of wt virus. the stability of gp ecto domain with virus passages was analyzed by rt-pcr analysis of isolated clones. after passages in tissue culture, % of the isolated clones contained the gp ecto sequence ( fig. b ) and expressed the corresponding mrna (data not shown), representing a modest increase of stability compared with gp full-length ( % stable) (fig. b) . unfortunately, in both cases, the heterologous gp sequences were lost at passage (data not shown), indicating that gp ecto long-term stability did not represent a sufficient improvement as compared to full-length gp . similar conclusions were extracted from immunofluorescence analysis of protein expression (data not shown). in a second step, an additional reduction in gp size was designed, by eliminating the predicted signal peptide of gp , whose cleavage is controversial (thaa et al., ; wissink et al., ) . the resulting amino acid fragment of gp protein (gp fr) was inserted in rtgev, leading to rtgev-s . -trs a -gp fr-trs n -m virus. this small domain contained the gp epitope critical in neutralization, glycosylation sites and the cysteine residue involved in the gp -m heterodimer formation. for gp fr detection a flag tag was fused at the carboxi-terminus ( fig. a ). this flag tag has been successfully used in cov protein tagging (alvarez et al., ) . the additional size reduction of the gp fragment cloned in rtgev led to an improvement in heterologous gene stability after passages in tissue culture, with % of the independent clones containing gp fr sequence (fig. b ) and expressing gp fr mrna (data not shown). moreover, long-term stability was significantly improved, with up to % of the isolated clones stably maintaining gp fr after passages in tissue culture (data not shown). studies on prrsv immunobiology have revealed that prrsv neutralizing antibodies recognized epitopes within the minor structural glycoproteins gp a, gp and gp (costers et al., ; oleksiewicz et al., ) . rtgevs were engineered expressing these proteins, alone or in various combinations, including the tricistronic expression of gp a, gp and gp . none of those proteins was stably expressed by rtgev vectors, with prrsv gp protein resulting extremely toxic for rtgev system, leading to its expression loss in early stages (m. becares, s. zuñiga and l. enjuanes, unpublished results). the recent identification of antigenic, linear domains in gp and gp (costers et al., ; vanhee et al., ) allowed the application of the small domain expression strategy to these proteins. fusion domains including gp or gp epitopes critical in neutralization, flanked by a few amino acids (table ) , and preceded by the corresponding signal peptide were designed. these peptidic domains consisting of and amino acids of gp (gp fr) and gp (gp fr), respectively, were fused to the flag tag at their c terminus end (fig. a) . the gp and gp fragments were cloned in tgev genome in the location previously occupied by non-essential genes a and b and their transcription was driven by the trs a . recombinant viruses rtgev-s . -trs a -gp fr and rtgev-s . -trs a -gp fr were recovered with titers similar to those of the wt virus. the stability of the recombinant viruses was analyzed after and passages in tissue culture, by studying plaque-purified clones by rt-pcr. all the clones maintained the heterologous gene sequence, and (gp ecto), and gp fragment (gp fr) that comprises the ectodomain lacking the signal peptide (sp). immunodominant epitope (ide) and epitope critical in neutralization (ecn), n-glycosylation sites (yellow), and the cysteine involved in gp -m heterodimer formation (red) are also shown. gp ecto and gp fr included an ha or flag tag, respectively, for their detection (tg, blue). (b) rt-pcr analysis of ten clones from plaque-purified passage rtgev-s . -trs a -gp -trs n -m (gp ), rtgev-s . -trs a -gp ecto-trs n -m (gp ecto) and rtgev-s . -trs a -gp fr-trs n -m (gp fr) viruses. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. lower size bands (indicated by red asterisks) correspond to deletion products from heterologous gene, meaning genomic instability. numbers on the right indicate the overall stability of each construct. expressed the corresponding mrna (fig. b ), indicating that both gp fr and gp fr were fully stable in the rtgev vector. protein detection using anti-flag antibody failed for gp fr, gp fr and gp fr, both in immunofluorescence and western blot assays (data not shown). altogether, these data revealed that heterologous gene size reduction led to a drastic increase in the stability of rtgev vectors. m protein is the most conserved structural protein among prrsv strains (kapur et al., ; murtaugh et al., ) and it is the main inducer of virus-specific t-cell response (bautista et al., ; jeong et al., ) . our results indicated that m protein was fully stable in rtgev (see above). therefore, we postulated that m protein could be used as a scaffold for the expression of small antigenic domains. as a proof of principle, the gp epitope critical in neutralization (ecn) domain was selected for expression fused to m protein. two exposed locations into m protein were predicted using tmpred transmembrane topology prediction algorithm (hofmann and stoffel, ) : the n-terminus and a loop comprising amino acids to . gp ecn was inserted at these m protein locations, leading to chimeric structures, gp ep-ntermm and gp ep-mloop, respectively (fig. a) . these chimeric genes were cloned into rtgev vector, and recombinant viruses rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop were rescued with titers similar to those of the parental virus. the stability of the recovered viruses was analyzed. after or passages in tissue culture independent clones were screened by rt-pcr. all the independent clones maintained the heterologous gene sequence (data not shown) after passages, whereas after passages % and % of rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop, respectively, contained the heterologous gene sequence and expressed the corresponding mrna (fig. b) . in order to evaluate stability and expression levels of the chimeric proteins, double immunofluorescence was performed on cells infected with passage rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop viruses. m protein scaffold was detected in % of infected cells in both cases (fig. c ). this detection level was similar to that observed in rtgev-s . -trs a -m expressing full-length wt m protein (see above). in contrast, flag epitope was detected in % and % of the rtgev-s . -trs a -gp ep-ntermm and rtgev-s . -trs a -gp ep-mloop infected cells, respectively (fig. c) . these results strongly suggested that m protein n-terminus was a more exposed location, and therefore better to present antigens. this data is in agreement with previous observations demonstrating that arterivirus m protein is tolerant to manipulations of its ectodomain (verheije et al., ) . in order to evaluate the protection provided by rtgevs expressing prrsv antigens, the rtgevs that showed an increased stability in cell culture were tested in vivo. for that purpose, rtgev-s . -trs a -m, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, and rtgev-s . -trs a -gp ep-ntermm were selected for in vivo experiments. two groups of twelve days-old piglets were inoculated with  pfu/animal of each rtgev-s . -trs a -m, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, and rtgev-s . -trs a -gp ep-ntermm (immunized group), or rtgev (non-immunized group), respectively, by three routes: oral, nasal and intragrastic. previous data from our group indicated that, in these conditions, the virus in the inoculum reached the target organs (respiratory and digestive tracts) and replicated to high titers (cruz et al., ; sanchez et al., ) . a boost was performed weeks after inoculation using the same conditions. two weeks later, a challenge was performed with  tcid of prrsv olot -like virulent strain. a control group was inoculated with  pfu/animal of rtgev and boosted two weeks later, but not challenged. pigs were monitored for clinical signs, focusing on respiratory symptoms such as tachypnoea, and abdominal breathing. prrsv infection resulted in moderate fever, depression and respiratory signs that persist from days to after challenge (fig. a) . the percentage of animals showing respiratory symptoms was significantly higher in the non-immunized group than in the immunized group (fig. a) . moreover, the average weight gain, which was reduced in challenged animals, was higher in immunized animals than in non-immunized animals between and days post-challenge (data not shown). lung damage was analyzed by histopathology of lungs from five randomly chosen piglets per group. lungs from challenged piglets exhibited features that are characteristic of prrsv infection such as pneumocyte hypertrophy and hyperplasia, and intra-alveolar accumulation of cell debris (fig. b, left panels) . the lungs from immunized animals showed a lower degree of lung damage than those from non-immunized piglets (fig. b, right panel) , indicating a certain degree of protection. the lower extent of lung inflammation observed in immunized animals was in agreement with the lower levels of proinflamatory cytokine il- observed in vaccinated animals' sera (fig. c) . immunized animals showed a moderate increase in il- by days post-challenge ( dpi, as shown in the figure), but levels rapidly returned to normal, while non-vaccinated animals showed a higher elevation of this cytokine, that continued at elevated levels during the experimental infection. altogether, these results suggested that rtgev vectors expressing prrsv antigens conferred partial protection against prrsv infection. to further analyze the protection conferred by rtgevs expressing prrsv antigens, prrsv viremia was analyzed by rt-qpcr at different times post-challenge. similar virus titers in serum were obtained in all challenged animals at the initial stages postchallenge ( - dpi) (fig. ) . interestingly, a significant reduction in virus titer was observed in the immunized group at - days post-challenge ( - dpi). in order to evaluate the potential of rtgevs stably expressing prrsv antigens as inducers of immunity against prrsv, the humoral response was analyzed at different times postinoculation. the antibody response against the tgev vector, prrsv virus and prrsv individual proteins expressed by rtgevs were determined by elisa. all the animals elicited a high humoral immune response against tgev indicating that the vector infected target tissues as expected, even though the piglets presented preexisting anti-tgev antibodies (data not shown). seroconversion against total prrsv was observed in all challenged animals by day after infection with the virulent virus, while for individual gp , gp and m protein it was detected by day after challenge. in all the above-mentioned cases, no differences were observed between immunized and non-immunized animals (data not shown). the humoral response against prrsv n protein followed identical pattern to that obtained for anti-prrsv antibodies (data not shown). these data indicated that anti-prrsv total antibodies response was most likely directed against n protein and did not play a role in protection, in agreement with previous reports showing an early non-neutralizing antibody response obtained after prrsv infection (mateu and diaz, ) . interestingly, a higher and faster antibody response against gp protein was found from day post-immunization in immunized animals as compared to the non-immunized ones (fig. a) . the neutralizing antibody response was evaluated in sera from immunized and non-immunized animals at , and days postchallenge. non-immunized animals showed higher levels of prrsv neutralizing antibodies than the immunized ones (fig. b ). this data, suggested a less effective prrsv infection in the immunized piglets, supporting partial protection against prrsv infection. in this study rtgev was engineered for the expression of small protein domains relevant in immune response against prrsv. previous results from our group indicated that instability of certain heterologous gene expression by rtgev might represent an important limitation for its use as an immunogenic vector. the expression of small protein domains was used as a strategy to improve heterologous gene stability. stable expression of protein antigenic domains contained in highly unstable full-length proteins was achieved. additionally, as full-length prrsv m protein was stably expressed by rtgev, it was used as a scaffold for the generation of chimeric proteins that exposed other prrsv antigens, resulting in highly stable expression. therefore, size reduction of the heterologous genes inserted in cov-derived vectors resulted in a promising strategy to achieve stable expression. protection experiments showed that rtgev, stably expressing prrsv antigenic structures, elicited partial protection against prrsv, with a reduction of clinical signs and lung damage in immunized piglets. the potential of cov-derived vectors as systems for gene delivery has been limited due to its restricted stability. in general, genetic stability is highly dependent on the nature of the foreign gene, with some inserts maintained at least twenty passages whereas others are lost at passage two (de haan et al., ; enjuanes et al., ; shen et al., ; sola et al., ) . in addition, other factors affect genetic stability of recombinant covs, such as the insert size (de haan et al., ) , and the genomic location in which it is inserted (bentley et al., ) . the maintenance of the inserted genes will also depend on the recombination rate, conditioned both by the insert size and the presence in the foreign sequence of regions showing homology with the virus genome, favoring homologous recombination (wang et al., ) . furthermore, other heterologous gene or protein characteristics may affect insert stability, leading to loss of the inserts harmful for the infected cell or virus replication. therefore, it is very difficult to predict the specific insert stability in advance, before a highly effort-consuming process to generate the cov-derived vectors expressing the heterologous antigen has been accomplished. prrsv gp and m genes have similar lengths ( and nucleotides, respectively) and small ectodomains exposed between three transmembrane domains. nevertheless, m protein was fully stable in rtgev vectors, while gp protein resulted toxic and its expression was lost after - passages of the virus in cell culture. interestingly, prrsv hydrophobic m protein resulted highly toxic in other expression systems, including bacteria and insect cells (jeong et al., ; plana-durán et al., ) . as deletion of the heterologous genes could be due to homologous recombination between the heterologous gene and tgev genome because of sequence identity, this possibility was analyzed. no statistically significant sequence identity was identified between prrsv gp sequence and tgev genome. in fact, analysis of the deletions observed in the unstable recovered viral clones did not show a common pattern of recombination. in constrast, random deletions were observed ranging from small deletions affecting trs, to larger ones covering most prrsv gp gene sequence (data not shown), that in all cases led to the loss of protein expression. additionally, the gp gene, when expressed alone in rtgev vectors, was lost at very early passages while it was stably maintained until passage when it was co-expressed with m protein, most likely due to the formation of gp -m heterodimers (cruz et al., ) . these data suggested that the instability was caused by protein toxicity affecting host cell viability or viral life cycle, rather than a negative effect of the heterologous gene sequences on virus genomic stability. this toxicity would confer selective advantage to those viral clones that did not express gp protein. in this work we showed that size reduction of the foreign insert significantly improved heterologous gene stability. even in the case of highly toxic inserts, such as prrsv gp protein, size reduction led to % stability. therefore, heterologous gene size reduction is a promising strategy to achieve stable expression in tgev-derived vectors and in general in covs. this effect could be due to a decrease of the probability of non-homologous recombination in shorter sequences, or to the elimination of protein domains toxically affecting the host cell or rtgev life cycle. this result is in agreement with previous studies showing that gene size affected foreign gene stability in cov vectors, as the larger firefly luciferase gene resulted less stable than the shorter renilla luciferase gene, when expressed by feline infectious peritonitis virus (fipv) vectors (de haan et al., ) . in this paper we used prrsv m protein, which we have shown that displays a high stability in rtgev vectors as a scaffold for the expression of small antigenic domains. this approach may be useful to modify the trafficking and accumulation of small protein domains expressed alone. in fact, protein detection using anti-flag antibody failed for gp fr, gp fr and gp fr, both in immunofluorescence and western blot assays (data not shown), probably due to low accumulation of those peptides inside the cell. interestingly, gp ep-ntermm was detected at high levels, indicating a higher accumulation in the infected cell of the chimeric protein. long-term stability of cov-derived vectors has not been systematically addressed. few proteins have been reported to be stably expressed by covs. among these, gfp was stable for more than or passages in tissue culture when expressed by mhv or tgev, respectively (sarma et al., ; sola et al., ) , but it showed instability in ibv-derived vectors (bentley et al., ) . prrsv m protein was also stably expressed by rtgev for more than passages (this manuscript). proteins such as luciferase expressed by mhv and ibv-derived vectors (bentley et al., ; de haan et al., ) , prrsv gp protein expressed using tgev virus vectors (cruz et al., ) or gus when expressed by tgev minigenomes (alonso et al., b) were lost at early passages. in our experience, using rtgev vectors, only around % of the heterologous genes were stably expressed for more than passages in tissue culture [(alonso et al., b; cruz et al., ; sola et al., ) , and unpublished results]. this instability is a key limiting factor in the use of cov-based vectors for the expression of full-length proteins. to improve the stability and efficacy of cov-derived vectors it would be essential to understand the factors that control the high recombination frequency of covs. to this end, a detailed analysis of cov proteins involved in genetic recombination is needed. several enzymes involved in cov rna synthesis, such as nsp (helicase), nsp (endonuclease), nsp (exonuclease), nsp and nsp (rna processivity components), or n protein could modulate recombination in covs. the engineering of recombination defective cov mutants by knocking-down one or several genes involved in the recombination process could be the first step to achieve stable expression of large heterologous genes. in this study, three prrsv protein domains from gp , gp and gp proteins, recognized by neutralizing antibodies were expressed by rtgev and used as immunogens (costers et al., ; plagemann, ; vanhee et al., ; wissink et al., ) , and the humoral response elicited by these rtgevs was measured. after challenge, a faster response against gp protein was observed in immunized piglets. in contrast, the response against gp and gp was similar in immunized and non-immunized piglets. these data suggested that gp fragment was immunogenic, while gp and gp domains were antigenic but had a reduced immunogenicity. immunization of piglets with a combination of rtgev expressing prrsv antigens led to a clear reduction of clinical symptoms after challenge, a lower degree of lung damage and a faster viremia reduction. these results represent an improvement over previous vaccination experiments using rtgev vectors expressing prrsv antigens (cruz et al., ) . prrsv correlates of protection remain to be identified (kimman et al., ) , what represents an additional limitation for the development of new vaccine candidates. neutralizing antibodies seem relevant for preventing prrsv infection (lopez and osorio, ) , but not enough to provide full protection (murtaugh and genzow, ) . t-cell responses seem also required in prrsv clearance (mateu and diaz, ) . in the present study, immunized animals showed a significant faster recall antibody response against gp protein, which is generally considered the main target of neutralizing antibodies (kim and yoon, ; ostrowski et al., ) . non-immunized animals developed a higher neutralizing response after challenge with a virulent prrsv strain, what is considered as an indication of higher infection, whereas the immunized animals were significantly, although partially, protected against prrsv infection. with the available data, it is not possible to determine whether the observed protection was due to an undetectable neutralizing antibody response before challenge [even commercial available vaccines have been reported to fail in the induction of detectable levels of neutralizing antibodies before challenge (geldhof et al., ) ] to immune cell responses [most likely directed to m protein present in the immunization cocktail], or both. the higher gp protein specific antibody response was observed from day post-challenge, while significant differences in neutralizing antibodies between immunized and non-immunized animals were observed between and days post-challenge, correlating with the differences in viremia, what suggests that the observed neutralizing response was due to a higher infection of nonimmunized swine. the relative contribution to protection of the humoral and cellular responses has not been determined. when a correlation between protection and induction of specific cytokines was analyzed, il- levels were significantly different between immunized and non-immunized piglets, with levels consistently higher in non-immunized animals. the exacerbated il- response elicited in non-immunized animals correlated with the higher lung damage observed in these animals. this result was in agreement with previous studies showing that piglets with more severe symptoms, including viremia and lung lesions, had a continuous elevation of il- in serum, while in animals with milder symptoms il- levels returned to normal by dpi (petry et al., ) . higher, but not significant, levels of ifn-α were also observed in immunized animals compared to non-immunized animals (data not shown), at day post-prrsv challenge. this result suggested that immunized animals developed a higher innate immune response, which nevertheless did not seem strong enough to induce a higher adaptative immune response. the construction of rtgevs expressing small antigenic domains has considerably improved the stability of the expression vectors. nevertheless, some of these small antigens may have limited immunogenicity. therefore, the expression of full-length antigens by engineering cov vectors with decreased recombination rate deserves further attention to definitely launch covs as efficacious vaccine vectors for animal and human health. experiments involving animals were performed in strict accordance with eu ( / /ue) and spanish (rd / and / ) guidelines. all the protocols were approved by the in site ethical review committee. baby hamster kidney (bhk- ) cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) (delmas et al., ) were grown in dulbecco's modified eagle's medium (dmem) supplemented with % fetal calf serum (fcs) and g ( . mg/ml) as a selection agent. recombinant tgev viruses obtained in this work were grown in swine testis (st) cells (mcclurkin and norman, ) . tissue culture adapted prrsv olot (genbank kc ) strain was grown and titrated in monkey kidney marc- cells (kim et al., ) . challenge was performed with a virulent prrsv strain homologous to prrsv olot (prrsv-olot -like). prrsv-olot -like was propagated in porcine macrophages differentiated from fresh peripheral blood mononuclear cells (pbmcs) as previously described (enjuanes et al., ) . briefly,  pbmcs isolated from fresh blood by centrifugation were seeded in -mmdiameter plates in roswell park memorial institute medium (rpmi) supplemented with % heat-inactivated swine serum. after h, non-adherent cells where removed and attached macrophages, that showed % of confluence, were infected with tcid of the parental prrsv-olot -like. at h postinfection (hpi), when cytopathic effect was clear, supernatant was collected and centrifuged. virus was titrated in porcine alveolar macrophages (pams) as previously described (duan et al., ) . fusion products gp fr, gp fr, gp fr gp ep-mloop and gp ep-ntermm were chemically synthesized and purchased from gen-eart (germany). the prrsv olot protein sequences forming the fusion products are summarized in table . gp ecto sequence was amplified by pcr using the forward primer ( -gcaggtcctatgtacccctacgacgtgcccgactacgccatgagatg-ttctcacaaattggggc- ) and the reverse primer ( -gcgctcagct-caggtctcgactgcccaatcaaaatg- ), which included ppumi and blpi restriction sites (underlined), respectively. m sequence was amplified using the forward primer ( -gcaggtcctatgggaagcc-tagacgatttttg- ) and reverse primer ( -gggctaagcttacc-ggccatacttgacgagg- ), which included ppumi and blpi restriction sites (underlined), respectively. in both cases, plasmid psl-trs a -orf -trs n -orf (cruz et al., ) was used as a template. prrsv sequences, both chemically synthesized or pcr amplified, were digested with restriction endonucleases ppumi and blpi and cloned into the same sites of plasmid psl-tgev-s . - ab including tgev genomic sequence from nt to . prrsv sequences replaced non-essential genes a and b, leading to intermediate plasmids psl-trs a -gp fr, psl-trs a -gp fr, psl-trs a -gp fr, psl-trs a -gp ep-mloop, psl-trs a -gp fr-ntermm, psl-trs a -gp ecto and psl-trs a -m. for the generation of the dicistronic vectors psl-trs a -gp ecto-trs n -m and psl-trs a -gp fr-trs n -m, the sequence of m protein preceded by the optimized synthetic trs n (alonso et al., a) was obtained from psl-trs a -orf -trs n -orf by digestion with restriction endonuclease blpi and cloned into the same site of psl-trs a -gp ecto and psl-trs a -gp fr. finally, all intermediate plasmids containing prrsv sequences were digested with avrii. the resulting fragments were cloned into the same sites of plasmid pbac-tgev-s . (c.m. sanchez, m. becares, s. zuñiga and l. enjuanes, unpublished results) . this plasmid was derived from the original pbac-tgev fl (almazan et al., ) , containing restriction sites paci and mlui flanking s gene (ortego et al., ) . cloning steps led to plasmids pbac-s . -trs a -gp fr, pbac-s . -trs a -gp fr, pbac-s . -trs a -m, pbac-s . -trs a -gp ep-ntermm, pbac-s . -trs a -gp ep-mloop, pbac-s . -trs a -gp ecto-trs n -m and pbac-s . -trs a -gp fr-trs n -m. all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. transfection and recovery of infectious rtgevs from cdna clones bhk-papn cells were grown to % confluence in -mmdiameter plates and transfected with μg of the corresponding pbac and μl of lipofectamine (invitrogen), according to the manufacturer's specifications. after h of incubation at c, cells were trypsinized and plated over a confluent st monolayer grown in -mm-diameter plate. after a -day incubation period, the cell supernatants were harvested (passage ). rtgevs were cloned by three plaque purification steps. rtgevs were grown and titrated as previously described (jimenez et al., ) . two clones of each rtgev were serially passaged, independently, in st cells every h. at passage and ten viral clones were plaque purified. rna from recombinant viruses was purified from infected st cells grown to overconfluence on -well plates. total intracellular rna was extracted at hpi using the rneasy mini kit (qiagen) according to the manufacturer's recommendations. reverse transcription was performed with high capacity rna-to-cdna™ kit (life technologies) according to the manufacturer's instructions. pcrs were performed to analyze the size and sequence of viral genomic rna (grna), at the locus where the heterologous genes were inserted, and heterologous mrna size and sequence synthesis. the primers used and the expected pcr fragment sizes are shown in table . subconfluent st cells grown on glass coverslips were mock infected or infected at a multiplicity of infection (moi) of . with each rtgev. at hpi cells were washed with phosphate-buffered saline (pbs), fixed with % paraformaldehyde, permeabilized with . % triton x- in pbs and blocked in pbs with % fcs. monoclonal antibodies specific for flag (flag m , : , sigma), prrsv m protein (em e c , : , kindly provided by inge-nasa), or a polyclonal rabbit serum specific for tgev ( : ) were used. bound primary antibody was detected with a alexa fluor or -conjugated antibodies specific for mouse or rabbit, respectively ( : , invitrogen). cell nuclei were stained with , -diamidino- -phenylindole (dapi) ( : , sigma). confocal microscopy was performed using a leica sp laser scanning microscope, and images were collected and processed with las af software (leica, wetzlar, germany). the percentage of infected cells expressing prrsv antigens was estimated by the analysis of independent microscopy fields, which represent an average of more than cells. forty-five twelve days-old non-colostrum-deprived piglets, born from prrsv seronegative sows, were inoculated with rtgev by three different routes (oral, gastric and intranasal) following standard procedures (sanchez et al., ) . piglets were divided into three -animal groups. piglets of group were inoculated with a mix of  plaque forming units (pfu)/animal of each of rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr, rtgev-s . -trs a -gp fr-m, rtgev-s . -trs a -m and rtgev-s . -trs a -gp ep-ntermm. piglets of groups and were inoculated with  pfu/animal of rtgev-s . . two weeks after the first immunization, all piglets were boosted in the same conditions, and two weeks later piglets of groups and were challenged with tcid of prrsv-olot -like per animal by intranasal route. infected animals were monitored daily to detect symptoms of disease, and body weights were determined every days. blood samples were taken at days , , , , , , , and post-first inoculation. five and ten animals per group were euthanized and necropsied at days and , respectively. lung macroscopic lesions were evaluated, and lung samples were collected frozen and in % buffered-formalin. five piglets per group were randomly chosen for histopathological study. lung representative sections were fixed with % paraformaldehyde and stored in % ethanol at c. paraffin embedding, sectioning and hematoxylin-eosin staining were performed by the histology service in the national center of biotechnology (cnb-csic, spain). samples were examined with a zeiss axiophot fluorescence microscope. determination of the lung damage score was obtained from unbiased observation of microscopy fields per animal, scoring from to attending to interstitial, peribronchiolar, and perivascular inflammation (page et al., ) . quantification of porcine il- β, il- , ifn-α, ifnγ, tnfα, il- and il- in serum samples was carried out using swine cytokine magnetic -plex panel (life technologies, tm), and the luminex is analyzer, according to the manufacturer's instructions. three serum samples, corresponding to the same experimental group and the same date of extraction, were randomly pooled and analyzed in a single well. data were calculated by xponent software using a fiveparameter model derived from the known reference cytokine concentrations supplied by the manufacturer. the sensitivity of this assay allowed the detection of cytokine concentrations with the following limits of detection: il- β ( . pg/ml), il- ( . pg/ ml), ifn-α ( . pg/ml), ifnγ ( . pg/ml), tnfα ( . pg/ml), il- ( . pg/ml), il- ( . pg/ml). viral rna was isolated from μl of serum using magmax™ viral rna isolation kit (life technologies) according to the manufacturer's instructions. prrsv rna quantity was measured by rt-qpcr analysis using a custom taqman assay detecting prrsv n rna (taqman probe -fam-acggcttttaatcaaggc-mgb; forward primer -ttccctctgcttgcaatcg- ; reverse primer -ggatgaaagcgacgcagttc- ), and the agpath-id one-step rt-pcr kit (life technologies) according to the manufacturer's instructions. the data were acquired with an abi prism sequence detection system and analyzed with abi prism sds version . . software (applied biosystems). viremia levels were expressed as the rt-qpcr cycle threshold (ct) values. antibodies induced against tgev and prrsv viruses or prrsv purified proteins were detected by elisa as described before (sambrook and russell, ) . prrsv gp , gp , gp , m and n proteins were expressed using the baculovirus-insect cell system. recombinant proteins were purified to near homogeneity by metal chelate affinity chromatography using ni-nta agarose (sigma-aldrich, madrid, spain) as previously described (nogales et al., ) . elisas were performed using partially purified tgev ( . μg per well) and prrsv ( . μg per well) viruses, or prrsv purified proteins gp ( . μg per well), gp ( . μg per well), gp ( . μg per well), m ( . μg per well) and n ( . μg per well). antigens were bound to -well microplates, saturated with % bovine serum albumin (bsa) in pbs for h at c and incubated with serial dilutions of the serum sample in wash buffer ( . % bsa, . % tween in pbs) for min at c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a (biorad) diluted : in pbs with . % bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky) for min table analysis of rtgevs stability by rt-pcr. expected product size and primers used for the analysis of viral grna and heterologous mrna expressed by rtgevs. expected size (bp) grna a mrna b reverse primer ( - ) a pcr for grna analysis was performed with the forward primer ( -attacgaaccaattgaaaaagtgc- ) and the reverse primer ( -ccgcctga-gaaaaggctgcattg- ) in all cases. b in all cases, forward primer ( -gtgagtgtagcgtggctatatctcttc- ), complementary to the viral leader sequence was used. c mrna size shown in the table corresponds to gp fr or gp ecto. at room temperature. reactions were stopped with . m h so , and the absorbance was read at nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio¼(od of sample À od of negative control)/(od of positive controlÀ od of negative control)]. serial dilutions of heat-inactivated serum were incubated for h at c in the presence of pfus of prrsv-olot in dmem containing % fcs. the mixtures were added to confluent marc- cells in -well plates. after one hour incubation at c medium was removed and ml of dmem containing % fcs and . % agar was added. after h, cells were fixed with % formaldehyde in pbs, stained with a crystal violet solution, and lysis plaques were counted. a positive control serum, obtained from a prrsv infected pig at dpi led to - % of virus neutralization at a sera dilution of : . in contrast, a non-immune control serum led to a neutralization of to % in the same experimental conditions. the neutralization index of each serum sample was expressed relative to the one obtained with the negative control serum in the experiment [neutralization index ¼ % À (pfus serum sample/pfus negative control)  ]. two-tailed, unpaired student t tests were used to analyze difference in mean values between groups. all results were expressed as means the standard deviations of the means. chisquare test was used to analyze statistical significance of differences in percentages of immunized and non-immunized groups. p values o . were considered significant (noymer, ) . interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus two types of non-homologous rna recombination in brome mosaic virus construction of a sars-cov infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate engineering the largest rna virus genome as an infectious bacterial artificial chromosome coronavirus reverse genetic systems: infectious clones and replicons transcription regulatory sequences and mrna expression levels in the coronavirus transmissible gastroenteritis virus in vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes the envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein and is ubiquitinated t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus infectious bronchitis virus as a vector for the expression of heterologous genes nonsegmented negative-strand viruses as vaccine vectors characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism reverse genetics system for the avian coronavirus infectious bronchitis virus manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects gp of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro coronavirus gene counteracts host defenses and modulates virus virulence vectored vaccines to protect against prrsv coronaviruses as vectors: stability of foreign gene expression determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site coronaviruses: an rna proofreading machine regulates replication fidelity and diversity the structural biology of prrsv virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) protein production: feeding the crystallographers and nmr spectroscopists titration of african swine fever (asf) virus the nidovirales coronavirus reverse genetics and development of vectors for gene expression expression vectors based on coronavirus genomes genetic variability: the key problem in the prevention and therapy of rnabased virus infections discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges tmbase-a database of membrane spanning protein segments comparative measurement of cell-mediated immune responses of swine to the m and n proteins of porcine reproductive and respiratory syndrome virus critical epitopes in transmissible gastroenteritis virus neutralization novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line molecular assessment of the role of envelope-associated structural proteins in cross neutralization among different prrs viruses challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology recombination in large rna viruses: coronaviruses the molecular biology of coronaviruses role of neutralizing antibodies in prrsv protective immunity porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine reverse genetics of the largest rna viruses gene n proximal and distal rna motifs regulate coronavirus nucleocapsid mrna transcription the challenge of prrs immunology studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the u.s.a. and europe immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) genetic variation in the prrs virus immunogenic characterization and epitope mapping of transmissible gastroenteritis virus rna dependent rna polymerase alpha, significance level of test, encyclopedia of survey research methods pocine b-cells recognize epitopes that are conserved between the structural proteins of american-and european-type porcine reproductive and respiratory syndrome virus generation of a replicationcompetent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection differential immunity in pigs with high and low responses to porcine reproductive and respiratory syndrome virus infection gp ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain lelystad virus baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain olot/ . involvement of orf and orf protein in protection transmissible gastroenteritis virus (tgev)-based vectors with engineered murine tropism express the rotavirus vp protein and immunize mice against rotavirus mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease transmissible gastroenteritis molecular cloning: a laboratory manual bacterial systems genetic evolution and tropism of transmissible gastroenteritis coronaviruses targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity signal peptide cleavage from gp of prrsv: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector the major envelope protein, gp , of a european porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its n-terminal ectodomain reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a we thank h. nauwynck and m. vanhee for sharing information about prrsv epitopes and ingenasa for kindly providing us with anti-m protein monoclonal anitbodies. we also thank m. gonzález, s. ros and r. fernández for technical assistance.this study was supported by grants from the ministry of science and innovation of spain (bio - ), and the european community's seventh framework programme (fp / (fp / - key: cord- -e nsol y authors: chang, yung-chi; nizet, victor title: siglecs at the host–pathogen interface date: - - journal: lectin in host defense against microbial infections doi: . / - - - - _ sha: doc_id: cord_uid: e nsol y siglecs are sialic acid (sia) recognizing immunoglobulin-like receptors expressed on the surface of all the major leukocyte lineages in mammals. siglecs recognize ubiquitous sia epitopes on various glycoconjugates in the cell glycocalyx and transduce signals to regulate immunological and inflammatory activities of these cells. the subset known as cd -related siglecs is principally inhibitory receptors that suppress leukocyte activation, and recent research has shown that a number of bacterial pathogens use sia mimicry to engage these siglecs as an immune evasion strategy. conversely, siglec- is a macrophage phagocytic receptor that engages gbs and other sialylated bacteria to promote effective phagocytosis and antigen presentation for the adaptive immune response, whereas certain viruses and parasites use siglec- to gain entry to immune cells as a proximal step in the infectious process. siglecs are positioned in crosstalk with other host innate immune sensing pathways to modulate the immune response to infection in complex ways. this chapter summarizes the current understanding of siglecs at the host–pathogen interface, a field of study expanding in breadth and medical importance, and which provides potential targets for immune-based anti-infective strategies. sialic acid-binding immunoglobulin-type lectins (siglecs) are cell surface receptors belonging to the immunoglobulin (ig) superfamily. the extracellular domains of these receptors comprise one ligand-binding v-set domain and a variable number of c -set domains, with high-sequence similarities to the variable and constant region of antibodies, respectively. siglecs are mostly located on the cell surface of hematopoietic cells, with the exception of siglec- (schwann cells) and siglec- (epithelial cells). until now, human siglecs and murine siglecs have been identified with different preferences for binding to terminal sialic acids (sia) on glycan structures in a linkage-sensitive fashion. the arginine residue in the siglec v-set domain is critical for contacting the carboxyl group of the sia to form a salt bridge that stabilizes the binding interaction. phylogenetically, siglec family members can be subdivided into two groups: first, there are siglecs that are conserved among different species, but showing low-sequence identity to one another, including siglec- , siglec- , siglec- , and siglec- . the other group comprises the so-called cd (siglec- )-related siglecs (cd rsiglecs), which show low gene conservation but possess higher degrees of sequence identity among the subfamily members. both conserved siglecs and cd rsiglecs include activating and inhibitory receptors. the inhibitory siglecs contain an immunoreceptor-based inhibition motif (itim) in their intracellular domain, conferring the ability to antagonize immune signaling pathways through the recruitment of the shp phosphatases. conversely, activating siglecs have an aspartic acid residue in their trans-membrane domain that associates with immunoreceptor-based activation motif (itam)-containing adaptor dap (dnax-activation protein of kda) to promote signaling and immune responses. lastly, siglec- does not possess a functional intracellular domain and is not known to signal directly, but rather plays a role in cell-cell and cell-microbe interactions. glycans are ubiquitous on eukaryotic cell surfaces via their incorporation in glycoproteins, glycosphingolipids, and glycerophosphatides. in mammalian cells, sia is usually the outmost sugar residue on the oligosaccharide chains of cell surface or serum glycoconjugates, where it functions in recognition and anti-recognition phenomena ranging from the regulation of complement activation to the control of cell-cell apposition (varki ) . like their host cells, bacteria have also evolved complex biosynthetic pathways to produce a diverse array of carbohydrates that form the building blocks of capsular polysaccharides (cps), lipopolysaccharides (lps), lipooligosaccharides (los), and peptidoglycans. these specialized bacterial glycans play pivotal roles in a number of biological processes, particularly mediating microbe-host interactions during the onset and development of infectious disease. it has been long known that the bacterial cps represents a key virulence factor for most encapsulated bacterial pathogens by protecting them from immune clearance within the host. some bacterial capsules interfere with the binding and activation of complement factors on the bacterial surface by inhibiting the c b convertase or recruiting inhibitory complement factor h (cross and kelly ) . bacterial capsules exerting this immune resistance mechanism usually contain sialic or polysialic acids or hyaluronic acid (ha), structurally identical or similar to the polysaccharides found in mammalian tissues, as exemplified by escherichia coli k , neisseria meningitidis types b and c, or group a or group b streptococcus (stevens et al. ; wessels et al. ; foley and wood ; dale et al. ) . envelopment in these host-like capsular structures confers resistance to complement-mediated killing and phagocytic uptake by neutrophils and macrophages, increasing the chance of bacterial survival and dissemination into host tissues. in addition to these passive mechanisms of protection, an emerging hypothesis has been raised based upon the discovery of inhibitory members of the siglec protein family on immune cells. researchers have explored whether these host-mimicking pathogens can blunt activation of antimicrobial responses via engagement of inhibitory siglecs using the sia in their cps. in parallel, studies have asked how hosts have evolved to better recognize and destroy such camouflaged pathogens. the interplay between a particular sialylated human pathogen, group b streptococcus (gbs), and host immune responses serves as a good first example to illustrate the role of siglecs and bacterial expression of sia in the pathogenesis of infection. gbs is a leading cause of neonatal pneumonia, sepsis, and meningitis and is increasingly recognized as a pathogen in elderly and immunocompromised adult populations (heath and schuchat ; thigpen et al. ; skoff et al. ). gbs can be classified into ten serotypes varying in structural and antigenic features, but which share in common the presence of a terminal sia residue that closely resembles the one presented throughout the abundant surface glycocalyx of all human cells (cieslewicz et al. ) . sialylated cps from different gbs serotypes interacts with several human siglecs in a sia-and serotype-specific manner (carlin et al. ) . through this sia molecular mimicry, gbs engages inhibitory human siglec- receptors on neutrophils, resulting in reduced production of reactive oxidative species (ros) and neutrophil extracellular traps (nets), which together impair bactericidal activity (carlin et al. b) . recently, gbs sia engagement of siglec- on human platelets to suppress their activation and release of antimicrobial peptides (amps) was shown to contribute to gbs resistance to platelet killing (uchiyama et al. ) . corroborating these findings, transgenic mice expressing a soluble siglec- receptor, which acts as a decoy to prevent neutrophil suppression, were more resistant to gbs infection in vivo (saito et al. ) . linkages to the underlying sugar chain as well as the substitution at certain carbon positions of sias are critical in determining their binding specificity for siglecs. one study used isogenic mutants with different o-acetylation phenotypes to show that sia o-acetylation protects gbs cps from enzymatic removal by microbial sialidases produced by commensal microbes occupying in the same mucosal niche as gbs (weiman et al. ). however, the same modification markedly reduced gbs binding to human siglec- , such that highly o-acetylated gbs mutants were less able to accomplish sia-mediated neutrophil suppression and showed reduced virulence in vivo (weiman et al. (weiman et al. , . these observations suggest that gbs must balance competing evolutionary selective pressures to fine tune the o-acetylation level of cps sias to maximize its survival advantage in the host. the role of gbs cps sia engagement of inhibitory siglecs in the context of in vivo infection was first addressed in siglec-e deficient mice due to the similarity of function and cellular distribution between human siglec- and murine siglec-e. upon lower dose gbs challenge intranasally or intravenously, siglec-e deficient mice showed increased production of several inflammatory cytokines and had reduced dissemination of the pathogen to the brain (gbs meningitis). however, exaggerated inflammatory mediators and reduced anti-inflammatory cytokine il- production were observed in the siglec-e deficient mice during a high-dose lethal challenge with gbs (chang et al. a ). thus, the sum consequence of the gbs molecular mimicry and inhibitory siglec engagement is likely to vary based upon the site, stage, and magnitude of infection. importantly, in addition to sia-dependent siglec engagement, certain gbs strains can use the surface-anchored β protein to bind human siglec- , another inhibitory siglec preferentially expressed on macrophages and neutrophils. this protein-mediated engagement increased bacterial attachment to the macrophage surface, but simultaneously paralyzed macrophage killing functions, leading to a net reduction of phagocytosis, ros production, net formation, and bactericidal activity (carlin et al. a) . to counteract inhibitory siglec hijacking strategies of bacterial pathogens, host siglec- (sialoadhesin, cd ) plays a crucial role in limiting bacterial dissemination. this receptor, uniquely expressed in the marginal metallophilic and subcapsular sinus macrophages, recognizing the same key sia epitope on the gbs surface, however lacks an intracellular itm motif (crocker and gordon ; crocker et al. ) . siglec- binding to gbs cps sia promoted phagocytic and bactericidal activity of macrophages in vitro and restricts gbs dissemination in vivo (chang et al. b ). loss of siglec- expression not only affected the macrophage sampling and trapping capabilities but also the production of anti-gbs antibodies, suggesting a key role in optimization of antigen presentation and subsequent adaptive immune response against sialylated pathogens (chang et al. b) . another evolutionary adaption of the host to defeat pathogen siglec hijacking is the emergence of activating siglecs with the potential to counteract inhibitory siglec-mediated immune suppression. in , angata et al. discovered siglec- , which possesses nearly identical siabinding domain as inhibitory siglec- through gene conversion, but is coupled with dap , an itam motif bearing adaptor (angata et al. ). thus siglec- and - represent paired receptors with opposite signaling effects. for example, on neutrophils and amniotic epithelium, β protein-expressing gbs can bind to siglec- and siglec- , with the latter engagement stimulating p map kinase and akt signaling to promote more efficient bacterial clearance. notably, a siglec -null polymorphism is present in some humans, caused by fusion between siglec and siglec genes, resulting in functional deletion of siglec- expression (yamanaka et al. ) . a genetic survey of the siglec -null polymorphism and gbs colonization and premature delivery found that siglec -null allele is associated with higher gbs colonization in mothers and more frequent premature birth of infants from gbs-positive pregnancies (ali et al. ). in addition to gbs, the pathogens escherichia coli k and neisseria meningitidis serotype b, important agents of bacterial meningitis in infants and children, possess sialylated capsules. the cps produced by these two bacteria resembles the same poly-α , -sia (psa) structure abundantly expressed on neurons and the glia cells of the developing central nervous systems (cns) is critical for neuron development and function (finne ; devi et al. ; rutishauser ) . the human-specific siglec- shows a unique expression pattern in human microglia and selective binding preference to α , -linked sias (angata et al. ; hayakawa et al. ) . engagement of siglec- by endogenous host psa exerts protective effects against inflammation-mediated neurotoxicity (wang and neumann ) . the mimicry of mammalian psa by e. coli k and n. meningitidis type b cps may, therefore, target siglec- expression on microglia to blunt immune responsiveness and facilitate neuroinvasion, consistent with the high rates of mortality and serious neurological sequelae seen with these pathogens (robbins et al. ; kaper et al. ) . interestingly, the activating siglec- , which shares over % of sequence identity to the first two ig-like domain of siglec- but is coupled with itamcontaining dap , was later discovered to be a paired receptor of siglec- allowing fine-tuning of immune responses (cao et al. ) . pathogenic e. coli k engages inhibitory siglec- through their psa capsule to inhibit macrophage anti-bacterial functions. in contrast, activating siglec- expressing macrophages recognizes the same epitope to promote elimination of pathogen (schwarz et al. ) . in sum, macrophage engagement of gbs β protein or e. coli k psa capsule illustrates an evolutionary dynamic in which the activating member of the siglec receptor pair may override the immune suppressive responses generated by pathogen mimicry of the inhibitory siglec member. in addition to the cps, sialylation of lps and los is also a prominent binding target of siglecs. for example, siglec- and siglec- bind to the sialylated lps of n. meningitidis, and cells expressing either siglec internalized meningococci in a siglec-and sia-dependent manner (jones et al. ) . strains of campylobacter jejuni express various monosialylated and disialylated los with α , or α , / , linked sia residues, respectively, which perfectly mimic host neural gangliosides gm , gd a, gd , or gt a. colonization with c. jejuni strains possessing sialylated los is epidemiologically associated with higher risk of developing a postinfectious autoimmune neuropathy termed guillain-barré syndrome (jacobs et al. ; willison et al. ; willison and yuki ) . several human siglecs recognize these particular sialylated los structures, although the functional consequences of this interaction require further investigation. siglec- , which is expressed on natural killer (nk) cells, monocytes, and dendritic cells (dcs), can mediate specific sia-dependent interaction with c. jejuni los (avril et al. ) . c. jejuni strains recognized by siglec- express terminal disialylated residues mimicking host gq blike epitopes, including gd c and gd . the siglec- binding signal of c. jejuni correlates with its ability to elicit anti-gq b antibodies, and strains recognized by siglec- were particularly associated with oculomotor weakness in guillain-barré syndrome and its so-called miller-fisher variant (heikema et al. ). on the other hand, c. jejuni with α , -linked sia on the los chain showed strong interaction with siglec- , which facilitated bacterial uptake and induce higher macrophage production of the pro-inflammatory cytokine il- (heikema et al. ) . a key role of siglec- in recognition of sialylated c. jejuni los was confirmed in siglec- deficient animals. bone-marrow-derived macrophages from mice lacking siglec- showed greatly reduced phagocytosis of sialylated c. jejuni, coupled with reduced production of proinflammatory cytokines and type i interferon responses (klaas and crocker ) . sialylated los is important in dc and macrophage activation as well as subsequent t cell polarization and b cell activation. removal of sia from the c. jejuni los causes reduced myeloid cell activation and subsequent b cell responses (kuijf et al. ; huizinga et al. huizinga et al. , . varied sialylated los structures differentially modulate dc-mediated t cell polarization in a siglec-dependent manner, wherein the gd a/gm a mimic induced a more pronounced th skewing, while the gd c mimic preferentially stimulated th responses (bax et al. ) . these observations suggest that targeting distinct dc-expressed siglecs may represent a potential strategy for manipulating th cell differentiation programs and forestalling autoimmune disease post c. jejuni infection. in addition to modulating host dc functions via its sialylated los, c. jejuni triggers il- production of dcs via engaging siglec- through the pseudaminic acid residues on its flagella (stephenson et al. ). these abundant sia and sia-like c. jejuni surface structures (e.g. pseudaminic acid) help mediate a complicated interaction with host immune cells that may impact c. jejuni human disease associations, ranging from autoimmune neuropathies to asymptomatic colonization in individuals with repeated exposure to campylobacter spp. in addition to the well-studied microorganisms mentioned above, a growing list of bacterial species have been discovered to display sialoglycoconjugates on their surfaces, such as pseudomonas aeruginosa (khatua et al. (khatua et al. , , klebsiella pneumoniae (lee et al. ) , and nontypeable haemophilus influenzae (nthi) (kalograiaki et al. ) . p. aeruginosa recruits host sialoglycoproteins and displays them on the bacterial surface, and these absorbed sias can enhance bacterial survival by reducing complement deposition and engaging inhibitory siglec- to suppress neutrophil bactericidal machinery (khatua et al. (khatua et al. , . k. pneumoniae exhibiting a hypermucoviscosity phenotype possess abundant sia cps; blocking of the pathogen's ability to engage inhibitory siglec- enhances neutrophil bactericidal activity (lee et al. ) . nthi los also contains a terminal sia residue that interacts with siglec- , which enhances inflammatory cytokine production in siglec- -expressing macrophages correlating to copd exacerbation in human patients (angata et al. ) . all these observations suggest that sia molecular mimicry by many medical important bacteria can interplay with various siglecs to affect infectious risk and clinical disease manifestations. glycosaminoglycans (gags) are another family of complex carbohydrates ubiquitously present on mammalian host cell surfaces and in the extracellular matrix, regulating a wide range of biological functions from cell adhesion and cell migration to tissue repair and immune responses (linhardt and toida ) . in another instance of molecular mimicry, gag structures have been discovered in several gram-positive and gram-negative bacterial capsules (wessels et al. ; jann and jann ; deangelis ) . the gag ha is structurally identical in animals and in the capsule of group a streptococcus (gas) where it is serving as a molecular camouflage to evade host immune responses (wessels et al. ; dale et al. ) . recently, gas was shown to use its ha capsule to target siglec- on neutrophils, thereby blocking net formation and oxidative burst to inhibit bactericidal function (secundino et al. ) . it is an interesting example of convergent evolution that two structurally unrelated carbohydrates, sia of gbs and ha of gas, each target distinct epitopes in the v-set domain of inhibitory siglec- to dampen immune responses and promote pathogen survival in the human host (secundino et al. ). although fundamentally distinct from the sia molecular mimicry strategies employed by bacterial pathogens to engage inhibitory siglecs for immune evasion, several lines of evidence indicate viruses can themselves take advantage of sia-siglec interactions for cell targeting, spreading, and trans-infection. human immunodeficiency virus- (hiv- ) exploits a sia-siglec axis to facilitate its entry into myeloid cells and trans-infection into cd + t cells. notably, siglec- possesses a unique extended ig-like extracellular domain structure extending out from the surface glycocalyx in an un-masked state which makes it an ideal surface entry target. moreover, in contrast to infected cd + t cells, myeloid cells are relatively resistant to hiv- -induced cytopathic effects and there is no obvious depletion of myeloid cells in hiv- -infected patients. to escape from host immune surveillance, hiv- may have evolved to hijack this siglec- -mediated cellular recognition pathway for its own benefit, to hide within infected macrophages and trans-infect cd + t cells for efficient viral spread. in the manner of many enveloped retroviruses, the hiv- envelope glycoprotein gp is heavily glycosylated, and gp mutations that remove n-linked glycan sites on hiv (and simian immunodeficiency virus siv) impair virus attachment and entry (auwerx et al. ) . a direct interaction of hiv- and siglec was first reported by rempel et al. in , where expression levels of siglec- were correlated to hiv- viral load, and siglec- was proven to bind hiv- in a sia-dependent manner and facilitate infection to a permissive reporter cell line (rempel et al. ) . later gp was proven to serve as viral ligand for siglec- and for several cd rsigecs, including siglec- , - , - , and - , with varying avidity and hiv- strain dependency. moreover, this siglec-gp interaction facilitates virus infectivity to macrophages and t cells (zou et al. ; varchetta et al. ) . mature dcs (mdcs) capture hiv- and viral membrane gangliosides, then transfer the virus to bystander cd + t cells via the established immunological synapses between these two immune cell types. host cellderived α , sialylated glycosphingolipid (gsl) on the hiv- particle membrane is the ligand for siglec- on mdc required for triggering mdc-mediated transinfection of cd + t cells in lymphoid organs (yu et al. ; izquierdo-useros et al. ; puryear et al. ) . a population of cervical dcs at the lamina propria of the ectocervix and the endocervix that express siglec- may be particularly important, and ex vivo studies suggest that cd + t cell trans-infection from these cells can be blocked by addition of anti-siglec- antibodies (perez-zsolt et al. a) . a loss-of-function variant (glu ter) of siglec gene was recently identified from the exome aggregation consortium genetic database to be present in % of the human population. monocytes isolated from individuals with this specific variant completely lack siglec- expression and have reduced hiv capture and transinfection phenotypes ex vivo. however, individuals carrying this truncated siglec- protein do not show marked difference in hiv- acquisition and aids outcomes in vivo, which suggests an indispensable (and perhaps dominant) role of the classical hiv- infectious routes in the hiv- dissemination within the infected individuals (martinez-picado et al. ) . the importance of siglec- -mediated viral spreading has also been confirmed in vivo in murine retroviral infection models. murine leukemia virus (mlv) is recognized by murine siglec- through its sialylated gangliosides (erikson et al. ; sewald et al. ) . mlv captured by siglec- -expressing sinus-lining macrophages and subsequent trans-infection into b- cells through their synaptic contacts was directly demonstrated by time-lapse intravital -photon laser scanning microscopy (sewald et al. ) . virus capture and efficient mlv infection at the lymph node and spleen were significantly reduced by siglec- -targeting antibodies as well as in siglec −/ − mice (sewald et al. ) . these data confirm a pivotal role of siglec- in initial retroviral capture and suggest that siglec- could represent a therapeutic target to reduce viral laden macrophage reservoirs and to prevent trans-infection. however, in a murine model of the splenomegaly-inducing retrovirus friend virus complex (fvc) infection, siglec- -expressing macrophages capture of incoming blood-borne retroviruses limited their spread to erythroblasts in the red pulp where fvc manifests its pathogenesis (uchil et al. ) . in this case, siglec- -mediated fvc capture was beneficial, and further activated dcs and promoted cytotoxic cd + t cell responses, promoting efficient clearing of fvc-infected cells (uchil et al. ) . another well-documented example of siglec exploitation for viral entry and immune modulation is the example of porcine reproductive and respiratory syndrome virus (prrsv). prrsv infection is one of the most economically devastating diseases in the global pork industry. prrsv has a narrow cell tropism, primarily targeting cells of the monocyte/macrophage lineage (duan et al. ) . two cellular proteins, cd and siglec- have been identified as the primary targets for prrsv binding and internalization (duan et al. ; vanderheijden et al. ). α - and, to a lesser extent, α - -linked sias on the prrsv virion mediate viral attachment and infection alveolar macrophages, with siglec- serving as the primary entry receptor responsible for sia-dependent binding through its n-terminal v-set domain (delputte and nauwynck ; delputte et al. ). siglec- neutralization blocks prrsv infection in a dose-dependent manner, and overexpression of siglec- in non-permissive cells enhances prrsv cell attachment and internalization (duan et al. ; vanderheijden et al. ) . in subsequent studies, sias on the viral envelope structural protein m/gp heterodimer were identified as the binding target of siglec- (van breedam et al. ) , and the interaction of prrsv with siglec- interferes with macrophage phagocytic activity, which may increase the incidence and severity of secondary bacterial infections complicating primary prrsv disease (de baere et al. ) . since siglec- and cd are the two key receptors for prrsv entry and internalization into porcine alveolar macrophages, neutralization of these two viral receptors has been explored as a potential target to control prrsv infection in pigs. two recent reports support this therapeutic concept. neutralization of prrsv infectivity for porcine alveolar macrophages was achieved by addition of soluble siglec- and cd fc fusion proteins, or by recombinant adenovirus-or exosome-delivered microrna that specifically targeted cd and siglec- (chen et al. b; zhu et al. ). in addition, pigs that received recombinant adenovirus-delivered soluble cd plus siglec- had reduced pprsv viral loads and fecal viral emission, concurrent with improved clinical scores and higher survival rates from the contagious infection (xia et al. ) . it is notable that an in vivo study conducted in siglec knockout pigs found that the absence of siglec- expression does not alter to the clinical course and histopathology of prrsv infection (prather et al. ) , implying a potential redundant function of cd and siglec- in prrsv infection and the importance of dual targeting for potential pharmacological interventions. although siglec- and cd are general recognized as the key entry mediators for prrsv infection, a recent report demonstrated that certain prrsv strains can infect siglec- -deficient cells by exploiting siglec- in the presence of cd (frydas and nauwynck ; xie et al. ). siglec- showed a higher affinity to type prrsv vs. type prrsv and mediates attachment and endocytosis of prrsv in a sia-dependent manner, while transfection of siglec- into non-permissive cells also restored the prrsv infectivity. these findings indicate that prrsv can use several siglecs to enter macrophages and may influence strain differences in pathogenesis (xie et al. ) . of note, prrsv lung infection induces minimal production of type i interferon and inflammatory cytokines compared to infections caused by swine influenza virus and porcine respiratory coronavirus (van reeth et al. ) . like other inhibitory siglecs, porcine siglec- contains one itim and one itimlike motif possibly, likely counteracting key immune activation pathways induced by viral infection . budding from the gsl-enriched domain is a conserved feature of other enveloped viruses. incorporation of host gsl into the viral envelope is found in hendra and nipah viruses, two enveloped rna viruses of the family paramyxoviridae, and these viruses can utilize the sia-siglec- axis to potentiate mdc-dependent capture and trans-infection of t cells . ebola viruses cause lethal hemorrhagic fever in humans, with many recent outbreaks on the african continent. recently, ebola virus entry into activated dcs was shown to involve siglec- recognition of sialylated gangliosides anchored to ebola virus membranes (perez-zsolt et al. b ). blockade of siglec- by specific monoclonal antibodies interrupted ebola viral uptake and cytoplasmic entry, providing cross-protection against other gsl-containing viruses including hiv- (perez-zsolt et al. b ). this finding suggests that incorporation of gsls in virus particle membranes facilitates siglec- binding to mdc and may be a conserved mechanism for enveloped rna viruses to exploit mdc for systemic virus dissemination. finally, siglec- modulates t cell-mediated anti-viral responses during human respiratory syncytial virus (rsv) infection, with key differences between newborns and adults. upon rsv infection, expression of siglec- is upregulated on monocytes from both newborns and adults, whereas expression of siglec- ligand, cd , is only highly upregulated on adult cd + t cells (van den berg et al. ; jans et al. ). this finding is consistent with observations that siglec- inhibits ifn-γ production by adult cd + t cells but not newborn cd + t cells, although the detailed mechanism remains unknown (jans et al. ). unlike many bacterial and viral infections, parasitic diseases caused by protozoa and helminths are often chronic, lasting months to years or even lifetimes. animal parasites have developed remarkable strategies to interfere with or avoid immune clearance mechanisms to establish a chronic infection in their vertebrate hosts, including several examples of sia-siglec-mediated interactions that help achieve this immune evasion phenotype. the protozoan parasite trypanosoma cruzi, the causative agent of chagas disease, illustrates well how sia can be exploited by animal parasites to escape host immune surveillance, from the very first encounter with innate immune cells to the late-mounted adaptive immune responses. although t. cruzi cannot synthesize sias de novo, it uses its trans-sialidase (tcts) enzyme to transfer sias from host sialylglycoconjugates to its own surface mucins (pereira ; pereira et al. ). these highly sialylated mucin-like coats generated on the t. cruzi surface confer resistance to complement-mediated killing by impeding c convertase assembly, and block potential lytic effects of host anti-α-gal antibodies that would recognize the otherwise exposed α-galactosyl epitopes on the mucin-like glycoproteins (pereira-chioccola et al. ; tomlinson et al. ) . moreover, t. cruzi sialyl-glycoconjugates facilitate its binding to siglec- on host macrophages, and this interaction may be involved in the initiation of trypomastigote infection (monteiro et al. ) . t. cruzi can also use its surface sialyl-glycoconjugates to engage siglec-e on dcs and actively suppress their production of proinflammatory cytokine il- , impairing generation of protective th responses (erdmann et al. ). presence of α - and α - linked sialoglycans has also been discovered on another flagellated protozoa, leishmania donovani, the causative organism of indian visceral leishmaniasis (chatterjee et al. ) , such that high sia-containing virulent strains bind both siglec- and siglec- . the sia-siglec- interaction promotes macrophage uptake of the parasite promoting dissemination to other sites within the body, whereas the sia-siglec- binding suppresses ros, no, and th -dominant cytokine responses in infected macrophages by counteracting mapk and pi k/akt signaling pathways (roy and mandal ) . lastly, vaginitis caused by the fungal pathogen candida is common and frequently recurrent condition in women's health. recently, a combined global genetic and immune profiling study of clinical populations identified polymorphisms in siglec as candidate genetic predispositions involved in candida vaginitis susceptibility (jaeger et al. ) . a particular siglec polymorphism was associated with great inflammasome signaling and il- β production in response to candida in vitro, and in vivo silencing of siglec in a murine vaginitis model was associated with increased fungal burden and neutrophilic inflammation (jaeger et al. ). recent evidence indicates that siglecs functionally intersect with other host innate immune sensing pathways to modulate the response to viral and bacterial infection in important ways. for example, the rna virus vesicular stomatitis virus (vsv) upregulates siglec-g expression in macrophages through intracellular nucleic acid sensor rig-i-or nf-κb-dependent mechanisms (chen et al. ) . subsequent recruitment of shp- phosphatase to the siglec-g intracellular domain initiates a pathway for rig-i degradation, thus suppressing anti-viral immunity. in corroboration, inactivation of siglec-g protects mice against lethal vsv infection (chen et al. ) . vsv infection also results in upregulation of siglec- in macrophages, which triggers a negative regulation pathway for tbk degradation via the ubiquitin ligase trim , suppressing type i interferon production and allowing the virus to escape immune elimination (zheng et al. ) . siglec-g expression is low in cd α + dcs, and siglec-g deficient mice generate more antigen-specific cytotoxic t cell responses to inhibit intracellular bacterial infection (ding et al. ) . one line of evidence developed in recent years suggests a potential for direct interactions between siglec receptors, e.g. human siglec- or - and murine siglec-e and -f, and various toll-like receptors (tlrs) to downregulate the inflammatory pathways downstream of their pattern recognition (chen et al. a ). consequently, siglec-e deletion was seen to boost dc responses to a broad range of microbial tlr ligands. in this model, activation of the mammalian sialidase neu to the cell surface disrupts siglec-e-tlr interaction, suggesting it can serve to derepress and allow positive feedback of tlr activation during infection (chen et al. a) . caution is warranted, however, by another study applied quantitative proteomics to three different strains of siglec-e-deficient mice. quantitative proteomics found no consistent differences in tlr signaling or tlr endocytosis in response to lps, nor did macrophages from the siglec-e-deficient mice exhibit significant differences in uptake or killing of salmonella enterica in vitro (nagala et al. ). siglecs are important and broadly distributed lectin receptors of leukocytes uniquely poised to detect sias and its perturbation during homeostasis and in disease states. the importance of siglecs in immunopathology was proposed as early as the identification of siglec- (cd ) and siglec- (cd ) as biological markers of myeloid leukemias and b cell lymphomas, respectively. mounting evidence supports the concept that sia molecular mimicry serves as a virulence mechanism to subvert host innate immunity or to infect permissive target cells through an interplay with various siglecs. as our understanding of siglec influences on glycans-mediated host-pathogen interactions is now rapidly expanding and deepening, there is considerable interest in exploiting siglecs for immunotherapy and disease prevention. design of glycan-based therapeutics and their requirements for potency and specificity will likely provide a new biotechnological approach to effectively intervene in immunological processes to reduce the incidence and severity infectious diseases. virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cellmediated capture and transfer of hiv- and henipavirus siglec- and siglec- are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group b streptococcus cloning and characterization of human siglec- . a recently evolved signaling molecule that can interact with shp- and shp- and is expressed by tissue macrophages, including brain microglia discovery of siglec- , a novel sialic acid receptor undergoing concerted evolution with siglec- in primates loss of siglec- reduces the risk of chronic obstructive pulmonary disease exacerbation glycan deletions in the hiv- gp v /v domain compromise viral infectivity, sensitize the mutant virus strains to carbohydrate-binding agents and represent a specific target for therapeutic intervention sialic acid-binding immunoglobulin-like lectin mediates selective recognition of sialylated glycans expressed on campylobacter jejuni lipooligosaccharides campylobacter jejuni lipooligosaccharides modulate dendritic cell-mediated t cell polarization in a sialic acid linkage-dependent manner siglec encodes a dap -associated receptor expressed in macrophages that evolved from its inhibitory counterpart siglec and has functional and non-functional alleles in humans group b streptococcal capsular sialic acids interact with siglecs (immunoglobulin-like lectins) on human leukocytes group b streptococcus suppression of phagocyte functions by protein-mediated engagement of human siglec- molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil siglec- and dampen the innate immune response group b streptococcus engages an inhibitory siglec through sialic acid mimicry to blunt innate immune and inflammatory responses in vivo role of macrophage sialoadhesin in host defense against the sialylated pathogen group b streptococcus identification and characterization of adsorbed serum sialoglycans on leishmania donovani promastigotes induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation broad and direct interaction between tlr and siglec families of pattern recognition receptors and its regulation by neu additive inhibition of porcine reproductive and respiratory syndrome virus infection with the soluble sialoadhesin and cd receptors structural and genetic diversity of group b streptococcus capsular polysaccharides mouse macrophage hemagglutinin (sheep erythrocyte receptor) with specificity for sialylated glycoconjugates characterized by a monoclonal antibody sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with immunoglobulin-like domains siglecs and their roles in the immune system bacteria-phagocyte interactions: emerging tactics in an ancient rivalry hyaluronate capsule and surface m protein in resistance to opsonization of group a streptococci interaction of the european genotype porcine reproductive and respiratory syndrome virus (prrsv) with sialoadhesin (cd /siglec- ) inhibits alveolar macrophage phagocytosis microbial glycosaminoglycan glycosyltransferases porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is dependent on the sialic acid-binding activity of the n-terminal immunoglobulin domain of sialoadhesin - )-alpha-n-acetylneuraminic acid] are elicited by immunization of mice with escherichia coli k conjugates: potential vaccines for groups b and c meningococci and e. coli k the lectin siglec-g inhibits dendritic cell cross-presentation by impairing mhc class i-peptide complex formation effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages sialylated ligands on pathogenic trypanosoma cruzi interact with siglec-e (sialic acid-binding ig-like lectin-e) mouse siglec- mediates trans-infection of surface-bound murine leukemia virus in a sialic acid n-acyl side chain-dependent manner occurrence of unique polysialosyl carbohydrate units in glycoproteins of developing brain studies on the pathogenicity of group a streptococci. ii. the antiphagocytic effects of the m protein and the capsular gel replication characteristics of eight virulent and two attenuated genotype and porcine reproductive and respiratory syndrome virus (prrsv) strains in nasal mucosa explants a human-specific gene in microglia perinatal group b streptococcal disease characterization of the specific interaction between sialoadhesin and sialylated campylobacter jejuni lipooligosaccharides siglec- specifically recognizes campylobacter jejuni strains associated with oculomotor weakness in guillain-barre syndrome and miller fisher syndrome sialylation of campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice sialylation of campylobacter jejuni endotoxin promotes dendritic cell-mediated b cell responses through cd -dependent production of ifnβ and tnfα siglec- is a novel dendritic cell receptor that mediates hiv- trans-infection through recognition of viral membrane gangliosides campylobacter jejuni infections and anti-gm antibodies in guillain-barre syndrome a systems genomics approach identifies siglec as a susceptibility factor in recurrent vulvovaginal candidiasis capsules of escherichia coli, expression and biological significance siglec- inhibits rsv-induced interferon gamma production by adult t cells in contrast to newborn t cells recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake combined bacteria microarray and quartz crystal microbalance approach for exploring glycosignatures of nontypeable haemophilus influenzae and recognition by host lectins pathogenic escherichia coli sialic acids acquired by pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated host-cell recognition sialoglycoproteins adsorbed by pseudomonas aeruginosa facilitate their survival by impeding neutrophil extracellular trap through siglec- sialoadhesin in recognition of self and non-self tlr -mediated sensing of campylobacter jejuni by dendritic cells is determined by sialylation sialic acid involved in hypermucoviscosity phenotype of klebsiella pneumoniae and associated with resistance to neutrophil phagocytosis role of glycosaminoglycans in cellular communication identification of siglec- null individuals infected with hiv- increased association of trypanosoma cruzi with sialoadhesin positive mice macrophages expression of siglec-e alters the proteome of lipopolysaccharide (lps)-activated macrophages but does not affect lps-driven cytokine production or toll-like receptor endocytosis a developmentally regulated neuraminidase activity in trypanosoma cruzi lectin receptors as markers for trypanosoma cruzi. developmental stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigote cells mucin-like molecules form a negatively charged coat that protects trypanosoma cruzi trypomastigotes from killing by human anti-alphagalactosyl antibodies dendritic cells from the cervical mucosa capture and transfer hiv- via siglec- anti-siglec- antibodies block ebola viral uptake and decrease cytoplasmic viral entry an intact sialoadhesin (sn/siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus interferon-inducible mechanism of dendritic cell-mediated hiv- dissemination is dependent on siglec- /cd sialoadhesin expressed on ifn-induced monocytes binds hiv- and enhances infectivity escherichia coli k capsular polysaccharide associated with neonatal meningitis leishmania donovani utilize sialic acids for binding and phagocytosis in the macrophages through selective utilization of siglecs and impair the innate immune arm polysialic acid in the plasticity of the developing and adult vertebrate nervous system a soluble form of siglec- provides a resistance against group b streptococcus (gbs) infection in transgenic mice paired siglec receptors generate opposite inflammatory responses to a human-specific pathogen host and pathogen hyaluronan signal through human siglec- to suppress neutrophil activation retroviruses use cd -mediated trans-infection of permissive lymphocytes to establish infection increasing burden of invasive group b streptococcal disease in nonpregnant adults pseudaminic acid on campylobacter jejuni flagella modulates dendritic cell il- expression via siglec- receptor: a novel flagellin-host interaction restricted complement activation by escherichia coli with the k- capsular serotype: a possible role in pathogenicity bacterial meningitis in the united states role of sialic acid in the resistance of trypanosoma cruzi trypomastigotes to complement a protective role for the lectin cd /siglec- against a pathogenic murine retrovirus dual actions of group b streptococcus capsular sialic acid provide resistance to platelet-mediated antimicrobial killing the m/gp( ) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages sialic acid-binding ig-like lectin- interacts with hiv- gp and facilitates infection of cd pos t cells and macrophages biological roles of oligosaccharides: all of the theories are correct alleviation of neurotoxicity by microglial human siglec- genetic and biochemical modulation of sialic acid o-acetylation on group b streptococcus: phenotypic and functional impact o-acetylation of sialic acid on group b streptococcus inhibits neutrophil suppression and virulence definition of a bacterial virulence factor: sialylation of the group b streptococcal capsule effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group a streptococci peripheral neuropathies and anti-glycolipid antibodies guillain-barre syndrome recombinant adenovirus-delivered soluble cd and sialoadhesin receptors protected pigs from porcine reproductive and respiratory syndrome virus infection molecular cloning of porcine siglec- , siglec- and siglec- , and identification of siglec- as an alternative receptor for porcine reproductive and respiratory syndrome virus (prrsv) deletion polymorphism of siglec and its functional implications glycosphingolipid-functionalized nanoparticles recapitulate cd -dependent hiv- uptake and trafficking in dendritic cells siglec suppresses antiviral innate immune response by inducing tbk degradation via the ubiquitin ligase trim inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus-and/or exosome-delivered the artificial micrornas targeting sialoadhesin and cd receptors siglecs facilitate hiv- infection of macrophages through adhesion with viral sialic acids key: cord- - ikzg authors: pan, xiaocheng; zhang, nianzhi; wei, xiaohui; jiang, yinan; chen, rong; li, qirun; liang, ruiying; zhang, lijie; ma, lizhen; xia, chun title: illumination of prrsv cytotoxic t lymphocyte epitopes by the three-dimensional structure and peptidome of swine lymphocyte antigen class i (sla-i) date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ikzg to investigate ctl epitope applications in swine, sla- (*) -restricted peptide epitopes matching porcine reproductive and respiratory syndrome virus (prrsv) strains were explored by crystallography, biochemistry, and the specific pathogen-free (spf) swine experiments. first, nine predicted prrsv peptides were tested by assembly of the peptide-sla- (*) (psla- (*) ) complexes, and the crystal structure of the sla- (*) complex with one peptide (nsp -tmp ) was determined. the nsp -tmp peptide conformation presented by psla- (*) is different from that of the peptides presented by the known psla- (*) and psla- (*)hs complexes. two consecutive pro residues make the turn between p and p of nsp -tmp much sharper. the d pocket of psla- (*) is unique and is important for peptide binding. next, the potential sla- (*) -restricted peptide epitopes matching four typical genetic prrsv strains were identified based on the peptide-binding motif of sla- (*) determined by structural analysis and alanine scanning of the nsp -tmp peptide. the tetrameric complex of sla- (*) and nsp -tmp was constructed and examined. finally, taking nsp -tmp as an example, the ctl immunogenicity of the identified prrsv peptide epitope was evaluated. the spf swine expressing the sla- (*) alleles were divided into three groups: modified live vaccine (mlv), mlv+nsp -tmp , and the blank control group. nsp -tmp was determined as a prrsv ctl epitope with strong immunogenicity by flow cytometry and ifn-γ expression. our study developed an integrated approach to identify sla-i-restricted ctl epitopes from various important viruses and is helpful in designing and applying effective peptide-based vaccines for swine. the development of new viral vaccines should be increasingly focused on biosafety, especially to remove the viral genetic material and to avoid the possibility of recombinant viruses developing due to the use of vaccines. in view of this central idea, diverse viral vaccines, such as cytotoxic t lymphocyte (ctl) and b cell epitope vaccines, have been experimentally researched in animals for the ongoing control of viral diseases and immunological deficiency diseases ( ) . porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine pathogens and has caused significant economic losses in the swine industry worldwide for two decades ( ) . prrsv is an enveloped positive-strand rna virus with a viral genome of ∼ kb in length and contains open reading frames (orfs) ( ) . orfs a and b are situated ′proximal to the polycistronic genome and encode two large non-structural replicase polyproteins, pp a, and pp b, which are processed into at least non-structural proteins (nsps). eight relatively small genes following orf in the ′ to ′ direction encode four membrane-associated glycoproteins, three membrane proteins, and a nucleocapsid protein. progress has been made in identifying nsp function related to rna synthesis (nsp and nsp ), subgenomic mrna synthesis regulation (nsp ), membrane-rearrangement (nsp and nsp ), replicative endonuclease (nsp ), major virulence factors (nsp - ) , and viral pathogenesis and host immunity (nsp , nsp , nsp , nsp , and nsp ) ( ) . the frequent mutation and recombination of the prrsv rna genome have resulted in the emergence of numerous variants ( , ) . these phenomena can cause the emergence of some virulent strains, such as the highly pathogenic prrsvs that are causing enormous economic losses in asia ( ) , and have led to the failure of vaccines against new emerging prrsvs. there are considerable challenges and specific requirements in the development of novel vaccines to prevent prrs, such as the ctl-epitope vaccine ( , ) . the greatest challenge is that prrsv can markedly suppress the swine immune defense system ( ) . the current evaluation of the prrsv vaccine is based on its induced antibody response. although high antibody titers can be produced after immunization, protection is not ideal because the key neutralizing antibodies (nabs) against prrsv appear late, typically > days post-infection (dpi), and usually at low levels ( ) . furthermore, nabs are usually specific for the homologous prrsv strain and confer little cross-protection against heterologous strains ( , ) . regarding ctl-mediated immunity, specific ctl responses have been observed in prrsv ( , , ) . the virulent type (lena) prrsv resulted in increased il- α production and a higher percentage of cd + t cells and ifnγ-producing cells compared with controls. cross-reactivity against divergent prrsv is also associated with cytotoxic cd +ifnγ and cd -ifnγ+ cells to a different extent ( ) . prrsv-specific t cells could be observed as early as weeks after infection, with the viral loads decreasing in persistent infection ( ) . modified live vaccines (mlvs) could induce ctl immune responses and confer better protection against heterologous prrsv strains than inactivated prrs vaccines ( ) . these findings indicate that specific cd + ctl immunity may play an important role in controlling prrsv infection. however, there is limited clear and direct evidence of ctls eliminating prrsv infection, and more basic immune reagents, such as the tetramer of swine major histocompatibility complex (mhc) class i with prrsv peptide epitope, are required to address these important issues ( ) . swine mhc class i has been referred to as swine lymphocyte antigen (sla-i). there are three classical sla-i loci (sla- , sla- , and sla- ) in the swine genome, and all are dominantly expressed ( ) . sla-i molecules can present viral peptide epitopes to swine cd + t cells and induce the ctl response to kill the infected cells ( ) . similar to human mhc (also known as human leukocyte antigen, hla), sla-i molecules are a highly polymorphic gene superfamily whose peptide-binding specificities are significantly influenced by highly variable sites ( ) . thus, far, more than sla-i genes have been cloned (ipd; http://www.ebi.ac.uk/ipd/index.html), and two three-dimensional ( d) structures of peptide-sla-i (p/sla-i) molecules have been determined, revealing the peptide presentation characteristics of sla-i molecules in swine ( , ) . thus, the situation is favorable for the design and development of a novel viral ctl vaccine against swine prrs based on these d structures of sla-i molecules. in an attempt to identify anti-prrsv ctl epitopes in this study, first, predicted peptide epitopes derived from prrsv were synthesized, and a trimolecular complex, the structure of the epitope from prrsv-nsp (tmppgfely, termed nsp -tmp )-bound sla- * (psla- * ), was solved. next, the potential sla- * -restricted peptide epitopes matching four typical genetic prrsv strains were identified. finally, the immunogenicity of the ctl epitope was identified. our results provide a novel strategy, i.e., the use of the mhc-restricted structural mechanism, to identify and validate ctl epitopes that could be used to develop a peptide-based vaccine against swine prrs. peptide epitopes were predicted by the netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/) based on the whole protein sequences of four typical prrsv strains (vr , genbank accession no. ef . ; hb- . , genbank accession no. eu . ; jxwn , genbank accession no. ef . ; and chsx , genbank accession no. kp . ). these potential non-apeptides were predicted using the sla- * allele (genbank accession no. hq ) and purified to > % purity by analytical reverse-phase high-performance liquid chromatography (hplc) (scilight biotechnology) ( table ) . these peptides were stored in lyophilized aliquots at − or − • c after synthesis and were dissolved in dimethyl sulfoxide (dmso) before use. refolding of the sla- * complex to assemble the psla- * complexes with each non-apeptide (table ) , sla- * heavy chain (hc) and swine β m(sβ m) inclusion bodies were refolded (in a : : molar ratio) via the gradual dilution method we described previously ( , ) . the sla- * hc and sβ m inclusion bodies were also refolded without peptides or with non-combined peptides as negative controls. in addition, the sla- * hc and sβ m inclusion bodies were refolded with a positive peptide (amino acid sequence nsdtvgwsw) as a positive control. after h of incubation at • c, the remaining soluble portion of the complex was concentrated and then purified via chromatography in a superdex / column, followed by resource-q anion-exchange chromatography (ge healthcare), as previously described ( ) . crystallization and data collection of psla- * the purified complex ( kda) of psla- * with the nsp -tmp peptide (amino acid sequence tmppgfely, derived from residues - of the prrsv non-structural protein) was dialyzed against crystallization buffer ( mm tris-hcl ph . , mm nacl) and concentrated to mg/ml. the sample was then mixed with reservoir buffer at a : ratio and crystallized via the hanging-drop vapor diffusion technique at and k. index kits (hampton research, riverside, ca) were employed to screen the crystals. with a protein concentration of mg/ml, crystals of psla- * were obtained in - days from index solution no. ( . m bis-tris ph . , . m ammonium acetate, % peg ) at • c. diffraction data were collected at a resolution of . Å (psla- * ) with an in-house x-ray source (rigaku micro-max desktop rotating anode x-ray generator with a cu target operated at kv and ma) and an r-axis iv ++ imaging plate detector at a wavelength of . Å. the crystals were first soaked in reservoir solution containing % glycerol as a cryoprotectant and then flash-cooled in a stream of gaseous nitrogen at − • c ( ). the collected intensities were indexed, integrated, corrected for absorption, scaled, and merged by using the hkl package ( ) . the structures of psla- * with nsp -tmp were solved via molecular replacement using the molrep program with hla-a * (pdb code, q ) as the search model. extensive model building was performed by hand with coot ( ), and restrained refinement was performed with refmac . additional rounds of refinement were conducted by using the phenix.refine program implemented in the phenix package ( ) with isotropic atomic displacement parameter (adp) refinement and bulk solvent modeling. the stereochemical quality of the final model was assessed with the procheck program ( ) . data collection and refinement statistics are listed in table . determination of the circular dichroism spectra and thermal unfolding of psla- * the thermostability of sla- * with six mutant peptides was examined via circular dichroism (cd) spectroscopy. cd spectra were measured at • c in a jasco j- spectropolarimeter equipped with a water-circulating cell holder. far-uv cd spectra ( - nm) were collected at a protein concentration of . mg/ml in mm tris (ph . ) buffer in a cuvette with a length of mm at . -nm spectral resolution. the ellipticity at nm was continuously recorded during heating. thermal denaturation curves were obtained by monitoring the cd value at nm in a cell with an optical path length of mm as the temperature was raised from to • c at a rate of • c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ ) by the standard method: the unfolded fraction (%) is expressed as (θ -θ n )/(θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states. the midpoint transition temperature (tm) was determined by fitting the data to the denaturation curves by using the origin . program (originlab), as described previously ( ) . the tetrameric psla- * complex was constructed according to a previously described method ( ) . briefly, a sequence containing a bira enzymatic biotinylation site was added to the c-terminus of the sla- * hc via pcr. the pcr primers and conditions were as described previously ( ) . then, the entire construct was cloned into the pet- a(+) vector, which was subsequently transfected into escherichia coli strain bl (de ) for protein expression. the inclusion bodies of recombinant sla- * hc containing the bira site and of sβ m were refolded with the nsp -tmp peptide as described above. the psla- * complex was then purified and biotinylated by using the bira enzyme (avidity aurora, co). finally, the complex was purified and tetramerized by mixing psla- * -bsp with pe-labeled streptavidin (biosource international, camarillo, ca) at a molar ratio of : , after which the samples were separated by using kda millipore tubes. sds-page electrophoresis was used to determine the efficiency of tetramerization. a total of nine specific pathogen-free (spf) swine ( kg, - weeks old). beijing center of spf swine breeding and management) expressing the sla- * alleles were divided into three groups: mlv, mlv+nsp -tmp , and a blank control group. for initial immunization, the mlv and mlv+nsp -tmp groups were injected with an attenuated prrsv vaccine according to the manufacturer's instructions (boehringer-ingelheim, ingelvac). after seven days, for the second immunization, the mlv + nsp -tmp group was injected with the nsp -tmp peptide mixed with complete freund's adjuvant (cfa, : emulsification). the mlv group was injected with the mlv peptide mixed with cfa. seven days later, peptide mixed with incomplete freund's adjuvant (ifa, : emulsification) was injected into the mlv+nsp -tmp group. the mlv group was injected with mlv mixed with ifa. the immune dose of the peptide was . mg/kg body weight. the control group was injected with phosphate-buffered saline (pbs), deionized water mixed with cfa ( : emulsification), and deionized water mixed with ifa ( : emulsification) at the same time as the immunization group. equivalent volumes were used in the immunization group and the control group. blood was collected from the anterior vena cava, and peripheral blood mononuclear cells (pbmcs) were isolated by the kit according to the manufacturer's instructions (solarbio). the pbmcs were incubated for min at • c in staining buffer (pbs with . % bsa and . % sodium azide) containing the pe-labeled tetrameric complex and the fitc-labeled anti-cd monoclonal antibody. the cells were then washed once with staining buffer and detected via flow cytometry. more than cell events were acquired for each sample. cells stained with pe-labeled tetramers and a fitc-labeled anti-cd monoclonal antibody were counted as ctl response cells ( ) . the results for fluorescence-activated cell sorting (facs) data are presented as the mean ± standard error of the mean (sem) for the three animals in each group. statistical analysis was performed using graphpad prism (https://www.graphpad.com) for windows. significant differences (p < . ) between means were tested by two-tailed student's t-test. immunization with nsp -tmp one week after immunization with nsp -tmp or deionized water, pbmcs were collected from immunized and control groups. these pbmcs were stimulated with nsp -tmp peptide at a concentration of µg/ml. pha was added at the same concentration to each positive control group, while an equivalent volume of pbs was added to the negative groups. swine ifn-γ in the supernatant was detected via an elisa kit according to the manufacturer's instructions (invitrogen) after the cells had been incubated at • c for h. six sla-i alleles were cloned from landrace pigs. sla- * showed better prrsv peptide-binding ability than the others (table s ) according to in silico prediction (http://www.cbs.dtu.dk/services/netmhcpan). nine prrsv peptides, all of which could be presented by sla- * , were synthesized to test this prediction ( table ) . all nine peptides could form complexes with sla- * and swine β m (psla- * ) by in vitro refolding. the stable psla- * complexes were further used to screen the crystal structures. d structure of psla- * sla- * in complex with nsp -tmp was crystallized in the p space group with a high resolution of . Å ( table ) . one asymmetric unit contains only one sla- * molecule. the psla- * complex displays a canonical p/mhc i structure, including the α , α , and α domains of the hc and the light chain sβ m. nsp -tmp is located in the peptide-binding groove (pbg) formed by the α and α domains ( figure a) . the root mean square differences (rmsds) between sla- * and two other solved p/sla i structures (sla- * , pdb code: qq ; sla- * hs , pdb code: h ) were found to be . and . , respectively, indicating similarities among the overall structures of the p/sla i molecules. the nsp -tmp peptide is fixed by hydrogen bonds with residues in the n-and ctermini of the pbg, and no hydrogen bonds were observed in the middle portion (p -p ) (figure b) . based on the surface model, the p and p residues are located outside the pbg, and their side chains are solvent accessible, especially the p residue, which is at the top position of the nsp -tmp peptide conformation ( figure c ). pocket composition of psla- * bound to nsp -tmp peptide the compositions and polarities of the six pockets of psla- * are shown in figure , and the interactions between the nsp -tmp peptide and these pockets are listed in table . the pockets of psla- * , p/sla- * , and p/sla- * hs are compared in figure . the a pocket of psla- * , composed of leu , tyr , phe , tyr , glu , tyr , leu , ser , and tyr , fixes p -thr via hydrogen bonds and strong van der waals forces (vdws) (figure a ; table ). the residues forming the a pockets of sla i molecules, including ser , are highly conserved (figure ) . in most mhc i molecules of other (figure b ; table ). the residue composition of sla- * is similar to that of sla- * , and only the residue at position (lys/val) is different (figure ) . the c, d, and e pockets usually form a large cavity in the middle portion of the pbg. the amino acid compositions of these three pockets in sla- * are shown in figures c-e. no hydrogen bonds or salt bridges were found in these structures; instead, many vdws were observed between the three pockets and the nsp -tmp peptide ( table ) . the d pocket is critical for the peptide selection of sla- * and sla- * hs because of the charged residue at position ( , ) . the non-polar met causes the d pocket of sla- * to be hydrophobic, in contrast to the charged d pocket of sla- * or sla- * hs (figure ) . the f pocket of psla- * consists of asn , ala , leu , tyr , leu , asp , tyr , ile thr , and lys and shows numerous interactions with p -tyr, reflecting a key anchoring site ( figure f ). p -tyr can form hydrogen bonds and many vdws with the residues of the f pocket ( table ). the f pockets of both psla- * and p/sla- * can accommodate p -tyr, and only two different residues (asn/gly and ala/thr ) were found between the two f pockets (figure ) . the nsp -tmp peptide conformation presented by sla- * is different from that of the peptides presented by sla- * and sla- * hs (figure ) . because of the two consecutive pro residues, the turn between p and p of the nsp -tmp peptide is much sharper than that in the other two peptides (figures a,b) . previous studies on sla- * and sla- * hs showed that the residue at position plays a key role in peptide binding by fixing the p residue with a salt bridge or hydrogen bond (figures c,d) . in contrast, no salt bridge or hydrogen bond forms between the p -pro of nsp -tmp and met of sla- * . to determine the peptide-binding motif of sla- * , the peptide nsp -tmp was mutated by alanine scanning ( ) , and cd spectra were used to test the stability of psla- * complexes with these mutant peptides ( figure ) . the in vitro refolding and cd results showed that the binding stabilities of p -ala, p -ala, and p -ala mutant peptides are significantly lower than that of the wild-type nsp -tmp peptide. although p -pro cannot form a hydrogen bond or salt bridge with the d pocket of sla- * , its ala mutant still impairs the stability of the psla- * complex. according to these results, the p , p , and p residues are the primary anchor residues of the epitope peptides presented by sla- * . the b, d and f pockets accommodate these primary anchor residues and determine the peptide-binding motif of sla- * , similar to sla- * and sla- * hs . the b and f pockets accommodate the p and p anchor residues of the binding peptide, respectively, and their preference for p and p anchor residues is determined by their amino acid composition. figure shows the pocket composition of sla- * . figure shows that the amino acid composition of the b and f pockets of sla- * is very similar to that of sla- * . differential amino acids are found only at one or two individual sites and do not form direct contacts with the side chains of the p or p residues of the binding peptide. because of the similar b and f pockets, the p and p residues of the sla- * -binding peptides should be the same as in the sla- * peptides. the sla- * binding peptide ( ) and the sla- * binding peptide have a large overlap at the p and p residues ( table ). the b pocket of sla- * accommodates multiple uncharged residues, while the f pocket mainly binds phe, tyr and trp. the in vitro refolding results for the peptides supported this reasonable speculation ( table ) . the uncharged d pocket of sla- * might accommodate various uncharged p residues, unlike those of sla- * and sla- * hs . peptides with p -ala cannot provide sufficient affinity, unlike larger amino acids, such as l, m, f, s, n, and p ( table ). in summary, the preliminary peptide-binding motif of sla- * is expected to contain the following combination: x-(s/m/f/w/t/v/i/l)-(l/p/m/f/s/n)-x-x-x-x-x-(f/y/w). the predicted peptide epitopes derived from whole protein sequences of four typical prrsv strains were screened: the first isolated strain, vr ; the low-virulence strain hb- . ; the highly pathogenic strain jxwn ; and the chsx strain, which was responsible for a recent epidemic in china (figure ) . most sla- * -restricted prrsv peptides are located in the non-structural protein and the rna-dependent rna polymerase (rdrp) encoded by orf a and b. although numerous mer sla- * -binding peptides exist in each of these four prrsv strains (∼ peptides), only peptides were found to be completely conserved in all four strains (table s ) . rdrp contains conserved peptides, which is a much greater number than in the other proteins. identification of nsp -tmp as the ctl epitope by using the tetramer technique and the detection of swine ifn-γ the tetrameric psla- * complex was constructed (figures a-c) ( ) . six landrace pigs expressing the sla- * genes were used to check the immunogenicity of nsp -tmp peptide (figure ) . a total of , events were recorded by the flow. the ratio of psla- * tetramer and cd double-positive cells was at a rate of ∼ . - % in the mlv+nsp -tmp -immunized group, which was significantly higher than in the control group (p = . ). the mlvimmunized group was significantly higher than in the control group (p = . ); however, there was no significant difference between the mlv+nsp -tmp -immunized group and the mlv-immunized group (p = . ) (figure b) . additionally, swine ifn-γ expression in the peripheral blood of each pig was detected according to the methods used by kumar and walker ( , ) . secreted ifn-γ was detectable in all of the immunized pigs but was lower than the lowest detectable limit in all control pigs ( figure c) . these data indicated that nsp -tmp , as the ctl epitope, could stimulate specific ctl immunity in swine. ctl epitopes might be a requirement of optimal prrsv immunity for the control and treatment of prrsv infection ( , ) . in our study, a novel approach was used to select prrsv ctl epitopes, i.e., starting from a computer prediction for prrsv peptides, followed by in vitro complex refolding with sla- * , the analysis of the complex crystal structure, the identification of sla- * -restricted potential epitopes from whole genomes of different prrsv strains, and finally, verification of the immunogenicity of sla- * -restricted prrsv epitope. the crystal structure of sla- * is the third to be solved for an sla-i allele. the crystal structure of sla- * exhibits the typical structural characteristics of an mhc i complex. the structure of sla- * is very similar to those of sla- * and sla- * hs , indicating that the overall combination of heavy chains, light chains, and peptides in the swine sla-i complex is highly conserved. however, in terms of peptide binding, the sla- * structure not only reflects the common features of sla-i alleles but also exhibits unique allelic-specific characteristics. similar to the previously resolved sla- * and sla- * hs , the n-terminus of the sla- * pbg is open because the amino acid at position of the a pocket is a small ser (figure a) , but in other species such as humans and mice, the amino acid at this position is a large trp ( , ) . the peptide-binding motif of sla- * , like that of sla- * and sla- * hs , is determined by the three pockets b, d and f together, while the hla-i molecule is mostly determined by the two pockets b and f. these common characteristics indicate that sla-i has its own unique species features in binding peptides. in the b and f pocket composition, sla- * and sla- * are very similar, and only the non-critical amino acids in the individual positions are different (figure ) , resulting in a large overlap of the anchoring residues accommodated in their b and f pockets ( ) . in the structures of sla- * and sla- * hs , the d pocket plays a key role in fixing the bound peptides, with a strong salt bridge between the charged residue and the p residue of the peptides ( , ) . the uncharged met of sla- * cannot form strong charge interactions with p residues similar to those observed for sla- * and sla- * hs (figure ) . nevertheless, the d pocket is still important in determining the peptide binding of sla- * and prefers uncharged residues of a certain size to form sufficient vdws. the three p/sla i structures indicate that regardless of its properties, the d pocket is critical in determining the peptide-binding motif of sla-i, and this phenomenon is expected to be a common feature among different sla-i alleles. sla- * was predicted in silico to present more prrsv peptide epitopes than other sla-i alleles cloned from landrace pigs, and the in vitro refolding results confirmed that most of the predicted prrsv peptides could be bound by sla- * . four typical prrsv strains of the north american genotype were used to screen sla- * -restricted binding peptides. according to the summarized peptide-binding motifs, approximately peptides in each prrsv strain could be presented by sla- * . these peptides are unevenly distributed in different regions, with the nsp / / proteins encoded by orf a, nsp / / encoded by orf b and gp / exhibiting most of the candidate peptide epitopes. approximately one out of three peptides are conserved among the four prrsv strains, and approximately half of these peptides are encoded by orf b of rdrp. ctl-epitope-based vaccines present advantages in terms of safety, specificity, and usability and are successfully used to control many viruses, such as hiv, hpv, and dengue virus ( ) ( ) ( ) ( ) . although studies aimed at developing an anti-prrsv epitope-based vaccine have been performed, no mature product is currently available ( ) ( ) ( ) . our data indicated that rdrp (especially nsp / / ) may be the best target for developing a prrsv vaccine to induce a ctl response to genetically heterologous strains. tetramers of p/mhc i alleles are basic reagents that are used in immunological studies ( , , ) . however, the absence of sla-i tetramers limits effective and convincing research on swine antiviral ctl responses, especially regarding accurate quantitative research. in this study, the crystallized nsp -tmp peptide was used to produce the tetramer for evaluating sla- * -restricted ctl responses. the nsp -tmp epitope could induce cd and tetramer double-positive ctls at a rate of ∼ . - % in mlv+nsp-tmp -immunized pigs, similar to the results obtained for other known efficiently protective viral ctl epitopes found in humans and mice by facs ( ) ( ) ( ) . our results also showed that the mlv used (produced by the vr strain) could induce a ctl response specific to prrs. somewhat disappointingly, we do not have live prrsv with which to challenge these swine groups and evaluate the protection of mlv and nsp-tmp epitope. however, nsp -tmp was identified as an immunogenic epitope that could stimulate the proliferation of specific cd + ctls and the expression of ifn-γ in spf landrace pigs bearing sla- * alleles. immunization enhancement with nsp -tmp produces a specific ctl response similar to that of immunization with mlv, indicating that the peptide vaccine can produce effective immunoprotection and thus that it is feasible to develop an effective prrsv polypeptide epitope vaccine. in conclusion, we solved the crystal structure of sla- * and described its prrsv peptide-binding map according to its preliminary peptide-binding motif determined via biochemical analyses. using the tetramer of sla- * , the immunogenicity of nsp -tmp was identified by facs and the expression of ifn-γ. the results increase our understanding of how to acquire a viral ctl vaccine against swine prrs disease. in addition, this study provides a complete and credible method for identifying sla-i-restricted viral epitopes, demonstrating the feasibility of peptide vaccines in antiviral immunity of swine. based on our experimental results, we encourage and promote the development of a safe peptide vaccine that can effectively activate ctl immune protection, solve the safety problems figure | identification of the functional nsp -tmp ctl epitope. (a) immunization program for spf pigs. nine pigs expressing sla- * were divided into three groups: the mlv group, mlv+peptide group and blank control group. the mlv and mlv+peptide groups were injected with an attenuated prrsv vaccine as the first immunization. seven days later, the mlv+peptide group was injected with the nsp -tmp peptide mixed with cfa, and the mlv group was injected with mlv mixed with cfa as the second immunization. at day , the mlv+peptide group was injected with the nsp -tmp peptide mixed with ifa, and the mlv group was injected with mlv mixed with ifa as the third immunization. pigs in control group were injected with pbs, deionized water mixed with cfa, and deionized water mixed with ifa. (b) nsp -tmp -specific ctls stained with pe-labeled sla- * tetramer and fitc-labeled anti-cd monoclonal antibody were detected via flow cytometry. (c) the secreted ifn-γ contents of the control group and immunized group were measured via elisa kit. the secreted ifn-γ content of the control group was less than the lowest detectable limit ( pg/ml). caused by conventional attenuated vaccines, and provide new ideas for controlling not only prrs but also the extent of african swine fever. the coordinates and structural characteristics of psla- * have been deposited in the protein data bank under accession number ylx; the sequence of sla- * is available at the national center for biotechnology information (ncbi) database under accession number hq . the animal trials in this study were performed according to the chinese regulations for laboratory animals-the guidelines for the care of laboratory animals (ministry of science and technology of people's republic of china) and laboratory animal requirements for environment and housing facilities (gb - , national laboratory animal standardization technical committee). the license number associated with this research protocol is cau - , which was approved by the laboratory animal ethics committee of china agricultural university. the protocol adhered to the recommendations in the institute for laboratory animal research's guide for the care and use of laboratory animals. full protection of swine against foot-and-mouth disease by a bivalent b-cell epitope dendrimer peptide assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states the structural biology of prrsv the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins analysis of orf and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes and retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wildtype prrsv strains genetic characterization of porcine reproductive and respiratory syndrome virus isolates in south china from to emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system innate and adaptive immunity against porcine reproductive and respiratory syndrome virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (prrsv) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (pbmc) from pigs vaccinated and exposed to natural infection porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects polymorphism and peptide-binding specificities of porcine major histocompatibility complex (mhc) class i molecules molecular genetics of the swine major histocompatibility complex, the sla complex structural and biochemical analyses of swine major histocompatibility complex class i complexes and prediction of the epitope map of important influenza a virus strains crystal structure of swine major histocompatibility complex class i sla- and identification of pandemic swine-origin influenza a h n virus cytotoxic t lymphocyte epitope peptides complex assembly, crystallization and preliminary x-ray crystallographic studies of rhesus macaque mhc mamu-a * complexed with an immunodominant siv-gag nonapeptide complex assembly, crystallization and preliminary x-ray crystallographic studies of mhc h- kd complexed with an hbv-core nonapeptide macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography refinement and reliability of macromolecular models based on x-ray diffraction data coot: model-building tools for molecular graphics the phenix software for automated determination of macromolecular structures main-chain bond lengths and bond angles in protein structures a role for the p anchor residue in the thermal stability of mhc class ii molecule i-ab screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes phenotypic analysis of antigen-specific t lymphocytes hla-a * t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins major histocompatibility complex class i (fla-e * ) molecular structure in domestic cats demonstrates species-specific characteristics in presenting viral antigen peptides generation of mhc-peptide tetramers: a new opportunity for dissecting t-cell immune responses cell-mediated immunity to predict cytomegalovirus disease in highrisk solid organ transplant recipients ex vivo monitoring of human cytomegalovirus-specific cd + t-cell responses using quantiferon-cmv identification of cytotoxic t lymphocyte epitopes on swine viruses: multi-epitope design for universal t cell vaccine identification of cd + cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice synthetic peptides containing b-and t-cell epitope of dengue virus- e domain iii provoked b-and t-cell responses a phase i trial of a human papillomavirus (hpv) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are hpv positive safety and immunogenicity of a ctl multiepitope peptide vaccine for hiv with or without gm-csf in a phase i trial location and characterization of the antigenic portion of the fmdv immunizing protein assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity mhc-peptide tetramers to visualize antigen-specific t cells the numbers game for virus-specific cd + t cells identification of an hla-a * -restricted t-cell epitope on the mpt protein, a major secreted protein derived from mycobacterium tuberculosis, by mpt overlapping peptide screening detection, phenotyping, and quantification of antigen-specific t cells using a peptide-mhc dodecamer identification and structural definition of h -specific ctl epitopes restricted by hla-a * derived from the h n subtype of influenza a viruses we thank the shanghai synchrotron radiation facility (ssrf), shanghai, people's republic of china, for the crystal diffraction data. we thank dr. lei zhou for his help in collecting information on prrsv strains. we thank prof. george f. gao of the chinese academy of sciences. cx: design of the study. xp and nz: data collection. xp, yj, and ql: analysis and interpretation of data. cx, nz, and xw: drafting the article. cx, xp, rl, lz, and lm: critical revision of the article. xp, nz, xw, yj, rc, ql, rl, lz, lm, and cx: final approval of the version to be published. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -qkjjxr p authors: li, liwei; wei, zuzhang; zhou, yanjun; gao, fei; jiang, yifeng; yu, lingxue; zheng, hao; tong, wu; yang, shen; zheng, haihong; shan, tongling; liu, fei; xia, tianqi; tong, guangzhi title: host mir- a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type i interferons date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: qkjjxr p micrornas (mirnas) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. to identify host mirnas important to controlling porcine reproductive and respiratory syndrome virus (prrsv) infection, we screened mirnas that were previously implicated in innate immunity or antiviral functions. over-expression of the mir- family strongly inhibited prrsv replication in vitro, as shown by virus titer assays, western blotting, and qrt-pcr assays. mir- a inhibited the replication of both type and type prrsv strains. mutating the seed region of mir- restored viral titers. luciferase reporters showed that mir- a does not target the prrsv genome directly but instead affects the expression of type i interferon and the ifn-stimulated genes mx and isg during prrsv infection. these results demonstrate the important role of mir- a in modulating prrsv infection and also support the possibility of using host mir- a to achieve rnai-mediated antiviral therapeutic strategies. micrornas (mirnas) are small (∼ nucleotides) non-coding rnas that bind to complementary sequences in the untranslated regions of target mrnas and contribute to gene regulation by reducing mrna translation or destabilizing transcripts (grassmann and jeang, ; skalsky and cullen, ) . the commonly accepted mechanism of mirna regulation is that the seed region ( ∼ nucleotides at the end) of an mirna is complementary to the or untranslated region ( -or -utr) of an mrna, leading to mrna degradation or translational inhibition (bartel, ; gottwein and cullen, ) . recent work has shown the importance of mirnas in regulating host-pathogen interactions and innate immunity (lodish et al., ; scaria et al., ; tenoever, ) . host mirnas can affect viral replication by binding directly to viral rna (lecellier et al., ) or by indirectly modulating host factors to provide a less permissive environment for virus replication (triboulet et al., ) . as mirnas are small molecules without antigenic properties, they are considered to have potential efficacy in antiviral therapeutic applications. for example, human mir- is an essential component of the biology of hepatitis c virus replication (jopling, ; jopling et al., ) and therapeutic blocking of mir- suppresses hepatitis c viremia in non-human primates (lanford et al., ) . porcine reproductive and respiratory syndrome virus (prrsv), a member of the arterivirus family, is the causative agent of porcine reproductive and respiratory syndrome [prrs; (chand et al., ) ]. based on their genetic and antigenic differences, prrsv strains are classified into two distinct genotypes, north american (type ) and european (type ), represented by the vr- (benfield et al., ) and lelystad virus (lv) (wensvoort et al., ) , respectively. these two genotypes on two different continents share only approximately % nucleotide sequence identity (forsberg, ; hanada et al., ) . many strategies for controlling prrsv transmission have been proposed but have generally shown little success, which has stimulated the search for new ways to control prrsv transmission. prrsv can escape from innate immunity and cause persistent infections (miller et al., ) . in mammalian cells, viral infection is a potent trigger of the interferon (ifn) response (sadler and williams, ; sen, ) . type i interferons can initiate the activation of jak/stat signaling to induce the expression of hundreds of ifn-stimulated genes (isgs), which play an important role in antiviral activities (albina et al., ; katze et al., ; overend et al., ) . however, in contrast to porcine respiratory coronavirus, prrsv is a poor ifn-inducer (buddaert et al., ) . many mirnas regulate ifn production pedersen et al., ) , maintain mrna stability (li et al., ) , and regulate signals downstream of ifn to modulate antiviral immunity (wang et al., ; yoshikawa et al., ) . while most mirnas characterized to date decrease the production of ifns (alam and o'neill, ) , a few mirnas that upregulate type i ifns have been reported. recent research has revealed that mir- may play a positive modulatory role in ifn production during prrsv infection (zhang et al., ) . given the breadth of mirna-mediated regulation of mammalian immunity (grassmann and jeang, ) , the role of host mirnas in prrsv infection is of significant interest. here, we found that mir- a is an anti-prrsv host factor. over-expression of mir- a inhibited infection by both of the major prrsv genotypes in a dosedependent manner. we found that mir- a does not target the prrsv genome directly, but rather affects the expression of type i interferon and the ifn-stimulated genes mx and isg during prrsv infection. our study reveals an example of a mirna that affects viral propagation and highlights a host factor that may be important for future control measures against prrs. marc- cells were grown in mem (invitrogen) with % fetal bovine serum (fbs, gibco-brl, gaithersburg, md, usa) and were maintained with % fbs at • c in a humidified % co atmosphere as described previously (yuan and wei, ) . baby hamster kidney (bhk- , atcc ccl ) cells were cultured in emem (atcc, manassas, va, usa) supplemented with % fbs. porcine alveolar macrophages (pams) were harvested from the lungs of -week-old prrsv-negative piglets as described previously (wensvoort et al., ) and maintained at • c in rpmi (gibco) supplemented with % fetal bovine serum (fbs). vaprrs (genbank accession no. gq ) (yuan and wei, ) and vshe (genbank accession no. gq ) (tian et al., ) were rescued from paprrs and pshe, respectively. vjx (at passage ) was isolated from the serum of a dying piglet displaying the clinical sings of porcine high fever disease (phfd) in . vjxm (genbank accession no. gq ) was obtained through serial passages of the highly pathogenic prrsv vjx strain (eu ) in marc- cells (wang et al., ) . the infectious cdna clone pjx was derived from vjx (lv et al., ) . high-titer virus stocks were obtained by infecting marc- cells at low multiplicities of infection (mois). infected cell supernatants were harvested after an % cytopathic effect (cpe) appeared, then the viruses were stored at − • c as stocks for further use. virus titer was determined by standard tcid assay using marc- cells. mirna mimics (table ) , which are double-stranded -omethyl-modified rna oligonucleotides with sequence complementarity to mature mirnas were synthesized by genepharma (shanghai, china). the sense sequences of the mir- mimics were: mir- a- -uucaaguaauccaggauaggcu- ; mir- b- -uucaaguaauucaggauaggu- ; corresponding non-seed-mutated mir- mimics ( - a, - u, and - a u) or seed-mutated mir- mimics ( a-m, b-m, c-m) are listed in table (underlined letters are mutated bases). the negative-control (nc) mimic sequence was -uucuccgaacgugucacgutt- . . . transfection of mirna mimic and viral multi-step growth kinetics mirna or nc mimics were transfected into pams or marc- cells at a concentration of nm (except for dosedependence experiments) using x-tremegene sirna transfection reagent (roche). twenty-four hours after transfection, cells were infected with prrsv. for analysis of prrsv growth, supernatants ( . ml/well) from cell cultures were collected at different time points post-infection and frozen at − • c. for virus quantification at each time point, a viral titer was measured in marc- cells by standard tcid assay using the method of reed and muench (reed and muench, ) . indirect immunofluorescence assays (ifa) were performed as described previously (tian et al., ) for the detection of nucleocapsid (n) protein in prrsv vjx infected marc- cells or pams pre-transfected with mir- family or mutant mimics. cells were fixed with cold methanol followed by blocking with % bovine serum albumin (bsa) and then incubated for h with a monoclonal antibody (sr a, rural technologies) that specifically recognizes type prrsv n proteins. after washing with phosphate-buffered saline (pbs), the cells were incubated for h with alexa fluor -labeled goat anti-mouse secondary antibody (invitrogen). cell nuclei were counterstained with g/ml of , -diamidino- phenylindole (dapi) for min. after a final pbs wash step, cells were visually analyzed using an olympus inverted fluorescence microscope. marc- cells were transfected with mir- a or nc mimics prior to prrsv infection. cells were washed twice with pbs at h post-infection and lysed with lysis buffer in the presence of mm n-ethylmaleimide (nem). after incubation for min on ice, cell lysates were centrifuged at , × g for min at • c and the supernatants were collected. protein samples were prepared in reducing buffer ( mm tris, ph . , % glycerol, % sds, . % [wt./vol.] bromophenol blue, mm dtt). samples then were heated at • c for min, resolved on % sds polyacrylamide gels, and transferred to hybond-pmembranes (amersham biosciences). membranes were blocked with % nonfat dry milk in tbst ( mm nacl, mm tris, ph . , . % tween ) for h at room temperature. membranes were incubated overnight at • c with primary antibody ( ag ) that specifically recognizes both type and type prrsv n proteins (kindly provided by ingenasa co., madrid, spain). after washing with tbst, blots were incubated with horseradish peroxidase (hrp)conjugated goat anti-mouse secondary antibody (santa cruz) for h at room temperature, washed again with tbst, and developed using supersignal west pico or femto chemiluminescent substrate according to the manufacturer's instructions (thermo fisher scientific). total rna and mirna were extracted with trizol (invitrogen) following the manufacturer's instructions. primescript tm st strand cdna synthesis kit (takara) was used for reverse transcription. quantitative rt-pcr (qpcr) analysis was performed using a step-one plus real-time pcr system (applied biosystems) and a sybr premix ex taq tm (takara). for detection of endogenous mirnas, a commercial mircute mirna first-strand cdna synthesis was purchased from tiangen biotech (beijing, china) and used for polyadenylation and reverse transcription. a commercial mircute mirna qpcr detection kit was purchased from tiangen biotech (beijing, china) for measuring mirna abundance. marc- cells infected with prrsv at a moi of . were collected at the indicated time points and total rna was extracted using trizol reagent (invitrogen). one g of this total rna was then used for reverse transcription with an rt-primer. the abundance of the mirna of interest in the resulting cdna was determined by qpcr using a universal reverse primer and a mirna-specific forward primer. the pcr procedure comprised pre-denaturation at • c for min, and cycles of • c for s, • c for s. the ubiquitously expressed u small nuclear rna (tiangen) was used for normalization purpose. all primers used for mirna qpcr were included in the commercial kit. the levels of orf rna, ifn-␣/␤, mx , isg mrna were quantified using a sybr premix ex taq tm (takara). relative expression levels were analyzed using the ct method (bookout et al., ) , and glyceraldehyde- -phosphate dehydrogenase (gapdh) mrna was used as an endogenous control. universal type i interferon was purchased from pbl interferonsource. all pcr experiments were performed in triplicate. other primers are listed in table . the pgl -control luciferase reporter vector (promega) was used as the cloning vector for luciferase assays to analyze potential mir- a target regions in the prrsv genome. twenty cdna fragments encompassing the prrsv genome were amplified by pcr from prrsv pjx and subcloned into the pgl -control vector downstream of the luciferase orf. the primers used are listed table sequence of oligonucleotide primers used in this study. sequence table . all cdna constructs were verified by dna sequencing. plasmids and mirna mimics were transfected into cells using lipofectamine (invitrogen) according to the manufacturer s protocol. for luciferase reporter assays, subconfluent bhk- cells cultured in -well plates were co-transfected with ng/well of the indicated reporter plasmid and ng/well of prl-cmv (as an internal control to normalize transfection efficiency, promega) along with the indicated amount of mir- a mimic. cells were lysed h later for determination of firefly luciferase activities using the luciferase assay system (promega). data are presented as the relative luciferase activities in mir- a mimic-transfected cells relative to nc mimic-transfected controls and are representative of three independent experiments. to screen potential mirnas for their ability to inhibit prrsv replication, mimics of mirnas that are well-conserved among different species and have been previously implicated in innate immunity and/or antiviral functions (banerjee et al., ; foley and o'neill, ; huang et al., ; yoo and liu, ; pauley and chan, ; schulte et al., ; selvamani et al., ) were synthesized (table ) . marc- cells were transfected with individual mirna mimics ( nm) and then infected with prrsv (vaprrs) at an moi of . . supernatants from infected cells were collected at and h post-infection to determine viral titers. among the mirnas tested, over-expression of the mir- a mimic strongly reduced prrsv titers (fig. a) . transfection of mir- a/ b inhibitors demonstrated the opposite effects (fig. b) , indicating that mir- has antiviral activity against prrsv replication and that mir- a is a more efficient suppressor than mir- b. all the other mirna mimics tested had no demonstrable impact on prrsv titers in marc- cells (fig. a) . furthermore, immunofluorescence assays using a fitc-conjugated monoclonal antibody against the prrsv n protein were consistent with viral titer data (fig. c) . to rule out the possibility that this antiviral effect of mir- a was specific to an individual prrsv strain, we analyzed the viral growth curves of two type prrsv strains (vjx , vjxm ) and a type prrsv strain (vshe) in marc- cells transfected with nc or mir- a mimics. over-expression of the mir- a mimic, but not the nc mimic, reduced prrsv replication in multiple prrsv strains of differing genotypes ( fig. a) . to corroborate our findings with mir- a further, marc- cells were transfected with increasing concentrations of mir- a mimic ( , , , nm) and then infected with vaprrs. both prrsv growth and the amount of orf mrna level were inhibited as a function of the dose of mir- a mimic ( fig. b and c) . consistent with this, transfecting the mir- a mimic also reduced the accumulation of the prrsv nucleocapsid (n) protein in a dose-dependent manner (fig. d) . to exclude the possibility that reduced prrsv replication was due to potential toxicity of the mir- a mimic, marc- cells were transfected with the mir- a mimic at different doses ( nm, nm, and nm). no appreciable effect of the mir- a mimic (at up to nm) on cellular viability and morphology was observed (data not shown). collectively, these data show that mir- a reduces prrsv replication in multiple prrsv genotypes in a dose-dependent manner. we next investigated the effect of other mir- mirna and mutants on prrsv replication. because mir- a is highly conserved between monkeys and pigs, we conducted the subsequent investigations in pams, which are the target cells of prrsv infection in vivo. as previously reported, mirna-mrna interactions may require seed-matched sites at nucleotides - (bartel, ). thus, we mutated mir- a mimic at non-seed nucleotides , , or and and mir- at seed nucleotides - ( a-m and bm). both mir- a and mir- b had anti-prrsv activity in pams. pams transfected with mir- a or mir- b mimics yielded significantly lower prrsv titers and orf gene expression compared with cells transfected with the nc mimic ( fig. a and b) . three mir- a mutants with non-seed mutations retained their ability to inhibit prrsv progeny production and gene expression ( fig. a and b). by contrast, seed mutations at nts - abrogated the ability of mir- family members to repress prrsv replication and gene expression (fig. a and b) , showing that the seed region was essential for inhibiting prrsv replication. to investigate further inhibition effect of mir- family and mutants on prrsv infection, we used an immunofluorescence assay to detect the prrsv n protein in pams. n protein expression in pams was suppressed by both mir- a and mir- b (fig. c , top) and by mir- non-seed mutants (fig. c, middle) , but was not affected by mir- seed mutants (fig. c, bottom) . we then analyzed the growth dynamics of hp-prrsv isolate vjx in pams transfected with mir- family or nc mimics. viral growth was suppressed about -fold in pams transfected with mir- a and about -fold in pams transfected with mir- b at h postinfection (fig. d) . notably, mir- a was more efficient suppressing viral growth than mir- b. these results indicated that mir- family members, especially mir- a, can inhibit vjx replication in pams. we then analyzed the kinetics of mir- expression in prrsv infected pam cells. the relative expression of mir- was upregulated as a function of prrsv infection time (fig. e) . targeting a specific viral sequence represents an efficient strategy by which mirnas can inhibit viral replication (jopling, ; lecellier et al., ) . in recent studies, mir- and mir- were confirmed to reduce viral gene expression and viral growth due to direct targeting of prrsv genomic rna (guo et al., ; zhang et al., ) . we determined whether mir- a specifically targets the prrsv genome to exert its antiviral effect by constructing a range of firefly luciferase reporter pgl -control based plasmids, which contained the cdna fragments representing the utr, nsp -nsp , orf -orf , and the utr of the prrsv genome statistical significance was analyzed using t-tests; *, p < . ; **, p < . ; ***, p < . . c. pams were transfected with the indicated mirna mimics and then infected with prrsv vjx (moi = . ) for h. cells were fixed and immunostained with the mouse monoclonal sr a antibody against the viral n protein and fitc-conjugated goat anti mouse igg. cellular nuclei were counterstained with dapi ( mg/ml). d. prrsv growth in pams transfected with mir- family mimics. pams were transfected with mir- family or nc mimics for h and then infected with prrsv vjx at an moi of . . culture supernatants were collected at the indicated times and titrated. e. time-course of mir- a/ b expression after prrsv infection. pam cells infected with vjx at a moi of . were collected at the indicated times and qrt-pcr analysis was performed to detect mir- a/ b expression. relative mir- a/b expression refers to the change in mir- a/b expression levels in prrsv-infected pams relative to mock pams. downstream of the firefly luciferase gene (fig. a ). if the prrsv cdna insert contains a mir- a target sequence, luciferase reporter expression is expected to be subjected to mir- a-regulation. mir- a or nc mimics were co-transfected with the individual reporter vectors into bhk- cells, along with an internal control vector prl-cmv. relative luciferase activities were quantified h post-transfection. the relative luciferase activities for different vectors containing various prrsv cdna fragments were not significantly different between cells transfected with mir- a mimic as compared with cells transfected with the nc mimic. thus, mir- a does not appear to target directly the prrsv genome. we found that over-expression of mir- a increased ifn-␣/␤ expression during vjx infection (moi = . ) at h in pams as compared with over-expression of nc (fig. a ). the ifn-stimulated genes mx and isg were also significantly upregulated (fig. b) . transfecting the mir- a mimic into pams in the absence of prrsv infection also enhanced type i ifn expression ( fig. a and b) . ifn-␣ and ifn-␤ were induced about . -fold in un-infected pams, and about . -and . -fold in prrsv infected pams, respectively. isg and mx were increased about . -and . -fold in un-infected pams, and about . -and . -fold in prrsv infected pams, respectively. in marc- cells, over-expression of mir- a up-regulated ifn-␣ and isg more strongly (fig. c ). ifn-␣ and isg were induced about . -and . -fold in un-infected marc- cells, and about . -and . -fold in prrsv infected marc- cells, respectively. transfection of mir- a inhibitors did not increase the expression of ifn-␣ or isg (fig. c) , confirming that the induction of the innate immune response is specifically mediated by mir- a. there is a growing body of evidence that cellular mirnas are important regulators of innate and adaptive immune responses and the intricate networks of host-pathogen interactions. herein, we identified mir- a as an inhibitor of prrsv replication that does not directly target the prrsv genome ( figs. and ) . overexpressed mir- a reduced prrsv replication and viral gene expression (fig. ) , in not only marc- cells, but also in pams (fig. ) , the main target cell for prrsv replication in vivo, confirming the biological relevance of this finding. mir- a belongs to a broadly conserved mirna family with perfectly identical sequences among vertebrates (griffiths-jones et al., ) . previous studies of mir- a have shown that this mirna is an important regulator of cell proliferation and differentiation that targets the smad transcription factor (ezh ), a suppressor of skeletal muscle cell differentiation (lu et al., ; luzi et al., ; sander et al., ; zhang et al., ) . by infecting pams with prrsv strain vr- , liu et al. generated small rna expression profiles at , and h post-infection to identify alterations in mirna expression associated with prrsv (yoo and liu, ) . overall, cellular mirnas were differentially expressed during at least one time point in prrsv-infected pams. however, in this study, mir- a was not mentioned (yoo and liu, ) . contrary to the previous study, we found that the expression of mir- a was up-regulated about -fold at h post-infection (fig. e) . one mechanism by which host mirnas regulate viral replication is the direct targeting of viral sequences (jopling, ; lecellier et al., ) . however, prrsv is a fast-evolving rna virus (prieto et al., ) and the relatively high mutation rate may limit the application of this kind of rnai-mediated antiviral therapeutic. cellular mirnas can also indirectly modulate cellular pathways that perturb the viral life cycle. in particular, the activation or enhancement of innate antiviral immune pathways has been suggested to be responsible for the antiviral effect of certain mirnas (lecellier et al., ; pedersen et al., ) . in the current study, the reduction of prrsv replication by mir- a did not appear to involve direct targeting of the prrsv genomic rna (fig. ) . moreover, this reduction occurred in both type and type prrsv strains ( fig. a) although these two genotypes share only approximately % nucleotide sequence identity. these data led us to hypothesize that mir- a might act on a cellular factor to reduce prrsv replication. the results presented here support a link between prrsv replication and the altered expression of mir- a in targeting host innate immune responses (fig. ) . type i interferons (ifns) are potent antiviral cytokines whose expression is triggered through recognition of viral components by pattern recognition receptors via a cascade of signaling molecules . pams are the main target cells for prrsv infection, and many gene expression studies have explored the immune response of pams to prrsv. such studies have shown that the expression levels of mx , usp, ifn-␤, il- , and tnf-␣ are affected by prrsv infection (albina et al., ; luo et al., ; van reeth et al., ) . overall, these analyses suggest that prrsv subverts host defenses by inhibiting the expression of pro-inflammatory cytokines (van reeth et al., ) and stimulating weak production of ifn-␣ . our results showed that over-expression of mir- a was capable of inducing expression of ifn-␣/␤ and the ifn-stimulated genes isg and mx , which might result in activation of the ifn response and further lead to the inhibition of virus infection. the restoration of innate immune responses to produce type i ifns in pams seems to be mirna specific, because another mirna (mir- b) had no such effect (data not shown). thus, it is possible that mir- a-induced type i ifn expression can overcome prrsv interference, contributing to viral clearance. this mechanism provides a higher genetic barrier to the emergence of viral escape mutants, so the identification and characterization of mir- a as an inhibitor of prrsv replication may open new ways to control fig. . mir- a increases type i ifn expression during prrsv infection. qrt-pcr analysis of (a. type i ifn ␣/␤ and b. mx /isg ) expression in pams transfected with nc or mir- a mimics or left untreated (mock) for h, and then infected with vjx for h at an moi of . , or left untreated. data were normalized to gapdh expression and are the mean ± standard deviation of three independent experiments. c. qrt-pcr analysis of ifn-␣ and isg expression in marc- cells transfected with nc, mir- a mimics or inhibitors, and then infected with vjx for h at an moi of . . data were normalized to ␤-actin expression. statistical significance was analyzed using t-tests; *, p < . ; **, p < . ; ***, p < . . future prrs outbreaks, for which effective control measures remain scant. our results showed that mir- a also can mediate the activation of ifns in the absence of prrsv infection (fig. ). the possible causes may relate to recent studies about a new function of mirnas, which is independent of their conventional role in post-transcriptional gene regulation fabbri et al., ; lehmann et al., ) . mir- , mir- a, and let- b have dual functions; on one hand, they bind to argonaute proteins and guide the silencing of target genes, and on the other hand, they act independently of argonaute proteins by interacting directly with tlrs. although there is no current evidence, mir- a may also serve as ligands for tlrs and activate ifns. future studies will be necessary to unravel the diverse functions of mir- a. overall, we demonstrated that over-expression of mir- a inhibits prrsv replication. although clearly defining the target and physiological role of mir- a remains an unfinished task, our study provided evidence that over-expression of mir- a enhances ifn-␣/␤ expression during prrsv infection, suggesting that mir- a could be used as a potential target for antiviral development. micrornas and the resolution phase of inflammation in macrophages immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) mir- a- p regulates differential activation of macrophages and inflammation micrornas: target recognition and regulatory functions characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) highthroughput real-time quantitative reverse transcription pcr vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) pathogenesis of porcine reproductive and respiratory syndrome virus micrornas are ligands of toll-like receptors micrornas bind to toll-like receptors to induce prometastatic inflammatory response mir- : a toll-like receptor-regulated mirna dysregulated in obesity and type ii diabetes divergence time of porcine reproductive and respiratory syndrome virus subtypes viral and cellular micrornas as determinants of viral pathogenesis and immunity the roles of micrornas in mammalian virus infection mir-base: microrna sequences, targets and gene nomenclature increasing expression of microrna inhibits porcine reproductive and respiratory syndrome virus replication and has implications for controlling virus infection the origin and evolution of porcine reproductive and respiratory syndrome viruses pseudorabies viral replication is inhibited by a novel target of mir- targeting microrna- to treat hepatitis c virus infection modulation of hepatitis c virus rna abundance by a liver-specific microrna viruses and interferon: a fight for supremacy therapeutic silencing of microrna- in primates with chronic hepatitis c virus infection a cellular microrna mediates antiviral defense in human cells an unconventional role for mirna: let- activates toll-like receptor and causes neurodegeneration microrna- a inhibits hcv replication by restoring the innate immune response microrna- l inhibits antiviral innate immune response by targeting interferon-alpha micromanagement of the immune system by micrornas mir- a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of ezh porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the mir- a targeting of the smad transcription factor an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc- cells recombinant swine beta interferon protects swine alveolar macrophages and marc- cells from infection with porcine reproductive and respiratory syndrome virus micrornas and their emerging roles in immunology interferon modulation of cellular micrornas as an antiviral mechanism influence of time on the genetic heterogeneity of spanish porcine reproductive and respiratory syndrome virus isolates a simple method of estimating fifty percent endpoints interferon-inducible antiviral effectors myc stimulates ezh expression by repression of its negative regulator mir- a host-virus genome interactions: macro roles for micrornas differential activation and functional specialization of mir- and mir- in innate immune sensing chikungunya virus exploits mir- a to regulate nf-kappab pathway in human synovial fibroblasts viruses and interferons viruses, micrornas, and host interactions interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus rna viruses and the host microrna machinery chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype suppression of microrna-silencing pathway by hiv- during virus replication differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity inducible microrna- feedback promotes type i ifn signaling in antiviral innate immunity by targeting suppressor of cytokine signaling development of a differentiable virus via a spontaneous deletion in the nsp region associated with cell adaptation of porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus characterization of the micrornaome in porcine reproductive and respiratory syndrome virus infected macrophages silencing of microrna- enhances interferon-alpha signaling in the liver through regulating socs promoter methylation construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins pathologically decreased mir- a antagonizes apoptosis and facilitates carcinogenesis by targeting mtdh and ezh in breast cancer microrna- inhibits prrsv replication by directly targeting prrsv rna and possibly by upregulating type i interferons the study was supported by the grants from the national basic research program ( plan) (no. cb ), the national natural science foundation of china (no. , no. , no. ), and the natural science foundation of shanghai (no. jc ). key: cord- -qu o iu authors: vlasova, anastasia n.; butler, john e. title: editorial: porcine anti-viral immunity date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qu o iu nan the swine industry is an important part of agriculture in many countries, generating over $ billion annually in the us alone. viral diseases in pigs pose a serious risk for swine health and significantly impact the economy of the global swine industry. pigs also serve as zoonotic reservoirs for viruses transmittable to humans and other species, including influenza a virus, nipah virus, and hepatitis e virus ( ) . these problems are made worse by globalization and industrial livestock rearing ( , ) . unlike bacterial pathogens, antibiotics do not protect against viral infections and cannot be used to control viral outbreaks or epidemics. since viruses can alter their genome > times faster than their mammalian hosts ( ), the latter would have succumbed to microbial infection were it not for their adaptive immune system that uses somatic gene rearrangement and mutation to counter the rapid diversification of microbes. immediately following viral infection, neonatal survival depends on innate immunity and passive protection by lactogenic immune factors such as pathogen-specific antibodies, until an adaptive immune response can develop. thus, intervention against these moving targets, depends on availability of effective maternal and neonatal vaccines and strict biosecurity measures. wide-spread porcine reproductive and respiratory syndrome virus (prrsv) and swine influenza virus (siv) represent major health challenges in the large us swine production systems and possibly worldwide. in the us alone, economic annual losses due to prrsv are estimated to be million u.s. dollar (usd) ( ) . while the exact costs associated with siv and other endemic viruses, including rotavirus, are hard to evaluate, they are economically important due to their ubiquitous nature, which can reduce growth and increased morbidity ( ) . emerging and re-emerging coronaviruses in pigs are quite often associated with diarrheal disease and high morbidity and mortality ( ) . the emergence of seneca valley virus (svv) and increased incidence of svv-associated vesicular disease alarmed the swine industry in several countries, including the us, brazil, china, thailand, and colombia ( ) . finally, a deadly swine disease, caused by african swine fever virus (asfv), that has been plaguing africa for decades, has now spread to south eastern europe and asia, and has severely impacted the world's largest swine industries in china ( ) . in this special volume of frontiers in immunology, comparative immunology section, lager and buckley provide an overview of the importance of swine in the world food supply and a review of the major viral infection that threaten this species (lager and buckley). in introducing the topic of anti-viral immunity, we emphasize the genetic diversity of viruses, the virus life cycle and the pathology that viral infection can cause. a total of articles are contained in this volume to which researches have contributed. despite the advantage of internal offspring development and the provision for passive immune protection, fetuses and neonates of higher vertebrates still remain vulnerable to environmental pathogens. piglets in utero are protected from eukaryotic parasites and bacteria, but are vulnerable to viruses (including parvovirus, prrsv, and porcine circovirus ) since they are able to cross the placental barrier ( ) ( ) ( ) . these cause pathological injury and often fetal abortion. in a number of mammals, viruses infect the thymus and can also cause hypergammaglobulinemia. butler et al. discuss the implication of thymic atrophy and apoptosis of thymocytes and hypergammaglobulinemia with regard to the persistence of prrsv. however, apoptosis is also a host mechanisms used to eliminating virus-infected cells ( ) , but can also promote spread of the virus ( ) . fernandes et al. demonstrated that svv developed a c protease-dependent mechanism for late apoptosis that facilitates virus release from infected cells. thus, the role and importance of virus-induced apoptosis awaits further research. since the adaptive immune system of fetal piglets is underdeveloped, the innate immune system is very important. schäfer et al., emphasize its importance with the specific focus on porcine invariant natural killer t cells (inkt) and their role in the pathogenesis of asf and si. they discuss inkt agedependence, levels and distribution in relationship to various porcine viral infections. one of the first host responses to viral infection is the production of interferons, which are needed to drive other elements of innate immunity and adaptive immunity. likai et al. show how a porcine deltacoronavirus escapes from the immune system by suppressing ifn-α production, while shi et al. describe a novel immune evasive mechanism that depends on prrsv non-structural protein a which antagonizes tbk -irf -ifn signaling. while the initial antibody response depends on broadly specific natural igm antibodies, effective anti-viral immunity depends on an adaptive immune response that delivers igg and iga antibodies. since adaptive immune responses depend on stimulation through the innate immune system, viruses that impair or interfere with the innate immune response, also impair adaptive immunity. nedumpun et al. discuss how prrsv-induced il- ra downregulates innate immune responses, t lymphocyte differentiation and proliferation. piglets, unlike humans, but like the offspring of other artiodactyls and perissodactyls (horses), are especially dependent on lactogenic immunity since there is no transplacental transfer of passive antibodies in this group of mammals. thus, providing ways to deliver anti-viral antibodies via colostrum/ milk is important. this topic is the focus of studies by langel et al.. antibodies can intercept and neutralize a virus before it infects additional cells and can prevent further infection by blocking the viral receptor; these are called virus neutralizing (vn) antibodies. naturally, a critical step in viral vaccine design is the identification of viral epitopes targeted by effective vn antibodies. this become increasingly important in the case of prrsv in which effective vn antibodies are slow to appear and current vaccines are of questionable efficacy. this is also an ongoing challenge in the case of asfv which is rapidly spreading and for which no vaccine exists. goldeck et al. describe a novel b cell cloning procedure to identify these epitopes on several strains of prrsv. while vn antibodies can block or delay infection, the ultimate elimination of a virus is to kill the cells in which the virus replicates which is the job of cytotoxic t cells. portions of the virus are displayed on infected cells. these are called a t cell epitope, i.e., a molecular structure that binds to the t cell receptor (tcr). in this volume, pan et al. describe their efforts to identify such epitopes for their potential use in stimulating expansion of the cytotoxic t cells that recognize them. viral vaccines take various forms, the simplest being the use of killed virus. a more tedious procedure is to use only parts of the virus as the vaccine (subunit vaccines) that target the immune response to those viral epitopes that elicit vn antibodies. killed viruses and subunit vaccines typically generate immediate responses that wane faster, making it imperative for long-lived mammals like humans, to receive booster vaccinations. a second approach to vaccine development is use of live attenuated virus that has been genetically modified or cell culture adapted and cannot produce a disease in the host but can still replicate. these are often more likely to stimulate virus-specific cytotoxic t cells and to induce longer term immunity. sharma et al. developed a recombinant svv strain using reverse genetics and tested its immunogenicity and protective efficacy in pigs. toman et al. provide comparative data on four vaccines for prrsv, two killed and two modified live. we believe that veterinary immunovirology should place more emphasis on how each viral pathogen effects and/or avoids the host immune system and on the identification of viral epitopes that are effective targets of vn antibodies and t cell epitopes that can promote the action of cytotoxic t cells. jb wrote the first draft and then both co-authors contributed equally to the final draft. reservoir host immune responses to emerging zoonotic viruses current drivers and future directions of global livestock disease dynamics the future of pork production in the world: towards sustainable, welfare-positive systems what contemporary viruses tell us about evolution: a personal view assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers impact of health challenges on pig growth performance, carcass characteristics, and net returns under commercial conditions emerging and reemerging coronaviruses in pigs emergence of a novel recombinant seneca valley virus in central china african swine fever virus in asia: its rapid spread and potential threat to unaffected countries apoptosis and necroptosis as host defense strategies to prevent viral infection viruses and apoptosis fetal infections and antibody profiles in pigs naturally infected with porcine circovirus type (pcv ) effect of wean-tofinish management on pig performance transplacental infection and embryonic death following maternal exposure to porcine parvovirus near the time of conception the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © vlasova and butler. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -tudl k g authors: opriessnig, tanja; gauger, phillip c.; faaberg, kay s.; shen, huigang; beach, nathan m.; meng, xiang-jin; wang, chong; halbur, patrick g. title: effect of porcine circovirus type a or b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: tudl k g to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-γ) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded, positive-sense rna virus, is characterized by a high mutation rate with the potential of genetically diverse strains evolving over time (forsberg et al., ; hanada et al., ; pirzadeh et al., ; rowland et al., ) . in the past, prrsv isolates have emerged within the swine population with varying degrees of virulence (fang et al., ; han et al., ; nelsen et al., ) possibly due to a high degree of mutation and recombination (yuan et al., (yuan et al., , (yuan et al., , (yuan et al., , . more recently, attention has focused on the occurrence of high mortality in chinese swine herds which veterinary microbiology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] to determine differences in infection kinetics of two temporally and genetically different type porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type a or b infection, pigs were randomly assigned to one of seven groups: negative controls (n = ); pigs coinfected with a prrsv strain (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with vr- and pcv b (coi- - b; n = ), pigs coinfected with a prrsv strain (nc b) and pcv a (coi- - a; n = ), pigs coinfected with nc b and pcv b (coi- - b; n = ), pigs infected with vr- (n = ), and pigs infected with nc b (n = ). blood samples were collected before inoculation and at day post-inoculation (dpi) , , and and tested for the presence of prrsv antibody and rna, pcv antibody and dna, complete blood counts, and interferon gamma (ifn-g) levels. regardless of concurrent pcv infection, vr- initially replicated at higher levels and reached peak replication levels at dpi . pigs infected with vr- had significantly higher amounts of viral rna in serum on both dpi and dpi , compared to pigs infected with nc b. the peak of nc b virus replication occurred between dpi and dpi and was associated with a delayed anti-prrsv antibody response in these pigs. pcv coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. ß elsevier b.v. all rights reserved. was associated with novel prrsv isolates and described as porcine high fever disease in tong et al., ; wu et al., ) . the prrsv isolates involved in porcine high fever disease contained unique nucleotide differences compared to other isolates. specifically, a discontinuous, amino acid deletion was identified within the nsp region which was initially suggested to be correlated with the pathogenicity of the virus wu et al., ). however, more recent reports have concluded that this deletion is unrelated to virulence (zhou et al., ) in spite of the high mortality that was initially associated with this prrsv variant tong et al., ) . interestingly, analysis of samples from affected pigs resulted in the identification of prrsv, porcine circovirus type (pcv ) and classical swine fever virus as the most common co-infection pathogens, suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality (lv et al., ; wu et al., ). pcv is a small, circular, non-enveloped dna virus belonging to the circoviridae family in the genus circovirus. pcv can be further divided into several subtypes of which pcv a and pcv b are prevalent worldwide (patterson and opriessnig, ) . to date, experimental infections comparing pcv a and pcv b in gnotobiotic and conventional pigs have not demonstrated major differences in virulence (beach et al., ; fort et al., ; lager et al., ; opriessnig et al., b) . pcv is the cause of porcine circovirus associated disease (pcvad) with multiple clinical manifestations including respiratory disease . prrsv has become an important component of the porcine respiratory disease complex (prdc) with major economic impact on the swine industry (chae, ) . retrospective studies identified prrsv as the most common cofactor in cases of pcvad (pallaré s et al., ) . experimental coinfection with prrsv and pcv has yielded mixed results. one study completed in reported minimal clinical disease or death loss in conventional pigs coinfected with pcv and prrsv (rovira et al., ) . in contrast, in another study, severe clinical disease and death in of pigs between and days postinfection (dpi) was reported in dually infected, caesarianderived and colostrum-deprived (cdcd) pigs (harms et al., ) . despite differences in severity of clinical presentation, experimental coinfection of pigs with pcv and prrsv has consistently resulted in up-regulation of pcv replication (enhanced viremia and pcv tissue load) and increased severity of prrsv-induced lesions in lung tissues (allan et al., ; harms et al., ; rovira et al., ) . in north america, both prrsv and pcv b have been identified in pcvad outbreaks characterized by excessive mortality suggesting a synergistic relationship between these two viruses gagnon et al., ; horlen et al., ) . the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates in a conventional pig model under the influence of concurrent pcv a or pcv b infection. the severity of clinical disease, macroscopic and microscopic lesions, amount of prrsv and pcv antibodies and nucleic acids in sera, amount of prrsv and pcv antigen associated with lesions, and interferon gamma (ifn-g) concentrations in serum were measured and compared between groups. fifty-three colostrum-fed, crossbred pigs were derived from sows known to be free of pcv , prrsv and mycoplasma hyopneumoniae in two separate batches, pigs in batch (b ) and pigs in batch (b ). in addition, batch (b ) consisted of colostrum-fed crossbred pigs derived from sows free of prrsv and m. hyopneumoniae but seropositive for pcv . b and b pigs were challenged at the same age as b pigs but the experiment was conducted approximately months after b pigs. insufficient numbers of pigs were available from the source herd for singularly prrsv-infected groups to be included with the original experiment. the experimental design and group designations are summarized in table . all pigs were housed under the same conditions and treated in a similar way. all pigs were weaned at three weeks of age and transported to the livestock infectious disease isolation facility at iowa state university, ames, iowa. on the day of arrival, all b pigs were comingled and randomly assigned to one of five rooms each containing or pigs: negative controls (n = ); pigs coinfected with a isolate of prrsv (vr- ) and pcv a (coi- - a; n = ), pigs coinfected with prrsv vr- and pcv b (coi- - b; n = ), pigs coinfected with a isolate of prrsv (nc b) and pcv a (coi- - a; n = ) and pigs coi- - b pcv b nc b prrsv-i- none vr- prrsv-i- none nc b b -prrsv-i- none vr- b -prrsv-i- none nc b a batch and pigs were derived from the same source herd free of prrsv and pcv whereas batch pigs were derived from a different source herd seropositive for pcv . coinfected with prrsv nc b and pcv b (coi- - b; n = ). b pigs were randomly assigned to one of two rooms each containing or pigs which were infected with prrsv vr- (prrsv-i- ) and prrsv nc b (prrsv-i- ), respectively. similarly, b pigs were comingled and randomly assigned to one of two rooms each containing or pigs that were infected with prrsv vr- (b -prrsv-i- ) and prrsv nc b (b -prrsv-i- ), respectively. the pigs from the different batches were kept in different but identical rooms. each room had m of solid concrete floor space, separate ventilation systems and one nipple drinker. inoculation was conducted at approximately days of age. blood samples were collected from all pigs prior to inoculation and at dpi , , and in serum separator tubes ( . ml bd vacutainer, benton dixon, franklin lakes, nj, usa). the blood was centrifuged at  g for min at c and serum was stored at À c until testing. serum samples were analyzed for levels of anti-pcv igg antibody, anti-prrsv-igg antibody, ifn-g, pcv dna, and prrsv rna. in addition, edta tubes ( . ml monoject tm % edta liquid, tyco healthcare group lp, mansfield, ma, usa) were collected at dpi , , , and , stored at room temperature and used within h after collection to determine blood cell counts. all pigs were necropsied on dpi and tissues collected during necropsy were analyzed by immunohistochemistry (ihc) for the presence of pcv and prrsv antigens. the experimental protocol was approved by iowa state university institutional animal care and use committee (iacuc approval # - - -s). prrsv isolate vr- with a rflp pattern - - was recovered in from pig tissues obtained from a sow herd in southwestern iowa affected by severe respiratory disease in - -week-old pigs and high numbers of late term abortions (halbur et al., b; meng et al., ) . the passage virus of the original vr- isolate was used to inoculate pigs in as described previously . serum from the pigs infected with vr- in was used to re-isolate the virus followed by two subsequent passages in marc- cells to produce the virus stock of vr- for this study. prrsv isolate nc b with a rflp pattern - - was isolated in from a clinically affected -week-old pig with systemic pcvad from a group of pigs from north carolina with a history of severe respiratory disease in % of the pigs and approximatley % mortality in the group (gauger et al., ) . the passage virus of the original isolate was used to experimentally infect a set of prrsv-free conventional pigs (data not shown) and the lung tissues from these pigs collected two weeks after infection were used for reisolation of the nc b virus followed by two subsequent passages in marc- cells to produce the nc b virus stock for this study. the two inocula were separated in different aliquots, stored at À c, and virus of the same lot was used for all batches of pigs. on dpi , coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- groups received ml of prrsv isolate vr- at a dose of . median tissue culture infective dose (tcid ) per ml. all pigs in groups coi- - a, coi- - b, prrsv-i- , and b -prrsv-i- received ml of prrsv isolate nc b at a dose of . tcid per ml. inoculation was intranasal by holding the pig in the upright position and slowly dripping ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). two different pcv subtypes were used for the inoculation of pigs. pigs in groups coi- - a and coi- - a were inoculated with the pcv a isolate , which was recovered from an iowa farm in (fenaux et al., ) and has been well characterized genetically (fenaux et al., ) and in the conventional specific pathogen free (spf) pig model (opriessnig et al., (opriessnig et al., , a . both pcv a and pcv b viruses were produced as described previously (opriessnig et al., b) and used for inoculation in this study at a titer of . tcid per ml . pigs in groups coi- - b and coi- - b were inoculated with pcv b isolate nc which was isolated in from a pig farm in north carolina (opriessnig et al., b) . both, pcv b nc and prrsv nc b originated from the same tissues. the pcv groups were inoculated intranasally ( ml) and intramuscularly ( ml) with their respective pcv subtype by injecting ml of the inoculum intramuscularly into the right neck area and ml ( . ml per nostril) intranasally by holding the pig in the upright position and slowly dripping . ml of the inoculum into each nostril using a ml syringe (fisher scientific, inc.). edta-treated blood samples were analyzed for white blood cells using a multispecies hematology instrument (hemavet hv fs, drew scientific, inc.). the white blood cell (wbc) count was reported as actual numbers of neutrophils, lymphocytes and total wbc per ml of whole blood. in addition to wbc, a ratio was determined between the total neutrophil count and the total lymphocyte count reported as the n/l ratio. values from negative control pigs were considered as baseline for the infected pigs on each dpi. serum samples from all pigs were also tested for the presence of anti-prrsv antibodies by a commercial prrsv elisa (herdchek prrs virus antibody test kit xr, idexx laboratories inc. westbrook, ma, usa), according to the instructions of the manufacturer. samples were considered positive if the calculated s/p ratio was equal to . or greater. all serum samples were tested for the presence of anti-pcv igg antibodies based on an open reading frame (orf ) elisa (nawagitgul et al., ) . samples were considered positive if the calculated sample-to-positive (s/ p) ratio was equal to . or greater. on dpi , three samples were randomly chosen from each group and room and tested for the presence of swine influenza virus (siv) antibodies by an in house nucleoprotein ns elisa (richt et al., ) and for the presence of antibodies to porcine parvovirus (ppv) by hemagglutination inhibition (hi) assay (mengeling et al., ) . a commercial elisa kit (swine ifn-g; invitrogen, camarillo, ca, usa) was used to detect and quantify ifn-g concentrations in serum according to the instructions of the manufacturer. following prrsv/pcv coinfection, the pigs were monitored daily for respiratory disease (dyspnea, sneezing, coughing, nasal discharge). rectal temperatures and behavioral changes such as lethargy and inappetence/ anorexia were also recorded daily. the observers were aware (not blinded) to the treatment status. rna extraction on serum collected at dpi , , , , and was performed using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). the agpath-id prrsv multiplex reagent kit (applied biosystems, foster city, ca, usa) was used for the real-time, reverse transcriptase pcr (rt-pcr) on each extracted rna sample. all samples were run in duplicate. each pcr consisted of ml template rna and ml of pcr master mix. the pcr master mix contained . ml of  rt-pcr buffer, . ml  prrsv primer probe mix, . ml  multiplex rt-pcr enzyme mix, . ml of zenorna- internal control rna and . ml nuclease-free water. each reaction included eight progressive : dilutions of a known copy number of prrsv to generate a standard curve for quantification. each plate was run in the sequence detection system (geneamp sequence detection system, applied biosystems) using the agpath-id company specific conditions ( min at c, min at c, followed by cycles of s at c and s at c). samples were considered negative when no signal was observed within the amplification cycles. dna extraction on serum collected on dpi , , , , and days was performed using the qiaamp dna blood mini kit (qiagen, valencia, ca, usa) and subsequently used for detection of pcv dna by quantitative real-time pcr utilizing primers and a probe designed for pcv orf as described (opriessnig et al., ) . the real-time pcr reaction consisted of a ml pcr mixture containing . ml commercially available master mix (taqman universal pcr master mix, applied biosystems by life technologies), . ml dna, ml ( . mm) of each primer, and . ml ( . mm) probe. the reaction was run in a fast real-time pcr system (abi, foster city, ca, usa) under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. all samples were run in duplicate. serial dilutions of a recombinant pcv dna clone were included on each plate to generate a standard curve. viral concentrations were expressed as the dna copy numbers per ml of sample. samples were considered negative when no signal was observed within the amplification cycles. all dna extracts were also tested for presence of pcv a and pcv b dna by utilizing a forward primer ( -gcagggccagaattcaacc- ), a reverse primer ( -ggcggtggacatgatgaga- ), a probe specific for pcv a ( -cal fluor orange -ggggaccaacaaaatctcta-tacccttt-bhq- ), and a probe specific for pcv b ( -quasar -ctcaaacccccgctctgtgccc-bhq- ), which were designed in the pcv orf as described . the multiplex real-time pcr reaction consisted of a total volume of ml containing . ml of the commercially available master mix (applied biosystems), ml dna, . mm of each primer, and . mm of each probe. all samples were run in duplicate. the reactions were carried out under the following conditions: c for min, c for min, followed by cycles of c for s and c for min. the sensitivity and specificity of the real-time pcr reaction was evaluated using known pcv a and pcv b isolates as well as ppv, prrsv, and pcv type (pcv ) isolates. samples were considered negative when no signal was observed within the amplification cycles. open reading frame (orf) of one prrsv rt-pcr positive pig in each group was sequenced on dpi as previously described (gauger et al., ) . on dpi , all pigs were humanely euthanized by intravenous pentobarbital overdose (fatal-plus vortech pharmaceutical, ltd., dearborn, mi, usa). macroscopic lung lesions were estimated based on the percentage of the lung surface affected by pneumonia ranging from to % (halbur et al., b ). the scoring system was based on the approximate volume that each lung lobe contributes to the entire lung: the right cranial lobe, cranial part of the left cranial lobe, and the caudal part of the left cranial lobe contribute % each to the total lung volume, the accessory lobe contributes %, and the right and left caudal lobes contribute . % each (halbur et al., b) . additionally, lymph node size was scored ranging from (normal) to (four times the normal size) (opriessnig et al., ) . lungs were insufflated with fixative as previously described (halbur et al., b) . sections of lymph nodes (tracheobronchial, mesenteric, mediastinal, superficial inguinal, and external iliac), tonsil, thymus, ileum, kidney, colon, spleen, heart, liver, and brain were collected at necropsy and fixed in % neutral-buffered formalin and routinely processed for histological examination. microscopic lesions were evaluated independently by two veterinary pathologists (to, pcg) blinded to the treatment status. sections of lung were scored for the presence and severity of interstitial pneumonia ranging from (normal) to (severe, diffuse) (halbur et al., b) . sections of heart, liver, kidney, ileum, colon and brain were evaluated for the presence of lymphohistiocytic inflammation and scored from (none) to (severe). lymphoid tissues including lymph nodes (trachea-bronchial, mediastinal, mesenteric, external iliac and superficial inguinal), tonsil, spleen and thymus were evaluated for the presence of lymphoid depletion ranging from (normal) to (severe) and histiocytic inflammation and replacement of follicles ranging from (normal) to (severe) (opriessnig et al., ) . . . immunohistochemistry . . . prrsv detection of prrsv-specific antigen was performed by ihc staining on lung sections as previously described (halbur et al., a) . sections were scored for presence of prrsv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. ihc for detection of pcv -specific antigen was performed on sections of lung, lymph nodes (tracheobronchial, mediastinal, mesenteric, superficial inguinal and external iliac), tonsil, spleen, thymus and small intestine using a rabbit polyclonal antiserum (sorden et al., ) . sections were scored for presence and amount of pcv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. if the results obtained by the two pathologists on a certain tissue differed, the mean of the two scores was used. pcv scores ranged from (no antigen) to (more than % of the lymphoid follicles contain cells with pcv -antigen staining) (opriessnig et al., ) . any tissue or tissue pool with detectable staining was given at least a score of . for the purpose of determining prevalence rates, a score of was considered negative and scores of , and were considered positive. for data analysis, jmp software version . . and sas software version . . (both sas institute, cary, nc, usa) were used. summary statistics were calculated for groups to assess the distributional property and data that were not distributed normally (pcr data) were log transformed prior to analysis. as log transformation can only be applied to numbers above , a constant number ( ) was added to each number in the data set prior to log transformation. a linear mixed model with the random effects ''source'' (source a: b and b and source b: b ) and ''batch'' (b , b , b , nested within ''source'') and the fixed effects ''prrsv strain'' (none, vr- , nc b) and ''pcv subtype'' (none, pcv a, pcv b) was used first on all outcomes. from this, it was determined that the random effect ''source'' contributed to the overall variation whereas ''batch'' did not. to decrease the heterogeneity of the animals in the analysis, all data obtained from the second source, b , were removed from the analysis but were provided as supplemental information throughout the ''results'' and tables. the final model to analyze continuous data collected over time (rectal temperatures, blood cell counts, log transformed pcv and prrsv genomic copies, and elisa s/p ratios) was a repeated measures analysis of variance (anova), where prrsv strain, pcv subtype, dpi and their interactions were the fixed effects and pig was the subject of repeated measures. compound symmetry variancecovariance structure was used to model the within pig correlation. a one-way anova was used to analyze crosssectional data (macroscopic and microscopic lung lesions) where prrsv strain, pcv subtype, and their interaction were the fixed effects. differences among the interacting groups (prrsv strain  pcv subtype) in the repeated measures anova or the one-way anova were assessed using tukey's t-test. a p-value of less than . was considered significant. differences in prevalence of prrsv and pcv antigen between groups (ihc staining) were determined by fisher's exact test. mild, transient lethargy and inappetence were observed in all inoculated groups, although coughing or sneezing was not a feature. pigs in all inoculated groups regardless of coinfection status developed a transient to persistent fever ranging from . c to . c between dpi and dpi . the mean rectal temperature time by group interaction after inoculation was significant (p < . ). all six inoculated groups had rectal temperatures significantly higher than the negative controls at dpi and dpi . by dpi , the mean group rectal temperatures in the prrsv-i- , prrsv-i- , coi- - a, coi- - b and coi- - a groups were significantly (p < . ) higher compared to the negative controls. when the effect of ''prrsv strain'' was evaluated across groups, no differences were found. compared to pigs infected with prrsv alone, coinfected pigs had higher mean rectal temperatures at dpi , and . when the effect ''pcv subtype'' was evaluated among coinfected groups, pcv a pigs had significantly (p < . ) higher rectal temperatures on dpi compared to pcv b pigs (data not shown). b pigs (b -prrsv-i- and b -prrsv-i- ) had similar rectal temperatures as b (prrsv-i- and prrsv-i- ) pigs. hematology results are summarized in table . there was an effect of ''prrsv strain'' on white blood cell counts at dpi with pigs infected with nc b having significantly (p = . ) higher levels of white blood cells compared to pigs infected with vr- ( . ae . versus . ae . ). also, there was a significant effect of ''pcv '' (p < . ): pcv -infected pigs had higher levels of white blood cells at dpi and compared to non-pcv -infected pigs ( . ae . versus . ae . and . ae . versus . ae . ). there was no effect of ''prrsv strain'' on numbers of neutrophils; however, there was a significant effect of ''pcv '' on mean group neutrophil counts at dpi , , and with pcv -infected pigs having elevated levels compared to pigs not infected with pcv ( . ae . versus . ae . , . ae . versus . ae . , and . ae . versus . ae . , respectively). differences in mean group lymphocyte counts were only observed on dpi (table ) and there was an effect of prrsv strain (p = . ): pigs infected with nc b had lower levels of lymphocytes compared to pigs infected with vr- ( . ae . versus . ae . ). additionally, pigs coinfected with pcv had higher levels of lymphocytes (p = . ) compared to pigs infected with prrsv alone ( . ae . versus . ae . ) suggesting an effect of ''pcv ''. all pigs in all groups were negative for prrsv-specific antibodies at dpi and negative control pigs remained negative for anti-prrsv antibody throughout the study. prevalence and group mean s/p ratios are summarized in table . overall, there was a significant effect of ''prrsv strain'' and ''pcv '' on the anti-prrsv antibody response at dpi . specifically, pigs infected with vr- had a significantly (p = . ) higher anti-prrsv antibody response compared to those infected with nc b. similarly, coinfected pigs had significantly (p = . ) higher anti-prrsv s/p ratios compared to pigs singularly infected with prrsv. there was no effect of ''pcv subtype'' on the magnitude of the anti-prrsv-antibody response among coinfected groups. all pigs in b and b were negative for pcv -specific anti-igg antibodies at dpi and the negative controls and b pigs remained pcv seronegative throughout the trial. in the pcv coinfected groups, seroconversion was observed at dpi . the prevalence and mean group anti-pcv -igg s/p ratios table mean group leukocyte values ( /ml of whole blood except for ratios) in the different treatment groups on days post-inoculation (dpi) , , , and . data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. hematology a negative controls (n = ) wbc . ae . . ae . are summarized in table . there was no effect of ''prrsv strain'' or ''pcv subtype'' on the magnitude of the anti-pcv -antibody response. b pigs were seropositive for pcv at the time of arrival (mean pcv elisa s/p ratio: . ae . ) and the s/p ratios remained at a similar level for the duration of the study (data not shown). at termination of the study, pigs randomly selected from each batch tested negative for antibodies against ppv, siv h n and siv h n (data not shown). prevalence of ifn-g positive samples and mean group ifn-g concentrations are summarized in table . there was no effect of ''prrsv strain'' or ''pcv '' on the ifn-g levels and no differences were found among groups; however, analysis of an effect of ''pcv subtype'' among coinfected groups revealed that on dpi , pcv b-inoculated pigs had significantly (p = . ) higher levels of ifng compared to pcv a-inoculated pigs ( . ae . pg/ml versus . ae . pg/ml). all pigs were negative for prrsv-rna in serum at dpi and the negative controls remained negative for prrsv rna throughout the study. the prevalence of prrsv rna positive pigs and group mean genomic copy numbers/ml are summarized in table . sequencing of the orf gene of prrsv and comparison with the original inocula confirmed that the correct prrsv isolates were present in the table prevalence of anti-prrsv antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups on days post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. different superscripts (a, b, c) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of anti-pcv igg antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups except prrsv-i- and prrsv-i- on day post-inoculation (dpi) , , , and . data presented as prevalence (mean s/p ratio ae se). grey shaded areas indicate the presence of pcv seropositive pigs (s/p ratio > . ) within a treatment group. different superscripts (a, b) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. prevalence of ifn-g and mean group concentration (pg/ml) in the different treatment groups on day post-inoculation (dpi) , , , and . data presented as prevalence (mean log group concentration ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. respective groups and rooms. when results were analyzed for a possible effect of ''prrsv strain'', a significantly (p < . ) higher amount of prrsv rna was detected in pigs infected with vr- at dpi and compared to pigs infected with nc b ( fig. ) . when the pigs infected with prrsv alone were removed from the analysis, coinfected pigs with nc b had significantly higher amounts of prrsv rna in serum compared to pigs infected with vr- on dpi ( . ae . versus . ae . ) and dpi ( . ae . versus . ae . ), respectively. an effect of ''pcv '' or ''pcv subtype'' on prrsv replication was not evident. all pigs were negative for pcv -dna in serum at dpi and the negative controls and b and b pigs remained pcv dna negative throughout the study (data not shown). at dpi , / pcv -inoculated pigs were positive for pcv -dna, and all pcv -inoculated pigs were positive for pcv -dna by dpi and remained positive until dpi . the log group mean pcv dna amounts are summarized in fig. . when results were analyzed for a possible effect of ''prrsv strain'' it was found that there was a significantly higher amount of pcv dna in pigs infected with vr- ( . ae . ) compared to pigs infected with nc b ( . ae . ) on dpi . there was a significant effect of ''pcv subtype'' on dpi ; groups infected with pcv b had significantly higher amounts of pcv dna in serum compared to groups infected with pcv a ( . ae . versus . ae . , respectively). an effect of ''pcv subtype'' was not evident in the later stages of infection. all pigs in the pcv a or pcv b groups were correctly infected with their respective subtype as determined by multiplex real-time pcr (data not shown) and crosscontamination between groups and rooms was not detected. table prevalence of prrsv and group mean log prrsv genomic copies per ml in the different treatment groups on days post-inoculation (dpi) , , and . data presented as prevalence (log prrsv rna ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included into the analysis. group negative controls / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a / ( . ae . ) a coi- - a / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b coi- - a / ( . ae . ) b,c / ( . ae . ) b,c / ( . ae . ) b / ( . ae . ) b coi- - b / ( . ae . ) b,d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) d / ( . ae . ) b / ( . ae . ) b / ( . ae . ) b prrsv-i- / ( . ae . ) c / ( . ae . ) c / ( . ae . ) b / ( . ae . ) b b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) b -prrsv-i- / ( . ae . ) / ( . ae . ) / ( . ae . ) / ( . ae . ) different superscripts (a,b,c,d) within columns indicate significant (p < . ) differences in mean group s/p ratios among groups. fig. . log transformed mean prrsv rna genomic copies (aese) in vr- and nc b infected pigs regardless of coinfection status on day postinoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by asterisks. the lines indicate the linear trend for pigs infected with vr- (gray, dashed) or nc b (black, solid). macroscopic lesions were characterized by mild-tomoderate enlargement of lymph nodes (especially tracheobrochiolar lymph nodes and mediastinal lymph nodes) and mottled-tan lungs with varying degrees of the lung surface affected by visible pneumonia lesions. the group mean lung lesion severity scores are summarized in table and were significantly (p < . ) lower for negative controls compared to all coinfected groups. there were no significant differences in lung lesions severity between the negative controls and the pigs infected with prrsv alone. there was an effect of ''pcv '' on the mean group macroscopic lung lesion scores as evidenced by the coinfected pigs having more severe macroscopic lung lesions compared to pigs infected with prrsv alone. however, there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed macroscopic lung lesions. lung tissues had multifocal-to-diffuse, mild-to-severe, lymphohistiocytic interstitial pneumonia. the mean microscopic lung lesion scores, which are summarized in table , were significantly (p < . ) lower in the negative controls compared to the four coinfected groups; however, the scores in the negative controls were not significantly lower than observed in the groups singularly infected with prrsv. there was a significant effect of ''pcv '' (p < . ) on microscopic lung lesions but there was no effect of ''prrsv strain'' or ''pcv subtype'' on the severity of the observed microscopic lung lesions. lymphoid lesions were either not observed or were characterized by mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups. significant differences in lymphoid lesion scores were not observed among the groups (data not shown). . . . prrsv all control pigs were negative for prrsv antigen by ihc on sections of lung. the prevalence of prrsv antigen in lung sections was / pigs in the nc b-inoculated group (b : / pigs) compared to / pigs in the vr- -inoculated group (b : / pigs). there were no significant differences in the prevalence rates of prrsv fig. . log transformed group mean pcv dna amounts (aese) in the pcv -prrsv coinfected groups on day post-inoculation (dpi) , , , and . significant (p < . ) differences between groups within a dpi are indicated by different superscripts (a, b). mean group macroscopic (percentage of lung surface affected by lesions) and microscopic (interstitial pneumonia ranging from = normal to = severe, diffuse) lung lesions (mean group amount ae se). data obtained from b -prrsv-i- and b -prrsv-i- pigs (gray shaded area) were not included in the analysis. significant (p < . ) differences between groups are indicated by different superscripts (a, b, c). macroscopic lung lesions ( - %) antigen in lungs between the virus-inoculated groups. the prevalence of prrsv antigen in lungs was independent of ''prrsv strain'' or ''pcv subtype''. all control pigs and all b and b pigs were negative for pcv antigen by ihc. low-to-high amounts of pcv antigen in lung sections were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pcv a-inoculated pigs and / pcv b-inoculated pigs. moreover, pcv antigen was detected in / vr- -inoculated pigs and in / nc b-inoculated pigs. the prevalence of pcv antigen in lung tissues was independent of ''prrsv strain'' or ''pcv subtype''. in lymphoid tissues, low-tohigh amounts of pcv antigen were detected in / coi- - a pigs, / coi- - b pigs, / coi- - a pigs and in / coi- - b pigs which corresponds to / pigs inoculated with pcv a and / pigs inoculated with pcv b, as well as / pigs inoculated with nc b and / pigs inoculated with vr- . the prevalence of pcv antigen in lymphoid tissues was independent of ''prrsv strain'' or ''pcv subtype''. the objective of this study was to characterize the infection dynamics and pathogenicity of two different type prrsv isolates recovered from pigs in and in a conventional pig model. to mimic field conditions where coinfections frequently occur, the pigs were concurrently infected with either pcv a or pcv b. the effect of each prrsv isolate was also evaluated in singularly inoculated pigs. however, due to limitations in numbers of available pigs from the source herd, the experiments with singularly prrsv-inoculated groups were conducted separately but under the same study conditions, using the same inocula and assays to analyze the samples. the prrsv isolate vr- used in this experiment has been well-characterized in the cdcd and the conventional pig models and is considered a relatively highly pathogenic prrsv isolate from the s (halbur et al., b meng et al., ) . in contrast, nc b represents a more recent prrsv isolated from an outbreak of respiratory disease on a farm characterized by high morbidity and mortality in (gauger et al., ) . the orf - sequence homology between vr- (genbank accession pru and pru ) and nc b (genbank accession hq ) was approximately . %. the orf region demonstrated the least nucleotide and amino acid homology at . % and . %, respectively (gauger et al., ) . in the past, dual infections with prrsv and porcine respiratory coronavirus (prcv) or prrsv and siv were studied using conventional pigs (van reeth et al., ) and gnotobiotic pigs (van reeth and nauwynck, ) and in general disease was found to be more pronounced in dually inoculated pigs. interestingly, in gnotobiotic pigs the effect of the coinfection appeared additive rather than synergistic (van reeth and nauwynck, ) . more recent studies have shown that prrsv modifies the innate immune response, induces immunosuppression and enhances the inflammatory response to prcv in pigs (jung et al., ; renukaradhya et al., ) . in another study, dual infection of specific pathogen-free pigs with prrsv and pseudorabies virus (prv) resulted in more severe clinical signs and increased pneumonia in pigs inoculated with both viruses compared to pigs infected with prrsv or prv alone (shibata et al., ) . it is also well recognized that pcv replication is enhanced by concurrent prrsv infection in both cd and conventional pigs compared to singularly inoculated pigs (allan et al., ; rovira et al., ) . to the authors' knowledge, the pathogenicity of genetically different prrsv isolates in the presence of concurrent viral infection has not been evaluated in vivo. the combination of prrsv and pcv is one of the most common coinfections associated with swine respiratory disease under field conditions (dorr et al., ; pallaré s et al., ) . both prrsv isolates used in the current study were isolated from field cases of high mortality and experimental infection of pigs with prrsv vr- has resulted in severe lesions and high levels of viremia (halbur et al., b . the two pcv isolates were initially recovered from typical field cases of pcvad in iowa and north carolina and have been characterized in the conventional pig model side by side without identifiable differences between pcv subtypes (opriessnig et al., b; sinha et al., ) . in the current study, clinical disease in the treatment groups was characterized by variable numbers of infected pigs experiencing transient, mild lethargy, mild respiratory disease and inappetence. coinfected groups had significantly higher mean rectal temperatures compared to pigs infected with prrsv alone and the negative controls. interestingly, when organized by coinfection status and analyzed by pcv subtype, pigs inoculated with pcv a had significantly higher mean group rectal temperatures compared to pigs inoculated with pcv b on dpi which was associated with an anti-pcv -antibody response in . % ( / ) of the pcv ainoculated pigs on dpi whereas a delayed antibody response was seen in pcv b-inoculated pigs ( . %; / ). it is well documented that pathogenic differences between type prrsv isolates exist (halbur et al., b . the uniqueness of the current study is the utilization of two temporally and genetically different prrsv isolates both from cases of high morbidity and mortality in the field but isolated years apart. in a separate in vitro study comparing phenotypic traits of the two prrs viruses, nc b demonstrated reduced growth characteristics compared to vr- (gauger et al., ) . nc b plaque sizes were slightly smaller than vr- and the peak viral titer demonstrated by nc b was approximately -fold lower than the vr- peak titer. this is in contrast to the in vivo growth characteristics demonstrated in this report. there were clear differences in initial replication between the two prrsv isolates used in this study. the vr- -inoculated pigs had significantly higher levels of prrsv rna in serum on dpi and . moreover, nc b replicated at higher levels at dpi and dpi compared to vr- which was associated with significantly lower levels of lymphocytes at dpi and a significantly lower n/l ratio at dpi . these results suggest that highly pathogenic prrsvs may replicate more efficiently in vivo in contrast to their decreased growth properties in vitro as previously suggested (johnson et al., ; wang et al., ) . this is further supported by the data obtained from the pigs infected with prrsv alone (b and b ) which clearly show an increase in replication in pigs infected with nc b in the later stages of the in vivo study. similar to other pcv -prrsv coinfection studies (allan et al., ; harms et al., ) , macroscopic and microscopic lesions in coinfected groups were enhanced compared to pigs singularly infected with prrsv. recently, it has been shown that pigs infected with vr- had significantly prolonged (until dpi) pcv viremia and shedding in prrsv-pcv coinfected pigs (sinha et al., ) . a similar approach using prrsv nc b, which replicated differently from vr- in the early stages of infection, could potentially offer new insights on viral interactions in pigs. in the current study, pcv b replication was significantly up-regulated shortly after initiation of the study at dpi compared to pcv a. furthermore, the coi- - b group had significantly higher quantities of pcv b in the serum compared to coi- - a (dpi and ) and coi- - b (dpi ) which was associated with a higher prevalence of pcv antigen in tissues ( . % versus . %) indicating a synergistic relationship between prrsv- (vr- ) and pcv . unlike previous studies where the average trial length ranged from to days (allan et al., ; rovira et al., ) , this trial was terminated at dpi to evaluate prrsv-induced lung lesions which tend to be most severe between dpi and dpi . it remains unknown if the observed trend would have resulted in a difference in clinical disease in the later stages of infection. as expected, and similar to a previous study , the pathological lesions associated with pcv were either not present or they were mild; however, pcv antigen was detected in most tissues in coinfected pigs. in this study, pcv naïve pigs were utilized, thus the relevance of the model to actual field situations is limited considering the majority of young pigs have high levels of passively acquired anti-pcv antibodies (opriessnig et al., b) . therefore, the impact of anti-pcv immunity on the pcv infection could not be ascertained in the experiment; however, this was not a major concern as we know from several experiments that pigs with passively derived antibodies, although protected from clinical pcv associated disease, can still be infected with pcv (mckeown et al., ; opriessnig et al., a) . therefore, we believe that a pcv naïve pig model increases the ability to identify trends and associations between prrsv and pcv . in the current study, prrsv-pcv coinfection was administered intranasally on the same day. this model of simultaneous dual inoculation does not fully mimic the population dynamics due to the variability in timing of exposure to these two pathogens within and between herds in field situations. on many conventional farms, endemic exposure and seroconversion to prrsv often occurs earlier than exposure to pcv . infection of pigs with prrsv prior to pcv may contribute to the manifestation of more severe pcv -induced clinical disease and lesions. prrsv is immunosuppressive, primarily infecting porcine alveolar macrophages (drew, ) , which decreases the pig's ability to clear subsequent infections. in contrast, prior prrsv infection may induce an immunostimulatory effect on the host immune response that serves to enhance pcv replication and lesions (krakowka et al., ) . it is possible that amino acid mutations acquired during serial passaging of prrsv on marc- cells could result in attenuation as reported previously (allende et al., ; an et al., ) . while this is also applicable to the current study, we attempted to minimize this risk, by using a relatively low passage of both viruses with a pig passage followed by only two in vitro passages in marc- cells. inoculation was completed two days after weaning and transport of the pigs to the research facility. it is also possible that the stress from weaning, transport, new socialization, and adjusting to a new environment may have affected the ability of the pigs to respond to concurrent prrsv-pcv infection and influenced the level of prrsv replication in the pigs. however, the data obtained from pigs infected with prrsv alone indicate that this was not the case and that the ability of the pigs to develop a humoral immune response was normal. overall, the data indicate no significant differences between the two prrsv isolates based on clinical signs, gross pathology, histology or hematology even though the prrsv isolates we utilized in this study were isolated from geographically separated herds (vr- from iowa and nc b from north carolina) over a period of years. differences in in vivo replication kinetics were identified. vr- initially replicated more quickly and to higher levels and peaked at dpi and the amount of vr- rna steadily declined thereafter. in contrast, pigs infected with nc b had lower levels of prrsv rna in serum initially and this steadily increased through termination of the study at dpi . concurrent pcv viremia was enhanced by prrsv vr- infection but not by concurrent prrsv nc b infection. a higher prevalence of pcv antigen was demonstrated in the lungs of pigs coinfected with vr- ( . %) compared to pigs coinfected with prrsv nc b ( . %). this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens on prrsv kinetics. additional investigations are necessary to further elucidate the specific mechanisms of the pcv -prrsv interaction in pigs. comparative genomic analysis of five pairs of virulent parental/attenuated vaccine strains of prrsv experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype novel chimeric porcine circovirus (pcv) with the capsid gene of the emerging pcv b subtype cloned in the genomic backbone of the nonpathogenic pcv is attenuated in vivo and induces protective and cross-protective immunity against pcv b and pcv a subtypes in pigs a review of porcine circovirus -associated syndromes and diseases detection of two porcine circovirus type genotypic groups in united states swine herds epidemiologic assessment of porcine circovirus type coinfection with other pathogens in swine a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus diversity and evolution of a newly emerged north american type porcine arterivirus: analysis of isolates collected between genetic characterization of type porcine circovirus (pcv- ) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of north america and development of a differential pcr-restriction fragment length polymorphism assay to detect and differentiate between infections with pcv- and pcv- a molecular clock dates the common ancestor of european-type porcine reproductive and respiratory syndrome virus at more than years before the emergence of disease porcine circovirus type (pcv ) vaccination of conventional pigs prevents viremia against pcv isolates of different genotypes and geographic origins the emergence of porcine circovirus b genotype (pcv- b) in swine in canada genetic and phenotypic characterization of a united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model replication and expression analysis of prrsv defective rna the origin and evolution of porcine reproductive and respiratory syndrome viruses three cases of porcine respiratory disease complex associated with porcine circovirus type infection experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus a cluster of farms experiencing severe porcine circovirus associated disease: clinical features and association with the pcv b genotype pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (prrsv) are related to viral load in acute infection porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus- (pcv- ) mortality in pigs given porcine circovirus type subgroup and viruses derived from dna clones an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome effects of porcine circovirus type (pcv ) maternal antibodies on experimental infection of piglets with pcv molecular cloning and nucleotide sequencing of the -terminal genomic rna of the porcine reproductive and respiratory syndrome virus characterization of a high-virulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses modified indirect porcine circovirus (pcv) type -based and recombinant capsid protein (orf )-based enzyme-linked immunosorbent assays for detection of antibodies to pcv porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv effects of the timing of the administration of mycoplasma hyopneumoniae bacterin on the development of lesions associated with porcine circovirus type influence of maternal antibodies on efficacy of porcine circovirus type (pcv ) vaccination to protect pigs from experimental infection with pcv porcine circovirus type (pcv )-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs differences in virulence among porcine circovirus type isolates are unrelated to cluster type a or b and prior infection provides heterologous protection experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with mycoplasma hyopneumoniae and porcine circovirus type effect of vaccination with selective bacterins on conventional pigs infected with type porcine circovirus derivation of porcine circovirus type -negative pigs from positive breeding herds porcine circovirus type (pcv- ) coinfections in us field cases of postweaning multisystemic wasting syndrome (pmws) epidemiology and horizontal transmission of porcine circovirus type (pcv ) genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope gp glycoprotein porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs vaccination of pigs against swine influenza viruses by using an ns -truncated modified live-virus vaccine experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type (pcv ) subtypes pcv a and pcv b by prolonging pcv viremia and shedding development of a polyclonal-antibody-based immunohistochemical method for the detection of type porcine circovirus in formalinfixed, paraffin-embedded tissue emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome proinflammatory cytokines and viral respiratory disease in pigs dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china porcine circovirus type (pcv ) distribution and replication in tissues and immune cells in early infected pigs complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence the authors thank the iowa livestock health advisory council for funding of this study. key: cord- -j ye ed authors: loemba, h. d.; mounir, s.; mardassi, h.; archambault, d.; dea, s. title: kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: j ye ed the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally- and experimentally-exposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the - or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus ande. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. summary. the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally-and experimentallyexposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the -or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus and e. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. the porcine reproductive and respiratory syndrome virus (prrsv) is an enveloped rna virus which has been found to be responsible for reproductive failure in sows of any parities (late-term abortions, increased numbers of stillborn, mummified and weakborn pigs) and respiratory problems affecting pigs of all ages [ , ] . this new porcine virus has been provisionally classified h.d. loemba et al. within the genus arterivirus, according to its biochemical and morphological characteristics [ , ] . the viral genome is a positive single-stranded rna molecule approximately kb in length that contains eight open reading frames (orfs) similarly organized to that of the coronaviruses [ , ] . the virion contains three major structural proteins: a nucleocapsid protein n of kda, an unglycosylated membrane protein m (matrix protein) of - kda and a glycosylated membrane protein e (envelope associated glycoprotein) of - kda which are encoded by orfs, , and , respectively [ ] . clinical symptoms and production losses may vary widely among herds, but the vast majority of prrsv infections are subclinical [ , ] . presently some field and experimental evidence exists that indicates persistent lifelong viremia during prrsv infection [ , ] . an antibody-dependent enhancement mechanism has been reported to explain the persistent infection of porcine alveolar macrophages and circulating monocytes [ , ] . so far, little is known about the immunological status of prrsv-infected pigs. some investigators suggest that prrsv causes immunosuppression because of the recrudescence of secondary bacterial infections among the infected pigs [ ] . previous data indicated that antibodies to prrsv may be detected as soon as to weeks post-exposure using indirect immunofluorescence [ , ] and the immunoperoxidase monolayer assay [ ] . virus neutralizing antibodies were reported to appear later during the infection, usually when clinical signs are resolved [ , ] . the purpose of the present study was to evaluate the humoral immune response of prrsv-infected pigs. the kinetics of the appearance of specific igm and igg antibody titers during prrsv infection, in addition to viral structural protein specificities of the antibodies and duration of viremia have been investigated in pigs naturally-and experimentally-exposed to prrsv. the qu bec cytopathic strain iaf-klop of prrsv [ ] was propagated in marc- cells [ , a highly permissive cell line to prrsv kindly provided to us by j. kwang, u. s. meat animal research center, clay center, nebraska. serological identification of the cell culture-adapted iaf-klop strain was confirmed by indirect immunoftuorescence (iif) using monoclonal antibody (mab) sdowl [ ] , kindly provided by d. a. benfield and e. nelson, department of veterinary science, south dakota state university. this mab was found to be directed to an epitope of the n protein shared by both the north american and european isolates of prrsv [ , ] . homologous hyperimmune sera to the iaf-klop strain of prrsv were produced in pigs and rabbits, as previously described [ ] . in the first experiment, seven spf piglets were placed in a clinically prrsvinfected herd, whereas two similar piglets were introduced in a clinically-healthy neighboring barn [ ] . the spf pigs were selected from a farm free of atrophic rhinitis, mycoplasma hyopneumoniae, transmissible gastroenteritis virus, swine influenza virus, encephalomyocarditis virus, s. hyodysenteriae, sarcoptic mange, salmoneltosis, a. pleuropneumoniae and prrsv. in the second experiment, four five-week old spf piglets were intranasally inoculated with l s tcidso of the iaf-klop strain of prrsv. clinical signs of both the above piglets and a simultaneously mock-infected group of three pigs were monitored daily. blood samples were collected at days and p.i., then weekly during -day and day observation periods for sentinel piglets and experimentally-infected pigs, respectively. among the principal clinical signs observed in both naturally-and experimentally-infected pigs were intermittent raising of body temperature ( - °c), anorexia, lethargy, periocular oedema, conjunctivitis and blue discoloration of the ears. coughing and dyspnea (abdominal thumping) were usually noticed by the end of the first week of exposure [ , ] . no symptoms were observed in control piglets. for each group, one representative animal was necropsied at the end of the first week p.i. macroscopic and histopathological lesions of nonsuppurative interstitial pneumonitis were only demonstrated in the two infected pigs [ , ] . at the end of the -day observation period, no significant macroscopic and histologic lesions could be observed in pigs that had been infected experimentally. however, naturally-exposed sentinel pigs still manifested mild to moderate macroscopic and histologic lung lesions days p.i., as previously described [ ] . to attempt virus isolation, marc- cell monolayers were inoculated with sera samples collected from prrsv-infected pigs and monitored daily for the appearance of a cytopathic effect (cpe) or the presence of specific fluorescent foci [ ] . for all pigs tested (naturally-exposed and experimentally-infected) the virus could be isolated from sera samples collected by the end of the first week of exposure till the end of the observation periods (data not shown). at no time, could prrsv be recovered from serum samples collected from the control pigs. the iif test was carried out using acetone fixed prrsv-infected marc- cells, and fluorescein-conjugated anti-pig igm (kirkegaard & perry laboratories inc.) or anti-pig igg (sigma), as previously described [ ] . the iif antibody titers were expressed as the reciprocal of the highest serum dilution giving specific cytoplasmic fluorescence (table ). in both naturally-and experimentally-infected pigs, igm antibodies to prrsv were detected as early as days p.i. (iif titers of to ) and maximal titers were reached by day p.i. (iif titers of ). then, specific igm antibody titers drastically decreased until day or day p.i. specific igg antibodies were also detectable by the end of the first week of exposure, maximal titers being obtained around day to p.i. (iif titers of to ). thereafter, there was generally little change in igg antibody titers to prrsv of sera collected till the end of the -or -week observation period. the modified procedure described by yoon et al. [ ] was used for in vitro detection of virus neutralizing (vn) antibodies to prrsv. the vn antibody titers were expressed as the reciprocal of the highest dilution of sera that neutralized cpe induced in marc- cell monolayers by a constant dose of tcid o of virus. specific vn antibodies to prrsv could be detected in sera samples from only half of the infected piglets (data not shown). antibody titers > were not observed until - weeks p.i., with maximal titers ranging between ---. to study the reactivity of pig sera to prrsv specific proteins, immunoblotting experiments were performed using sucrose-gradient purified virus, as previously described [ ] . briefly, replicas of viral proteins, separated by sds-page and electrophoretically transferred onto nitrocellulose membranes, were incubated for h at °c in the presence of : dilution of the tested porcine sera. after washing in a . m tris-buffered saline (tbs) solution containing . % tween , the membranes were further incubated in the presence of : dilution of an alkaline phosphatase-conjugated anti-porcine igg (sigma). the immune complexes were revealed using a commercial alkaline phosphatase conjugate substrate kit (biorad, mississauga, ont.), containing nitroblue tetrazolium and -bromo- -chloro- -indolyl phosphate in tbs buffer. under reducing conditions, the three major prrsv structural proteins n ( kda), m ( kda) and e ( . kda) were consistently identified with the tissue cultureadapted iaf-klop strain of prrsv using homologous porcine hyperimmune serum [ ] . in the immunoblotting experiments with sera from both naturallyand experimentally-infected pigs (fig. ) , seroconversion to the m and n proteins of prrsv could not be demonstrated before the end of the first two weeks of infection. on the other hand, seroconversion to the e glycoprotein was generally obtained by the end of the third or fourth week p.i. viral protein specificity of the humoral immune response of infected pigs was further confirmed by western immunoblotting analyses using e.coli-expressed orfs to gene products. genomic rna was extracted from purified virus by the one-step guanidinium isothiocyanate-acid phenol method [ ] , then respective viral genes were amplified by rt-pcr, as previously described [ , ] . six oligonucleotide primers, containing ecori (sense primers) and bamhi (antisense primers) restriction sites at their '-end, were designed according to the sequence of the iaf-exp. strain of prrsv (embl/genebank accession number l ) [ ] . the pcr amplified products were purified using the geneclean ii as illustrated in fig. , with clinical sera samples no. and (naturallyinfected pigs), antibodies directed against the recombinant e protein could be detected by the end of the first week of infection, whereas antibodies directed against the recombinant n and m proteins could only be detected by the end of the second week of the prrsv infection. no reaction has been noticed with mbp protein (fig. c) and gst protein (data not shown) alone. in the present study, prrsv-infected piglets generally developed mild clinical signs of a respiratory disease within the first week post-exposure to the virus that lasted not more than to days. however, all the animals tested remained viremic till the end of the or -day observation period. our data are in agreement with previous observations by others who demonstrated that prrsv may persist for many weeks, even months in the infected pigs, despite their relatively high iif antibody titers [ , , ] . noteworthy, our results on the detection of igg antibodies to prrsv in infected pigs sera are also compatible with previous findings that specific igg antibodies can be detected by iif from - days p.i. up to several weeks (more than - months) after exposure to the virus [ , ] . in general, igg antibody titers detected by iif peaked by the end of the third or fourth week post-exposure. more recently, nelson et al. [ ] demonstrated that peaked igg antibody titers to prrsv (iif titers > ) may persist in experimentally-infected pigs for more than months, then decrease progressively to reach very low levels (iif titers < ) after more than days p.i. consequently, detection of igg antibodies in pig sera may indicate that the animals have been infected by prrsv in a recent or distant past. since no correlation could be demonstrated between the levels of igg antibody titers determined by iif and the viremic status of the animals, one cannot precisely pin point when the exposure to the virus had occurred or whether the pigs had been carrying the virus for a long time. interestingly, our results indicate that detection by iif of igm antibodies to prrsv may provide more precise information in the serological diagnosis of prrsv infection. indeed, the short-term persistence of circulating specific igm antibodies may be considered in the differentiation between acute and chronic prrsv infections in pigs. also in agreement with previous findings by other authors, antibodies with in vitro vn activity were relatively slow to appear and were not detected until - weeks p.i. [ , , ] . although the vn test has been reported to be less sensitive than the iif tests and immunoblotting [ ] , the long term persistence of prrsv in the experimentally-or naturally-infected animals despite high levels of antibodies to prrsv, raises the question as to what role the humoral immune response has in the protection to prrsv infection. also, it is possible for antibodies to enhance viral infection of fc-receptor-positive cells such as macrophages, by forming immune complexes that use the fc receptor to bind to the macrophages [ ] . thus, the putative protective role of neutralizing antibodies in prrsv infection needs to be further documented. western blotting analyses demonstrated that the immune response of experimentally-and naturally-infected pigs was directed to the three major viral structural proteins n, m and e, as previously reported in cases of pigs that have been experimentally-infected with the reference atcc-vr us strain [ ] . these preliminary observations were further confirmed by testing the reactivity of the porcine anti-prrsv sera against e. coli-expressed orfs to gene products. in light of our observations, it appears that antibody development to the various viral proteins in prrsv-infected pigs is chronological, initial detection of antibodies to the n, m and e proteins varied among pigs, ranging from to days p.i. these results are in agreement with previous findings by others [ , ] . however, it has been postulated that the stronger signal observed to the n and m proteins may reflect a higher molar ratio of these proteins in the virion relative to the e protein, or it may suggest a greater immunogenicity of these proteins in the pigs [ , ] . the results obtained using e. coil-expressed orfs to are in favor of the first hypothesis. indeed, antibodies to the recombinant mbp-e protein could be demonstrated as early as days pi, whereas antibodies to the n and m proteins could only be detected by the end of the second week p.i. this finding was not expected in case of the recombinant mbp-e protein since reactivity towards the viral envelope glycoprotein in immunoblotting experiments, using purified virus, could not be detected until the end of the third or fourth week p.i. this discrepancy may be due to the fact that when dealing with recombinant proteins, comparable amounts of the various proteins could be transferred onto the nitrocellulose membranes, whereas with purified viral preparations, the n and m proteins are expected to be present at a higher molar ratio comparative to the e glycoprotein [ , ] . indeed, in the case of equine arteritis virus it has been demonstrated that with the exception of gs (small envelope glycoprotein), the proteins are approximately equal in abundance, being present at a molecular ratio of (n): (m): (gi: large envelope glycoprotein) [ ] . the gs protein, which is expressed at a level similar to that of m in infected cells, is strikingly underrepresented in virus particles ( to %) [ ] . thus, it can be expected that for prrsv, the immunogenicity of the e glycoprotein in the pig is likely comparable to that of the n and m proteins. from results obtained with recombinant proteins, as well as with purified virus, there was no clear correlation between the appearance of circulating antibodies as detected by vn and iif tests, viremia and protein specificities of the circulating antibodies after various time p.i. the neutralization process may be the result of virus interaction with antibodies directed against epitopes located on different viral proteins. recently, we have been able to demonstrate that individually recombinant n, m and e proteins expressed by e. coli cells can induce the production of specific antibodies in rabbits and pigs; although iif antibody titers to iaf-klop strain ranged between to , the antisera are devoid of neutralization activity. expression of recombinant viral proteins in eucaryotic vectors should permit us to demonstrate if the conformation and glycosylation of viral envelope proteins are essential requirements for the expression of epitopes involved in virus neutralization. comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation antibody-dependent enhancement of sirs virus replication single-step method of rna isolation by acid guanidium thiocyanate-phenol-chloroform extraction molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group structural proteins of equine arteritis virus porcine reproductive and respiratory syndrome enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line antigenic comparison of canadian and u.s. isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to the nucleocapsid protein porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of quebec isolates associated to acute and chronic outbreaks of prrs detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between canadian and european strains by reverse transcription and pcr amplification molecular analysis of the orfs to genes of porcine reproductive and respiratory syndrome virus, quebec strain iaf-exp kinetics of humoral immune response to structural proteins of prrsv diagnosis of porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav characterization of proteins encoded by orfs to of lelystad virus differentiation of us and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus antibody production and blastogenic response in pigs experimentally infected with prrs virus mystery swine disease in the netherlands: the isolation of lelystad virus morrison rb (t ) an indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera persistent and contact infection in nursery pigs experimentally infected with porcine reproductive and respiratory syndrome virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection we thank louise wilson, micheline ch nard, and nicole sawyer tbr their technical assistance. this work was supported in part by grants received from the national research council of canada (strategic grant # strgp- ), the minist re de t'agriculture, des p~cheries et de l'alimentation du qu bec, la f d ration des producteurs de porcs du quebec, and vetrepharm research inc., london, ontario, canada. received august , key: cord- - e u authors: paploski, igor adolfo dexheimer; corzo, cesar; rovira, albert; murtaugh, michael p.; sanhueza, juan manuel; vilalta, carles; schroeder, declan c.; vanderwaal, kimberly title: temporal dynamics of co-circulating lineages of porcine reproductive and respiratory syndrome virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e u porcine reproductive and respiratory syndrome virus (prrsv) is the most important endemic pathogen in the u.s. swine industry. despite control efforts involving improved biosecurity and different vaccination protocols, the virus continues to circulate and evolve. one of the foremost challenges in its control is high levels of genetic and antigenic diversity. here, we quantify the co-circulation, emergence and sequential turnover of multiple prrsv lineages in a single swine-producing region in the united states over a span of years ( – ). by classifying over , prrsv sequences (open-reading frame ) into phylogenetic lineages and sub-lineages, we document the ongoing diversification and temporal dynamics of the prrsv population, including the rapid emergence of a novel sub-lineage that appeared to be absent globally pre- . in addition, lineage was the most prevalent lineage from to , but its occurrence fell to . % of all sequences identified per year after , coinciding with the emergence or re-emergence of lineage as the dominant lineage. the sequential dominance of different lineages, as well as three different sub-lineages within lineage , is consistent with the immune-mediated selection hypothesis for the sequential turnover in the dominant lineage. as host populations build immunity through natural infection or vaccination toward the most common variant, this dominant (sub-) lineage may be replaced by an emerging variant to which the population is more susceptible. an analysis of patterns of non- synonymous and synonymous mutations revealed evidence of positive selection on immunologically important regions of the genome, further supporting the potential that immune-mediated selection shapes the evolutionary and epidemiological dynamics for this virus. this has important implications for patterns of emergence and re-emergence of genetic variants of prrsv that have negative impacts on the swine industry. constant surveillance on prrsv occurrence is crucial to a better understanding of the epidemiological and evolutionary dynamics of co-circulating viral lineages. further studies utilizing whole genome sequencing and exploring the extent of cross-immunity between heterologous prrs viruses could shed further light on prrsv immunological response and aid in developing strategies that might be able to diminish disease impact. porcine reproductive and respiratory syndrome virus (prrsv), the etiological agent of prrs, is one of the most important endemic viruses affecting the swine industry in the united states (holtkamp et al., ) and globally (stadejek et al., ; vanderwaal and deen, ) . the economic impact of the disease in the united states has been estimated at $ million annually (holtkamp et al., ) . clinical signs in affected farms vary by viral variant and according to the farm's production stage (e.g., breeding or growing herd), herd management, immune status, and other factors (goldberg et al., ) . premature farrowing can occur in - % of sows in an affected farm, and up to % of piglets are stillborn during an outbreak (christianson and joo, ) . piglets may be born with low weight and can present with lethargy and anorexia, which can lead to a mortality of more than % among piglets (pejsak et al., ) . prrsvinfected pigs are also susceptible to secondary infections leading to poor average daily gain and feed conversion, further increasing production loss (solano et al., ; xu et al., ) . up to % of united states breeding herds experience outbreaks annually (tousignant et al., a) and control of the disease in the united states, europe, and globally is challenging due to high levels of antigenic variability and its rapidly expanding genetic diversity (frossard et al., ; brar et al., ; guo et al., ; smith et al., ) . porcine reproductive and respiratory syndrome virus was first recognized almost simultaneously in europe (wensvoort et al., ) and north america (collins et al., ) in the late s and early s, but genetic differences suggested a much earlier evolutionary divergence between the north american and european viral types. thus, prrsv is divided into two major phylogenetic clades, prrsv type (more prevalent in europe) and type (more prevalent in north america) (shi et al., a,b; stadejek et al., ) . within each clade, high levels of genetic and antigenic diversity exist and cross-protection is only partial (roberts, ; kim et al., ; correas et al., ) . genetic similarities between prrsv isolates have been used as a tool to understand disease transmission and epidemiology (kapur et al., ; wesley et al., ) , and several different strategies have been used for classifying isolates of prrsv into epidemiologically meaningful groups. for prrsv type , the most commonly used classification system is based on restriction fragment length polymorphisms (rflp) and sequencing, both of which are typically based on the open reading frame (orf ) portion of its genome (kapur et al., ; wesley et al., ) . the orf gene encodes for the major envelope protein (gp ), which plays a role in inducing virus neutralizing antibodies and cross-protection among prrsv variants (dea et al., ; kim et al., ) . rflps have been broadly adopted by the u.s. swine industry despite shortcomings, such as the fact that the genetic relationship between different rflp types is unclear, the potential for two distantly related viruses to share the same rflp type, and the instability of rflp-typing when assessing isolates related to each other by as few as animal passages (cha et al., ) . in , a classification system based on the phylogenetic relatedness of the orf portion of the virus's genome was proposed (shi et al., a,b ). this classification system aggregates isolates into phylogenetic lineages based on the ancestral relationships and genetic distance among isolates. using this system, nine different lineages were described within prrsv type , each of which was estimated to have diverged between and (shi et al., b) . phylogeny-based classification of organisms is seen as the most powerful and robust instrument for distinguishing between variants of a viral population (hungnes et al., ) and has been used in the study of other viral diseases (liu et al., ) . phylogeny-based classification of prrsv, rather than rflp profiling, is expected to provide fewer ambiguities and more insight into the evolutionary relatedness amongst different variants. while the existence of prrsv lineages is well established, the dynamics of their cocirculation within a given region has not been well documented. vaccination is often used as a tool to mitigate clinical impact and viral shedding (holtkamp et al., ) . although specific practices vary across farms, gilts are typically vaccinated before entering the herd, and sometimes the sow herd is mass vaccinated during the year. most commercial prrsv vaccines currently sold in the united states are considered "modified live vaccines" (mlv), which means that the vaccine is an attenuated live virus. vaccines against prrsv show different degrees of protection against homologous and heterologous challenges (cano et al., ; díaz et al., ; geldhof et al., ) ; the exact definition of what constitutes a homologous or heterologous challenge is often not clear, especially taking into consideration the genetic diversity existing within prrsv type (shi et al., b) . five major prrsv vaccines are commercialized in the united states, each developed using a different wild prrsv isolate (lineages , , , and , with the lineage vaccine being the most widely used historically). porcine reproductive and respiratory syndrome virus is known to possess a high mutation rate (hanada et al., ; brar et al., ) . genetic mutations for prrsv are thought to result from rna polymerase errors (murtaugh et al., ) and from the lack of proofreading (kappes and faaberg, ) . coupled to that, genetic recombination events can contribute to prrsv diversity (forsberg et al., ) . thus, the emergence of new variants of prrsv is expected to occur potentially through both mutation and recombination. viral variants can quickly emerge in animals (goldberg et al., ) even after inoculation with a single variant (chang et al., ) . thus, the viral population within an animal can be referred to as a viral cloud or swarm (lauring and andino, ) , which suggests that mutation has a considerable impact in virus diversification even on short time scales. in addition, it is assumed that the immune response removes genetic variants of the virus that it recognizes with high specificity, potentially creating selection pressure favoring antigenically divergent prrsv variants (murtaugh et al., ) . hypervariable portions of the viral genome may be subject to immune selective pressure (chen et al., ) ; variation in proteins coded by those sites may play a role in evasion of host immune defenses (ansari et al., ; darwich et al., ) . prrsv vaccines are known to diminish the severity of clinical signs once an infection occurs, but not to prevent an infection from occurring (lyoo, ) . at the population scale, it can be expected that most animals have some level of immunity because of the high prevalence of natural infection and widespread use of vaccine. this creates the potential for immune-mediated selection to be a driver of prrsv diversification and evolution (murtaugh et al., ) . the identification of point mutations that are undergoing positive selective pressure is often interpreted as evidence of increased evolutionary fitness (kryazhimskiy and plotkin, ) . one way to identify such sites is to evaluate dn/ds ratios, which measure the rate at which substitutions at non-synonymous sites (dn) occur relative to substitutions in synonymous sites (ds). substitutions in synonymous sites are thought to be mostly neutral, but a higher occurrence of substitutions in nonsynonymous sites can be interpreted as evidence of selective processes that favor changes in the protein sequence (kosakovsky pond and frost, ) . positive selective pressure in sites that code for epitopes recognized by the host immune system are of special interest, because they suggest that the origin of such selective pressure, if present, could be driven by the host immune response. the rapid evolution of prrsv coupled with the periodic emergence of new and sometimes more virulent viral variants creates a need to continually update our knowledge on circulating prrsv variants. reports that show the waxing and waning of different viral types in the whole north america (shi et al., b) are helpful when understanding continent-wide status of prrsv lineages. however, understanding viral dynamics on a regional scale could provide important insights into local evolutionary and ecological dynamics of prrsv, including an improved understanding of how often new variants emerge or re-emerge within the region. here, we describe the temporal dynamics of prrsv occurrence in a swine-dense region of the united states, characterizing these patterns according to orf genetic lineages and sub-lineages. we quantify the contemporary occurrence of each lineage, investigate the temporal dynamics and turnover of lineages, identify emerging sub-lineages, and examine evolutionary patterns for evidence of positive selective pressures. monitoring project (mshmp) were used for this analysis. briefly, mshmp is an ongoing voluntary producer-driven nation-wide monitoring program for endemic swine diseases that affect the u.s. swine industry. based at the university of minnesota (umn), this program collects weekly reports on the infection status of sow farms from participating swine-producing companies, veterinary practices, and regional control programs, which serves to capture the occurrence of infectious diseases in the country (tousignant et al., a,b; perez et al., ) . infection status data classifies farms into the following categories (holtkamp et al., ) : status : positive-unstable, status : positive-stable, either through use of live virus inoculation ( lvi) or use of vaccines ( vx); status : provisional negative; and status : negative. the main difference between positive-unstable (status ) and positive-stable (status vx or lvi) is that unstable herds have an active clinical outbreak and are weaning prrsv rt-pcr positive piglets. in contrast, prrsv may be still present in positive-stable herds (through use of field virus inoculation or modified live vaccine) but clinical disease is controlled and piglets weaned from such farms are prrsv-negative as a result of herd immunity, decreased shedding, and maternal antibodies (holtkamp et al., ) . mshmp collects farm-level data from approximately . million sows, which represents approximately . % of the united states breeding herd population (national agricultural statistics service [nass] , agricultural statistics board, and united states deparment of agriculture [usda], ). specific production systems (companies involved in pig production) participating in the project also share the orf prrsv sequences identified on their farms as part of routine veterinary management. for example, samples may be submitted by veterinary practitioners to determine if circulating prrsv on the farm is the same or different from the vaccine virus or a previous variant present on the farm. for this analysis, we analyzed , sequences reported between and from mshmp participants located in a relatively isolated swine-dense region in the united states with an approximate area of thousand square kilometers. production systems operating in this region account for ∼ % of the united states sow population. approximately % of farms within this region participate in mshmp and in this project in particular. sequences used in this study came mostly from sow ( . % of sequences), nursery ( . %) and finisher farms ( . %), followed by boar stud farms ( . %) and sequences without a description of their origin ( . %). sequences shared with us by project participants were sequenced according to standardized protocols adopted by laboratories at sdsu (animal disease research and diagnostic laboratory et al., ), isu (zhang et al., ) and eurofins genomics. of the orf gene sequences used in this analysis, seven had fewer than nucleotides. these were deemed incomplete and were excluded from further analysis. we also included orf gene sequences previously classified into nine different genetic lineages (shi et al., a,b) and added these to the collection of mshmp sequences. these sequences, assembled from a database of sequences that spanned from to , were used as guides to classify the mshmp sequences into the previously described genetic lineages, and will be referred to here as "anchor" sequences. we also obtained the orf gene sequences for five vaccines (ingelvac prrsv atp -genbank id dq . , ingelvac prrsv mlv -genbank id af . (both from boehringer ingelheim), fostera prrsv from zoetis -genbank id kp . , prime pac prrsv rr from merck -genbank id dq . , and prevacent, from elanco -genbank id ku . ). the ingelvac prrsv atp and fostera vaccines use isolates belonging to lineage , while ingelvac prrsv mlv uses a lineage isolate, prime pac a lineage isolate and prevacent a lineage isolate. we also obtained two prrsv prototypes (lelystad -genbank id nc_ . , and vr -genbank id ef . , which represent the prototypical european type and north american type viruses, respectively). the sequence dataset used here is sequences were aligned using the muscle algorithm implemented in aliview (larsson, ) using default settings. the alignment was then examined for the presence of recombinants using the recombinant detection program version (martin et al., ) , followed by removal of potential recombinants. in addition, duplicated sequences (with % nucleotide similarity) were identified and set aside for the allocation of sequences into lineages. the aligned and cleaned dataset was imported into mega (kumar et al., ) , where the genetic pairwise distance was measured as a percentage nucleotide difference. using stata (statacorp, ), each of the mshmp sequences were assigned to the lineage that had the smallest genetic distance to an anchor. after sequences were classified into lineages, the duplicated sequences were allocated to their respective lineage group according to the sequence with % similarity that was kept in the lineage classification process. a flow-chart of these steps can be seen in figure . a maximum likelihood phylogenetic tree illustrating genetic relatedness of sequences was constructed based on , bootstraps, adopting the tamura-nei model for substitution of amino acids (tamura and nei, ; kumar et al., ) . clusterpicker software was used to further stratify the most abundant lineage into sub-lineages (ragonnet-cronin et al., ) , in a matter that seemed consistent with the tree main branches while still returning epidemiological meaningful sublineages. the phylogenetic tree was then colored according to the lineage classification and source of sequences (anchor versus mshmp) using microreact (argimón et al., ) . traditional bootstrap support is estimated based on resampling and replication, which tends to yield low support particularly on deep branches and in large trees with hundreds or thousands of sequences (lemoine et al., ) . branch support on the phylogenetic tree thus was evaluated using the bootstrap support by the transfer method (lemoine et al., ) . this method circumvents issues of traditional bootstrapping by assigning a gradual "transfer" index to each clade within the tree rather than a binary presence/absence index for the presence of a clade in each bootstrap (i.e., a clade is considered absent in the bootstrap replicate if the sequences found within the clade is different by even a single member). temporal changes in the frequency of different lineages was tabulated by quarter of the year. graphs representing the relative frequency of prrsv lineages over time were constructed using stata . the frequency with which each lineage occurred over different years was compared using trend analysis for proportions (using the ptrend command) in stata (statacorp, ) . for this test only, lineages with fewer than sequences overall were grouped. the ratio of synonymous to non-synonymous mutations (dn/ds) for all sites in the orf gene region was calculated using the single-likelihood ancestor counting protocol (kosakovsky pond and frost, ) , implemented on the datamonkey webserver . because the analysis can only be performed on sequences at a time, the analysis was repeated on ten random subsets of sequences (after removal of % identical sequences). sites were considered under positive selective pressure if the p-value associated with a higher rate of non-synonymous versus synonymous mutations was smaller than . . the dn/ds (re-scaled for branch length) of all sites from different runs were averaged and the percentage of runs in which each codon was identified as under significant positive selection was calculated. after removal of the seven inadequately sized and two recombinant sequences from the mshmp data, the remaining , mshmp sequences were classified in five different lineages. . % ( , sequences) were classified as lineage , . % ( ) as lineage , . % ( ) as lineage , . % ( ) as lineage , and . % ( ) as lineage . a group of . % ( ) of the mshmp sequences were genetically closer to the european prototype (lelystad) reference, and were thus classified as type prrsv sequences. lineage was further separated into five sub-lineages (a to e). out of the total , sequences in lineage , . % ( ) were classified in lineage a, . % ( ) in lineage b, . % ( ) in lineage c, . % ( ) in lineage d and . % ( ) in lineage e. the phylogenetic tree with all sequences used in the analysis can be seen on figure . using the booster method (lemoine et al., ) , branch support on main branches (lineages and sub-lineages) was above %. the within-and between-lineage nucleotide pairwise genetic distance is shown in table . in general, between lineage/sub-lineage distances are higher than within lineage variation. the distances between sublineages of lineage seem to be smaller between them than between other lineages. broad tree topology was similar when the tree was constructed using nucleotides or amino acids alignment (supplementary figure s ) . on average, the total number of sequences reported to mshmp increased by each year (supplementary table s ), and there was a clear seasonal pattern (figure b ). the first quarter of each year (january -march) was the one with highest number of sequences reported in all but year. the relative frequency of each lineage changed through time ( figure a and supplementary table s ), and specific patterns are noteworthy. first, the absolute and relative occurrence of lineage decreased over time from . % ( sequences) in to < % ( sequences) in the years - . as lineage occurrence to determine whether changes in sampling effort across time impacted general patterns observed here, we repeated the analysis five times, each time randomly sampling orf sequences per quarter. general patterns of lineage occurrence did not change, suggesting that patterns of lineage occurrence were not affected by sampling effort in each quarter (supplementary figure s ) . the visual patterns and turnover of lineages apparent in figure a were shown to be statistically significant. the increase in the frequency of lineages a, , , and type (p < . ) was significant, and changes in the grouped frequency of other lineages (a sum of lineages d, e, and , p = . ) was also significant, but with a difficult interpretation since this is an aggregate of several uncommon lineages. lineages b and c increased in frequency and then decreased (p < . ). lineage frequency decreased over time (p-value < . ), while lineage occurrence remained unchanged (p-value = . ). a total of sites were identified as under positive selection in at least one single-likelihood ancestor counting run (figure ) . some sites were identified as under positive selection in all runs, while others were only identified in some runs. those identified in all runs (with the largest p-value across all runs), were sites (p-value = . ), (p-value = . ), (pvalue < . ), (p-value < . ), (p-value < . ), (p-value < . ), (p-value = . ), and (p-value = . ). a list of all sites identified as under positive selection in at least one run can be found in the caption of figure . most of the sites positively selected were located in the first third of the prrsv orf . the infection status of farms part of mshmp in the studied area over the study time span is shown in figure . this data show two periods in which vaccine usage increased, the first one in mid- , and a second in approximately mid- . not all farms that reported its status to mshmp contributed to sequences to this analysis. we documented the circulation, emergence and sequential turnover of multiple prrsv lineages in a single united states swine-producing region over a span of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . by classifying over , prrsv orf contemporary sequences into phylogenetic lineages based on pre- data (shi et al., a,b) , we illustrated the continual diversification and temporal dynamics of the prrsv population. through further stratifying lineage into three main sub-lineages, we also describe the rapid emergence of a sub-lineage ( a), which was absent in the pre- analysis even though that dataset was based on > sequences from across the world (including the region in which we collected our samples) (shi et al., b) . we also identified sites within prrsv orf gene and resultant orf protein that showed evidence of positive selective pressure, indicating that non-synonymous mutations that lead to amino acid changes in the protein at these sites are favored. from to , lineage was the most prevalent genetic group observed in our dataset. shi et al., a,b showed that lineage was rapidly increasing in genetic diversity, which is a proxy for the effective population size of the virus, from to , and reached a peak from to . our data suggests that, at least for our study region, the occurrence of lineage peaked pre- , after which it rapidly declined and was replaced mostly by lineage variants. from to , three different major sub-lineages within lineage emerged, two of those being the most prevalent lineage in certain years ( c from to , a from to ). the emergence of sublineage a, beginning in and peaking in was perceived by veterinarians in the studied area as being a noteworthy event coinciding with the spread of the - - rflp-type. in our dataset, . % of the sequences belonging to the a sub-lineage were rflp-typed as - - (followed by . % of sequences with rflp - - and less than % of - - , - - , - - , - - and several others with less than % -see supplementary table s ) . while the failure to achieve consistent and reliable prrsv control and prevention through vaccination demonstrates gaps in our understanding of prrsv immunology (murtaugh, ) , based on current understanding, prrsv vaccines are expected to better protect against wild viral variants that have a higher degree of similarity to the original parental isolate used for vaccine development (cano et al., ; díaz et al., ; geldhof et al., ) . despite our limited understanding of heterologous cross-protection for prrsv, the emergence and sequential dominance of different variants leading to lineages and sub-lineages is consistent with the theory of multi-strain dynamics (gupta et al., ; kucharski et al., ) . immune responses, whether originating from human interventions or accumulation of immunity toward wild variants, can exert selective pressure that can ultimately lead to the emergence of new pathogen sub-populations (gupta et al., ) . as a virus evolves, immune responses generated against a past variant are expected to become less effective, resulting in a highly complex system, with different lineages interacting through the partial cross-immunity that they generate in the host population (gupta et al., ; kucharski et al., ) . theory predicts that due to frequency-dependent selection amongst co-circulating viral variants, rare antigenic variants are expected to spread more widely in the host population but then subsequently decline as herd immunity rises. such dynamics have been more thoroughly understood for influenza a (webster et al., ; mccullers et al., ; ferguson et al., ; nelson et al., ) and hiv (mcmichael et al., ) . for prrsv, recent research demonstrates that antibodies can exert a strong selective pressure to viral pathogens by targeting specific viral sub-populations, while allowing for the establishment of other sub-populations (wang, ) . when comparing prrsv genetic diversity before and after vaccine adoption in south korea, prrsv vaccination was suggested to increase viral genetic heterogeneity and the emergence of new glycosylation sites in viral populations (kwon et al., ) . however, the extent in which prrsv immunity, whether from natural infection or vaccination, can potentially drive the evolution of the virus in the field remains largely unanswered. our data does show a dominance of non-vaccine related lineages over time, which leads to speculation that these lineages have partially escaped the immunity induced by commercial vaccines or natural infection by variants in other lineages. prrsv vaccines do not protect against infection (scortti et al., ) , but diminish clinical signs and improve animal performance (cano et al., ) . since our project did not evaluate clinical signs of animals, it is difficult to assess the effects of vaccination in that regard. however, despite high region-wide vaccine usage from onward (figure ) , lineage a spread widely in the studied region, suggesting that vaccination and other biosecurity measures were insufficient to limit the transmission of lineage a. lineages shown (figure ) and discussed here and elsewhere are based on phylogenetic relationships in the orf region, and might not be predictive of cross-protection and immunological responses developed by hosts when faced with viruses belonging to different lineages. despite that, the lineage classification protocol used in this study did reveal temporal patterns consistent with what is expected based on epidemiological theory related to the spread of disease in immunologically naive populations. for example, epidemic-shaped curves of occurrence of different prrsv populations were seen, a pattern consistent with the spread of new pathogens (or subtypes) within a naive population. new (sub-) lineages may potentially be able to become the dominant prrsv in the population if they are sufficiently immunologically distinct to overcome herd immunity, and herds with different levels of immunity induced by pre-exposure protocols or natural infections might create selective pressure that changes how fast a new viral variant is selected in that population. for prrsv, it is apparent that protection against homologous prrsv is more robust than against heterologous variants, though the definition of what constitutes a heterologous virus is highly variable (cano et al., ; díaz et al., ; geldhof et al., ) . at the same time, genetic distance has not been shown to correlate with cross-protection, perhaps because pairwise nucleotide identity fails to capture key mutations that impact cross-protection. studies that further explore the immunological cross-reactivity among prrsv lineages are needed. with the data available in this study, it was not possible to investigate the occurrence of specific lineages with vaccination use and more precisely to which vaccine each farm/system used or to which virus was circulating previously on a specific farm. mshmp data of farms from systems that contributed sequences to this paper ( figure ) show two periods in which vaccine usage increased. the first increase in mid- , and a second in approximately mid- . the second spike in vaccine usage coincided with when lineage a began spreading in the study area. it is possible that this second spike in vaccine usage was a reaction to the shift in circulating lineages (more specifically, to the emergence of lineage a prrsv). it is also possible that the increased use of vaccines onward (shown on figure ) and the occurrence of lineages b and c (shown on figure ) immunologically selected sequences in a manner that allowed for the emergence of lineage a in . by mid- , a proportion of farms began using live virus inoculation (lvi). this strategy refers to the use of controlled exposure in gilts through inoculation with live virus isolated from recent clinical outbreak(s) at the farm (desrosiers and boutin, ) . the rationale is that by exposing gilts to virus found in a farm, gilts will mount "homologous" immunity to that specific wildtype virus and contribute to herd immunity and thus stability. according to veterinarians in the area, the increased use of lvi was due to the circulating virus being "different enough" from the viruses used in commercial vaccines. the practice of lvi in the systems here reported began primarily in (figure ). it is difficult to assess the impact that lvi might have on immunologically selecting for specific viral populations within specific lineages, especially with the aggregated data used in this analysis. while the inability of vaccination to control the spread of prrsv lends credence to immunological selection as a driver of prrsv diversification (murtaugh et al., ) , the impacts that immune-driven selection could have on long term prrsv evolution remain unknown. recording exposure procedures (lvi or vaccine use) within farms is crucial when trying to interpret longitudinal patterns of occurrence of prrsv. in future research aimed at more robustly testing hypotheses about immunity as a driver of evolutionary change, this crucial information would allow for investigation of frequencies in which specific lineages occur in farms pre-and post-vaccine/lvi adoption. within orf , we found sites under positive selective pressure within or near two hypervariable regions (figure ; hanada et al., ; delisle et al., ) located near the principal neutralizing epitope (pne). the pne is located between amino acids - and forms an ectodomain which triggers antibodies development during prrsv infection (plagemann et al., ; hanada et al., ) . the flanking hypervariable regions can be linked to the development of an immune response that block accessibility of antibodies to the pne (popescu et al., ) , including n-linked glycosylation sites such as n , n , and n (ansari et al., ) . in general terms, glycosylation may modulate protein-protein interactions, whether these proteins involve the humoral or cellular immune response of the host (lisowska, ) . in prrsv, there is evidence that these glycosylation sites play a role in glycan shielding, which is an important mechanism by which the virus evades neutralizing immune responses (vu et al., ) . while our findings do not explicitly explain the change in lineage, it does raise one hypothesis of the mechanism behind such change. further studies on how specific portions for the genome, both within orf and the whole genome, modulate immune recognition and possibly selective pressure are needed. we also consistently identified positive selective pressure within the pne region, specifically for amino acid . the identification of positive selective pressure in this region suggests that viral variants with different amino acid composition in that region may experience higher fitness and thus are favored. since this region seems to be the primary binding site of neutralizing antibodies developed during prrsv infection (plagemann et al., ; kim et al., ) , this suggests that the reason for such selective pressure could be immune in nature. such a scenario has been considered as a possible explanation for long-term evolution of rna viruses (domingo et al., ; pérez-sautu et al., ) . additional in vitro research is necessary to further clarify the immunological importance of sites identified in our analysis. however, our results suggest the plausibility of a scenario where prrsv variants with mutations in key immunological regions are able to evade immune responses and thus persist and spread within host populations with partial immunity (figure ) . further studies to investigate the role of an incomplete immunity on the evolution of prrsv are required. other mechanisms that might change the ability of the virus to infect hosts have also been proposed. non-muscle myosin heavy chain (myh ) is a molecule that has been shown to be an essential host factor for prrsv infection (gao et al., ) . myh interacts with prrsv glycoprotein (coded for by orf ), changing cell susceptibility to infection. further studies that investigate the contribution that molecules such as myh have on the infection of different orf prrsv variants are needed. additionally, non-neutralizing antibodies can delay the induction of neutralizing antibodies (ostrowski et al., ) in prrsv infection. indeed, the mean level and duration of viremia in pigs was greater among animal injected with sub-neutralizing prrsv-specific igg antibodies (yoon et al., ) , suggesting the existence of an antibody-dependent enhancement (ade) effect in prrsv. the extent in which prior exposures to the virus can elicit such effect, and how this may relate to emergence of new viral variants, also remains uncertain. as an epidemiologic study relying on secondary data generated at the population level, this study has several limitations. our sequence data were generated by different production systems that differ in number of farms, number of samples submitted, management practices, and health monitoring protocols. because of that, information may be incomplete and interpretation of data might not always be straightforward. for example, the reason for sample collection (clinical outbreak or routine monitoring), sample composition (single versus pool of animals) and type of sample (serum or tissues) is not always clear. the lack of a denominator (total amount of animals sampled in a farm, total number of farms tested) does not allow for the calculation of risk indicators for disease occurrence. data contribution by each system also varies with time. however, restricting the data to only the periods in which all systems contributed to the dataset would limit our ability to visualize long-term trends. additionally, the production system that was responsible for % of all sequences was present in the study for the entire study period. therefore, we believe that biases introduced by this issue were likely small and would not have changed the conclusions of our work. in this united states region, systems that participate in the mshmp represent approximately % of the swine farms. the remaining % of farms belong to smaller systems in the area or independent farmers. by having data from systems that represent the vast majority of farms in this region, we expect our data to be reasonably representative of prrsv occurrence in the region as a whole. additionally, despite the shortcomings mentioned above, the usage of mshmp data allows us to work with data directly from the systems, which might suffer less bias toward diseased animals than usual veterinary diagnostics laboratories data do. another limitation of this analysis involves the data generation process for the sequences analyzed here. production systems usually collect samples and send them to different diagnostic laboratories. laboratory details on quality of sequence reads were not available. these sequences most likely represent a consensus of viral sub-populations present within the host (goldberg et al., ; lauring and andino, ) , but further information that could help in assessing the quality of the read and the variability of sub-populations is not available. the sequences used here are from the orf gene alone and may not fully represent evolutionary dynamics elsewhere in the genome, since the orf gene represents approximately % of the whole genome of prrsv. studies that further explore whole genome sequencing as a tool to understand prrsv epidemiological and evolutionary patterns are required. factors affecting prrsv dynamics in specific farms are not clearly understood. we show overall temporal dynamics of prrsv in a swine-producing region of the united states, however, we have limited farm-level information. thus, we have limited ability to track turnover of viral variants within farms, though we expect this to be influenced by management practices, such as the vaccination protocol adopted by farms, the movement figure | we hypothesize that prrsv evolution is partially driven by immune-mediated selective pressure. immune-mediated pressure (either within an animal or during transmission between animals/farms) selects for escapee viral variants (inset). over time, the selection of escapees may allow for emergence of a heterologous viral populations (i.e., strains, genetic groups, or lineages) which are able to spread within the host population. in scenarios in which some method of pre-exposure is adopted, prevalence of immunity against specific types of prrsv is high (often artificially through vaccination or live virus inoculation) despite high population turnover, possibly favoring the occurrence of immune-mediated selection. of animals and personnel to and between farms, the proximity to other swine producing farms, how neighboring farms manage their animals, etc. pig production in the u.s. swine industry is characterized by multi-site pig production, which refers to segregating the breeding herd from the growing herd such that animals in each stage of production are housed at separate locations. multi-site production results in the movement of animals between different production sites, which can be located in different states within the united states (valdes-donoso et al., ; kinsley et al., ) . the role of animal movement in shaping the temporal dynamics of prrsv lineages is outside the scope of this study, but is an area of active research. in addition, the commingling of animals from different sources, which might have been previously exposed to different viral populations, may allow for the introduction of viral types prevalent in other parts of the country and also exacerbate the potential for recombination of viral populations. still, in our dataset we found evidence for recombination in only two mshmp sequences. immune interaction between infections of differing prrsv isolates remains poorly understood in swine. the vast adoption of control protocols that rely on imperfect immune response aimed mostly at reducing severity of upcoming infections (such as pre-exposure protocols with commercial vaccines or with lvi) suggests that a better understanding of the cross-immunity generated by infection with different isolates of the virus would be valuable to the industry as a whole. prospective studies that obtain sera from sow farms under different pre-exposure regimens and follow the farms through time recording prrsv occurrence would provide valuable information of potential cross-immunity in field conditions. of interest also is the better understanding of how the spread different lineages/sublineages are related to epidemiological data, for example, animal movement data and farm proximity. this might allow for a better comprehension of drivers for prrsv transmission while allowing for the evaluation of the effectiveness of practices aimed at reducing prrsv risk (dead animals disposal, manure composting, filtering the air of farms, to name a few). this study reflects data from a single united states region, which possibly does not reflect prrsv diversity and temporal dynamics of the whole swine industry in the country (shi et al., b) . that being said, the data presented here reflects a substantial portion of the u.s. swine industry in a region that is relatively spatially discontinuous from other swine producing regions in the united states. in addition, the general pattern of emergence and turnover of different lineages over time observed here describe an evolutionary phenomenon that is expected to also occur in other united states regions. a better understanding of the natural history of prrsv can provide insights that can potentially aid in mitigating the impact of the emergence of new viral variants as well as serving as a basis for further work exploring the evolution of prrsv and the effect this has on disease control, management and impact on the industry. here, we describe the occurrence of prrsv over years in a single united states region. we identified the emergence and turnover of different lineages and sub-lineages in the commercial pig population. such rapid turnover in the dominant lineage through time suggests that temporal patterns of prrsv occurrence are characterized by multi-strain dynamics, where different prrsv variants potentially interact through immune-mediated competition or selection. however, cross-immunity between different prrsv lineages elicited by natural or intentional infection is not fully understood, which hinders the effectiveness of disease control. more research is needed on drivers of evolution and emergence of new sub-lineages in order for the industry to be able to predict, prevent, and mitigate the impacts of prrsv. ongoing surveillance for prrsv using molecular epidemiological methods is invaluable to characterize the evolution of the virus but also to identify recent and historical trends that help understanding the natural history of prrsv in the united states. the sequence dataset used here is available in genbank under the accessions numbers mn -mn . ip and kv analyzed, conceptualized, and designed the study. cc, js, cv, and kv contributed to acquisition of the data. ip, cc, ar, js, cv, ds, and kv interpreted the data. mm aided in early interpretation of data. all authors but mm were involved in drafting the manuscript and revising it critically for intellectual content and have given final approval of the version to be published. dna sequencing of prrsv using orf influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies microreact: visualizing and sharing data for genomic epidemiology and phylogeography genomic evolution of porcine reproductive and respiratory syndrome virus (prrsv) isolates revealed by deep sequencing evolutionary diversification of type porcine reproductive and respiratory syndrome virus effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs instability of the restriction fragment length polymorphism pattern of open reading frame of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs orf of porcine reproductive and respiratory syndrome virus (prrsv) is a target of diversifying selection as infection progresses from acute infection to virus rebound porcine reproductive and respiratory syndrome: a review isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype i strains current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates porcine reproductive and respiratory syndrome virus diversity of eastern canada swine herds in a large sequence dataset reveals two hypervariable regions under positive selection an attempt to eradicate porcine reproductive and respiratory syndrome virus (prrsv) after an outbreak in a breeding herd: eradication strategy and persistence of antibody titers in sows characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection basic concepts in rna virus evolution ecological and immunological determinants of influenza evolution the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe porcine reproductive and respiratory syndrome virus: genetic diversity of recent british isolates myh is an essential factor for porcine reproductive and respiratory syndrome virus infection comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection associations between genetics, farm characteristics and clinical disease in field outbreaks of porcine reproductive and respiratory syndrome virus the prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in china: a molecular epidemiological perspective chaos, persistence, and evolution of strain structure in antigenically diverse infectious agents the origin and evolution of porcine reproductive and respiratory syndrome viruses assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers terminology for classifying the porcine reproductive and respiratory syndrome virus (prrsv) status of swine herds molecular epidemiology of viral infections. how sequence information helps us understand the evolution and dissemination of viruses. review article prrsv structure, replication and recombination: origin of phenotype and genotype diversity. virology - genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states significance of genetic variation of prrsv orf in virus neutralization and molecular determinants corresponding to cross neutralization among prrs viruses characterization of swine movements in the united states and implications for disease control not so different after all: a comparison of methods for detecting amino acid sites under selection the population genetics of dn/ds capturing the dynamics of pathogens with many strains mega : molecular evolutionary genetics analysis version . for bigger datasets differential evolution of antigenic regions of porcine reproductive and respiratory syndrome virus before and after vaccine introduction aliview: a fast and lightweight alignment viewer and editor for large datasets quasispecies theory and the behavior of rna viruses renewing felsenstein's phylogenetic bootstrap in the era of big data the role of glycosylation in protein antigenic properties panorama phylogenetic diversity and distribution of type a influenza virus porcine reproductive and respiratory syndrome virus vaccine does not fit in classical vaccinology rdp : detection and analysis of recombination patterns in virus genomes reassortment and insertion-deletion are strategies for the evolution of influenza b viruses in nature the immune response during acute hiv- infection: clues for vaccine development what we know about the primary immune response to prrsv the ever-expanding diversity of porcine reproductive and respiratory syndrome virus agricultural statistics board and united states deparment of agriculture multiple reassortment events in the evolutionary history of h n influenza a virus since identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain clinical signs and economic losses caused by porcine reproductive and respiratory syndrome virus in a large breeding farm monitoring the spread of swine enteric coronavirus diseases in the united states in the absence of a regulatory framework hepatitis a virus vaccine escape variants and potential new serotype emergence the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain datamonkey: rapid detection of selective pressure on individual sites of codon alignments gp of porcine reproductive and respiratory syndrome virus (prrsv) as a target for homologous and broadly neutralizing antibodies automated analysis of phylogenetic clusters using "dna finger printing" to monitor the prrs viruses infecting a sow herd reproductive performance of gilts following vaccination and subsequent heterologous challenge with european strains of porcine reproductive and respiratory syndrome virus molecular epidemiology of prrsv: a phylogenetic perspective phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses phylogenetic analysis of porcine reproductive and respiratory syndrome virus isolates from northern ireland porcine reproductive and respiratory syndrome virus (prrsv) interaction with haemophilus parasuis molecular evolution of prrsv in europe: current state of play stata statistical software: release . college station estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees comparison between the - and - annual porcine reproductive and respiratory syndrome virus epidemics in a cohort of sow herds in the united states temporal and spatial dynamics of porcine reproductive and respiratory syndrome virus infection in the united states using machine learning to predict swine movements within a regional program to improve control of infectious diseases in the us global trends in infectious diseases of swine role of animal movement and indirect contact among farms in transmission of porcine epidemic diarrhea virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein immunological selection as a driver of porcine reproductive and respiratory syndrome virus evolution and ecology of influenza a viruses mystery swine disease in the netherlands: the isolation of lelystad virus differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from north american field strains by restriction fragment length polymorphism analysis of orf secondary infection with streptococcus suis serotype increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs high-throughput whole genome sequencing of porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology we gratefully thank the contributions that emily smith and andres perez made on early stages of the project. we would like to acknowledge the industry partners who contributed to data for this analysis and to shic and mshmp in general, especially to emily geary, involved in the mshmp data curation. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -fudeixy authors: xu, kui; zhou, yanrong; mu, yulian; liu, zhiguo; hou, shaohua; xiong, yujian; fang, liurong; ge, changli; wei, yinghui; zhang, xiuling; xu, changjiang; che, jingjing; fan, ziyao; xiang, guangming; guo, jiankang; shang, haitao; li, hua; xiao, shaobo; li, julang; li, kui title: cd and papn double-knockout pigs are resistant to prrsv and tgev and exhibit decreased susceptibility to pdcov while maintaining normal production performance date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: fudeixy porcine reproductive and respiratory syndrome virus (prrsv) and transmissible gastroenteritis virus (tgev) are two highly infectious and lethal viruses causing major economic losses to pig production. here, we report generation of double-gene-knockout (dko) pigs harboring edited knockout alleles for known receptor proteins cd and papn and show that dko pigs are completely resistant to genotype prrsv and tgev. we found no differences in meat-production or reproductive-performance traits between wild-type and dko pigs, but detected increased iron in dko muscle. additional infection challenge experiments showed that dko pigs exhibited decreased susceptibility to porcine deltacoronavirus (pdcov), thus offering unprecedented in vivo evidence of papn as one of pdcov receptors. beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit papn or cd for entry. porcine reproductive and respiratory syndrome (prrs) is a highly infectious viral disease characterized by reproductive disorders including premature birth, late abortion, stillbirth, weak and mummy fetuses, and respiratory dysfunction in piglets and in growing pigs (wensvoort et al., ) . since its discovery in the united states in , prrs has rapidly spread worldwide, with frequent outbreaks causing large economic losses (holtkamp et al., ) . three surface receptors on porcine alveolar macrophages (pams) have been shown to function in prrsv invasion in vivo: heparin sulphate (hs), sialoadhesin (sn), and cd (calvert et al., ; crocker and gordon, ; jusa et al., ) . multiple studies have reported that cd is an essential receptor for prrsv infection, with scavenger receptor cysteine-rich domain (srcr ) serving as the core domain for virus recognition (calvert et al., ; van gorp et al., ; patton et al., ) . gene editing technology has been emerging as an important approach of livestock animal and plant germplasm improvement. the technology makes possible for precise modification of more than one gene simultaneously, which is particularly desirable for obtaining important economic traits that are controlled by multiple genes. in , prather's group was the first to use crispr/cas technology to generate srcr domain-targeted cd knockout pigs. they demonstrated that a cd knockout line was completely resistant to genotype prrsv infection (whitworth et al., ) . subsequently, several laboratories have generated anti-prrsv pigs targeting cd . for example, the cd srcr domain was replaced with human cd -like srcr domain to generate prrsv genotype resistance (wells et al., ) . wei et al., reported homozygous geneedited large white pigs with a bp deletion in exon of the cd gene (wei et al., ) that are fully resistant to genotype prrsv. there are also examples of deletion of the srcr domain seeking resistance to both prrsv genotypes (burkard et al., ) , or introducing a premature termination in the cd srcr domain to generate hp-prrsv (highly pathogenic prrsv)-resistant duroc pigs . deleting the srcr lbp region has also been reported to generate a prrsv genotype resistant pigs . all these studies demonstrate that prrsvresistant pig breeds can be generated by editing the cd gene, enabling alleviation of the severity of prrsv. in addition to prrsv, transmissible gastroenteritis virus (tgev), an acute high-contact infectious virus, is known to frequently occur to co-infect with other porcine diarrhea-associated viruses such as porcine epidemic diarrhea virus (pedv), porcine rotavirus (porv) (zhang et al., ) . tgev is globally distributed and causes tremendous economic losses in pork production (gerdts and zakhartchouk, ) . characterized by vomiting, severe diarrhea, and dehydration, the mortality rate of tgev-infected piglets under the age of days approaches %. tgev is a single-stranded, positive-sense rna coronavirus which targets pig intestinal epithelium for infection (brierley et al., ; wesley and lager, ) . studies have shown that the papn protein acts as a receptor in mediating tgev infection. the viral glycoproteins bind to papn receptors on the surface of small intestinal epithelial cells and mediate membrane fusion, thus resulting in the virus entering into elife digest pig epidemics are the biggest threat to the pork industry. in alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. the porcine reproductive and respiratory virus (prrs virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. two coronaviruses -the transmissible gastroenteritis virus (or tgev) and the porcine delta coronavirus -cause deadly diarrhea and could potentially cross over into humans. unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. traditionally, breeding pigs to have a particular trait is a slow process that can take many years. but with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. when viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the prrs virus relies a protein called cd , and tgev uses papn. xu, zhou, mu et al. used gene editing technology to delete the genes that encode the cd and papn proteins in pigs. when the animals were infected with prrs virus or tgev, the nonedited pigs got sick but the gene-edited animals remained healthy. unexpectedly, pigs without cd and papn also coped better with porcine delta coronavirus infections, suggesting that cd and papn may also help this coronavirus infect cells. finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. these experiments show that gene editing can be a powerful technology for producing animals with desirable traits. the gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale. epithelial cells (delmas et al., ; hansen et al., ) . inhibition or direct knockout of papn in small intestinal epithelial cells can mitigate tgev infection zhu et al., ) . papn knockout pigs are resistant to tgev (luo et al., ; whitworth et al., ) . pdcov is a highly virulent porcine coronavirus discovered in that causes watery diarrhea and vomiting in sows and piglets, with piglet mortality rates of % to % woo et al., ) . there is controversy about whether or not papn is a functional receptor for pdcov. wang et al., showed that papn functions as a receptor to promote pdcov entry into cells , while zhu et al., confirmed its involvement but showed that papn was an unnecessary important functional receptor for pdcov infection (zhu et al., ) . li et al., suggested that pdcov infection may require a co-receptor, in addition to papn . using cells isolated from papn knockout pigs, however, stoian et al., showed that these pig cells were still susceptible to pdcov infection in vitro. it was suggested that papn may be one of the receptors for pdcov, and an unknown receptor or factor may compensate for papn function in the absence of papn (stoian et al., ) . however, whether papn knockout pigs may be resistant to pdcov infection in vivo remains unknown. although gene-edited cd knockout (prrv resistant) pigs and papn knockout (tgev resistant) pigs have been previously generated, respectively, pigs that are resistant to the infection of both viruses are lacking. our objectives in the present study were ( ) to knockout cd and papn simultaneously using a gene editing approach; ( ) to verify if the resultant dko pigs are simultaneously resistant to infection by prrsv and tgev; ( ) to use the dko pigs as an in vivo experimental model to test for potential papn-mediated resistance to pdcov infection. we report successfully generated gene-edited large white pigs with both cd and papn gene knockouts using crispr/cas and somatic cell nuclear transfer (scnt). through viral challenge experiments, we found that these dko pigs exhibit complete resistance to genotype prrsv and tgev, and exhibit decreased susceptibility to pdcov infection. in addition, with the exception of meat color score and iron content, no differences in the production performance, reproductive performance, or pork nutrient content were observed between dko pigs and wt pigs. thus, in addition to demonstrating that our dko pigs are robustly resistant to both prrsv and tgev without suffering deleterious effects for production performance, our study also provides insights into ongoing controversy about the papn protein as a potential receptor for pdcov infection of pigs. in order to generate cd and papn dko cloned pigs, we constructed sgrna delivery plasmids targeting these genes, and selected successful dko pig fetal fibroblasts (pefs) as nuclear transfer donors ( figure a ). for cd , the srcr domain-binding site for prrsv in exon (van gorp et al., ; ma et al., ) was selected as the sgrna recognition site. to inactivate the papn protein, a sgrna target site in exon two immediately downstream of the atg start codon was selected ( figure b) . successful dko colonies were cultured as donor cells for scnt (supplementary file ). the cloned pigs generated in this experiment were obtained via both primary and secondary clonings. for primary cloning, the selected dko cells are used as donors for nuclear transplantation. for secondary cloning, the ear-derived fibroblasts of the primary cloned pigs are re-cloned, which rapidly provided a large number of high-quality dko donor cells, thus improving cloning efficiency and resulting in many genotypically identical pigs. in our primary cloning, a total of reconstructed embryos were transplanted into surrogate sows, of which two were pregnant and gave birth to eight live piglets. of these piglets, four survived after weaning ( figure c and supplementary file ). we determined the cd and papn genotypes of the four surviving piglets using pcr and sanger sequencing. the genotypes of the three piglets (# , # , and # ) matched that of cell colony # , which had an bp deletion on both copies of cd near the target site, and a copy of papn carrying a bp deletion on one copy and a bp deletion on the other, both resulting in frameshift mutations or premature termination after the target site ( figure d ). papn protospacer pam in order to generate more dko pigs for viral challenge experiments, we collected ear tissue samples from three piglets (# , # , and # ) and isolated ear-derived fibroblasts. a total of reconstructed embryos generated from ear-derived fibroblasts of # were transplanted into nine surrogates. four sows successful gave birth to a total of live piglets, among which survived post-weaning (supplementary file ). the genotypes of these piglets matched that of # , and the three dko primary clones used for subsequent experiments. we used flow cytometry and western blotting for cd , immunohistochemistry (ihc) and western blotting for papn, and confirmed that expression of both proteins was undetectable in dko pigs but detectable in wt pigs of the same age and breed ( figure e -g). we designed multiple pairs of amplification primers for the px vector backbone to confirm that no random integration of px vector fragments were in cloned pigs (figure -figure supplement ). we also tested for off-target modifications in dko pigs using potential off-target sites for each of the two sgrnas and found no alteration in any of these predicted sites in the cloned pigs (supplementary file ). this data demonstrates that clones of sus scrofa line with multiple gene-edited can be generated through primary and secondary cloning with high efficiency and no off-target detected. for testing of prrsv resistance in pams derived from dko pigs, we selected the highly pathogenic genotype prrsv strain wuh to challenge dko and wt pams at a multiplicity of infection (moi) of . . qrt-pcr and western blot analyses were used to assess prrsv proliferation in pams. at hr post-infection (hpi), dko pams carried a significantly lower prrsv load compared with wt pams, and no viral rna or prrsv-n protein was detected thereafter in dko pams (figure a and b). the low level of prrsv rna that was initially detectable in the dko line at hpi is likely attributable to the adsorbed prrsv independent of the existence of cd , as cd is thought to be primarily responsible for the uncoating and viral rna release processes of prrsv infection van gorp et al., ) . we next sought to examine if dko pigs are resistant to prrsv in vivo. four -day-old dko pigs and six wt control pigs of the same age were challenged with the prrsv strain wuh . nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml) were used to infect both experimental groups. the phenotypic data of body temperature, feed intake, respiration, defecation, and mental condition were recorded daily after infection. as shown in figure c , while fever (over ˚c) began at day post-infection (dpi) and persisted throughout the remainder of the experimental period in the wt group, the body temperature of the dko pigs stayed normal throughout the days of the post-viral challenge observation period. scoring for other clinical symptoms of prrsv at dpi showed that wt pigs exhibited decreased appetite, shortness of breath, cough, malaise, drowsiness, and difficulty walking, whereas the dko group displayed no abnormalities except for a brief cough and diarrhea in two pigs at dpi and dpi, respectively ( figure d ). the body weight of the dko pigs increased, throughout the day post viral challenge observation period: the detected body weights of the wt pigs were all lower than dko pigs after dpi ( figure e ). of the six challenged wt pigs, one was slaughtered at dpi to harvest pams, and the five remaining wt pigs died within dpi. in sharp contrast, all four pigs in the dko group remained healthy, and survived for the entire duration of the -day experiment ( figure f ). among the dead and slaughtered wt pigs, the lungs were swollen, with severe bleeding, and obvious lesions, while the lung tissues of dissected dko pigs did not exhibit lesions or any other distinct symptoms associated with prrsv ( figure g ). hematoxylin and eosin (h and e) staining showed thickening of the alveolar walls and infiltration of a large number of inflammatory examination of prrsv antigens in lung tissue via ihc, it was revealed that the viral antigens were present in the lungs of the wt group, but not that of the dko pigs ( figure h , lower panel). moreover, we measured the prrsv viral load in the serum of both groups at , , , , and dpi and found that in the wt group, the prrsv load increased rapidly and significantly by dpi, reaching its maximum at dpi. in agreement with other experiments showing viral resistance, the prrsv viremia in the dko group remained negative throughout the challenge ( figure i ). we also tested the prrsv viral load in pams, lung tissues, and tonsil tissues of the two groups of pigs after viral challenge. whereas a high titer of prrsv was detected in all tissues examined in the wt group, prrsv was almost undetectable in dko pigs ( figure j ). from dpi, the amount of prrsv-specific elisa antibodies in the serum of wt pigs increased significantly, and antibody levels were positive (s/ p! . ) at and dpi, while such antibodies in dko pigs remained consistently negative (s/p< . ) ( figure k ). taken together, these results provide compelling in vitro and in vivo evidence that the dko pigs are resistant to prrsv infection. following characterization of prrsv resistance, we next sought to determine if double knockout of cd and papn also conferred resistance against tgev. four -day-old dko pigs and six wt control pigs of the same age and breed were fed under the same conditions and infected with tgev. a total of ml of tgev (  tcid /ml) were orally administered to each pig in two doses (day and day , ml/day). at dpi, one dko pig and one wt pig were slaughtered to collect intestinal tissues for pathological examination, and the remaining pigs were housed under regular husbandry conditions until slaughter, and tissues were sampled at dpi. body temperature was recorded daily beginning at day , prior to inoculation, and piglet weighing and blood sampling for serum separation were conducted at , , and dpi. during the viral challenge period, no abnormalities were observed among the pigs, with the exception of two wt pigs that had diarrhea. there was no significant difference in weight gain between the two groups (data not shown). detection of tgev-specific neutralizing antibodies in serum showed no neutralizing antibodies in the dko pigs throughout the experiment, while two of the wt pigs were positive for neutralizing antibodies at dpi, and all wt pigs were positive by dpi ( figure a) . all slaughtered pigs from both wt and dko groups (sampled at dpi and dpi) were dissected to examine potential lesions in small intestine tissues. for the dko group, no lesions were found in the small intestine samples collected at either dpi or dpi ( figure b ). in marked contrast, wt group tissues collected at dpi demonstrated a thin and yellowing small intestine wall, with hemorrhages typical of tgev clinical symptoms, and by dpi there were notable duodenum, jejunum, and ileum hemorrhages, accompanied by intestinal wall thinning and enlarged mesenteric lymph nodes ( figure b ). pathological examination of small intestine tissue sections revealed pathological changes, including necrosis and shedding of intestinal mucosal epithelial cells, intestinal villi fusion, plasma cells accumulating in the lamina propria, and infiltration of eosinophils in the duodenum, figure continued in serum. wt: to dpi, n = ; dpi, n = ; dpi, n = . dko: to dpi, n = . data are expressed as means ± sd. statistical significance was determined by student's t test; ns, p> . ; *p< . ; **p< . ; ***p< . . the online version of this article includes the following source data for figure : source data . the qrt-pcr detection of prrsv load in pams. source data . rectal temperatures of pigs. source data . clinical symptoms scores of pigs. source data . body weights of pigs. source data . the survival rate of pigs. source data . the qrt-pcr detection of prrsv load in serum. source data . the qrt-pcr detection of prrsv load in pams, lung tissues and tonsil tissues. source data . prrsv-specific antibodies in serum (s/p ration). jejunum, and ileum of wt pigs at dpi and dpi, while the same small intestine tissues in dko pigs appeared healthy ( figure c ). we also analyzed the ratio of intestinal villus height (vh) to the crypt depth (cd). the smaller the ratio, the more severe the intestinal villi atrophy. we found that compared with the mock group, the three intestinal segments of the wt group had significant intestinal villous atrophy, and the intestinal villi of these intestinal segments in the dko group did not show atrophy; that is, the degree of intestinal villous atrophy in the three intestine segments in the wt group was significantly higher than that in the dko group ( figure d) . these results consistently demonstrate that our cd /papn dko pigs exhibit strong resistance to tgev infection. pdcov is a highly pathogenic virus that has recently been shown to cause diarrhea in newborn piglets, although the functional receptors for pdcov have not yet been confirmed stoian et al., ; zhu et al., ) . whether papn functions as a receptor or co-receptor in pdcov infection of pigs remains controversial. to test the hypothesis that papn may functionally mediate pdcov infection, we tested the susceptibility of our dko pigs to this virus. two -day-old dko pigs and four wt pigs of the same age and breed were challenged with pdcov. a total of ml of pdcov ( .  tcid /ml) was orally administered to each pig in two doses (day and day , ml/day). during the days of pdcov challenge study, both the dko and wt pigs appeared normal, with no distinct differences in body temperature or weight (data not shown). blood was collected at , , and dpi to assay for levels of virus-specific antibodies. at and dpi, wt pigs were all antibody-positive, while the dko pigs were all antibody-negative at dpi, but carried antibody levels comparable to that of the wt group by dpi ( figure a ). this suggests that the double-gene knockout led to a delayed onset of humoral immunity in pigs, possibly due to delayed-immune system exposure to the virus. all pigs were slaughtered at dpi, and the small intestine tissues were collected to evaluate disease severity. it was found that the intestinal wall of the wt had become thinner, with watery fluid in the small intestine, and mesenteric hyperemia, none of which was observed in the small intestine of the dko pigs ( figure b ). pathological examination of small intestine tissue sections revealed significant lesions in the small intestine tissues of both of the wt and dko groups, which included intestinal villi fusion, infiltration of lymphocytes in the intestinal mucosa, with many lesions in the intrinsic membrane in the duodenum and jejunum tissues. in the ileum, there were signs of necrosis and shedding of intestinal mucosal intraepithelial cells and naked lamina propria. the extent of lesions in the wt pigs was more severe than that of the dko pigs ( figure c ). we also detected the ratio of intestinal villus height to the crypt depth, and found that compared with the mock group, the three intestinal segments of both of the wt group and the dko group had intestinal villous atrophy, but the degree of villous atrophy in the ileal tissue in the dko group was lower than that of the wt group ( figure d) . in addition, we tested the resistance of pams derived from dko pigs to pdcov. dko and wt pams were infected with pdcov, and indirect immunofluorescence assays (ifa), tissue culture infectious dose (tcid ) assays, qrt-pcr, and western blot analyses to assess pdcov proliferation in pams all indicated that dko pams exhibit significantly decreased susceptibility of pdcov infection compared to wt pams (figure -figure supplement ) . these data suggest that although the dko line is still susceptible to pdcov infection, the viral invasion and damage to the small intestines was partially inhibited compared to that of the wt line. we next evaluated the growth and performance indices of dko pigs. three -month-old dko large white boars and three wt large white boars of the same age were selected for slaughter testing. the live weight at slaughter, carcass weight and length, dressing percentage, ham percentage, lean rate, loin eye area, average backfat thickness, muscle ph, marbling, and drip loss were determined. as shown in table , with the exception of meat coloring score, dko pigs showed no difference in comparison with wt pigs for these indices. in addition, there was no significant difference in birth weight or in the average daily gain between wt and dko pigs (supplementary file ). most notably, the meat color score in the dko pigs ( . ± . n= ) was significantly higher than that of wt pigs ( . ± . n= ), although both were within the normal range of to according to the guideline of 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) ( table and figure a -b). since the cd protein is known to play a role in the degradation of haemoglobinhaptoglobin (hb-hp), and considering that fe is an important component of haemoglobin, we reasoned that the increased meat color score (redness) may be due to the decreased hb metabolism as a consequence of cd knockout, and subsequently mild accumulation of fe containing hb in the meat. to test this hypothesis, the meat fe level was analyzed, it was found that the concentration of fe was significantly higher in dko pigs compared to wt pigs ( figure c ). we also tested the serum haptoglobin (hp) content and found that the hp content in dko pigs was significantly higher than that of wt control pigs ( figure d ). evaluation of the nutritional components of pork such as total protein, total fat, ash, moisture, specific minerals, and amino acid content was also performed. as shown in table and supplementary file , no differences in these indices were observed between the two groups. in order to test the reproductive performance of the dko boars, semen from dko male pigs (n = ) and that of wt pigs (n = ) of the same age and breed were analyzed. it was revealed that the concentration, motility, and velocity distribution of the sperm from dko boars did not differ from wt boars (table ) . furthermore, there was no difference in the litter size between the two genotypes: dko litters were . ± . (n = , litter size from to ) and the wt litters were . ± . (n = , litter size from to ). in addition, these three dko pigs did not show any growth abnormalities or disease phenomena during the -month rearing process, and no abnormalities were observed in the main tissues and organs after slaughter (data not shown). taken together, with the exception of slight meat coloring score increase, these results show that the simultaneous, editing-based disruption of the cd and papn loci, does not affect the normal growth and reproductive performance of the resultant dko pigs. conventional breeding for complex traits using molecular marker-assisted selection is a lengthy process, requiring multiple rounds of crosses and backcrosses to introgress each individual gene. crispr/cas gene editing not only allows bypassing of this long process, but also provides a possible means to obtain multiple beneficial genotypes in a single generation while also avoiding gene penetration from donor species, thus maintaining the desirable qualities of the original species. zhou et al., first used crispr/cas in combination with scnt to generate knockout of park and pink genes, whose dysfunction are known to contribute to the early onset of parkinson's disease in humans . huang et al., got the pig model with metabolic disorder successfully by editing apolipoprotein e and low density lipoprotein receptor genes simultaneously . our study is the first report on how multiple gene edits can be combined in livestock animal to offer simultaneous resistance to two major viral infection. similar to the previous reports above, double knockout efficiency using crispr/cas -mediated dual gene editing method without any drug or flow cytometry screening was high in our study, reaching . % ( dko cell colonies out of cell colonies). in this experiment, we quickly generated a large number of dko pigs by re-cloning. we found that the re-cloning efficiency ( . %, / ) was much higher than the primary cloning efficiency ( . %, / ). a possible reason for this elevated efficiency could be that the monoclonal cells used for the primary cloning must be cultured in vitro for a long time, which has been reported to inhibit cloning efficiency (li et al., ; magnani et al., ; mastromonaco et al., ) . the donor cells used in re-cloning were ear-derived fibroblasts isolated directly from dko pigs, eliminating the requirement for a long-term, in vitro screening process. our findings support the notion that the efficiency of this approach is not gene specific, and may be applicable to the knockout of other genes that allow improving disease resistance or animal production. in , calvert et al. first discovered that cd functions as a prrsv receptor protein during pams infection, which has since been confirmed by several studies (calvert et al., ; van gorp et al., ; guo et al., ; patton et al., ) . structural studies of cd revealed that the srcr domain corresponding to cd exon seven is necessary to mediate prrsv infection (van gorp et al., ) . in recent years, several groups have successfully generated prrsv-resistant gene-edited pigs by targeting exon of the pig cd gene (burkard et al., ; the online version of this article includes the following source data for the online version of this article includes the following source data for table : source data . comparison of the concentration, motility, and velocity distribution of the sperm between dko and wt large white pigs. ; whitworth et al., ; yang et al., ) . in the present study, we used a single sgrna targeting exon of cd , generated an bp double-stranded deletion that terminated protein translation near the target site. our finding on the complete resistance to prrsv genotype in our knockout line is consistent with those previous reports. cd is known to play a role in promoting the clearance of plasma free haemoglobin (kristiansen et al., ) . our finding that the dko pigs have higher meat fe content and have elevated serum hp levels is consistent with this idea, and may explain the observed darker red color in our dko meat. interestingly, and consistent to our finding, wells et al., also reported that the serum hp levels are elevated in cd knockout pigs (wells et al., ) . despite the slight color score increase, no abnormal growth or reproductive performance was observed in our dko pigs, and the meat color of both dko pigs and wt pigs were within the normal range. production performance evaluations and identification of pork nutritional components showed that our dko pigs were indistinguishable from that of the wt pigs in growth rate and reproductive performances, except for the meat color score and iron content. however, the number of dko pigs tested by us is still small, and the production performance of dko pigs still needs to be verified in large populations in the future. apn is known to be a receptor for many coronaviruses, and studies have shown that separate domains function in virus recognition vs. hydrolase catalytic activity (reguera et al., ) . two research groups have recently demonstrated that papn knockout pigs block tgev but not pedv infection (luo et al., ; whitworth et al., ) . our data showing that papn knockout can completely prevent tgev virus infection are consistent with these recently published findings. in addition to tgev and prrsv, we also determined if papn deletion conferred protection against pdcov. apn is a receptor for multiple coronaviruses and is abundantly expressed on small intestinal epithelial cells, which has led to the speculation that papn may also be a receptor for pdcov. wang et al., and li et al., proposed that papn functions as a receptor in mediating pdcov infection wang et al., ) . however, another study found that knockout of papn in ipi- i cells inhibited but did not completely block pdcov infection, suggesting that papn was not essential for viral recognition (zhu et al., ) . taken together, these studies suggest that papn may be involved in pdcov infection, but pdcov may also be able to enter cell through other pathway(s). our results on the delayed pdcov-specific neutralizing antibodies production, and a reduced extent of gross and histopathological lesions on small intestine in dko pigs compared to wt pigs are consistent with this previous suggestion that papn may play a role but is not the only path for pdcov cell entry. interestingly, a recent study showed that pams, but not lung fibroblast-like cells, from papn knockout pigs showed resistance to pdcov infection (stoian et al., ) , a finding consistent with our in vitro experiments showing that dko pams exhibit decreased susceptibility to pdcov infection. in addition, papn knockout pigs are susceptible to pdcov when virus levels were detected using qrt-pcr, and virus neutralization activity was measured, although the extent of tissue lesions between the ko and wt groups was not compared (stoian et al., ) . our findings are in line with this study reporting that papn knockout pigs are still susceptible to pdcov. however, as reflected by the delay in neutralizing antibody response, and much lighter intestine damage in the dko pigs, the susceptibility of the papn knockout group to the virus is reduced compared that of the wt pigs, indicating the potential role of papn in mediating pdcov infection. additionally, the effect of cd knockout in the delayed adaptive immune response cannot be ignored. despite the important role of cd in innate immunity, an inhibiting effect of soluble cd on the adaptive immune system has also been reported (frings et al., ; o'connell et al., ) . it is thus possible that the delayed adaptive immune response we observed in pdcov-infected dko pigs may be associated with cd knockout-induced immunosuppression. in summary, the dko pigs generated in this study are simultaneously resistant to prrsv and tegv, and exhibit decreased susceptibility to pdcov, while maintaining the same growth and reproductive production traits when compared to wt animals. these pigs may offer breeding starting points for disease-resistant pig colony generation and will be a valuable model to help deepen our understanding of the role and mechanisms of these receptor proteins in the infection mechanisms of multiple viruses. the cd and papn genotypes of colonies and piglets born after nuclear transfer were detected by pcr and sanger sequencing. one third of the cells in the -well plate and the ear tissue were used to extract the genomic dna. the primer pairs cd -f/cd -r and papn-f/papn-r were used to amplify the sequences near the sgrna target sites in the cd and papn genes, respectively. the primer sequences are shown in supplementary file . the pcr products were genotyped by sanger sequencing. potential off-target sites were predicted using an online software: crispor (http://crispor.tefor.net/ ). we identified the potential off-target sites for each of the two sgrnas. twenty pairs of primers were designed to amplify the potential off-target sites from the genomic dna isolated from the dko pigs ( #, #, #). sanger sequencing was performed to determine whether any mutations occurred. the primer sequences are shown in supplementary file . the total protein extracted from lung tissue, liver tissue, and spleen tissue of non-challenged wt pigs and dko pigs was used to detect cd , and protein extracted from duodenal, jejunal, and ileal tissues were used to detect papn expression. whole cell lysates of prrsv-infected pams and pdcov-infected pams were used to quantify the expression levels of prrsv nucleocapsid (n) protein and pdcov nucleocapsid (n) protein, respectively. the protein samples were separated by % or % sds-page and transferred to a polyvinylidene fluoride membrane (millipore). the membrane was blocked with % skim milk for hr, and then incubated with primary antibody at ˚c overnight and secondary antibody at room temperature for hr. chemiluminescent signals were developed with supersignal west pico plus chemiluninescent substrate (thermos scientific) and captured with a tanon- (tanon). cd rabbit polyclonal antibody ( - -ap; proteintech) was used to detect porcine cd . apn polyclonal antibody (a ; abclonal) was used to detect papn, anti-prrsv-n antibody (made in our laboratory) was used to detect prrsv-n protein, anti-pdcov-n antibody (made in our laboratory) was used to detect pdcov-n protein, gapdh rabbit antibody ( ; cell signaling) or b-actin rabbit antibody (ac ; abclonal technology) was used to stain gapdh or b-actin as a loading control. hrp-conjugated affinipure goat anti-rabbit igg(h+l) (sa - ; proteintech) and hrp-conjugated affinipure goat anti-mouse igg(h+l) (sa - ; proteintech) were used as the secondary antibody. pams were isolated from dko piglets and wt piglets. the lungs were obtained from the euthanized piglets. the lung surfaces were rinsed with pbs, and pams were subsequently obtained by bronchoalveolar lavage with prmi- medium (gibco, usa). the collected lavage solution was dispensed into a ml centrifuge tube, centrifuged at g for min, and the supernatant was discarded. pams were washed again with prmi- medium and then frozen in cryopreservation solution containing % fbs and % dmso. for further in vitro infection experiments, pams were cultured in rpmi- medium with % fbs and  antibiotic antimycotic ( ; invitrogen) at ˚c/ % co , and then infected with a highly pathogenic prrsv (hp-rrsv) strain wuh (gen-bank accession number hm ) (li et al., ) at a dose of moi = . and pdcov strain chn-hn- (genbank accession number kt ) at a dose of moi = . the production of progeny prrsv was evaluated through western blot, and qrt-pcr assays, and the production of progeny pdcov was evaluted through ifa, tcid , qrt-pcr and western blot assays. pams were fixed in % formaldehyde for min at room temperature. the cells were then blocked with % bsa overnight at ˚c and incubated with mouse anti-pig cd mabs (mca pe; bio-rad) at ˚c for hr in the dark. after washing with pbs three times, pams were resuspended in pbs and immediately analyzed using a bd facsverse flow cytometer (bd biosciences, ca) and flowjo software (treestar, ca). all wt pigs used in the infection experiment were born from natural breeding, and they were matched by age and breed with the dko pigs. the four dko and six wt pigs used for prrsv wuh viral challenge were both about days old. viral inoculation was conducted by nasal intubation drip ( ml: tcid /ml) and intramuscular injection ( ml: tcid /ml). during the days of prrsv challenge, piglet rectal temperature and clinical symptoms data (feeding, breathing, defecation, mental state) were collected every morning. at the same time, piglet survival rate was recorded, blood was collected, and the piglets were weighed regularly. if any pigs died during the course of the prrsv challenge, pictures were immediately taken and samples were collected. all surviving pigs were slaughtered at days post-infection (dpi) and lung tissue was examined for disease symptoms. four dko pigs and six wt pigs were used for tgev challenge. pigs were inoculated with a total of ml of tgev strain wh- (genbank accession number hq ) (  tcid /ml) that were orally administered to each pig in two doses (day and day , ml/day). for the pdcov challenge, two dko pigs and four wt pigs were orally administered a total of ml of pdcov strain chn-hn- ( .  tcid /ml) divided into two doses delivered on day and day ( ml/day). for both the tgev and pdcov groups, the rectal temperature of the pigs was measured daily for the full day experiment and the diarrhea of the piglets was observed. blood was collected and the piglets were weighed regularly. in the tgev group, a dko pig and a wt pig were slaughtered on day , and the remaining pigs in the tgev group and all pigs in the pdcov group were slaughtered at dpi. after slaughter, the pigs were dissected to observe the gross lesions in small intestine tissue, to collect small intestine tissue samples, and to detect any pathological changes by h and e staining. meanwhile, during these days, wt pigs and dko pigs were reared under the same conditions without any virus infection, and these pigs were used as the mock group. lung tissues of pigs in the prrsv challenge group, and duodenum, jejunum, and ileum tissues in the tgev and pdcov groups were collected. the tissues were fixed in % paraformaldehyde fixative, dehydrated, embedded, and cut into ~ mm-thick sections. for histopathology, the sections were stained by h and e. for ihc, tissue sections were stained with antibodies specific to the corresponding protein antigens. tissue sections were then observed and photographed with a fluorescence microscope. the antibodies used to detect papn protein were purchased from abcam (ab ); the antibody used to detect prrsv-n protein was made by our laboratory. the blood tissues of three experimental groups of pigs were collected at different times after viral challenge and the sera were separated. for the prrsv group, the sera from all samples were subjected to prrs antibody detection by commercially available enzyme-linked immunosorbent assay (elisa) kit (idexx, me). the antibody level was determined to be negative or positive according to the s/p value. if s/p< . , the antibody is negative, and if s/p! . , the antibody is positive. in order to detect tgev-specific and pdcov-specific antibody levels in serum, we used a serum neutralization test (snt). briefly, sera were heat inactivated by min of incubation in a ˚c water bath. then serial -fold dilutions of serum samples in four replicates were mixed with tcid of tgev strain wh- in a : ration. after incubation, ml of the mixture was added into st cells (a swine testicular cell line permissive of tgev infection; atcc crl- ) at a confluence of~ %, seeded in well cell culture plates. appropriate serum, virus ( tcid , tcid , tcid , and . tcid ), and cell controls were included in this test. for about hr after incubation, the cells were monitored for tgev-specific cytopathic effects. neutralization titers were calculated as the reciprocal of the highest dilution resulting in complete neutralization. similarly, sera were diluted mixed with tcid of pdcov strain chn-hn- . in contrast, pdcov titers were assessed using llc-pk cells (a porcine kidney cell line permissive of pdcov infection; atcc cl- ) that were washed twice with dulbecco's modified eagle's medium (dmem) (invitrogen, ca), and supplemented with . mg/ ml trypsin (gibco, usa) prior to and after hr incubation with these mixtures. cells were then cultured in dmem supplemented with . mg/ml trypsin for approximately hr, and the neutralization titers of sera from pdcov group were calculated. to quantify the copies of prrsv and pdcov in the infected experimental group, we extracted prrsv rna from pams, serum, lung tissue, and tonsil tissue from both the challenge and the mockinoculated group, and extracted pdcov rna from dko pams and wt pams after infected with pdcov. rna extraction was performed using trizol reagent (omega bio-tek). the rna was reverse transcribed into cdna according to the instructions for a transcriptor first strand cdna synthesis kit (roche). the cdna was then amplified with sybr green real-time pcr master mix (applied biosystems) in an abi real-time pcr system (applied biosystems). rna copy numbers were calculated from a standard curve drawn from positive standards at different dilutions. the primers used for qrt-pcr are listed as follows: '-gcaattgtgtctgtcgtc- ' and '-cttatcctccctgaatc tgac- ' for prrsv; '-gccctcggtggttctatctt- ' and '-tccttagcttgccccaaata- ' for pdcov. dko pams and wt pams in -well cell culture plates were infected or mock-infected with pdcov at a multiplicity of infection (moi) of . at hpi, cells were fixed with % paraformaldehyde for min and permeabilized with methanol for min at room temperature. the cells were then blocked with bovine serum albumin ( %) diluted in phosphate-buffered saline (pbs) for hr, and incubated with a pdcov-n-protein-specific monoclonal antibody for hr and an alexa fluor -conjugated donkey anti-mouse igg for hr. the cell nuclei were counterstained with ', -diamidino- -phenylindole (dapi) for min at room temperature. after three washes with pbs, the stained cells were observed with an inverted fluorescence microscope (olympus ix , japan). pdcov-infected pams were frozen and thawed repeatedly to completely release viruses. next, llc-pk cells (a pig kidney cell line known to be highly permissive to pdcov infection) were seeded in -well plates and were infected with -fold serial dilutions of virus samples in eight replicates. at hpi, pdcov titers were calculated based on cytopathic effects and expressed as the tcid value per milliliter, using the reed-muench method. the amount of hp in serum was measured using an enzyme-linked immunosorbent assay (elisa) kit ( - , alpha diagnostic) specific to pig hp, as previously described . assays were performed in triplicate for each sample. the quality and performance of pigs related to slaughter were determined by a third-party testing center (the national breeding swine quality supervision and testing center (chongqing), ministry of agriculture and rural affairs of china). all testing followed the guidelines stipulated in the 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t - ) . briefly, dko pigs and control wt pigs were weighed before slaughter, euthanized after fasting for hr, and hairs, heads, hoofs, and internal organs were removed after carcass dissection. the weight of carcass, length of carcass, loin eye areas, thickness of skin, and backfat thickness of carcass were all measured. ham, skin, bone, lean, and fat were dissected from the left side of the carcass and their individual weights were determined. to evaluate meat quality, we measured muscle ph, meat color score, intramuscular fat, marbling, and drip loss of longissimus dorsi. for analysis of pork nutrition, total protein, total fat, ash, moisture, amino acid, and individual minerals, amino acids were analyzed for the longissimus dorsi. the nutritional content of the pork was tested by the beijing institute of nutritional sources. semen from dko pigs and wt control pigs were collected and returned to the laboratory in a ˚c incubator for testing their quality. the detection system was hamilton-thorne research ivos ii computer-assisted sperm analyzer to measure the concentration, motility, and velocity distribution of the sperm. all data are presented as the mean ± standard error of mean (sem). data from each of the two groups of pigs were compared with an unpaired t-test when a normal distribution was not obtained. the significance levels were set at . , . , and . , as indicated by *, **, ***, respectively. the data was analyzed with graphpad prism . . for windows (graphpad software, la jolla, california). animal experimentation: all experimental protocols related to animal work described in this study were reviewed and approved by the institutional animal care and use committee (iacuc) at the institute of animal sciences, chinese academy of agricultural sciences. all experiments were performed in accordance with the approved guidelines for animal care and management of research projects. (ias - ). decision letter and author response decision letter https://doi.org/ . /elife. .sa author response https://doi.org/ . /elife. .sa supplementary files . source data . amino acid content of dko lean meat and wt lean meat. . source data . birth weights and average daily gains of wt pigs and dko pigs from birth weight to slaughtering weight. table , table and table . quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat signaling pathway characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses generation of pigs resistant to highly pathogenic-porcine reproductive and respiratory syndrome virus through gene editing of cd properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev isolation, genomic characterization, and pathogenicity of a chinese porcine deltacoronavirus strain chn-hn- only the soluble form of the scavenger receptor cd acts inhibitory on phorbol ester-activated t-lymphocytes, whereas membrane-bound protein has no effect vaccines for porcine epidemic diarrhea virus and other swine coronaviruses modulation of cd expression by metalloprotease adam regulates porcine reproductive and respiratory syndrome virus entry highly efficient generation of pigs harboring a partial deletion of the cd srcr domain, which are fully resistant to porcine reproductive and respiratory syndrome virus infection the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers crispr/cas -mediated apoe-/-and ldlr-/-double gene knockout in pigs elevates serum ldl-c and tc levels aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells n-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus identification of the haemoglobin scavenger receptor effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses broad receptor engagement of an emerging global coronavirus may potentiate its diverse crossspecies transmissibility porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-b production aminopeptidase n-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus the crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine cd reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer monocytelymphocyte cross-communication via soluble cd directly links innate immune system activation and adaptive immune system suppression following ischemic stroke modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies highly efficient crispr/cas -mediated transgene knockin at the h locus in pigs the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus porcine coronavirus hku detected in us states porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry generation and propagation of cluster of differentiation biallelic gene edit ing pigs replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cd knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus occurrence and investigation of enteric viral infections in pigs with diarrhea in china generation of crispr/cas -mediated gene-targeted pigs via somatic cell nuclear transfer contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. for the cd gene, the sgrna was designed to target exon , and for the papn gene, the sgrna was designed to target exon . the sequences of the two sgrnas are as follows: ggaaacc-caggctggttggaggg (cd -sgrna) and gcatcctcctcggcgtggcgg (papn-sgrna). the pam is indicated in bold font. the two sgrna sequences were cloned into the px vector (addgene plasmid # ) and named px -cd and px -papn, respectively. two plasmids were extracted (tiangen, dp ) in large quantities and used to transfect the fetal fibroblasts of large white pigs. the fetuses of large white pigs at -day-old were used to isolate pefs, which were then cultured in dmem medium containing % fbs. when the cells grew to % confluence, approximately cells were transfected with px -cd ( . ug) and px -papn ( . ug) plasmids. a lonza b nuclear transfection system was used for transfection with nucleofector program t- . the entire transfection process was performed according to the kit instructions (lonza, vpi- ). cells were cultured for hr after transfection and then seeded into cm dishes at a density of cells/dish. the culture medium was changed every days, and cells were cultured for days to form singlecell colonies. single-cell colonies were transferred to -well plates for expansion culture. when cells in the -well plates reached confluence, / of the cells were taken for genotype identification, and the remaining cells continued to expand. cells with genotypes identified as double-gene mutations were cultured and frozen for scnt. the oocytes for scnt were derived from a nearby slaughterhouse, and the nuclear donor cells were the dko fibroblasts. the nuclear transfer donor cells were transferred into enucleated oocytes, and the reconstructed embryos were activated and cultured to develop into blastocysts. we then selected well-developed recombinant embryo clones to be surgically transferred into the oviduct of recipient gilts on the day after estrus was observed. after the embryo transfer, the technicians observed the estrus of the sow, and regularly checked the pregnancy by b-ultrasound. changli ge: is affiliated with shandong landsee genetics co., ltd. the author has no financial interests to declare. key: cord- - krp r authors: henao-diaz, alexandra; giménez-lirola, luis; baum, david h.; zimmerman, jeffrey title: guidelines for oral fluid-based surveillance of viral pathogens in swine date: - - journal: porcine health manag doi: . /s - - -w sha: doc_id: cord_uid: krp r recent decades have seen both rapid growth and extensive consolidation in swine production. as a collateral effect, these changes have exacerbated the circulation of viruses and challenged our ability to prevent, control, and/or eliminate impactful swine diseases. recent pandemic events in human and animal health, e.g., sars-cov- and african swine fever virus, highlight the fact that clinical observations are too slow and inaccurate to form the basis for effective health management decisions: systematic processes that provide timely, reliable data are required. oral fluid-based surveillance reflects the adaptation of conventional testing methods to an alternative diagnostic specimen. the routine use of oral fluids in commercial farms for prrsv and pcv surveillance was first proposed in as an efficient and practical improvement on individual pig sampling. subsequent research expanded on this initial report to include the detection of ≥ swine viral pathogens and the implementation of oral fluid-based surveillance in large swine populations (> , pigs). herein we compile the current information regarding oral fluid collection methods, testing, and surveillance applications in swine production. between and , the worldwide swine inventory increased from to million pigs (+ %), with the most marked growth in the developing regions of the world, i.e., africa + %, asia + %, and south america + % [ ] . over the same period, albeit with regional variations, the majority of pig production moved from smaller, farrow-to-finish enterprises into larger, multisite production systems that are highly dependent upon the interchange of animals, people, equipment, and sundries between production sites; a process that connects farms and moves infectious agents between them [ , ] . from an animal health perspective, larger pig populations combined with extensive interactions between production sites produce conditions that promote the circulation of infectious diseases within/between farms while simultaneously challenging our ability to control them [ , ] . from a business perspective, these changes have limited our ability to avoid or control the economic impact of adverse disease events and, thus, place producers at greater financial risk [ ] . in , calvin schwabe, responding to the emerging problem of multi-factorial "production diseases", noted that the simple causal models described by koch and pasteur no longer applied, i.e., predisposing causes, latency, carriers, opportunistic pathogens [ , ] . to adapt to these new conditions, he recommended on-going surveillance to establish baseline levels of disease "against which effects of intervention (control) efforts can be measured". no one followed schwabe's advice -perhaps because the serum-based surveillance methods of the time were too cumbersome and expensive for routine use in commercial swine herds. since the publication of schwabe's comments, a variety of diagnostic alternatives to serum have been described, e.g., tonsil scrapings [ ] , tonsil biopsies [ ] , tonsil swabs [ ] , blood swabs [ ] , nasal swabs [ ] , nasal wash [ ] , buccal swabs [ ] , probang samples [ ] , and oral fluids [ ] . these specimens offer new possibilities for surveillance, but are they diagnostically equivalent? direct comparisons are rare in the published literature, but it is broadly recognized that the concentration of diagnostic targets changes in various diagnostic specimens over the course of an infection with corresponding specimendependent changes in test performance. thurmond ( ) , described this process as "disease transition stages", and the corresponding changes in diagnostic performance for specific combinations of specimen and test as "diagnostic transition stages". thus, henao-diaz et al. ( ) showed that the probability of detecting porcine reproductive and respiratory syndrome virus (prrsv) ribonucleic acid (rna) in serum by reverse transcription polymerase chain reaction (rt-pcr) at days post infection (dpi) was~ % versus~ % in lymphoid tissues (tonsil) by bioassay [ ] . a complete discussion of the diagnostic performance issues intrinsic to various specimen is beyond the scope of this review, but these differences must be considered in designing sampling/testing protocols. with this caution in mind, the objective of this review is to present the fundamental concepts of oral fluid-based diagnostics and recommendations for their use in the field. saliva, mixed saliva, and oral fluid differ by composition and the method of collection [ ] . saliva is primarily produced by three pairs of salivary glands (parotid, submandibular, sublingual) and is collected using specific techniques, including cannulation of the salivary ducts. parotid glands produce a serous secretion; submandibular and sublingual glands produce seromucous secretions rich in proteins and other serum-derived components [ , ] . mixed or whole saliva, the fluid collected from the buccal cavity by spitting or drainage, is a mixture of saliva and other constituents, i.e., mucosa transudate, gingival crevicular exudate, cell detritus, tracheal-nasal secretions, food debris, gastrointestinal reflux, and serum-derived compounds [ , ] . oral fluid, as defined by atkinson et al. ( ) , is the liquid collected by placing an absorptive device in the buccal cavity. various commercial devices are available for collecting oral fluids from humans, but oral fluids are usually collected either from one individual pig or a group of pigs by suspending a length of cotton rope in the pen, then recovering the individual fluid or the aggregate fluid by compressing the rope [ ] . as reviewed by prickett and zimmerman ( ) , research on immunologic and diagnostic aspects of oral fluids from humans and domestic animals began roughly a century ago and has included work on some of the most impactful diseases of humans and livestock, e.g., poliovirus and food-and-mouth disease virus (fmdv). a key event in this timeline was the recognition that both human immunodeficiency virus (hiv) and hiv antibody ( ) were present in oral fluids from infected individuals; a finding that led to the development of commercial oral fluid hiv antibody tests ( ) [ ] . for both humans and domestic animals, there are two major issues for oral fluid diagnostic assays: ( ) antibody assays must account for the lower antibody concentration in oral fluid versus serum [ ] , and ( ) pcr assays must account for the unique characteristics of the oral fluid matrix. in sum, diagnostic assays are not directly interchangeable between specimens and must be adapted to the oral fluid matrix to provide the best diagnostic performance [ , ] . oral fluid is a dynamic and complex matrix consisting of water, hormones, metabolites, electrolytes, enzymes, antibodies, mucins, and an assortment of other proteins [ , ] . oral fluid necessarily contains components produced in buccal-associated tissues, e.g., saliva and antibodies produced by plasma cells located in salivary glands and tonsils [ ] . in addition, the oral cavity is covered with a protective layer of semipermeable mucosa tightly bound to the underlying connective tissue. the semipermeability of these tissues facilitates a continuous exchange between the circulatory/lymphatic systems and the buccal cavity by both passive and active processes: ) ultrafiltration of small molecules through intercellular gap junctions, e.g., water, ions, hormones, urea; ) transudation of components from capillaries associated with the mucosa; and ) selective and/or receptor-mediated transport of larger molecules and lipophilic compounds from capillaries, e.g., antibodies, hormones, and other proteins. likewise, a dynamic exchange of inorganic components, e.g., bicarbonate, chlorine, potassium, and phosphate, occurs as fluid moves through the salivary ducts, ultimately affecting the ph and tonicity of buccal fluids [ , ] . in addition to physiologically intrinsic constituents, oral fluids usually contain enteric micro-organisms, feed components, drug compounds and metabolites recovered by the pigs from their environment. that is, as a result of pigs' normal exploratory behavior, i.e., smelling, tasting, biting, and rooting, environmental diagnostic targets are collected in the buccal cavity [ ] -some of which are subsequently passed onto the rope and into the oral fluid specimen. this is not a negative attribute of the specimen; to the contrary, the presence of such material broadens the diagnostic utility of the oral fluid sample [ , ] . immunoglobulins (ig) m, iga, and igg reach the buccal cavity by passive diffusion and/or via receptor-mediated transportation from the circulatory and/or lymphatic systems [ , ] . in addition, all classes of antibody, including secretory iga, are produced by plasma cells located in tissues associated with the buccal cavity [ , ] . serum and oral fluid antibody isotype kinetics are similar, as demonstrated in studies on the antibody responses to african swine fever virus (asfv) [ ] , classical swine fever virus (csfv) [ ] , influenza a virus (iav) [ ] , porcine circovirus type (pcv ) [ ] , and prrsv [ ] . as observed in serum, igm is detected in oral fluids before iga and igg, but has a shorter half-life than other isotypes. iga (primarily secretory iga) appears earlier than igg but usually igg is the preferred target of oral fluid enzyme-linked immunosorbent assays (elisas) because its longer half-life and because usually provides for more diagnostically sensitive and specific assays [ , ] (fig. ) . importantly, the isotype-specific responses against each pathogen must be investigated during the process of assay development because there are exceptions to this general rule. thus pcr (or rt-pcr, depending on the genetic composition of the pathogen considered) is a general approach for detecting nucleic acids that has been adapted to a variety of diagnostic specimens, including oral fluids (table ) . most typically, the detection of viral nucleic acids in oral fluids is coincident with viremia or viral replication in buccal tissues and/or the upper respiratory tract, but viruses in the environment will also be detected. for example, pedv, porcine coronavirus (pcv ), and porcine delta coronavirus (pdcov) shed in feces were likewise detected in oral fluids [ , , ] . pcr is considered the best method for "early" detection of viral infections in oral fluids, but "early" varies among pathogens. for example, fmdv replicates in the soft palate, pharyngeal epithelium, and tonsils, and may be detected in oral fluids at ≥ dpe [ ] . pseudorabies virus (prv) first replicates in epithelial cells of the upper respiratory tract and was detected in oral fluids at ≥ dpe [ ] . csfv replicates in tonsils and lymphoid nodes has been detect in oral fluids at ≥ dpe [ ] . a further complication, is the variation in detection by pcr observed among strains of the same virus and even among specimens from the same animals. weesendorp et al. table ) . with mixed success, some of the key viral pathogens of swine (fmdv, svdv, iav, sva, and prrsv) have been isolated from oral fluids under research conditions [ , , , , ] . virus isolation is not routinely used in surveillance and will not be addressed in this review. if virus recovery is an objective, it should be attempted on optimally-collected specimens from clinically-affected individuals. similar … but not the same [ ] . similarly, differences in detection rates have been reported for pen-based oral fluids versus individual table provides examples of the detection of virus-specific nucleic acids and/or antibody in swine oral fluids, i.e., is not comprehensive b acronyms defined in the list of abbreviations and terms pig buccal or nasal swabs for animals inoculated with fmdv, iav, senecavirus a (sva), and swine vesicular disease virus (svdv) [ , , , ] . in each study, oral fluids provided higher rna detection rates than swabs (table ) . these results may be explained by the fact that swabs inherently collect a smaller amount of target and typical collection procedures further dilute the sample. in particular, placing the swab in - ml of medium for transport to the laboratory reduces the concentration of the target in the sample and, therefore, the probability of detection [ ] . in the case of buccal swabs, physiology also comes in to play. salivary glands, ducts, and small vessels are innervated by sympathetic (fight-or-flight) and parasympathetic systems. the process of restraining a pig to collect the buccal swab induces a stress response that includes vasoconstriction of vessels supplying the buccal mucosa, salivary glands, and salivary ducts. this response reduces the flow of fluids to the mouth ("dry mouth") and alters buccal fluid composition [ , , ] . oral fluid sample collection is possible because it is consistent with pigs' natural behavior. pigs are curious and will readily explore unfamiliar objects, e.g., a rope dangling in the pen, by biting and chewing [ ] . pig are also highly social and if one member of the group interacts with the rope other pigs will follow [ , ] . thus, because it is voluntary and does not require animal restraint, oral fluid sampling is a welfarefriendly process with no discomfort or stress to pigs or animal caretakers [ ] . numerous videos illustrating the oral fluids collection process are available on the internet [ ] , but the following comments may be helpful. oral fluid samples are readily collected from groups of pigs ≥ days of age by providing access to a suspended length of cotton rope for~ min [ , , ] . the majority of oral fluid research has been done on pens of pigs, in which case one rope will provide an oral fluid sample representing~ % of the animals in a min sampling [ , ] . samples are most easily collected in the morning when pigs first awake and prior to feeding, if on a feeding schedule [ , ] . allow to min of sampling time at the first collection for pigs to learn the process. thereafter, the pigs will remember and will respond quickly to the presence of the hanging rope and min should be sufficient to obtain the sample [ , , ] . to harvest the sample, remove the rope, place the wet portion of the rope inside a plastic bag, extract the oral fluid (by hand or wringer), and decant the sample into a container [ ] . oral fluids can likewise be collected from individual animals, as is done in sows, gilts, or some boar studs. however, sampling individuals tends to be less successful than group sampling, particularly older sows and boars, although "training" the animals (see description in trouble-shooting section) can be helpful [ , , ] . as opposed to nylon, hemp, or polyester, cotton rope provides the best overall diagnostic utility for antibody and nucleic acid detection [ , ] . three-stranded cotton rope~ . cm in diameter is optimal for oral fluid collection in animals ≥ kg, but younger animals prefer smaller diameter rope (≤ . cm). alternatively, individual strands of larger diameter rope can be used to meet the younger pigs' thinner rope preference [ , ] . in the field, ropes are commonly suspended from pen gates, thereby avoiding the need to enter the pen either to hang or collect the rope. however, providing additional space around the rope will increase the number of pigs interacting with the rope at any one time [ , ] . strategies for providing additional space include hanging the rope from a rafter or from a bracket extending from a side of the pen [ , ] . take care of the sample the stability of antibody in swine oral fluids is poorly described and the data available is limited to prrsv. antibody degradation in oral fluids is temperaturedependent, e.g., prrsv elisa s/p (sample-to-positive) ratios in samples stored at °c were stable for - days, with faster degradation coinciding with increasing temperature. no antibody was detected in oral fluid after h at °c [ , ] . freezing oral fluid samples for subsequent prrsv antibody testing is a safe option, i.e., antibody oral fluid is highly resistant to "freeze-thaw" degradation, as indicated by consistent prrsv elisa s/ p values in samples subjected to repeated "freeze-thaw" cycles (unpublished data). likewise, little quantitative information is available regarding the degradation of intact virus or viral nucleic acids in oral fluids. calculations based on published data [ ] estimated the half-life of rt-pcrdetectable prrsv rna in oral fluids as approximately h ( °c), h ( °c), and ≥ days (< °c). unlike antibody, nucleic acids are susceptible to freeze-thaw degradation. this effect is undoubtedly not uniform across viruses, i.e., iav rna is particularly susceptible to this effect (unpublished data). to achieve the best results on frozen samples, weiser et al. ( ) showed that thawing frozen oral fluids overnight at °c produced more prrsv rt-pcr positive results than thawing at °c ( % vs % on matched samples, respectively) [ ] . in summary, to maintain diagnostic targets (antibody and/or nucleic acids) when collecting oral fluids in the field, chill samples as soon as possible after collection by refrigeration ( °c) or by placing the samples in coolers containing crushed ice or ice packs. the cold chain should be maintained throughout transport and in the laboratory until tested. if it is not possible to maintain the cold chain and/or testing cannot be completed within days, samples should be frozen (≤ − °c). however, multiple freeze-thaw cycles should be avoided, particularly in the case of samples intended for pcr testing. it follows that, long-term storage in self-defrosting freezers must be avoided because of temperature fluctuations during the defrost cycle. attempts to stabilize oral fluids antibody using antimicrobials or oral fluids rna using nucleic acid stabilizers showed no improvement when compared to chilling ( °c) samples [ , , ] . the use of flinders technology associates (fta) cards to preserve oral fluids prrsv rna was effective, but showed significant loss of rt-pcr sensitivity [ ] . when collected under clean conditions, e.g., experimental settings, oral fluids are straw-colored and translucent. when collected under field conditions, oral fluids contain environmental contaminates, e.g., manure and feed, to varying degrees (fig. a-e) . sample contamination may be reduced by not allowing the rope to reach the floor, i.e., set the bottom of the rope at pigs' shoulder height [ , ] . in the laboratory, organic contaminants per se do not affect the diagnostic properties of the sample (nucleic acid or antibody detection), but cleaner samples are easier to process and more amenable to accurate pipetting. variable centrifugation protocols for swine oral fluids have been reported in literature, e.g., , g × h [ ] , , g × s [ ] , g × min [ ] . gibert et al. ( ) reported that centrifuging at g × min improved prrsv nucleic acids detection in spiked samples compared to no centrifuged samples [ ] . however, a "gentle" centrifugation protocol ( g × - min) should be helpful in eliminating large particles. efforts to fully remove suspended particulates, e.g., chemical [ ] , filtration [ ] , or prolonged centrifugation have shown no benefit in terms of improved diagnostic performance and may rise the sample processing time. some pigs, particularly younger pigs, may be reluctant to approach the hanging rope upon their first exposure. enticing pig participation by providing rope with flavored (fruits juice, sucrose,) [ ] , colored, or aromatic substances (garlic, unpublished data) has generally not been rewarding with the exception of one report showing a higher oral fluid collection success rate in suckling piglets exposed to rope treated with a commercial baby pig supplement [ ] . however, the initial reluctance to approach the rope can often be overcome by training the pigs, i.e., placing the rope on the floor for the pigs to investigate ( min), slowly dragging the rope to the spot where it will be hung (the pigs will follow the "tail" of the rope), and then hanging a new clean rope [ , ] . as is desirable for working with pigs, this process should be done quietly and without sudden movements so as to avoid startling the animals. some individually housed adults may also be trained (as described above) to oral fluid collection [ , ] . little information is available on collecting oral fluids from pigs housed with organic bedding or under free range conditions, i.e., situations in which the presence of a hanging rope may not attract the pigs' attention [ , ] . in free range animals, oral fluids have been collected using "samplers" consisting of short lengths (~ cm) of rope embedded with a cereal-based bait matrix. investigators have reported that the animals chewed the samplers, dropped them on the floor, and the next pig repeated the process, thereby providing an "aggregate" specimen [ , ] . under experimental conditions, this approach provided for better csfv or fmdv nucleic acids detection than oropharyngeal, nasal, or buccal swabs [ , , ] . several rope samplers can be provided to a group of pigs. samplers should be recovered while still moist to maximize oral fluid collection. as the proportion of infected pigs in a pen increases, the probability of a positive test result (pcr or elisa) likewise increases. olsen et al. ( b) quantified this relationship for prrsv by establishing prevalence ( to %) in pens of pigs using mlv prrsv vaccinated pigs ( days prior). oral fluids were then collected and tested for prrsv rna and antibody. for this review, data from olsen et al. ( b) were analyzed in a logistic regression model to establish the relationship between the positive oral fluid testing results (rna or antibody) and within pen prevalence (table ). data on detection by prevalence in pens of larger size is lacking. this is a concern because the industry is trending toward larger pens (up to pigs in some systems). if the population of interest is a pen(s), current information would suggest using one rope per pigs in order to optimize the proportion of pigs interacting with the ropes and, thereby, increase the probability of detection [ , , ] . the surveillance objective in larger populations is determining the status of the barn or the production site (multiple barns). rotolo et al. ( ) estimated the barn-level probability of prrsv detection as a function of sample size, sampling location within the barn, and prrsv prevalence (positive pens) [ ] . figure , derived from data in rotolo et al. ( ) , provides guidelines regarding the association between prevalence (number of positive pens) and the number of oral fluid samples required to detect prrsv in the barn population. for example, collecting pen-based oral fluids in a barn with~ pigs would provide for a~ % probability of prrsv detection when % of pens are positive. however, this probability increases to~ % when the number of positive pens increases to %. the original estimates were based on detection of rna, but similar estimates would be expected for antibody. likewise, a similar association would be expected for other viral pathogens. number of serum samples required to match the probability of detection for one oral fluid sample was estimated using a hypergeometric distribution when collected from a group of pigs, the aggregate and undiluted oral fluid samples represent such group of animals. further pooling oral fluid samples collected within barns or on a production site is not recommended because of the potential for creating false negatives by diluting low concentrations of nucleic acids or antibody below the assay limit of detection, as is known to occur in serum [ ] . given a limited budget for surveillance, collecting fewer samples and establish their status with confidence is preferable to collecting a higher number of samples and pooling them. regardless of the number of samples collected, rotolo et al., ( ) showed that fixed spatial sampling, i.e., spacing sampling points equidistant over the length of the barn, provided a higher probability of detection than random sampling. this reflects the spatially-dependent pattern of disease spread. that is, pathogens move from pig-to-pig and pen-to-pen; they are not randomly distributed within a barn [ ] . in commercial systems, on-going site-level surveillance based on collecting a limited number of samples from all barns at fixed time intervals is preferable to collecting many samples sporadically [ ] . a systematic approach provides real-time information on the dynamics of viral spread. for example, ramirez et al. ( ) collected oral fluids every weeks from the same pens in wean-to-finish barns on different production sites for weeks (n = oral fluid samples, representing , pigs). nucleic acid testing for pcv , torque teno virus (ttv) , ttv , iav, and prrsv showed the presence of various combinations of viral infections on a continuous basis in all barns; albeit, the timing of infections was highly variable, even among barns in the same production system [ ] . testing for surveillance is fundamentally different from testing to achieve a diagnosis. that is, diagnostic assays typically use cutoffs that find a balance between diagnostic sensitivity and diagnostic specificity (youden index). in contrast, surveillance assays must provide near-perfect diagnostic specificity (no false positives!) even at the cost of lower diagnostic sensitivity. this because false alarms trigger disruption in production and quickly poison the consumer's confidence in the surveillance system. in some cases, improvement in diagnostic specificity can be achieved with minimal impact on diagnostic sensitivity [ ] . for example, in an evaluation of a prrsv oral fluid elisa (n = oral fluid samples), henao-diaz et al. ( ) found that changing the elisa's cutoff from s/p ≥ . to s/p ≥ . lowered diagnostic sensitivity from . to . %, but raised diagnostic specificity from . to % [ ] . pragmatically, this lowered diagnostic sensitivity represented a < day delay in detection as the infected animals' antibody response reached s/p ≥ . . in the field, lower test sensitivity can be offset either by collecting more samples at each sampling or by using routine surveillance testing (every - weeks). there are no perfect tests, but some tests are better than others. thus, marked differences in diagnostic performance were reported among commercial prrsv oral fluid antibody elisas and among iav oral fluid rt-pcrs [ , ] (fig. ) . since testing cannot be perfect, the use of statistical process control charting can be used provide a historical context for interpreting test results. in addition, have a plan in place for dealing with unexpected results. the plan may include retesting the original sample (in duplicate) and/or [ ] . error bars represent the range of detection assuming assay diagnostic sensitivity of - % resampling the population. a robust confirmatory testing strategy can combine the strengths of both antibody and nucleic acid detection [ ] . from an animal health perspective, larger pig populations combined with the extensive movement of pigs, people, and material between production sites produce conditions that promote the circulation of infectious diseases within/between farms [ , ] . these circumstances challenge our ability to prevent, control, and/or eliminate impactful swine pathogens. disease management based on clinical observations is neither timely nor accurate; we require an active and systematic process that achieves the timely detection of pathogens and produces useful data that can guide management decisions. the last decade has seen significant advances in the routine use of aggregate specimens in surveillance, including oral fluids. further improvements may be anticipated as additional diagnostic assays specifically adapted to the oral fluid matrix emerge and statistical refinements in the application of oral fluid sampling to populations are achieved. food and agriculture organization of the united nations. faostat statistical database. fao. the influence of between-farm distance and farm size on the spread of classical swine fever during the - epidemic in the netherlands characterization of swine movements in the united states and implications for disease control the current epidemiological revolution in vet medicine the current epidemiological revolution in veterinary medicine. part i cost of porcine reproductive and respiratory syndrome virus at individual farm level -an economic disease model porcine reproductive and respiratory syndrome virus: routes of excretion a novel technique for the collection of ante mortem tonsil biopsies from alert swine longitudinal study of senecavirus a shedding in sows and piglets on a single united states farm during an outbreak of vesicular disease comparison of specimens for detection of porcine reproductive and respiratory syndrome virus infection in boar studs probability of detecting influenza a virus subtypes h n and h n in individual pig nasal swabs and pen-based oral fluid specimens over time oral fluids as a live-animal sample source for evaluating cross-reactivity and cross-protection following intranasal influenza a virus vaccination in pigs collection of oral fluids using cotton ropes as a sampling method to detect foot-andmouth disease virus infection in pigs the comparative utility of oral swabs and probang samples for detection of foot-and-mouth disease virus infection in cattle and pigs collection of oral fluid from individually housed sows understanding and interpreting prrsv diagnostics in the context of "disease transition stages guidelines for saliva nomenclature and collection proteomic analysis of porcine saliva saliva specimen: a new laboratory tool for diagnostic and basic investigation oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type infections the development of oral fluid-based diagnostics and applications in veterinary medicine effect of collection material and sample processing on pig oral fluid testing results detection of porcine reproductive and respiratory syndrome virus (prrsv) antibodies in oral fluid specimens using a commercial prrsv serum antibody enzyme-linked immunosorbent assay ring test evaluation of the detection of influenza a virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation a proteomic approach to porcine saliva salivary iga as a useful non-invasive marker for restraint stress in pigs principles of saliva secretion toward a better understanding of pig behavior and pig welfare bioaerosol and surface sampling for the surveillance of influenza a virus in swine exogenous source of prrsv antibody in positive oral-fluid elisa results saliva as a manifestation of the common mucosal immune system structure and function of immunoglobulins detection of african swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein (p ) dual matrix indirect elisa detection of classical swine fever virus (csfv) e and erns antibody (igg, iga) in oral fluid specimens from inoculated (ald strain) or vaccinated (lom strain) pigs kinetics of influenza a virus nucleoprotein antibody (igm, iga, and igg) in serum and oral fluid specimens from pigs infected under experimental conditions prolonged detection of pcv and anti-pcv antibody in oral fluids following experimental inoculation kinetics of the porcine reproductive and respiratory syndrome virus (prrsv) humoral immune response in swine serum and oral fluids collected from individual boars evaluation of three commercial porcine reproductive and respiratory syndrome virus (prrsv) oral fluid antibody elisas using samples of known status detection of total and prrsv-specific antibodies in oral fluids collected with different rope types from prrsv-vaccinated and experimentally infected pigs detection of porcine reproductive and respiratory syndrome virus (prrsv)-specific igm-iga in oral fluid samples reveals prrsv infection in the presence of maternal antibody porcine epidemic diarrhea virus (pedv) detection and antibody response in commercial growing pigs congenital infection with atypical porcine pestivirus (appv) is associated with disease and viral persistence detection of african swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription realtime polymerase chain reaction rope-based oral fluid sampling for early detection of classical swine fever in domestic pigs at group level detection of genome, antigen, and antibodies in oral fluids of pigs from pigs infected with foot-and-mouth disease virus development of a fmdv abc antibody elisa for swine oral fluid specimens molecular detection of porcine reproductive and respiratory syndrome virus, porcine circovirus and hepatitis e virus in oral fluid compared to their detection in faeces and serum detection of influenza a virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking elisa shedding of japanese encephalitis virus in oral fluid of infected swine. vector-borne zoonotic dis pathogenicity of nipah henipavirus bangladesh in a swine host real-time pcr detection patterns of porcine circovirus type (pcv ) in polish farms with different statuses of vaccination against pcv detection and genetic characteristics of porcine circovirus based on oral fluids from asymptomatic pigs in central china development and evaluation of antibody detection assay for pcv virus characterization and evolution of porcine deltacoronavirus in the united states porcine hemagglutinating encephalomyelitis virus: a review validation of a real-time reverse transcription pcr assay for detection of porcine kobuvirus (pkv) in porcine diagnostic samples. ames; detection, isolation, and in vitro characterization of porcine parainfluenza virus type isolated from respiratory diagnostic specimens in swine detection patterns of porcine parvovirus (ppv) and novel porcine parvoviruses through (ppv -ppv ) in polish swine farms evaluation of the serologic cross-reactivity between transmissible gastroenteritis coronavirus and porcine respiratory coronavirus using commercial blocking enzyme-linked immunosorbent assay kits diagnosis of the lelystad strain of porcine reproductive and respiratory syndrome virus infection in individually housed pigs: comparison between serum and oral fluid samples for viral nucleic acid and antibody detection detection of aujeszky's disease virus dna and antibody in swine oral fluid specimens vesicular disease in pigs inoculated with a recent canadian isolate of senecavirus a use of oral fluids for detection of virus and antibodies in pigs infected with swine vesicular disease virus efficient surveillance of pig populations using oral fluids proof of principle: non-invasive sampling for early detection of foot-andmouth disease virus infection in wild boar using a rope-in-a-bait sampling technique effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities dynamics of virus excretion via different routes in pigs experimentally infected with classical swine fever virus strains of high, moderate or low virulence sensitivity of oral fluids for detecting influenza a virus in populations of vaccinated and non-vaccinated pigs. influenza other respir viruses use of ropes to collect oral fluids from gestating sows housed in dynamic groups and fed via electronic sow feeder collecting oral fluid samples from due-to-wean litters the center of food security and publich health assessment of abattoir based monitoring of prrsv using oral fluids feasibility of pooled oral fluid collection from pre-weaning piglets using cotton ropes recommendations for pen-based oral-fluid collection in growing pigs individual and pen-based oral fluid sampling: a welfare-friendly sampling method for group-housed gestating sows detection of porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions behavioral aspects of oral fluid sample collection effect of chemical clarification of oral fluids on the detection of porcine reproductive and respiratory syndrome virus igg stability of porcine reproductive and respiratory syndrome virus and antibody in swine oral fluid effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time pcr effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time pcr effect of handling and storage conditions and stabilizing agent on the recovery of viral rna from oral fluid of pigs evaluation of flinders technology associates cards for collection and transport of samples for detection of porcine reproductive and respiratory syndrome virus by reverse transcription polymerase chain reaction porcine reproductive and respiratory syndrome virus (prrsv) surveillance using pre-weaning oral fluid samples detects circulation of wild-type prrsv comparison of protocols for the analysis of type porcine reproductive and respiratory syndrome virus by rt-pcr using oral fluids the effects of flavored rope additives on commercial pen-based oral fluid yield in pigs optimising oral fluid collection from groups of pigs: effect of housing system and provision of ropes novel rope-based sampling of classical swine fever shedding in a group of wild boar showing low contagiosity upon experimental infection with a classical swine fever field strain of genotype . probability of detecting porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a function of within-pen prevalence sampling guidelines for oral fluid-based surveys of group-housed animals feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by elisa field application of a commercial porcine reproductive and respiratory syndrome virus (prrsv) oral fluid antibody enzyme-linked immunosorbent assay (elisa) continual process improvement -the case of the prrs oral fluid elisa springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements ahd received support through becas al extranjero conacyt, mexico.authors' contributions ahd and jz wrote the main part of this paper. all authors have critically reviewed the manuscript, agreed fully to the content of the review, and approved the final manuscript. authors' information ahd is a ph.d. candidate focused in prrsv epidemiology and diagnostics;lgl is an associate professor with expertise in infectious diseases and diagnostics technology development; dhb is the serology section leader in not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare no conflicts of interest regarding this publication.received: june accepted: august key: cord- -vzbh k authors: zhang, weijun; lin, yan; bai, yu; tong, tiegang; wang, qun; liu, nihong; liu, guangliang; xiao, yihong; yang, tao; bu, zhigao; tong, guangzhi; wu, donglai title: identification of cd (+ )cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vzbh k twenty-seven nanopeptides derived from the matrix (m) protein of porcine reproductive and respiratory syndrome virus (prrsv) were screened for their ability to elicit a recall interferon-γ (ifn-γ) response from the splenocytes of balb/c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus rwr-prrsv-m. we identified two peptides (amino acid residues k( )fitsrcrl and f( )gymtfvhf) as cd (+ )cytotoxic t lymphocyte (ctl) epitopes. these peptides elicited significant numbers of ifn-γ secreting cells, compared with other m nonapeptides and one irrelevant nonapeptide. bioinformatics analysis showed that the former is an h- k(d)-restricted ctl epitope, and the latter is an h- d(d)-restricted ctl epitope. multiple amino acid sequence alignment among different prrsv m sequences submitted to genbank indicated that these two ctl epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to prrsv. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine viral pathogens, and has caused significant economic losses to the swine industry worldwide. characterization of field isolates suggested that prrsv are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. based on significant sequence differencesprrs viruses are grouped into two distinct genotypes, european isolate (lelystad virus, lv) and north american isolate (vr- ) [ ] prrsv has two major structural proteins, gp and m, encoded by orfs and , respectively. gp , the most important neutralizing antigen of prrsv, has the highest genetic diversity among isolates [ ] . and, recent studies in yorkshire × landrace crossed and outbred pigs, showed that there are two immuno-dominant t-cell epitopes derived from the gp protein: l aalicfvirlaknc and k grlyrwrspvii/vek [ ] . the m protein, which contains highly conserved amino acid sequences, also has very good immunogenicity and is associated with protection against prrsv infection. dna vaccinations have also revealed that m is the most potent inducer of t lymphocyte proliferation [ ] . at present, effective vaccination strategies for the prevention and control of prrsv infection are not available. vaccines based on inactivated prrsv virus have been ineffective at inducing protective immune responses. live-attenuated prrsv vaccines can provide protection against this pathogen, but have been observed to revert to virulence [ ] , restricting the application of this vaccination approach. the rational development of future prrsv vaccines will necessitate a systematic understanding of the protective humoral and cellular immune responses that occur during prrsv infection, and should aim to induce a broad immune responses that accommodates the plasticity of the major antigenic sites. recent research has indicated that cell-mediated immunity may play a very important role in the clearance of prrsv [ ] . major histocompatibility complex (mhc) i restricted cytotoxic t lymphocytes (ctls) can kill virus-infected cells and eliminate potential sources of new virus [ ] . hence, identification of ctl epitopes is crucial in the design of synthetic vaccines, and a number of studies have successfully identified pathogen-derived t cell epitopes [ ] [ ] [ ] [ ] . unlike many other pathogens, there is limited knowledge of the specific prrsv-derived peptides targeted by t-cells. in the current study, we report the identification of ctl epitopes from the prrsv (ch- a strain) m protein in a mouse model. we screened peptides derived from the prrsv m protein for their ability to induce interferon (ifn)-γ in splenocytes harvested from balb/ c mice following dna vaccination and a booster vaccination with recombinant vaccinia virus expressing m protein. the screen identified two peptides that elicited ifn-γ production in cd + cd + splenocytes of vaccinated mice. a multiple amino acid sequence alignment among different prrsv m proteins indicates that these two peptides are strongly conserved across multiple prrsv strains and therefore should be considered for further research. the prrsv ch- a strain, the vaccinia virus wr strain, and the akabane virus obe- strain were part of our laboratory collection. the former was propagated in marc- cells, and the latter two were propagated in bhk- cells, respectively, and these two cell lines were cultured in dmem (invitrogen) supplemented with % of fetal calf serum (fcs, invitrogen) in a humidified °c, % co incubator. total rna was extracted from the prrsv ch- a strain and the spleens of balb/c mice. full length cdnas were amplified based on the complete open reading frames (orfs) of m and ubiquitin (ub) following reverse transcription (genbank accession numbers ay and az ) using the m-f and m-r primers pair or the ub-f and ub-r primer pair ( table ) . the full length cdnas were cloned into the pmd -t vector (takara) and are referred to as pmd-m and pmd-u, respectively. an indirect enzyme-linked immunosorbent assay (ielisa) method was established to evaluate specific antibody against m protein. a truncated m protein ( - aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. the truncated m gene cdna was amplified from pmd-m using the truncated-m-f and truncated-m-r primers (table ) , ligated into pgex- p- (ge healthcare), and subsequently named pgex-m. for protein expression, pgex-m was transformed into e.coli bl (de ) and recombinant protein expression was induced with . mm iptg. the samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline (pbs, ph . ). after being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and western blot with anti-prrsv-m monoclonal antibody (our laboratory collection), the fusion-expressed truncated m protein was purified by gst tag according to the manufacturer's instructions (ge healthcare). finally, the concentration of the purified protein was determined using a bradford kit (bio-rad) according to the manufacturer's instructions. the ub and m genes were fused using splicing by overlapping extension pcr (soe-pcr). the ub and m genes were amplified from pmd-u and pmd-m with the primer pairs ub-f and fusion-m-ub-r and fusion-m-ub-f and m-r, respectively ( table ). the fusion gene product, referred to as ub-m, was amplified from the purified pcr products with ub-f and m-r and inserted into the eukaryocyte expression vector pvax , and was named pvax -ub-m. the transferring vector psc (our laboratory collection), which is composed of the early promoter p . and late promoter p of vaccinia virus, and the lacz and amp genes controlled by the promoter p as the reporter genes, was used in this study. to construct the transferring vector psc -m, the complete m gene was amplified with the psc -m-f and psc -m-r primer pair (table ) and inserted into the psc . the primer p . -f (table ) , derived from promoter p . sequence, was used for directional identification and the positive clones, which were subsequently named psc -m. homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid psc -m and the wr strain vaccinia virus into % confluent tk - cells cultured in mem medium containing μg/ml brdu (sigma). the viruses were collected after the appearance of cytopathic effect (cpe), and recombinant vaccinia virus was purified according to the expression of lacz gene. western blot analysis was performed as described previously [ ] . briefly, a bhk- cell layer was infected with rwr-prrsv-m recombinant vaccinia virus or the vaccinia virus wr strain at a multiplicity of infection (moi) of . . cells were harvested days post-infection, and total cell lysates were prepared with lysis buffer ( mm tris-cl ph . , mm mgcl , . % np , μg/ml dnase i). cell lysates were separated by sds-page and were subsequently transferred to a membrane for western blot analysis using an anti-prrsv-m monoclonal antibody, hrp-conjugated goat anti-mouse igg secondary antibody (bio-rad), and dab substrate. the positive recombinant virus was named rwr-prrsv-m. the expression of m was further confirmed by ifa. briefly, bhk- cells were infected with either rwr-prrsv-m or the vaccinia virus wr strain when the bhk- cells reached - % confluence in a -well plate. m protein expression was evaluated by ifa days post infection using the anti-prrsv-m monoclonal antibody followed by a fitc-conjugated rabbit antimouse igg secondary antibody (sigma). specific fluorescence was observed using a fluorescence microscope (leica dm ire ). the sequences of m were screened for potential h -k d / h -d d /h -l d -mer epitopes using the algorithms from the syfpeithi website [ ] , the hla peptide binding prediction website (bimas) [ ] and pred balb/c [ ] . the peptides (table ) , with highest binding score (bs) as predicted by each algorithm, were synthesized by scilight biotechnology llc (beijing, china) and purified to a purity > % using high performance liquid chromatography (hplc). all animal experiments were performed according to national and institutional guidelines. one hundred and ninety female balb/c mice (vital river laboratory animal technology co., beijing, china) were maintained in isolation cages at the experimental animal center of harbin veterinary research institute (harbin, china). mice were divided into three groups: the pvax -ub-m vaccination group (n = ), the pvax control vaccination group (n = ) and the pbs control vaccinated group (n = ). the plasmid dna used for immunization was purified using the endofree mega plasmid preparation kit (qiagen). the pvax -ub-m and pvax cohorts were intramuscularly (i.m.) vaccinated with μg pvax -ub-m or pvax plasmid dna in μl pbs, respectively. the pbs control group received an i.m. injection of μl pbs. each group was vaccinated four times at -week intervals. to enhance the specific ctl responses to m protein, the mice received an intraperitoneal (i.p.) injection containing . moi of rwr-prrsv-m on day following the fourth dna vaccination. at the same time, five mice from the pvax and pbs vaccination groups were also inoculated intraperitoneally with . moi of rwr-prrsv-m on day following the fourth vaccination in order to compare the specific antibody raised against m protein of different experimental groups following vaccination with rwr-prrsv-m. all procedures were conducted with the protocols approved by experimental animal center of harbin veterinary research institute (hvri) of the chinese academy of agricultural sciences (caas). to investigate the specific antibody response, serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m. an ielisa was performed according to methods described previously [ ] . the purified m protein was used as the coating antigen, the tested sera applied at a : dilution, and an hrp-conjugated goat anti-mouse igg antibody (bio-rad) used as the secondary antibody. the microplates were developed using orthophenylene diamine (opda, amersco) and h o for min, after which the reaction was stopped by the addition of m h so . finally, the optical density (od) was read at nm. an anti-prrsv-m monoclonal antibody was used as a positive control. serum containing antibodies against akabane virus and the sera of control group mice served as negative controls. isolated splenocytes were added to u-bottomed -well plates (corning inc) at cells/well in μl complete rpmi- medium supplemented with % fcs. the cells were then mixed with μl media containing note: the peptides with highest binding score (bs) as predicted by each algorithm are shown, starting with the peptide giving the highest score at the top for each protein. sequences of mers are given from bimas, syfpeithi and pred balb/c predictions, respectively. the n-terminal amino acid position is indicated for epitopes predicted from the whole m protein. a nonamer peptide derived from prssv m protein at μg/ml, or phorbol- -myristate- -acetate (pma ng/ ml) and ionomycin ( ng/ml) as a positive control. cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (ibv) h strain [ ] were used as negative controls. following a h incubation at °c, um monensin (sigma) was added and the splenocytes were incubated for an additional h at °c before staining. the number of cd + cd + t cells producing ifn-γ on days , and after boosting with rwr-prrsv-m was determined using flow cytometric analysis. ics analysis was performed using a facscalibur flow cytometer (bd) according to the methods described previously [ ] . twenty mice were tested from the group vaccinated with pvax -ub-m, and five mice from the groups vaccinated with pvax and pbs were tested at each time point. the splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with m protein-derived peptides, as detailed in section . . ten thousand cd + t lymphocytes were acquired per sample and the number of ifn-γ-secretion cd + cd + t cells were enumerated. the cd + cd + lymphocytes that expressed ifn-γ following peptide stimulation were considered to be peptide-specific ctls. specific ctl responses were evaluated as the increase in the number of cd + cd + ifn-γ + cells. reagents used include percp-conjugated cd e, pe-conjugated cd a and fitc-conjugated ifn-γ, and bd cytofix/cytoperm™, all purchased from becton, dickinson & co (bd). to further confirm the results of the ics, the other sixty mice from the group vaccinated with pvax -ub-m and thirty mice from the groups vaccinated with pvax and pbs were tested by the ifn-γ elispot assay at three different time points (as detailed in section . ). data are expressed as the number of ifn-γ-secreting cells per two hundred thousand splenocytes. peptide-specific ifn-γ elispot responses were considered to be positive if the response (minus media background) was ≥ fold above the media response and ≥ spot-forming cells (sfc)/ × splenocytes were registered. the ielisa results, the percentage of ifn-γ positive cd + cd + t lymphocytes and the number of spots per × splenocytes were analyzed using the analysis of variance (anova), and a probability value below . was considered significant. as the full-length m protein is difficult to express in vitro, we expressed a truncated version of m that lacks hydrophobic amino acids at the n-terminus. sds-page analysis showed that cells transformed with the pgex-m expression vector produced a large amount of protein with a molecular mass of approximately kda, consistent with the expected molecular weight of the truncated m protein fused with a gst tag (data not shown). western blot analysis using an anti-prrsv-m antibody confirmed the expression and identity of the truncated m protein fused with a gst tag (additional file , fig. s ). the ub proteasome pathway (upp) is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. in order to enhance the ub-mediated degradation efficiency of m protein, we expressed a ub-m fusion protein in bhk- cells using the eukaryotic expression plasmid pvax -ub-m, in which the ub coding sequence was fused in-frame with the prrsv m coding sequence. following transient transfection of bhk- cells with the pvax -ub-m plasmid, the ub-m fusion protein was expressed and accumulated predominantly in the cytoplasm (additional file , fig. s ). immunization strategies that prime and boost with recombinant dna vectors encoding antigens have been shown to elicit t-cell immunity against hiv in non-human primates [ ] , and more recently, in humans [ ] . therefore, we used a dna vector encoding the prrsv m protein to immunize mice in order to generate and characterize m protein-reactive cd + t cells. the recombinant vaccinia virus rwr-prrsv-m drove expression of an m protein with the expected molecular weight when transfected into bhk- cells (additional file , fig. s ). ifa confirmed expression of m protein following transfection of bhk- cells, and revealed m protein accumulation in the cytoplasm (additional file , fig. s ). m protein-specific serum antibody increased steadily from the second to the fourth dna vaccination with the pvax -ub-m vector which drives m protein expression in vivo (additional file , fig. s ), indicating that dna vaccination elicited m protein-specific immune responses as expected. a subsequent booster vaccination with the recombinant vaccinia virus, rwr-prrsv-m, elicited a further increase in prrsv-specific antibody (p < . , additional file , fig. s ). in contrast, control mice vaccinated with pvax -only or pbs-only did not show significant increases in m protein-specific antibody titers after the booster vaccination with rwr-prrsv-m (p > . , additional file , fig. s ). overall, mice vaccinated with pvax -ub-m generated significantly higher m protein-specific antibody titers compared to mice vaccinated with pvax or pbs (p < . , additional file , fig. s ). these results indicate that rwr-prrsv-m amplifies the protective effects of dna vaccination and reveals the advantage of this priming-boosting strategy. dna vaccination with pvax -ub-m likely drives the differentiation of memory b cells which are subsequently activated by rwr-prrsv-m following the booster immunization, resulting in increased m-specific antibody titers. mice vaccinated with pbs and pvax would not be expected to generate m protein-reactive memory b cells, accounting for a less pronounced increase in m protein-specific antibody titers following rwr-prrsv-m innoculation. in this study we identified potential ctl epitopes in balb/c mice vaccinated with pvax -ub-m and boosted with rwr-prrsv-m. the frequency and number of cd + cd + t cells that produced ifn-γ following stimulation with peptides derived from prssv m protein was enumerated using ics and elispot assays. using these approaches, we identified two peptides from prssv m protein that elicited ifn-γ production from the splenocytes of vaccinated mice. as shown in figure , intracellular ifn-γ staining following stimulation of splenocytes from vaccinated mice with the peptide k fitsrcrl and f gymtfvhf revealed a population of ifn-γ producing cd + t cells comprising - % of the total cd + splenocyte population. in contrast, unstimulated splenocytes and splenocytes exposed to an irrevelant peptide did not contain a population of ifn-γ producing cd + t cells (figure , panel b and c). consistent with the ics data, peptides k fitsrcrl and f gymtfvhf elicited ifn-γ production from splenocytes of vaccinated mice when measured by elispot, whereas unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide did not reveal ifn-γ producing cells (figure ). the k fitsrcrl and f gymtfvhf prssv m protein peptides were identified bioinformatically as h- k d and h- d d restricted ctl epitopes (table ) . specific increases in the number of cells producing ifn-γ following stimulation with the peptides "k fitsrcrl" and "f gymtfvhf" was observed by day after the booster vaccination with rwr-prrsv-m (figure and ) . furthermore, splenocytes from mice vaccinated with pvax -ub-m responded strongly to "k fitsrcrl" and "f gymtfvhf", but not other m protein-derived peptides, at all time points tested following the booster vaccination with rwr-prrsv-m. when the same pattern of reactivity on three different time points after boosting with rwr-prrsv-m within each vaccination group were analyzed statistically using anova analysis, statistically significant differences were noted for the peptides "k fitsrcrl" and "f gymtfvhf" when compared to the other peptides tested among mice vaccinated and boosted with pvax -ub-m and rwr-prrsv-m, respectively (p < . , figure a and b). conversely, and confirming the specificity of these responses, no difference among the stimuli was observed with mock-vaccinated (pvax ) mice and naïve mice (data not shown). it is important to recognize that the ics assay calculates the percentage of ifn-γ + cells among cd + t cells in the spleen ( - %), whereas the elispot assay assesses the number of ifn-γ + cells among all splenocyte cell types ( . %), and cannot definitively assign the production of ifn-γ to a particular cell type. thus, the two assays use different denominators in calculating the frequency of ifn-γ + production by splenocytes. importantly, each method clearly identified k fitsrcrl and f gymtfvhf as the only two peptides from a panel of that elited significant ifn-γ production from the splenocytes of vaccinated mice. in this study we used balb/c mice as a model system to identify ctl epitopes in the m protein of prssv to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. the identification of prssv ctl epitopes in balb/c mice allow for the future generation of reagents, such as mhc class i: peptide staining reagents, that will enable the in-depth investigations of cd + t cell responses during prssv infection and immuinization. whether these two epitopes can bind the sla class i molecules of pigs remains to be determined. in some cases, specific peptide epitopes are known to be recognized by cytotoxic t cells in different animal species. for instance, the core region of hcv contains an epitope that is recognized by cytotoxic t cells of both mice and humans [ ] . further research on the role of these peptide epitopes in different species is ongoing in our laboratory. in conclusion, we identified peptides "k fitsrcrl" and "f gymtfvhf" from the m protein of prssv as h- k d and h- d d restricted ctl epitopes, respectively. in this study, we also developed a mouse model of prrsv infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to prrsv. porcine reproductive and respiratory syndrome virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates identification of immunodominant t-cell epitopes present in glycoprotein of the north american genotype of porcine reproductive and respiratory syndrome virus t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations immunological responses of swine to porcine reproductive and respiratory syndrome virus infection hepatitis b virus immunopathogenesis understanding presentation of viral antigens to cd + t cells in vivo: the key to rational vaccine design identification of an h- d(b)-restricted cd + cytotoxic t lymphocyte epitope in the matrix protein of respiratory syncytial virus characterization of a new h- d(k)-restricted epitope prominent in primary influenza a virus infection dna immunization with c fmdv non-structural protein reveals the presence of an immunodominant cd +, ctl epitope for balb/c mice immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp / gp of porcine reproductive and respiratory syndrome virus and swine il- syfpeithi: database for mhc ligands and peptide motifs scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains predbalb/c: a system for the prediction of peptide binding to h d molecules, a haplotype of the balb/c mouse dna vaccination of pigs with open reading frame - of prrs virus localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus a dna/mva-based candidate human immunodeficiency virus vaccine for kenya induces multi-specific t cell responses in rhesus macaques enhanced t-cell immunogenicity of plasmid dna vaccines boosted by recombinant modified vaccinia virus ankara in humans submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • 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 additional file : elisa antibody response in mice after immunization following dna vaccination and a booster vaccination with recombinant vaccinia virus. fig.s . m protein-specific antibody responses in mice immunized with pbs, or pvax or pvax -u-m dna, and boosted with rwr-prrsv-m. serum samples was obtained from vaccinated mice days after each dna vaccination and days after boosting with rwr-prrsv-m, and were evaluated for reactivity to mprotein in an elisa based on coating with the truncated m protein fused with a gst tag. and, the day represents the day of the first dna immunization. statistically significant differences are indicated by "*" or "**" for p-values < . or < . , respectively, as determined by anova.authors' contributions dlw and gll gave me the idea of this study. wjz and yl participated in the design and conducted the majority of the experiments as well as drafted the manuscript. tgt helped revise the manuscript and participated in the first stage of the experiments. yb and qw participated in the prediction of ctl epitopes and analyzed the data. yhx, nhl and ty participated in the sequence alignment. zgb provided the expression system of recombinant vaccinia virus, and gzt provided the prrsv ch- a strain. all the authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -hx rsi authors: you, jae-hwan; howell, gareth; pattnaik, asit k.; osorio, fernando a.; hiscox, julian a. title: a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: hx rsi porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, in common with many other positive strand rna viruses, encodes a nucleocapsid (n) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing n protein. the dynamic trafficking of positive strand rna virus nucleocapsid proteins and prrsv n protein in particular between the cytoplasm and nucleolus is unknown. in this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (frap and flip), was used to investigate the trafficking of fluorescent labeled (egfp) prrsv-n protein. the data indicated that egfp-prrsv-n protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. further the nuclear import of n protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of n protein between the cytoplasm and the nucleolus. a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging introduction porcine reproductive and respiratory syndrome virus (prrsv) is a spherical, enveloped virus, which causes highly contagious, severe disease in the natural host, the swine, with a spectrum of disease states ranging from respiratory failure in neonates to abortions in sows and persistence (wills et al., ) . prrsv is endemic to most swine producing countries and imposes a heavy economic burden due to the high mortality associated with the disease. prrsv is a positive strand rna virus belonging to the arteriviridae family, order nidovirales. although the primary site of replication is the cytoplasm, the viral encoded nucleocapsid (n) protein (an rna binding protein) localises predominately in the cytoplasm and nucleolus during virus infection and when over-expressed in cell culture (rowland et al., ) . this property is common with related viruses including the equine arterivirus (eav) n protein (tijms et al., ) and the coronavirus n proteins wurm et al., ) with the possible exception of the severe acute respiratory syndrome coronavirus (sars-cov) n protein (li et al., ; rowland et al., ; timani et al., ; you et al., ; you et al., ) . localisation and trafficking of the avian coronavirus infectious bronchitis virus (ibv) n protein to the nucleolus (cawood et al., ) correlates with the stage in the cell cycle (g ) when virus protein synthesis and progeny virus production is most efficient harrison et al., ; li et al., ) . the nucleolus is a dynamic sub-nuclear compartment found principally during interphase in eukaryotic cells (andersen et al., ; boisvert et al., ; hernandez-verdun, ; matthews and olson, ) . solution of the nucleolar proteome (andersen et al., ) has indicated that the nucleolus has a number of functions ranging from ribosome subunit biogenesis to non-classical functions such as the stress response (mayer and grummt, ; rubbi and milner, ) and regulation of cell growth (hernandez-verdun and roussel, ) . virology ( ) there are over proteins associated with the nucleolar proteome (leung et al., ) and these include nucleolin, fibrillarin and b . . localisation of positive strand rna virus capsid proteins and replication proteins (and in one case partial rna synthesis) in the nucleus and/or nucleolus appears to be an emerging feature of positive strand rna viruses. the reason why positive strand rna virus capsid proteins localise to the nucleolus is unknown, and several hypotheses have been proposed including; as part of a cellular defense mechanism to sequester viral proteins away from sites of virus replication and assembly, to recruit nucleolar proteins to facilitate virus replication, to usurp cellular processes by disrupting the nucleolar proteome or because the viral protein contains motifs which mimic nucleolar localisation signals (nolss) (hiscox, (hiscox, , (hiscox, , uchil et al., ; weidman et al., ) . however, the majority of rna synthesis in positive strand rna viruses (ahlquist, ; de haan and reggiori, ; taylor and kirkegaard, ) including arteriviruses (posthuma et al., ; van hemert et al., in press ) is thought to occur on cytoplasmic membrane bound replication complexes. assembly also occurs in the cytoplasm; therefore capsid proteins are required in this cellular compartment to facilitate these processes. this would infer the need for appropriate trafficking signals to ensure import and export from the nucleus and several such motifs have been identified. mutational analysis and abrogation of nuclear/nucleolar localisation of viral proteins for the prrsv n protein (lee et al., ; pei et al., ) and the positive strand rna plant rna virus groundnut rosette virus orf (kim et al., a,b) have elegantly demonstrated the importance of nucleolar localisation in the biology of these viruses. for prrsv n protein, the motifs which direct nucleolar localisation are composed of a nuclear localisation signal (nls) acting in concert with a nols (lee et al., ; . whereas for ibv n protein an eight amino acid motif was shown to be necessary and sufficient to act as an nols (reed et al., ) . for nuclear export of the arterivirus and coronavirus n proteins, nuclear export signals (ness) have either been identified or inferred from molecular genetic and/or bioinformatic analysis, structural modeling or use of specific metabolic inhibitors of nuclear export tijms et al., ; you et al., ) . the dynamic trafficking of positive strand rna virus capsid proteins between the nucleolus and the cytoplasm or the mobility in the nucleolus is not clearly understood. previous studies which have been mainly based on preparations of fixed cells could not distinguish whether there were for example two populations of capsid protein; one which sequestered to the cytoplasm and one which sequestered to the nucleolus or whether protein trafficked between the two compartments. in order to distinguish between these possibilities and derive a model for prrsv n protein trafficking, this study used live cell confocal microscopy to investigate the trafficking of fluorescent labeled n protein in porcine and non-porcine cell lines which were permissive and non-permissive for prrsv. to investigate whether the localisation of n protein within live cells was equivalent to that previously observed in fixed cells and for subsequent trafficking analysis, the prrsv n gene was pcr cloned downstream of the enhanced green fluorescent protein (egfp) in the expression vector pegfpc . this created plasmid pegfp-prrsv-n, which when transfected into cells led to the expression of prrsv n protein fused to egfp. although several cell lines were used in this study, primarily, the continuous porcine monocytic cell line, d / , was used in the majority of experiments. although not permissive for prrsv infection, these cells were derived from porcine alveolar macrophages (which are permissive) following expressing of the sv large t antigen (weingartl et al., ) . to investigate the localisation of egfp-prrsv-n protein, d / cells were co-transfected with pegfp-prrsv-n and a vector which directed the expression of the nucleolar protein b . fused to dsred (dsred-b . ) (reed et al., ; you et al., ) to act as a marker for the nucleolus (fig. a) . two different localisation patterns of egfp-prrsv-n protein were observed: in d / cells which expressed lower amounts of egfp-prrsv-n protein, localisation was predominately nucleolar with some nuclear signal (fig. a, panel a) . in d / cells which expressed higher amounts of protein localisation was predominately either cytoplasmic and nucleolar (fig. a , panel c) or predominately nucleolar with some cytoplasmic and nuclear signal (fig. a, panel d) . this difference in expression was evident in that the fluorescent signal from egfp-prrsv-n protein in fig. a , panel a was captured in the linear range. however, using the same setting led to over-saturated pixels in cells expressing higher amounts of egfp-prrsv-n protein e.g. fig. a , panel b (red indicates that the image range is not linear). gain adjustment to ensure pixels were captured in the linear range restored image clarity (fig. a , panel c). nucleolar localisation of egfp-prrsv-n protein was confirmed due to colocalisation with the nucleolar marker protein dsred-b . (yellow signal). previously, n protein has been shown to interact with the nucleolar protein fibrillarin . there are several different mechanisms which may account for the concentration dependent localisation pattern of n protein. n protein could diffuse and/or be actively transported into the nucleus through the nuclear pore complex (npc), diffuse through the nucleoplasm and be permanently sequestered in the nucleolus. as the nucleolar concentration of n protein becomes saturating so excess protein could either diffuse or be actively transported through the npc to the cytoplasm. alternatively, n protein could be continuously exchanged between the nucleolus and the nucleus and the cytoplasm, with localisation being driven by possible differential relative trafficking rates between nuclear import and export. to investigate and discriminate between these possibilities dynamic live cell imaging using fluorescence loss in photobleaching (flip) and fluorescence recovery after photo-bleaching (frap) was used to study movement of egfp-prrsv-n protein. relative trafficking within the nucleolus and trafficking from the cytoplasm to the nucleolus was investigated using flip to continuously photo-bleach a defined portion of the nucleolus in d / cells expressing egfp-prrsv-n protein. this was compared to the trafficking of three nucleolar marker proteins (reed et al., ; you et al., ) ; egfp-nucleolin, egfp-b . and efp-fibrillarin (fig. b) . loss of fluorescence is then a result of the egfp-tagged fusion protein trafficking into and out of the photo-bleached area with time. the data indicated that by s, loss of fluorescence from cells expressing egfp-nucleolin and egfp-fibrillarin was greater than egfp-b . . in cells expressing lower amounts of egfp-prrsv-n protein, fluorescence was completely photo-bleached by s and in higher expressing cells egfp-prrsv-n protein fluorescence was completely photo-bleached in the nucleus and reduced in the cytoplasm by s (fig. b) . this indicated that prrsv n protein was potentially as mobile as the cellular nucleolar proteins examined. to compare the relative nucleolar mobility between egfp-prrsv-n protein and egfp-nucleolin, egfp-b . and efp-fibrillarin in d / cells, frap was used to photo-bleach a defined portion of the nucleolus ( fig. a ) and the time taken to refill the photo-bleached area was measured (fig. b ) and t / values were compared (fig. c ). the data indicated that egfp-prrsv-n protein had the lowest t / value and therefore had a slightly faster mobility than the three cellular nucleolar marker proteins used in this study. cellular nucleolar proteins can have different mobility rates and nucleolar localisation patterns depending on their functions (andersen et al., ) and can show a continuous rapid turn over between the nucleus and the nucleolusyet the overall structure of the nucleolus is maintained (misteli, ) . the diffusion coefficient (d) value of egfp-prrsv-n protein and egfp-nucleolin, egfp-b . and efp-fibrillarin was calculated from the recovery curves (phair and misteli, ) . this value is the rate at which a protein repopulates a photo-bleached area. this confirmed that egfp-prrsv-n protein had an equivalent (or slightly higher) mobility (d = . μm /s) than egfp-nucleolin (d = . μm /s), egfp-fibrillarin (d = . μm /s) or egfp-b . (d = . μm /s). in a previous study the half time of egfp-fibrillarin in bhk cells was reported to be approximately s with a d value of . μm /s (phair and misteli, ) , which is in similar agreement to this study. phair and misteli ( ) noted that the d values of egfp-fibrillarin and other fluorescent tagged nuclear/nucleolar proteins were times lower than that reported for free solutes in the nucleus and suggested that this was due to interactions with other nuclear components. therefore, similar to the cellular nucleolar proteins the mobility of egfp-prrsv-n protein indicated that n protein probably localised to the nucleolus by diffusing through the nucleoplasm until it located an appropriate binding site, possibly a nucleolar protein or ribosomal rna, and thus appeared to accumulate in this compartment. prrsv n protein has been shown to interact with fibrillarin . the precise mechanism of trafficking of n protein within a cell and to the nucleolus is unknown, although n protein has been shown to interact with and pull down fibrillarin and thus may potentially localise to the nucleolus via this interaction . for example, the nucleolin binding activity of hepatitis delta antigen is associated with nucleolar localisation (ning and shih, ) . prrsv n protein has also been shown to interact with importin-α and importin-β and is sensitive to leptomycin b (lmb), an inhibitor of crm -dependent export . several different nuclear-cytoplasmic trafficking pathways are utilized by viruses (whittaker et al., ) and have been described for a number of different virus nucleocapsids and nucleocapsid proteins. for example, in vesicular stomatitis virus nucleocapsids are trafficked via a microtubule mediated process (das et al., ) , as are the hantaan virus nucleocapsid protein (ramanathan et al., ) and the west nile virus capsid protein (chu and ng, ) . to investigate whether the trafficking of n protein between the cytoplasm and the nucleus/nucleolus was associated with microtubules or energy dependent, the mobility of egfp-prrsv-n protein was compared in d / cells incubated at °c in the presence and absence of nocodazole ( ng/ml nocodazole (sigma) for h), a microtubule inhibitor, or incubated at °c. in preparations of fixed cells, in the absence of nocodazole (fig. a ) microtubules were visualized (observed as filaments) using an anti-tubulin antibody. as before egfp-prrsv-n protein localised predominately to the nucleolus. in preparations of fixed cells in the presence of nocodazole (fig. b ) microtubule polymerization was inhibited (diffuse staining of tubulin), although similar to untreated cells, egfp-prrsv-n protein remained predominately localised in the nucleolus. the activity of nocodazole was also confirmed by analyzing the cell cycle stage of treated cells (using flow cytometry as described previously harrison et al., ) ), which demonstrated enrichment in the g /m stage (fig. b ) compared to untreated cells (fig. a) , and is a result of the inhibition of spindle formation in metaphase. to investigate the trafficking of egfp-prrsv-n protein in live cells, a nucleolus in d / cells expressing egfp-prrsv-n protein was continuously photo-bleached (flip) in the presence of nocodazole at °c (fig. c ) and in the absence of nocodazole in cells that had been pre-cooled to °c (fig. d) . in both cases, photo-bleaching resulted in the loss of fluorescent signal in both the cytoplasm (where appropriate) and nucleus and the non photo-bleached nucleolus, indicating that cytoplasmic to nuclear trafficking occurred, and was not inhibited by either the presence of nocodazole or cooling to °c. this suggested that the trafficking of n protein from the cytoplasm to the nucleus was not microtubule or energy dependent. to investigate trafficking within the nucleolus a defined portion of the nucleolus was photobleached (frap) in cells incubated at either °c, °c or °c in the presence of nocodazole (fig. a ) and the time taken to refill the photobleached area was measured (fig. b) . the data indicated that egfp-prrsv-n protein at °c had a t / value of . ± . which was similar to the t / value ( . ± . ) at °c and at °c in the presence of nocodazole (t / value of . ± . ). the mobility of egfp-prrsv-n protein between the nucleolus and the cytoplasm and vice versa was compared to egfp using frap and flip on the cytoplasm or the nucleus/nucleolus in d / cells. egfp is approximately kda and contains no known nls, nols or nes motifs and therefore the relative localisation of egfp between the nucleus and the cytoplasm would reflect the relative trafficking of the protein across the npc, most likely due to diffusion and concentration gradients, such that equilibrium would be reached depending on the relative balance between the rate of nuclear import versus nuclear export and the relative volume of the cytoplasm versus the nucleus. prrsv n protein is approximately kda (and with the addition of egfp approximately kda) and although the fusion protein has the potential to freely diffuse through the npc (which has a size exclusion limit for passive transport of approximately kda (macara, ; terry et al., ) ), clearly egfp-prrsv-n protein has a discrete localisation pattern being cytoplasmic and nucleolar (with some nuclear signal) (rowland et al., ) . without the presence of trafficking signals n protein would be predicted to display similar trafficking and localisation to egfp. the trafficking of egfp compared to egfp-prrsv-n protein between the nucleus and the cytoplasm and vice versa was analyzed by frap (fig. ) and the relative level of fluorescence between the nucleus and the cytoplasm was measured using imagej software (nih) as described for the d / cell line. this takes into account the reduction in fluorescence during photo-bleaching due to ongoing trafficking into and out of the bleach area. in egfp-expressing d / cells the pre-bleach level of egfp in the nucleus to that of the cytoplasm was . ± . to . ± . , respectively. to examine trafficking into the nucleus, this area was photo-bleached and subsequent recovery of fluorescence in the nucleus (due to import of egfp from the cytoplasm) indicated that after s the relative level of egfp between the nucleus and the cytoplasm was . ± . compared to . ± . , respectively, which was not significantly different from the pre-bleach levels (fig. a) . analysis by s was then used as a base line for comparison of trafficking of egfp and egfp-prrsv-n protein between the nucleus and the cytoplasm in frap analysis. in d / cells expressing egfp-pprsv-n protein with nucleolar/nuclear and cytoplasmic distribution, the prebleach level of egfp-prrsv-n protein between the total nuclear content and the cytoplasm was . ± . and . ± . , respectively. s after photo-bleaching, the nuclear to cytoplasm level was . ± . to . ± . , respectively (fig. b) , which was not significantly different from the pre-bleach level. to examine trafficking of egfp into the cytoplasm, this area was photo-bleached in d / cells and subsequent recovery of fluorescence in the cytoplasm (due to import of non photo-bleached egfp from the nucleus) was compared to levels before photo-bleaching (nucleus to cytoplasm level of . ± . to . ± . , respectively) to levels s after photo-bleach when the nuclear to cytoplasm ratio was . ± . compared to . ± . , respectively (fig. c ). this indicated that egfp had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. to examine trafficking of egfp-prrsv-n protein into the cytoplasm, this area was photo-bleached and subsequent recovery of fluorescence in the cytoplasm (due to import of egfp-prrsv-n protein from the nucleus) was compared before photo-bleach (nucleus to cytoplasm relative level of . ± . to . ± . , respectively) to s after photo-bleach, when the nuclear to cytoplasm ratio was . ± . compared to . ± . , respectively (fig. d ). (note that this experiment was performed on cells in which egfp-prrsv-n protein localised also in the cytoplasm). this data indicated that the ratio of egfp-prrsv-n protein had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. this indicated that the export of egfp-prrsv-n protein from the nucleus was potentially slower than import and given the difference in relative ratios, slower than the export of egfp from the nucleus to the cytoplasm. the relative difference between nuclear import and nuclear export observed with egfp-prrsv-n protein was investigated using flip in which either a defined area of the nucleus (not the nucleolus) was photo-bleached for either egfp (fig. a ) or egfp-prrsv-n protein (fig. b ) expressing d / cells or a defined area of the cytoplasm was photo-bleached for either egfp (fig. c ) or egfp-prrsv-n protein (fig. d ) expressing d / cells. again, comparison of the data from the two proteins indicated that more fluorescent signal of egfp-prrsv-n protein was lost during import than export, suggesting that the former process operates faster than the latter. reflecting the frap analysis, the relative trafficking of egfp-prrsv-n protein between the nucleus and the cytoplasm was in favor of nuclear import. the observed live cell trafficking patterns of egfp-prrsv-n protein may have been specific to d / cells. therefore, to investigate this possibility, the localisation and dynamic trafficking of this protein was compared to egfp in marc- cells (an african green monkey kidney cell line permissive for prrsv infection (kim et al., ) ) and hela cells (derived from a human cervical cell line, and non-permissive for infection). the data indicated that in both marc- and hela cell types egfp-prrsv-n protein localised predominately to the nucleus/ nucleolus compared to egfp which localised throughout the cell (figs. a and a, respectively). very few cells (less than %) exhibited more than % of the total fluorescence from egfp-prrsv-n protein in the cytoplasm. note that for egfp, the transmission phase image is presented to highlight the differentiation between the cytoplasm, nucleus and nucleolus. (such images were used in subsequent analysis for placement of photo-bleaching in the egfp fluorescent cell, where distinction between the nucleus/nucleolus/cytoplasm was not possible). the sub-cellular localisation patterns for both fluorescent proteins were similar to that observed in d / cells. investigation of the trafficking of egfp-prrsv-n protein compared to egfp between the cytoplasm and the nucleus and vice versa by continuous photo-bleaching (flip) a defined portion of the nucleus or the cytoplasm in both marc- cells (fig. ) and hela cells (fig. ) , indicated that photo-bleaching in the nucleus in cells expressing egfp resulted in apparent simultaneous loss of fluorescence from the nucleus and the cytoplasm (figs. b and b), indicating rapid trafficking of egfp between the two compartments. in comparison, little fluorescence (approximately~ % of total fluorescence) of egfp-prrsv-n protein was observed in the cytoplasm in both marc- and hela cells and photobleaching in the nucleus resulted in loss of fluorescence from the nucleoplasm and then the nucleolus (figs. c and c upper panels, respectively) . in approximately % of cells expressing egfp-prrsv-n protein fluorescence was observed in the cytoplasm, but only if imaging was adjusted so that the fluorescent signal in the nucleus/nucleolus was above the linear range (for example fig. c, lower panels) . in this case photo-bleaching in the nucleus resulted in loss of fluorescence from the cytoplasm which could be accounted by faster trafficking into the nucleus of non-photo-bleached protein which was then photo-bleached than the export of photo-bleached protein into the cytoplasm. photobleaching in the cytoplasm of either marc- cells or hela cells expressing egfp resulted in loss of signal from both the nucleus and the cytoplasm (figs. d and d, respectively) . in comparison, photobleaching in the cytoplasm of either marc- cells or hela cells expressing egfp-prrsv-n protein resulted in less loss of fluorescence when compared to egfp (figs. e and e) . the photo-bleaching analysis for egfp-prrsv-n protein also indicated that when the nucleus was photo-bleached loss of fluorescence in the cytoplasm was due to the trafficking of non photo-bleached protein into the nucleus rather than the rapid export of photo-bleached protein from the nucleus into the cytoplasm. thus the photo-bleaching analysis of egfp and egfp-prrsv-n protein in marc- and hela cells was similar to that found in d / cells. thus we propose a model of n protein trafficking in which the nuclear import of n protein is faster than the nuclear export and n protein is apparently localised in greater amounts in the nucleolus (fig. ) . the deficiency of the model is that the analysis was conducted in the absence of other viral proteins or rna (i.e. infected cells) which may associate with n protein and alter its relative trafficking rates. this may be certainly true for the cytoplasm, where n protein will complex with viral rna and possibly components of the replicase complex. although there is currently no published data that other prrsv macromolecules localise in the nucleus/nucleolus, the eav non-structural protein (nsp ) has also been observed in the nucleus (tijms et al., ) , and therefore prrsv nsp may have similar properties. prrsv n protein does not accumulate or is sequestered in the nucleus/nucleolus per se and in common with other cellular nuclear/nucleolar proteins is continuously being exchanged between the nucleolus and the nucleoplasm and indeed is as mobile as the cellular nucleolar proteins examined. the implication of this study is that all de novo synthesized n protein (when over-expressed) traffics to the nucleolus. together with molecular genetic analysis showing that virus replication is reduced when n protein nucleolar localisation is abolished (lee et al., ; pei et al., ) , suggests that the nucleolar trafficking of n protein is playing a crucial role in virus biology. what this role is remains to be elucidated. cell culture d / cells were grown at °c with % co in dmem and rpmi ( : ). marc- and hela cells were maintained in dmem supplemented with % foetal bovine serum and grown at °c with % co . mm glass bottom tissue culture dishes were seeded with × cells h prior to transfection. the plasmid pegfp-prrsv-n, which expressed prrsv n protein fused c-terminal to egfp, was constructed by amplifying the n gene using the amplicon fl (truong et al., ) (genbank accession number ay ) as template. primers were designed to the ′ and ′ end of the n gene and contained appropriate restriction enzyme sites; ′ hindiii and ′ ecori respectively. the amplification product was ligated into the topo vector pcr . and then cloned into hindiii and ecori restricted pegfp-c (clontech). the insert was sequenced in both directions to confirm authenticity and western blot analysis to confirm expression. plasmids which led to the expression of the nucleolar marker proteins egfp-nucleolin, egfp-b . , dsred-b . and efp-fibrillarin have been described previously (reed et al., ; you et al., ) . transfected cells were imaged at °c in co -independent media (gibco) supplemented with % fbs on an inverted lsm meta confocal microscope (carl zeiss) using a × objective and a factor zoom. five images were captured before a short period of photobleaching using conditions that had negligible effects on total cell fluorescence. photo-bleaching was performed on an area of by pixels (approx . μm ) with a mw argon laser at % power, bleaching took approximately . s. fluorescence was analyzed with lsm software and raw data was imported into microsoft excel for analysis and generation of graphs. raw data were averaged before normalizing. normalization of the data was performed in such a way as to ensure that the recovery profiles were set to zero at the initial time point post bleaching, and set to at the final time point; as previously described (marcelli et al., ; stenoien et al., ) . time to half life recovery (t / ) was performed on the mean raw data, determined by the equation (i e − i o ) / , where i e was the intensity at the final time point, and i o the intensity at the time point immediately following photo-bleaching. diffusion coefficient (d) values were calculated from the half life recovery (t / ) using the following diffusion equation d =(w / t / ) × . where w is the width of the bleach area, approx . μm, and a constant factor of . was used for a gaussian beam profile (axelrod et al., ) . data from experiments were used per construct and standard deviation was calculated in the usual manner using microsoft excel. transfected cells were imaged in glass base dishes as outlined above. imaging and photo-bleaching was performed with the same laser settings as detailed in the frap microscopy. in each flip experiment cells were imaged times followed by a period of photobleaching for a total time of min. photo-bleaching was performed on a defined area ( by pixels (approx . μm )) within the cell for iterations (mean bleach time . s). parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses nucleolar proteome dynamics directed proteomic analysis of the human nucleolus mobility measurement by analysis of fluorescence photobleaching recovery kinetics the multifunctional nucleolus cell cycle dependent localisation of the coronavirus nucleocapsid protein trafficking mechanism of west nile (sarafend) virus structural proteins visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells are nidoviruses hijacking the autophagy machinery? cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication characterisation of cyclin d down-regulation in coronavirus infected cells nucleolus: from structure to dynamics regulators of nucleolar functions brief review: the nucleolus -a gateway to viral infection? the interaction of animal cytoplasmic rna viruses with the nucleus to facilitate replication rna viruses: hijacking the dynamic nucleolus the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection cajal bodies and the nucleolus are required for a plant virus systemic infection mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication nopdb: nucleolar proteome database sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p transport into and out of the nucleus. microbiol quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility what is new in the nucleolus? workshop on the nucleolus: new perspectives. embo rep cellular stress and nucleolar function protein dynamics: implications for nuclear architecture and gene expression nucleolar localization of human hepatitis b virus capsid protein functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association high mobility of proteins in the mammalian cell nucleus formation of the arterivirus replication/transcription complex: a key role for nonstructural protein in the remodeling of intracellular membranes dynein-dependent transport of the hantaan virus nucleocapsid protein to the endoplasmic reticulum-golgi intermediate compartment delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences the localisation of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells disruption of the nucleolus mediates stabilization of p in response to dna damage and other stresses intranuclear ataxin inclusions contain both fast-and slow-exchanging components potential subversion of autophagosomal pathway by picornaviruses crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport nuclear localization of non-structural protein and nucleocapsid protein of equine arteritis virus model of the trafficking of prrsv n protein within the cell where the width of arrows denotes the relative movement of n protein and shading is relative concentration of the protein. trafficking from the cytoplasm to the nucleus and localisation to the nucleolus is fast compared to export of n protein from the nucleus to the cytoplasm which is slower nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus nuclear localization of flavivirus rna synthesis in infected cells the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor the interaction of cytoplasmic rna viruses with the nucleus continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility viral entry into the nucleus duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rnaassociated protein fibrillarin subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein this research was supported by a national pork board grant to jah. drs amanda stuart and t. david k. brown at the university of cambridge are thanked for providing the materials and reagents. key: cord- -geh aaf authors: wagner, judith; kneucker, annette; liebler-tenorio, elisabeth; fachinger, vicky; glaser, melanie; pesch, stefan; murtaugh, michael p.; reinhold, petra title: respiratory function and pulmonary lesions in pigs infected with porcine reproductive and respiratory syndrome virus date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: geh aaf pulmonary dysfunction was evaluated in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv, isolate vr- ) and compared to clinical and pathological findings. infected pigs developed fever, reduced appetite, respiratory distress and dullness at days post-inoculation (dpi). non-invasive pulmonary function tests using impulse oscillometry and rebreathing of test gases (he, co) revealed peripheral airway obstruction, reduced lung compliance and reduced lung co-transfer factor. prrsv-induced pulmonary dysfunction was most marked at – dpi and was accompanied by a significantly increased respiratory rate and decreased tidal volume. expiration was affected more than inspiration. on histopathological examination, multifocal areas of interstitial pneumonia (more severe and extensive at dpi than dpi) were identified as a possible structural basis for reduced lung compliance and gas exchange disturbances. porcine reproductive and respiratory syndrome virus (prrsv), a member of the family arteriviridae, genus arterivirus, causes porcine reproductive and respiratory syndrome (prrs), an important cause of production losses in pigs (rossow, ) . the consequences of prrsv infection have been well documented for the reproductive system and for systemic infection (christianson et al., ; botner et al., ; mengeling et al., ; lager et al., ; kranker et al., ) . numerous studies have evaluated the clinical, pathological and immunological features of the respiratory form of prrs (labarque et al., ; samsom et al., ; opriessnig et al., ) , but the effects on pulmonary function have not been investigated, even though respiratory dysfunction may have a significant impact on the clinical outcome of the disease. typical clinical signs of respiratory infection with prrsv are tachypnoea or dyspnoea that can be accompanied by an increased respiratory effort, lethargy, fever and occasionally coughing, sneezing and chemosis (rossow et al., ; opriessnig et al., ) . gross lung lesions include failure of the lungs to collapse, as well as moderately well demarcated, mottled, brown areas of pneumonia (opriessnig et al., ) . microscopic lung lesions are charac-terised by type ii pneumocytic hypertrophy and hyperplasia, septal infiltration with mononuclear cells and alveolar exudates (opriessnig et al., ) . no data are currently available on the pathophysiological features and derangements of pulmonary function induced by prrsv. since the lungs are important for oxygen supply to all tissues, pulmonary dysfunction might have significant clinical and subclinical systemic consequences. pulmonary function testing has been applied to pigs with bacterial respiratory infections by our group (reinhold et al., , . a combination of impulse oscillometry and rebreathing of test gases can be used to evaluate lung ventilation, respiratory mechanics and pulmonary gas exchange in spontaneously breathing pigs. since both methods are non-invasive and applicable to conscious animals, changes in pulmonary function can be evaluated over time. in this study, pulmonary dysfunction was characterised in relation to clinical signs and pathological changes in pigs with experimental prrsv infection. twenty-four german hybrid pigs from a closed specific-pathogen-free herd known to be free of prrsv were transported to our institute at - days of age and were enrolled in the study after a quarantine period of days. pigs were housed according to the guidelines for animal welfare and fed twice daily with a - /$ -see front matter Ó elsevier ltd. all rights reserved. doi: . /j.tvjl. . . commercial grower diet without antibiotics. water was supplied ad libitum. pigs were housed in two groups of animals each, with pens of six pigs being separated from each other. virus vr- , the prototype type ii prrsv isolated in in minnesota, usa, has been shown to be virulent for sows and piglets (rossow et al., ; opriessnig et al., ; nielsen et al., ) . sixth passage vr- , propagated and titrated on ma- cells, was prepared at a concentration of log % tissue culture infectious doses/ml for use as the inoculum. the study had a randomised, negatively controlled design (table ) and was performed at biosafety level . ethical approval was obtained from the commission for the protection of animals of the state of thuringia, germany (registration number - / ). twelve pigs were exposed to prrsv and pigs served as controls. on the day of challenge, pigs exposed to prrsv had an age of . ± . days (mean ± standard deviation, sd) and a body weight of ± kg, while controls were . ± . days old and weighed ± kg. pigs were inoculated intranasally ( ml per nostril) and intramuscularly ( ml per pig) with either prrsv vr- or . % saline (controls). for intranasal inoculation, a ml syringe was connected to a tube ( cm  . mm inner diameter feeding tube, rüsch sterile, ref , size no. . willy rüsch gmbh) which had been inserted approximately cm into the nose. one millilitre of virus solution, along with ml air, per syringe was administered into each nostril with manual pressure, followed by im injection of ml into the gluteal muscle. clinical observations were recorded twice daily and included general behaviour, feed intake, appetite, rectal temperature, respiratory rate (rr) and the presence or absence of clinical signs of respiratory disease or diarrhoea. to monitor for the presence of prrsv by pcr and for seroconversion, blood samples were collected three times before challenge (À , À days and À h) and six times after challenge ( , , , , and days post-inoculation, dpi). blood samples, nasal swabs, rectal swabs and tracheal swabs were also collected to exclude concurrent infections (table ) . pulmonary function tests (pfts) were performed twice before challenge (À and À days) and seven times after challenge ( , , , , , , and dpi) in eight pigs exposed to prrsv and in eight controls (table ) . body weight was deter-mined day before each pft and at postmortem examination. four pigs per group (without pft) were sacrificed at dpi and eight pigs per group (with pft) were sacrificed at dpi for gross pathological, histopathological and immunohistochemical examination (table ) . to quantify the prrsv load in blood, serum samples were analysed by real-time pcr for prrsv strain vr- and its attenuated form, ingelvac mlv. rna was extracted using qiaamp viral rna mini kit (qiagen). reverse transcription was carried out using the multiscribe rt enzyme kit (applied biosystems). reverse transcription conditions were °c for min, °c for min and °c for s. amplification was carried out in triplicate using the taqman universal pcr master kit (applied biosystems) and the prrs vr- -specific primers mlv f ( -gcagctcccatctacagctgatt- ) and mlv r ( -agacaatgtgagtca aaacgggaaagat- ). the probe was tet- -ttggctagctaacaaatttgattgggc agtggagagttt- -tamra. thermal cycling conditions were °c for min, °c for min, then cycles of °c for s and °c for min. prrsv cdna quantification was achieved by comparison of the unknown sample with a standard curve derived from known amounts of plasmid dna. a commercial prrsv elisa (herdcheck prrs elisa, idexx) was used to detect anti-prrs antibodies in the blood serum of pigs before and after challenge. samples with sample-to-positive (s/p) ratio p . were considered to be positive for antibodies against prrsv, as recommended by the manufacturer. routine bacteriological culture was performed on nasal, tracheal and faecal swabs for bordetella spp., pasteurella spp., haemophilus spp., actinobacillus pleuropneumoniae (app) and salmonella spp. (table ). samples were tested for mycoplasma spp. by indirect immunofluorescence and for chlamydia spp. by pcr. paired serum samples from each pig (blood collected at the beginning of the study and at postmortem examination) were used for serology (table ) . commercial elisa test kits were used to detect antibodies against mycoplasma hyopneumoniae (dako m. hyo elisa, oxoid), app (cypress diagnostics), swine influenza virus (siv; idexx), transmissible gastroenteritis virus (tgev; svanova) and porcine table study design for prrsv monitoring, additional microbiological analysis, pulmonary function testing and postmortem examination. m. hyopneumoniae, mycoplasma hyopneumoniae; app, actinobacillus pleuropneumoniae; pcv- , porcine circovirus type ; prcv, porcine respiratory coronavirus; siv, swine influenza virus; tgev, transmissible gastroenteritis virus; pft, pulmonary function tests ( pigs per group examined postmortem at dpi). a n = pigs per group. b n = pigs per group (with pft). c n = pigs per group (without pft). respiratory coronavirus (prcv; svanova). testing for antibodies against porcine circovirus type (pcv- ) was performed by an indirect fluorescent antibody test (fachinger et al., ) . approximately min prior to pft, each pig was sedated with diazepam ( . - . mg/kg body weight im; faustan, weimer pharma). the sedated animal was restrained in a canvas sling with openings for the limbs and wore a tightly fitting face mask, allowing spontaneous breathing. after an adaptation period of approximately min, two non-invasive lung function techniques were applied consecutively: ( ) impulse oscillometry system (ios; masterscreen ios; jaeger); and ( ) rebreathing system (masterscreen diffusion; jaeger). both systems were originally produced for human medicine and have been successfully applied to pigs previously (reinhold et al., , . ios was used to measure variables of respiratory mechanics based on a forced oscillation technique, as previously validated for pigs (klein and reinhold, ; klein et al., ) . externally generated test impulses given by a loudspeaker were superimposed on spontaneous airflow of the breathing animal. variations in pressure and flow signals were analysed using fast fourier transformation to calculate the complex respiratory impedance, which consists of both respiratory resistance (rrs) and respiratory reactance (xrs) (smith et al., ) . airflow was registered during spontaneous breathing using a lilly-type pneumotachograph with a mesh resistance of pa/(l/s) and used to calculate spirometric variables of spontaneous breathing (rr and tidal volume, vt). each test per day and animal consisted of three consecutive ios measurements free of any artefacts (e.g. coughing or irregular breathing pattern). each measurement lasted for s. three impulses were generated per second, leading to independent results per min. the sampling rate was set at hz (period between two sampling points of ms), selecting sampling points after each impulse. results of the three measures per pig and per time point were averaged for statistical analysis. the following variables of ventilation and respiratory mechanics were analysed: ( ) rr; ( ) vt; ( ) volume of minute ventilation (mv); ( ) both vt and mv related to body weight (vt/kg, mv/kg); ( ) rrs; ( ) xrs, each at , , and hz, and separated for inspiration and expiration at each frequency (rrs,in hz . . . rrs,in hz ; rrs,ex hz . . . rrs,ex hz ; xrs,in hz . . . xrs,in hz ; xrs,ex hz . . . xrs,ex hz ); ( ) resistance of proximal airways (r prox ); and resistance of distal airways (r dist ). using different test gases and a multiple breath approach (i.e. steady state method), the rebreathing system permitted the simultaneous measurement of two additional pulmonary function variables. the functional residual capacity (frc) of the lungs was measured by the helium (he) dilution technique (washin). the transfer factor of the lungs for carbon monoxide (tl co) was determined in order to evaluate the transfer of oxygen from the lungs into the blood. for assessing both frc and tl co, each pig inhaled the test gas mixture ( % he, . % co in synthetic air) from a reservoir bag. since animals were growing, the volume filled in the reservoir bag was l in the pre-challenge period and was adjusted for the increasing lung volume with growth to l from to dpi. the time to perform this test (i.e. rebreathing time) increased from ± s (mean ± sd) week before challenge (body weight of ± kg, mean ± sd) to ± s at dpi (body weight of ± kg) due to a significant correlation with increasing body weight (r = . ; r = . %; p = . ) or increasing frc (r = . ; r = . %; p = . ), respectively. frc was calculated per kg body weight (frc/kg) to avoid body weight being a confounding factor. data for tl co were corrected for the individual concentration of haemoglobin in the blood (tl co hb ), as determined before each pft using a haemoxymeter that allows species-specific analysis of haemoglobin fractions for pigs (osm , radiometer). to correct for body weight and metabolism, tl co hb was calculated per kg body weight (tl co hb /kg), as well as per kg metabolic body weight (tl co hb /kg . ). pigs were euthanased by intravenous injection of pentobarbital sodium (release, wdt) at p mg/ kg body weight. the trachea was exposed, severed distal to the larynx and the lungs were instilled with neutral buffered formalin (nbf) at a pressure of mm water column. after min of intrathoracic fixation, the trachea was closed and the lungs were removed from the thoracic cavity and placed in nbf for h. samples collected from proximal and distal parts of the cranial and caudal pulmonary lobes and from one area of the accessory lobe were embedded in low-melting paraffin ( - °c). paraffin sections were stained with haematoxylin and eosin for histological examination. immunohistochemistry prrsv antigen was detected by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase (apaap) method. paraffin sections collected on charged slides were pretreated with proteinase k ( . % in phosphate buffered saline, ph . ) for min. the monoclonal antibody sr -a (rural technologies) was used as the primary antibody and rabbit anti-mouse ig (dianova) was used as the secondary antibody to bind the apaap complex (dako). alkaline phosphatase was visualised using neufuchsin. slides were counterstained with methylene green. positive control sections (bioscreen evdmc) were included with each reaction. statistical analyses were performed to determine the relationship between prrsv infection and clinical signs, body weight or lung function variables. for statistical evaluation of clinical signs, the study period from days before inoculation until dpi was separated into nine sub-periods, each of three consecutive days. within each sub-period, data obtained per day and per animal were averaged. for calculating baseline data obtained by pft, all measures per pig before inoculation were averaged. numeric data are presented as medians, minima and maxima. the mann-whitney-wilcoxon-test (w-test; comparison of medians) was used to determine significant differences between groups at p . . for regression analyses a linear model was used (y = a + bx) and both coefficient of linear correlation (r) and coefficient of determination (r ) were calculated. pcr for quantification of viral load and elisa for antibody detection demonstrated that all pigs were negative for prrsv before challenge. controls remained free of prrsv infection throughout the trial. in pigs exposed to prrsv, infection was confirmed by increasing viral loads in serum. viraemia was detected in all pigs at dpi and reached a maximum dpi (fig. a) . antibody titres against prrsv were first observed at dpi in one pig and at dpi in all other pigs, reaching a maximum at dpi (fig. b) . none of the control pigs exhibited any clinical signs of respiratory disease during the study. pigs exposed to prrsv developed clinical signs initially observed at - dpi, including reduced appetite, dullness and fever. body temperature was significantly increased in challenged pigs compared to controls from to dpi and this increase showed a biphasic character, with maxima at - dpi and - dpi ( fig. a) . most prrsv challenged pigs remained dull until the end of the trial, whereas reduced appetite and/or diarrhoea was identified only sporadically from to dpi. the daily weight gain was ± g (mean ± sd) in controls and ± g in pigs exposed to prrsv; there was no significant difference between groups. coughing, dyspnoea and ocular discharge were detected in pigs exposed to prrsv. coughing was present from to dpi in / pigs and was most prominent from to dpi. rr increased significantly in prrsv challenged pigs compared to controls starting in the period to dpi and reached maximal values at - dpi (fig. b) . dyspnoea developed in / pigs in the period - dpi, with a maximal intensity at - dpi. one prrsv challenged pig exhibited ocular discharge in the period - dpi. interestingly, no nasal discharge and no vomiting was observed. compared to controls, vt/kg was significantly reduced in pigs exposed to prrsv after challenge (fig. a) . this decrease was most prominent in the period - dpi. in contrast, the volume of mv in relation to body weight (mv/kg) increased significantly in prrsv challenged pigs. compared to controls, the most marked increase in mv was seen between and dpi (fig. b) . in pigs experimentally exposed to prrsv, marked changes in respiratory impedance occurred at - dpi; the changes were more prominent during expiration than during inspiration. spectral curves of rrs and xrs within the frequency range of - hz at dpi are shown in fig. . rrs at and hz measured during expiration was significantly elevated in comparison with controls, leading to a significantly larger difference between inspiratory and expiratory resistance values. this increase in rrs,ex was statistically significant on days and after challenge for both rrs,ex hz and rrs,ex hz , as well as for rrs,ex hz alone on days and post-challenge (supplementary table ). in contrast, rrs at and hz measured during inspiration was not significantly different between groups (fig. , supplementary table ). both rrs,ex and rrs,in assessed at and hz were lower in pigs exposed to prrsv compared to controls (supplementary table ). due to significantly increased expiratory resistance data at low frequencies ( and hz) and significantly lower rrs,ex data at hz, the resistance curves during expiration became inversely frequency dependent after prrsv challenge, i.e. resistance decreased with increasing frequency (fig. ) . xrs at all frequencies ( - hz) was significantly lower in prrsv infected pigs compared to controls (fig. ) . this observed negativity of the total xrs curves was more prominent during expiration compared to inspiration, mainly from to days after exposure (supplementary table ). proximal and distal airway resistances are shown in table . after challenge, pigs exposed to prrsv had significantly higher distal airway resistance compared to controls. in contrast, r prox was significantly lower in prrsv challenged pigs than in control pigs. as shown in table , volumes of frc increased in growing pigs of both groups during the study (linear correlation between body body temperature (a) and respiratory rate (b) of pigs exposed to prrsv and controls. box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). days À to : n = per group; days - : n = per group. # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). weight and frc: control pigs: r = . ; r = . %; p . ; prrsv infected pigs: r = . ; r = . %; p . ). neither absolute volumes of frc nor frc/kg body weight (data not shown) differed significantly between groups at any time point. in controls, tl co hb ranged from . mmol/min/kpa (median; minimum-maximum: . - . ) days before challenge to . mmol/min/kpa (minimum-maximum: . - . ) at dpi. due to the increase in body weight of pigs during the trial (r = . ; r = . %; p . ) and because gas exchange in the lung physiologically adapts to metabolism, groups were compared on a basis of tl co hb data corrected for either body weight (fig. a ) or metabolic body weight (fig. b ). compared to controls, reduced gas transfer became evident in pigs challenged with prrsv from dpi until the end of the study. this decrease was statistically significant for tl co hb /kg at and dpi and for tl co hb /kg . at dpi. there were no gross changes in the lungs of prrsv infected or control pigs. systemic enlargement of lymph nodes (including the tracheobronchial lymph nodes) was evident at and dpi in the prrsv inoculated group. histopathological examination of the lungs of inoculated pigs revealed mild to moderate multifocal interstitial pneumonia characterised by thickening of interalveolar septa, septal infiltration with mononuclear cells, hyperplasia and hypertrophy of type ii pneumocytes and alveolar exudates of macrophages, necrotic debris and occasionally multinucleated cells (fig. a) . pulmonary lesions were more frequent and more severe in cranial lobes compared to caudal lobes and in the proximal part of lobes compared to the distal part. at dpi, mild to moderate interstitial pneumonia was evident in / pigs. at dpi, lesions were milder, but all eight pigs were affected. hyperplasia of the tracheobronchial lymph nodes was evident in / pigs at dpi. in the lungs of control pigs, there were mild to moderate peribronchiolar and perivascular lymphocytic infiltrates, along with mild lymphocytic infiltrates in interalveolar septa and mild multifocal clusters of alveolar macrophages in alveolar spaces. there was no thickening of interalveolar septa (fig. b ). prrsv antigen was detected only in pigs inoculated with prrsv. there was a close correlation between pulmonary lesions and the presence of viral antigen. prrsv antigen was present in the cytoplasm of alveolar macrophages and occasionally in multinucleate cells within alveoli (fig. c) . it was also seen in mononuclear cells within thickened interalveolar septa and in type ii alveolar epithelial cells. prrsv antigen was also detected in one pig which did not develop interstitial pneumonia. at dpi, the highest amount of viral antigen was found in two pigs with moderate pulmonary lesions. there was less prrsv antigen present in the lungs at dpi compared to dpi. all pigs were free of app, bordetella spp., mycoplasma spp., pasteurella spp., siv and tgev. chlamydia spp. were detected in rectal swabs of all pigs. all animals had antibodies against pcv- and prcv. haemophilus parasuis was detected in a nasal swab from / pigs in the control group before challenge. salmonella spp. were detected sporadically in rectal swabs before and after challenge in both groups. this study characterised respiratory dysfunction induced by the well-characterised prototypical na-type prrsv isolate vr- (rossow et al., ; opriessnig et al., ; nielsen et al., ) . moderate clinical disease was induced that allowed consecutive pulmonary function testing until weeks after challenge without any spontaneous deaths before the end of the study. this is the first simultaneous within-subject study of clinical, functional and morphological changes in pigs experimentally infected with prrsv. in the absence of other major infectious causes of respiratory disease, changes in clinical status, pulmonary dysfunction and pathology in pigs in this study can be attributed to prrsv. prrsv viraemia and seroconversion were evident in all challenged pigs. fig. . tidal volume per kg body weight (vt/kg; a) and minute ventilation per kg body weight (mv/kg; b) of pigs exposed to prrsv (n = ) and controls (n = ) as measured with the impulse oscillometry system. box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (wtest, p . ). challenge with vr- induced reproducible and representative respiratory disease consistent with previous studies using the same strain (rossow et al., ; opriessnig et al., ) . prrsv viraemia was detected at dpi and coincided with an increase in rectal temperature. an increase in rr was evident at dpi and lasted until the end of the study, i.e. until at least days after infection. other clinical signs included dyspnoea and coughing and were most prominent from to dpi. pulmonary dysfunc- fig. . medians of spectral respiratory impedance within the frequency range of - hz separated for respiratory impedance during inspiration (a) and respiratory impedance during expiration (b) of pigs exposed to prrsv (n = ) and controls (n = ) dpi. rrs, respiratory resistance; xrs, respiratory reactance, both during expiration (rrs,ex, xrs,ex, respectively) and inspiration (rrs,in, xrs,in, respectively) . # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). distal airway resistance (r dist ) and proximal airway resistance (r prox ) in pigs exposed to prrsv (n = ) and controls (n = ). control prrsv a.i. represents data measured prior to inoculation with prrsv (averaged per pig). # indicates significant difference between pigs exposed to prrsv and controls (w-test, p . ). functional residual capacity (frc) in pigs exposed to prrsv (n = ) and controls (n = ). tion was evident earlier ( dpi) and was most prominent at dpi. microscopic lung lesions were more severe at dpi than at dpi which is in accordance with opriessnig et al. ( ) . some authors have reported coughing as a clinical sign of prrs (done et al., ; beyer et al., ) , whereas others have attributed coughing to secondary infections (e.g. m. hyopneumoniae, b. bronchiseptica, pcv- ) (thacker et al., ; brockmeier et al., ; thacker and thanawongnuwech, ) . we observed a spontaneous dry cough in most of the pigs exposed to prrsv, but not in controls. due to the absence of other major respiratory pathogens in the present study and because there was no evidence of tracheitis, bronchiolitis or airway mucus on pathological examination, we interpret the observed cough as being induced by bronchospasms as a component of the respiratory illness caused by prrsv. this interpretation is supported by the presence of peripheral airway obstruction, as evaluated by pulmonary function testing. the pattern of ventilation, as indicated by spirometric data determined during spontaneous breathing, includes rr, vt and mv. in our study, rr increased significantly in pigs exposed to prrsv, with maximal values at - dpi ( % compared to baseline data). increased rr may be a response to hyperthermia, as well as an attempt to compensate for reduced vt and/or arterial oxygen deficiency caused by reduced gas transfer from the lungs into the blood. relative vt (vt/kg) was significantly reduced (< ml/kg) in pigs exposed to prrsv, while vt/kg was always > ml/kg in healthy controls. reduced vts indicate changes in the pattern of breathing caused by obstructive or restrictive disorders and/or by disorders in gas exchange. pulmonary function tests in prrsv challenged pigs indicate both obstructive and restrictive disorders (confirmed by increased rrs at frequencies hz and decreased xrs), as well as disorders in gas exchange (confirmed by decreased tl co hb ). the latter correlates with the pathological findings of thickened interalveolar septa and the presence of alveolar exudates. the observed increases in mv in prrsv challenged pigs were caused by increases in rr and indicate compensation for reduced vts. pigs infected with prrsv had shorter breathing cycles and shallower inspiration. this pattern of breathing, however, is pathophysiologically linked to a higher percentage of dead space venti-lation compared to alveolar ventilation and consequently carries the risk of alveolar hypoventilation. in addition, the energy requirement for breathing increases due to increased effort of respiratory muscles. impulse oscillometry measures complex respiratory impedance, which consists of rrs and xrs (smith et al., ) . while rrs reflects mainly airway resistance, xrs is determined by inertive and capacitive components of the respiratory system. pressure impulses generated by a loudspeaker in a frequency range of - hz are used as test signals. low frequencies ( - hz) penetrate deep into the respiratory tract and provide a representation of the peripheral airway system, whereas high frequencies ( - hz) provide a representation of the upper and central airway systems. results at frequencies > hz were disregarded, since they are mainly influenced by mechanical properties of the face mask, which acts as a confounding factor for respiratory impedance measurements in animals (reinhold et al., ; klein et al., ) . in addition to the spectral curves of rrs and xrs, two model-derived resistances (i.e. r dist and r prox ) were analysed to differentiate between the effects of prrsv on either proximal airways (nasal cavities, larynx and pharynx) or distal airways (lung periphery), respectively. all variables of respiratory mechanics were significantly different in pigs challenged with prrsv compared to controls. rrs at - hz, r dist and xrs are important variables with respect to the lung periphery, including peripheral airways. rrs at frequencies hz was significantly elevated in pigs exposed to prrsv and this increase was only evident during expiration. this phenomenon indicates airflow limitation predominantly in the peripheral airway system, since peripheral airway obstruction affects expiration much more than inspiration. the presence of peripheral airway obstruction is emphasised by significant increases in model-derived resistance of distal airways (r dist ). due to the absence of mucus and bronchial wall oedema, peripheral airway obstruction was most likely to have been caused by bronchospasms. morphological correlates for bronchospasms, however, are lacking, since contractions or spasms of airway muscles do not persist after euthanasia and the fixation method used in this study (intratracheal instillation with formalin) opens airways and fills alveolar regions, irrespective of the ventilation status . transfer factor of carbon monoxide corrected for haemoglobin in relation to body weight (tl co hb /kg; a) and metabolic body weight (tl co hb /kg . ; b) in pigs exposed to prrsv (n = ) and controls (n = ). box-and-whisker plot represents median value, % and % percentiles (box), range, outlier values (o) and extreme values (Ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (w-test, p . ). during normal breathing. decreases in xrs indicate reduced compliance of the lung. this might be caused by restrictive disorders due to both stiffness of obstructed airways and pulmonary tissue components. the lungs of pigs challenged with prrsv had thickened interalveolar septa and septal infiltration with mononuclear cells, which may be responsible for reduced compliance. findings with respect to central and/or upper airways are reflected by both rrs at - hz and r prox . after prrsv challenge, proximal airway resistance in exposed pigs was significantly lower compared to controls. this phenomenon might be interpreted as 'upper airway opening', an attempt to compensate for peripheral airway obstruction by increasing the diameters of upper airways (nasal cavities, larynx and pharynx). due to the functional character of this phenomenon, no morphological equivalent is visible at postmortem examination. in humans, enlargement of the aperture of the glottis is recognised during a high frequency, low vt breathing pattern (stȃnescu et al., ) . a further approach to enlarge the upper airway diameter is reduction of mucosal thickness by changes in blood flow. in rats, cervical sympathetic nerve stimulation induces a reduction in upper airway resistance, which is most likely induced by a-adren-ergic vasoconstriction of the upper airway mucosal vasculature, resulting in reduced mucosal thickness (o'halloran et al., ) . stimulation of cervical sympathetic nerves, however, can be caused by systemic hypoxia and hypercapnia (matsumoto et al., ) . prrsv challenged pigs showed a significantly increased partial pressure of co compared to controls in venous blood gas analysis (data not shown). other factors contributing mainly to proximal airway resistance are anatomical peculiarities in the upper airways and head position. while performing pfts, head position was standardised as recommended for pigs (klein et al., ) . the anatomical structure of the nasal cavities, however, might contribute to different upper airway resistances between individuals and could explain significantly different rrs data p hz between groups even before exposure. influence of prrsv on functional residual capacity frc represents the volume present in the lung at the end of spontaneous expiration. therefore, it might be an indicator of 'trapped air', hyperinflation or emphysema. in our trial, no signifi- cant changes in frc could be identified, indicating that airway obstruction was not severe enough to induce 'air trapping' or obstructive emphysema. this is in accordance with a lack of emphysema on pathological examination in our study, as well as studies of others (pol et al., ; halbur et al., ; rossow, ) . in the present study, both tl co hb in relation to body weight (tl co hb /kg) and metabolic body weight (tl co hb /kg . ) revealed significant decreases in the diffusion of oxygen from the lungs into the blood in pigs exposed to prrsv compared to controls. limiting oxygen diffusion from the alveoli to the blood can be explained by increased diffusion distance due to infiltration of alveolar walls by mononuclear cells. in addition, hyperplasia and hypertrophy of the alveolar epithelial cells, as well as the presence of alveolar exudates, decreases the amount of gas within alveoli. alveolar hypoventilation due to a rapid and superficial pattern of breathing may also contribute to decreased oxygen exchange. in the pig, which has no collateral airways (mclaughlin et al., ) , each airway obstruction results in regional heterogeneity of alveolar ventilation (robinson, ) . consequently, obstruction of ventilation in pigs exposed to prrsv most likely resulted in imbalances between ventilation and perfusion, leading to reduced oxygen transfer from the lungs into the blood. this would result in respiratory acidosis characterised by reduced ph and increased partial co pressure in venous blood, indicating reduced alveolar ventilation. this is the first study evaluating disorders of lung function caused by prrsv in pigs. significant imbalances in respiratory mechanics, lung ventilation and pulmonary gas exchange were correlated with clinical signs and pathological findings. prrsv isolate vr- alone was capable of inducing respiratory distress for at least weeks after exposure. studies using other prrsv strains would be useful to identify differences in the pathogenesis of infections caused by different prrsv isolates. evaluation of therapeutic and vaccination strategies will be supported by combining the functional approach of this study with knowledge generated from animal studies focussing on inflammatory markers and the immune response. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. porcine reproductive and respiratory syndrome virus (prrsv): kinetics of infection in lymphatic organs and lung isolation of porcine reproductive and respiratory syndrome (prrs) virus in a danish swine herd and experimental infection of pregnant gilts with the virus effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs experimental reproduction of swine infertility and respiratory syndrome in pregnant sows porcine reproductive and respiratory syndrome (prrs): a review, with emphasis on pathological, virological and diagnostic aspects the effect of vaccination against porcine circovirus type in pigs suffering from porcine respiratory disease complex comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus analysis of respiratory mechanics by impulse oscillometry in non-sedated and diazepam-sedated swine respiratory mechanics in conscious swine: effects of face mask, head position and bronchoconstriction evaluated by impulse oscillometry experimental inoculation of swine at various stages of gestation with a danish isolate of porcine reproductive and respiratory syndrome virus (prrsv) effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine a study of the subgross pulmonary anatomy in various mammals cervical preganglionic sympathetic nerve activity and chemoreflexes in the cat comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure generation of an infectious clone of vr- , a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus influence of cervical sympathetic nerves on ventilation and upper airway resistance in the rat comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr- ), atcc vr , and two recent field isolates of prrsv pathological, ultrastructural and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) validation of impulse oscillometry in friesian and blue belgian calves with respect to changes in extrathoracic upper airway resistance evaluation of lung function in pigs either experimentally or naturally infected with chlamydiaceae an experimentally induced chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation some functional consequences of species differences in lung anatomy experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs porcine reproductive and respiratory syndrome changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for cd (+) cells forced oscillation technique and impulse oscillometry glottis opening and airway resistance mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia porcine respiratory disease complex (prdc) the authors are very grateful to annelie langenberg, sylke stahlberg, ines lemser and to all colleagues working in the team of the animal house (fli jena, germany) for their skilful assistance during the study. furthermore, the authors are thankful to colleagues of the institute of bacterial infections and zoonoses (ibiz) in the 'friedrich-loeffler-institut' (jena, germany), namely dr. ulrich methner and his team for salmonella spp. diagnosis and dr. astrid raßbach and co-workers for performing bacterial screening for pasteurella spp., bordetella spp., haemophilus spp. and app. in addition, the authors thank dr. konrad sachse and staff of the oie reference laboratory for chlamydiosis for chlamydia spp. testing and renate haß for assaying mycoplasma spp. this work was funded by the non-profit organisation 'akademie für tiergesundheit e.v.' (germany), which provided a scholarship for judith wagner. supplementary data associated with this article can be found, in the online version, at doi: . /j.tvjl. . . . key: cord- -lg gvgwt authors: zhou, shaochuan; ge, xinna; kong, can; liu, teng; liu, aijing; gao, peng; song, jiangwei; zhou, lei; guo, xin; han, jun; yang, hanchun title: characterizing the prrsv nsp deubiquitinase reveals dispensability of cis-activity for replication and a link of nsp to inflammation induction date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: lg gvgwt the papain-like cysteine protease (plp ) within the n-terminus of the porcine reproductive and respiratory syndrome virus (prrsv) nsp replicase protein specifies a deubiquitinating enzyme (dub), but its biochemical properties and the role in infection have remained poorly defined. by using in vitro assays, we found that the purified plp could efficiently cleave k and k linked polyubiquitin chains ub - in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. the subsequent mutagenesis analyses revealed that the requirement for plp dub activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., d r, d r, etc.) that largely ablated the dub function also blocked the cis- but not trans-proteolytic cleavage of nsp / polyprotein. moreover, the analyses identified key mutations that could differentiate dub from plp cis- and trans-cleavage activities. further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the dub activity were all lethal to the virus, (ii) a point mutation t g that selectively blocked the cis-cleavage activity of plp did not affect viral viability in cell culture, and (iii) an e q mutation that did not affect either of the plp activities led to rescue of wt-like virus but displayed significantly reduced ability to induce tnf-α production. our findings support the possibility that the plp dub activity, but not cis-cleavage activity, is essential for prrsv replication. the data also establish a strong link of nsp to pro-inflammatory cytokine induction during infection that operates in a manner independent of plp dub activity. protein ubiquitination is an important post-translational modification that regulates a variety of cellular biological processes [ ] [ ] [ ] , i.e., signaling transduction [ ] , protein turnover [ ] , cell cycle progression [ ] , and the immune and inflammatory responses [ , , ] , etc. this modification, however, can often be reversed by deubiquitinases (dubs) through removing various ubiquitin molecules from substrates [ ] . not surprisingly, many dna and rna viruses have evolved to encode dubs to manipulate diverse cellular pathways [ ] . the currently discovered viral dubs largely resemble their cellular counterparts in structure and generally fall into two major classes: ubiquitin-specific preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b, lane ) . moreover, this fragment in the sonicated supernatants could be subsequently purified to homogeneity by one-step affinity purification ( figure b , lane ) with a yield of - mg per ml culture. thus, we have successfully developed a strategy to realize high-level soluble expression of prrsv plp . the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( μg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b and c) . overall, when the total amount of processed ub monomer and dimers (ub+ub ) more recently, prrsv plp was shown to possess deubiquitinating activity in transfected ft cells and can antagonize interferon signaling through inhibiting activation of nf-κb [ , , ] , a feature that is similar to the counterparts from eav and nairoviruses of the family of bunyaviridae [ , , ] . further, deaton et al. have reported the dub activity of prrsv plp by in vitro assay and found that the e. coli purified recombinant plp (aa. - ) is able to cleave both k and k poly-ubiquitin [ ] . on the other hand, although prrsv plp was shown to have deisgylation activity in transfected human cells [ ] , the recombinant plp showed little in vitro deisgylating activity toward isg of porcine origin [ , ] , leaving in question whether it can actually efficiently cleave swine isg conjugates in primary macrophages. in any case, it is clear now that the prrsv plp possesses at least cis-, trans-cleavage, and dub activities [ , , ] . however, their biochemical properties and the contribution to viral infection have remained poorly defined. in this study, we started with characterizing the plp dub activity of hp-prrsv strain jxwn by using in vitro and cell-based assays. by using site-directed mutagenesis, we were able to identify mutations that can differentiate the dub activity from cisand trans-cleavage activities and assess their roles in the context of prrsv infection by reverse genetics analysis. our results revealed novel biochemical aspects of prrsv plp and showed that the plp dub activity, but not the cis-cleavage activity, is likely important for prrsv replication. unexpectedly, we also revealed a strong link of prrsv nsp to virus-induced inflammation that occurs in a dub-independent manner. the findings further advance our understanding of prrsv nsp function and it is the regulation of host immunity and has implications in antiviral drug and vaccine development. marc- and hek ft cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen, carlsbad, ca, usa) with % fbs (invitrogen, ca, usa). primary porcine pulmonary alveolar macrophages (pams) were obtained from specific-pathogen-free (spf) pigs as previously described [ ] and cultured in rpmi medium (invitrogen, ca, usa) containing % fetal bovine serum (fbs) (invitrogen, ca, usa). the chinese highly pathogenic prrsv strain jxwn infectious cdna clone was described previously [ ] . mouse anti-flag monoclonal antibody (f ), mouse anti-ha monoclonal antibody (h ), mouse anti-β-actin monoclonal antibody (a ), and rabbit anti-myc polyclonal antibody (c ) were all purchased from sigma-aldrich (st. louis, mo, usa). horseradish peroxidase (hrp)-conjugated goat anti-mouse polyclonal antibodies and hrp-conjugated goat anti-rabbit polyclonal antibodies were purchased from zsgb-bio (beijing, china). all the restriction enzymes used in this study were from new england biolabs inc. (beverly, ma, usa). the prokaryotic expression plasmids expressing plp ( - )-strepii and plp ( - )-strep ii were constructed by cloning the respective region coding for nsp aa. - and aa. - from hp-prrsv strain jxwn (accession number: ef ) into the vector pet- a (novagen, madison, wi, usa) at the sites of nco i and xho i with a strep ii tag attached to c-terminus for purification purpose. the mammalian plasmid expressing plp ( - )-myc was made by cloning the same plp sequence into the vector pegfp-n (clonetech, ca, usa) at the sites of bamh i and xho i in frame with c-myc epitope tag coding sequence. the plasmids pnsp -myc, pflag-ha-nsp - , and pflag-ha-nsp - ∆ - were generated by fusion expression of the indicated epitope tags with the corresponding coding region for nsp , nsp - or nsp - lacking the first amino acids in the vector pegfp-n (clonetech, mountain view, ca, usa) at the sites of bgl ii and bamh i. all genes in the created eukaryotic plasmids are under the control of cmv promoter, and a kozak core sequence is also included to allow optimal expression. for expression of the derivatives of plp , nsp , or nsp - , the point mutation mutants were created by quikchange site-directed mutagenesis. all the constructs were generated by standard recombinant dna procedure and verified by dna sequencing. bacterial strain bl (de ) containing the plasmid for plp ( - )-strepii or its derivative was cultured overnight at • c and then inoculated at : into ml of yeast extract-tryptone medium culture ( xyt). when the bacterial density at nm reached . to . , protein expression was induced • c for h by addition of iptg (isopropyl-d- -thiogalactopyranoside) (sigma) at the final concentration . mm. the cells were harvested by centrifugation at rpm for - min and then resuspended in pbs with the protease inhibitor cocktail (sigma, p ). the cells were sonicated and lysed for min at • c on ice with % triton x- (sigma, t ). the cell lysates were cleared by centrifugation at , rpm for - min. the supernatants were incubated with ul streptactin sepharose (ge healthcare, - - ) at • c overnight on the rocker. the streptactin sepharose beads were collected by centrifugation and washed six times with tris buffer ( mm tris, ph . ). the target protein was eluted with ul elution buffer ( mm tris, . mm desthiobiotin, % glycerol, ph . ). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, µm ub-amc (boston biochem, u- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a -well black microplate. the fluorescent intensity (excitation, nm, emission, nm) of each well was observed by infinite m pro plate reader (tecan, inc). the dub activity of purified prrsv plp was assayed in µl reaction containing indicated amount of purified plp protein, . µg k -(boston biochem, uc- ) or k -linked polyubiquitin chains ub - (boston biochem, uc- ), and × reaction buffer ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and mm dtt) in a µl volume ep tube. the mixture was incubated at • c for h and then subject to sds-page analysis on % tris-hcl gel. hek t cells grown to %- % confluence in six-well plates were transfected plasmids encoding ha-ubiquitin or plp -myc or nsp , nsp - or derivatives either singly or in combination via lipofectamine plus (invitrogen, ca, usa). at h post-transfection, the cells were treated with mg- (selleck, s ) at µg/ml for h before collection. the cells were washed by pbs for twice and lysed by ripa buffer on the shaker at • c for min. the cell lysates were subject to sds-page and western blot analyses with antibodies to ha or myc epitope. the amount of actin served as a loading control. for the trans-cleavage assay, hek t cells were transfected to express plp -myc, or nsp d n, or its derivatives together with the substrate nsp - ∆ - or nsp - ∆ - g g via lipofectamine plus (invitrogen, ca, usa). for cis-cleavage assay, plasmids coding for nsp - or its derivatives were transfected singly into t cells. at h post-transfection, the expression of enzyme plp or nsp was subject to western blot analysis by using the cell lysates, whereas the cleavage of substrate was analyzed by immunoprecipitation coupled with western blot. briefly, the cells were rinsed with cold pbs and lysed with ripa buffer. the cell debris was removed by centrifugation at , rpm for min and the supernatants were precleared with the protein a/g agarose (santa cruz, ca, usa) on the shaker at • c for min. after that, the cleared supernatants were incubated with ug antibodies to flag epitope and ul protein a/g agarose (santa cruz, ca, usa) at • c overnight. the immunocomplexes were washed for thrice by cold pbs, and the proteins were separated on sds-page and transferred to pvdf membrane. for western blot analysis, the membrane was blocked by pbst with % skimmed milk for h at room temperature and incubated with indicated primary antibody (antibody to ha epitope) at • c overnight. after being washed for thrice by pbst, the membrane was incubated with indicated secondary antibody for h at room temperature. after being washed three times, the membrane was developed with ecl western blot analysis system. for mutagenesis of the infectious clone of hp-prrsv strain jxwn (ef ), the mutations were first introduced into shuttle plasmid peasy-blunt (transgen, beijing, china) contain the fragment a [ ] by site-directed quikchange mutagenesis. after verification by sequencing, digested fragment a was transferred to the infectious clone backbone as described previously [ ] . marc- cells grown on six-well plates were transfected with plasmids of either wt or prrsv mutants. the virus-induced cpe was monitored daily and the cell culture was harvested at - days post-transfection. the cell lysates were passaged blindly onto fresh monolayers for three passages. the viability of mutants was determined by rt-pcr targeting orf and immunofluorescence to n protein. the rescued viruses of passage were titrated on marc- cells. for growth kinetics analysis, marc- cells or primary pams in six-well plates were infected with plp mutants and parental virus at an moi of . . for growth on marc- cells, after h of incubation at • c, the cells were first washed with acid buffer ( mm nacl, mm kcl, mm citric acid, ph . ), followed by once rinse with dmem. at indicated time points of infection, the whole culture was harvested and freeze-thawed three times to release cell-associated viruses. samples were then titrated on marc- cells by using the standard endpoint dilution assay according to the reed-muench method [ ] . primary pams in -well plates were infected with nsp mutants or parental virus at an moi of . . at h post-infection, total rnas were extracted from lysates of infected pams with trizol reagent (invitrogen, ca, usa) according to manufacturer's protocols and dissolved into rnase free water. the concentration of rna in samples was measured with a nanodrop lite spectrophotometer (thermol scientific, usa). µg of total rnas per sample was used for cdna synthesis by fastquant rt kit (tiangen, beijing, china). we conducted qpcr assays using real-time sybr master mix kit (applied biosystem, usa) on an ab real-time pcr system (applied biosystem, usa). primary pams in -well plates were infected with nsp mutants or parental virus at indicated moi. at indicated time points post-infection, tnf-α in cell culture supernatants was quantified using a commercial sandwich enzyme-linked immunosorbent assay (elisa) according to the manufacturer's protocol (cusabio, wuhan, china). the i-tasser online service tool was used to model the structures of prrsv strain jxwn plp [ ] . the plp structures were referred to eav plp ( ium) [ ] , and then its interaction with ubiquitin was analyzed by pymol and coot software. the in vitro cleavage efficiency of k and k -linked polyubiquitin chains was expressed as the ratio of the amount of ub plus ub over total ub (ub+ub +ub +ub +ub +ub +ub ) by measuring the band density in each lance of sds-page images. the ratio of ub over ub was calculated by band density of ub over ub ). the relative dub activity of plp mutants was measured by the corresponding band density of polyubiquitin conjugates and then normalized against actin and expression level of plp . all the data are shown as mean± sem with independent experiments. statistical significance was evaluated by using a two-way analysis of variance (anova). statistical analyses were performed using graphpad prism software (version . ). the quantitation of each protein band is measured by image j software (version . . ). flanking sequence is critical for the yield and solubility of prrsv plp protease domain in e. coli the n-terminal residues of prrsv jxwn nsp was recently reported to exhibit dub activity when expressed and purified from e. coli bl cells [ ] . this fragment, however, was expressed at a very low level in our hands, preventing further efficient purification. since the downstream flanking sequence (nsp aa. - ) is critical for prrsv nsp function during infection [ ] , we hypothesized that this region might be critical for the folding of plp domain, and if so, a c-terminal extension might improve the solubility and yield of plp . accordingly, we made two additional constructs to include prrsv strain jxwn nsp region aa. - and aa. - ( figure b ). these proteins were tagged with a strep ii epitope tag at the c-terminus to facilitate purification. when expressed in e. coli bl cells, plp ( - )-strep ii mostly existed in the pellet, preventing it from efficient affinity purification in large scale ( figure b , lane ). in contrast, plp ( - )-strepii was well expressed, and a substantial amount was presented in the supernatants ( figure b the dub activity of purified plp was subsequently investigated in a series of in vitro assays. we first tested its ability to hydrolyze ub-amc, an ubiquitin conjugated aminomethylcoumarin fluorophore (amc). by monitoring the release of amc, the dub activity was measured. as shown in figure a , the purified plp exhibited dub property in vitro with better activity achieved at a higher concentration ( µg). next, we examined the cleavage of a specific type of polyubiquitin chains ( figure b ,c). overall, when the total amount of processed ub monomer and dimers (ub+ub ) was calculated, prrsv plp exhibited similar efficiency in cleaving both k -and k -linked polyubiquitin chains ub - (% cleavage efficiency = ub +ub /ub - ) ( figure b ,c). however, prrsv plp display a differential activity converting k and k linked ubiquitin dimers into monomers. the results showed that the cleavage into monomer of k -linked ub - was relatively inefficient ( figure c ) with the reaction taking place in a dose and time-dependent manner ( figure c ). for example, at a lower dose of plp ( µg), k -linked ub - was mainly cleaved into ub dimers ( figure c , lane ), even if the incubation time was extended to two hours ( figure c , lane ). however, when the dose increased ( µg), plp could hydrolyze k ub - efficiently into monomers within h ( figure c , lane ). the difference in kinetics toward cleaving k versus k ub - was further investigated in a more detailed time-dependent manner from to min at a concentration of µg for plp . the result showed that plp was able to quickly cleave both k and k ub - within minutes ( figure d ,e), but the conversion from ubiquitin dimer to monomer was several fold faster for plp k dub activity ( figure d -f). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a ). to identify critical residues that are potentially important for plp dub activity, we modeled the structure of plp core domain with the online program i-tasser [ ] by using eav plp (pdb id: ium) as a model. the homology remodeling revealed that prrsv strain jxwn plp (aa. - ) has an overall similar structure to that of the counterpart eav plp , except for two minor structural differences as indicated by the red arrows ( figure a) . the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged cterminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub cterminus ( figure c ). the bioinformatics analysis also identified an acidic cluster (d dwatded ) that has potential contact with the positively charged c-terminus of ubiquitin ( figure b ). in particular, the ub residues r and r form a positively charged surface, whereas prrsv plp residue d , d , d , e and d form a negatively charged region. the modeling showed that the charged c-terminus is inserted into a grove formed by negatively charged residues d , e , and d of plp ( figure b ). further, a closer look into the contact sites revealed that both plp d and d can potentially interact with r and r respectively through potential hydrogen bond and electrostatic interactions ( figure c ). in contrast, d and d are in a position that is far away from ub c-terminus ( figure c ). for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. for this identified acidic cluster, the residues d , d , w , a , d , and d are highly conserved among various prrsv strains, whereas the residues t and e are highly conserved among type ii prrsv strains but are replaced with the respective residues s and y in type i prrsv strains ( figure s ) [ ] . among these residues, both w and d have been shown to be critical for the trans-cleavage of nsp / [ ] . thus, we, therefore, selected other residues (d , d , t , e , d ) for further mutational analysis with a purpose to distinguish dub from its trans-cleavage activity. the acidic residues were changed to either the residue asparagine (n) or to the opposite charge residue arginine (r), whereas t was replaced with either serine (s), glycine (g), or arginine (r). the corresponding mutants were expressed and purified from e. coli bl cells, and their enzymatic activities were tested by the in vitro dub assay ( figure a ) and the quantified relative activity was shown at the right of corresponding figures. the results showed that the mutations of the residues d and e (e.g., d n, d r, e r, e q, etc.) did not have much effect on plp dub activity, as the corresponding mutants could efficiently cleave k or k polyubiquitin chains into monomers ( figure a , lanes , , , and ). in contrast, mutation of the residue d to either n or r largely blocked the cleavage of ub - with only a very small portion cleaved into ub dimer or monomer ( figure a , lane and ). the mutational effect of d was amino acid-dependent. the d r mutation largely blocked the plp dub activity ( figure a , lane ), whereas the mutation d n did not affect the cleavage much ( figure a, lane ) . for the residue t , substitution with r (t r) largely ablated the dub activity ( figure a , lane ), whereas the mutation to serine (t s) did not have an effect ( figure a, lane ) . the t g mutation did not have a large effect on the ability of plp to process the k polyubiquitin chains ( figure a , lane , bottom panel), but it partially crippled the ability of plp to cleave the ubiquitin dimer ( figure a , lane , top panel), suggesting a differential sensitivity. we next used a cell-based assay to further check on the mutational effect on plp dub activity within mammalian cells ( figure b ). at the same time, the mutants plp c a and d n ( figure b , lanes and ), which have been shown to be defective of trans-cleavage activity [ ] , were used as negative control. the ha-tagged ubiquitin was transiently expressed in hek t cells together with plp or plp -derived mutants. at h post-transfection, the cell lysates were collected to assess the level of ubiquitination by western blot analysis with antibodies to ha epitope ( figure b ). the results showed that the mutants d r, d n, t s, e q, and d n were dub active ( figure b ). in contrast, the mutants d r, d n, t r, and d r lost the dub activity, together with the control mutants plp c a and d n ( figure b ). on the other hand, the mutations e r and t g partially crippled the plp dub activity ( figure b, lanes and ) . overall, the cell-based results are largely consistent with the in vitro dub assay. in addition, the results of mutagenesis studies are in accordance with the bioinformatics prediction, except for several mutations (e.g., t r, e r, and d r), which will be discussed in the discussion section. we next investigated whether the same mutation affects the plp cisor trans-cleavage activity, which can be important for the processing of prrsv polyprotein nsp - during infection. to address this question, we performed the cell-based assay by co-expressing plp or its derivatives with the substrate nsp - ∆ - lacking the plp domain in transfected ft cells ( figure a ). for a negative substrate control, the plp cleavage site at the nsp - junction was mutated to make the construct nsp - ∆ - g p ( figure a ). as shown in figure a , except for the control d n, all other plp mutants were capable of efficiently processing the nsp - polyprotein, suggesting that the mutations did not affect the trans-cleavage activity of plp . thus, the mutations (e.g., d n, d r, e r, d r, t r, etc.) that affected the dub activity can be used to differentiate the dub activity from trans-cleavage activity. viruses , , x for peer review of we next investigated whether the same mutations affect the plp cis-cleavage activity. since cis-cleavage is essentially an intramolecular interaction, the mutational effect was evaluated by using the full-length nsp - polyprotein precursor as reported previously [ ] . in addition, since both cisand trans-cleavage activities can direct the nsp - cleavage [ ] , the trans-cleavage has to be silenced in the first place before proceeding to test the effect on cis-activity. here, we took advantage of the d n mutation, which is able to decouple the nsp cisfrom trans-cleavage activity by using prrsv strain vr- [ ] . consistently, the d n mutation strongly reduced the trans-cleavage activity of plp to process nsp - ∆ - in trans in the cotransfection assay ( figure a, lane ) , and the cleavage efficiency was only about - %. to make sure the full-length nsp carrying d n mutation also behaves the same as seen for the plp fragment ( figure a, lane ) , we introduced this mutation into the full-length nsp of prrsv strain jxwn . as expected, the full-length nsp carrying d n mutation also failed to process in trans the polyprotein nsp - lacking the protease domain (nsp - ∆ - ) in the co-transfected cells ( figure b, lane ) , suggesting the trans-cleavage activity is blocked. when the same mutation was introduced into the nsp - precursor (nsp - d n) , it, however, did not affect the processing in cis of nsp from nsp - d n ( figure c, lane ) . thus, this result is in agreement with the previous report based on prrsv strain vr- [ ] . next, we introduced the corresponding acidic cluster point mutations into the background nsp - d n ( figure c ), which allows only cis-processing of nsp . to our surprise, the mutations (e.g., d n, d r, t r, and d r) that largely ablated the dub activity also blocked the cis-activity of plp ( figure c , lanes , , , and , respectively). in contrast, neither d n nor d r affected the cis-cleavage activity ( figure c , lane and ). notably, the mutation t g could selectively block the plp cis-cleavage activity ( figure c, lane ) , as the mutant plp t g was still equipped with trans-cleavage activity ( figure a , lane ) and largely dub activity ( figure a, lane ) . together, these results suggest that the requirement for plp dub activity somehow is more closely related to that for cis-cleavage activity, rather than the trans-cleavage activity. in addition, t g is a critical mutation that can differentiate the cis-cleavage activity from dub and trans-cleavage activity. the mutational effect on viral replication was tested by introducing the corresponding point mutations into the infectious cdna clone of hp-prrsv strain jxwn in a dna-launched system. the recombinant plasmids were transfected into marc- cells for virus recovery. our analyses by reverse genetics of plp mutants led to the findings as follows (table ). (i) mutations (d n, d r, t r, and d r) that largely blocked the dub activity in cell-based assay were all lethal to the virus, (ii) mutations (d r, d n, t s, and e q) that did not have an effect on dub activity did not affect viral viability, (iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and (iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a ). interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). slightly blocked viral viability, iii) the point mutation t g that selectively blocked the plp cis-activity did not affect viral viability in cell culture, and iv) the mutation d n and e r, which did not affect either of the plp activities, somehow did not allow the rescue of viable virus. together, our results strongly suggest a dispensable role of the plp cis-activity for prrsv viability. on the other hand, the fact that the cis-cleavage activity is not required for prrsv replication suggests that the lethal phenotype of several point mutations (d n, d r, t r, and d r), which blocked both dub and cis-activity, is not due to a block in cis-cleavage activity, but may be linked to the inactivation of plp dub activity. four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus，except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h postinfection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). largely blocked - : active, -: blocked, nd: not determined, asmids were transfected into marc- cells for virus recovery. our analyses by plp mutants led to the findings as follows (table ) . i) mutations (d n, d r, at largely blocked the dub activity in cell-based assay were all lethal to the virus, , d n, t s, and e q) that did not have an effect on dub activity did not affect e point mutation t g that selectively blocked the plp cis-activity did not affect l culture, and iv) the mutation d n and e r, which did not affect either of the ehow did not allow the rescue of viable virus. together, our results strongly ble role of the plp cis-activity for prrsv viability. on the other hand, the fact activity is not required for prrsv replication suggests that the lethal phenotype tations (d n, d r, t r, and d r), which blocked both dub and cis-activity, ck in cis-cleavage activity, but may be linked to the inactivation of plp dub le . the association between various plp activity and virus viability. ced production of tnf-α and il- β is strongly associated with nsp that is dub activity tants of passage (p ) were chosen for growth kinetics analysis in both marc-) and primary pams ( figure b ). all the mutants tested showed relatively similar to the parental virus，except for the mutant t g, which exhibited reduced cells by about half a log and a slight decrease in pams although being icant ( figure b ). to test the stability, we sequenced the plp mutation region at nts or p viruses. the results showed that the mutations were stable ( figure c) . not see any other compensating mutations arising within nsp . since prrsv nsp onizing host innate immunity and the plp mutants did not have an apparent rimary pams, we tested whether mutations have an effect on inflammatory n rather than on interferon signaling. in particular, we focused on the expression f-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this s were infected with wt or mutant viruses with an moi of . . at h postlevel of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr peptidylprolyl isomerase a (ppia), a molecule that has been previously used for - ] . the results showed that prrsv strain jxwn significantly upregulated the of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was ure a). interestingly, the mutant e q exhibited greatly reduced ability to f both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a ). four viable mutants of passage (p ) were chosen for growth kinetics analysis in both marc- cells ( figure a ) and primary pams ( figure b ). all the mutants tested showed relatively similar growth properties to the parental virus, except for the mutant t g, which exhibited reduced growth in marc- cells by about half a log and a slight decrease in pams although being statistically insignificant ( figure b ). to test the stability, we sequenced the plp mutation region at the end of experiments or p viruses. the results showed that the mutations were stable ( figure c ). in addition, we did not see any other compensating mutations arising within nsp . since prrsv nsp is involved in antagonizing host innate immunity and the plp mutants did not have an apparent growth defect in primary pams, we tested whether mutations have an effect on inflammatory cytokine production rather than on interferon signaling. in particular, we focused on the expression and secretion of tnf-α, il- β, and il- ( figure ) , the most potent inflammatory cytokines. to this end, primary pams were infected with wt or mutant viruses with an moi of . . at h post-infection, the mrna level of tnf-α, il- β, and il- were measured by relative quantitative rt-pcr normalized against peptidylprolyl isomerase a (ppia), a molecule that has been previously used for internal control [ ] [ ] [ ] . the results showed that prrsv strain jxwn significantly upregulated the mrna expression of both tnf-α and il- β ( figure a ). on the other hand, the effect on il- was not impressive ( figure a) . interestingly, the mutant e q exhibited greatly reduced ability to induce expression of both tnf-α and il- β, and to a lesser extent by the mutant t g ( figure a) . similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands. to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a ). similarly, none of the mutants affected the level of il- ( figure a ). we also measured the secretion of these cytokines in the infected supernatants by elisa. consistent with a reduction at the mrna level, we observed a significant drop of tnf-α in the cell culture supernatant from primary pams infected with the mutants t g and e q ( figure b ). for il- β and il- , we tried several commercial kits, and unfortunately, they did not work well in our hands (data not shown). to test whether the effect was contingent on the infection dose, we chose e q and t g for further analysis (figure ) . the results showed that either moi of . , . , or could significantly reduce the secretion of tnf-α in the infected supernatants ( figure a ). when compared with wt, this reduction is by more than - % for the mutants e q and t g at h post-infection or later, moreover, the e q mutant had a greater ability to reduce the production of tnf-α ( figure a) . finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b, lane ) , and nsp t g was largely dub-active ( figure b, lane ) . in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a cterminal extension dramatically improved the plp yield and solubility ( figure b) . our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we finally, to further evaluate whether the observed effect on pro-inflammatory cytokine production is associated with altered plp dub activity, we introduced the corresponding mutations (e q, t g, d r, and t r) into the full-length nsp rather than merely into the plp fragment ( figure b ). the deubiquitinating ability of nsp or its derivatives was then tested in transfected t cells by co-expression with ha-ubiquitin. consistent with the phenotype exhibited by the truncated nsp , the mutant nsp e q ( figure b , lane ) behaved just like wt nsp ( figure b , lane ), and nsp t g was largely dub-active ( figure b , lane ). in contrast, the catalytic site mutation c a blocked the dub activity of the full-length nsp ( figure b, lane ) . together, the above data provided strong evidence on a critical role of prrsv nsp in inducing tnf-α and il- β during infection, and also this property of nsp is independent of the plp dub activity. in this report, we developed a strategy to obtain high-level soluble expression of prrsv strain jxwn plp in e. coli bl cells for assaying its dub activity in vitro. the in vitro and cell-based assays uncovered important biochemical properties of plp , including efficient cleavage ability towards both k -linked and k -linked polyubiquitin chains and a surprisingly shared requirement for both cis-cleavage and dub activity. moreover, we identified mutations that could distinguish the three activities of plp . further reverse genetics analysis revealed dispensability of cis-activity for prrsv replication. in contrast, viruses carrying point mutations that largely blocked the dub activity were not viable, pointing to a potentially critical role of plp dub in prrsv replication. during characterizing the viral mutants, we also unexpectedly discovered a dub-independent regulation of pro-inflammatory cytokine production by prrsv nsp . together, these findings further deepen our understanding of the biological properties and function of prrsv plp and nsp in virus life cycle and have implications in understanding viral pathogenesis and in vaccine development. the relevant significance or insights are discussed below. it is now known that prrsv plp possesses at least cis-, trans-cleavage, and dub activities and that the trans-activity requires the nsp region aa. - [ ] . in this report, we went much further to reveal several important biological aspects of this particular protease. beginning with the in vitro purification, we found that the downstream flanking sequence (nsp aa. - ) was critical for the yield and solubility of plp domain in the prokaryotic expression system ( figure b) . although a previous study reported that the prrsv strain jxwn plp (nsp aa. - ) is sufficient for dub activity in vitro, but it did not provide description about the expression level and purification of this fragment [ ] . in our hand, this fragment was unfortunately poorly expressed in e. coli bl cells despite attempts for optimization of expression conditions. although truncation is usually the way for protein expression, we used an unconventional approach to solve the problem, making a c-terminal extension dramatically improved the plp yield and solubility ( figure b ). our results suggest that the flanking sequence likely plays an important role in the folding of plp core domain, and this is consistent with our previous finding that deletion of nsp aa. - is lethal to prrsv strain vr . the realization of high-level soluble expression also paves the way for future structural studies and in vitro anti-plp drug screening. previous studies have shown that prrsv plp possesses differential dub activity toward cleaving k and k ubiquitin dimers into monomer. our results are in general in agreement with this finding [ ] . different from the previous study, we found that prrsv plp is able to cleave ubiquitin dimer but just in a dose and time-dependent manner. this difference is likely attributed to the plp flanking sequence (aa. - ) (figure ), which can promote the folding of plp core domain, leading to increased cleavage efficiency toward k polyubiquitin chains. in addition, we found that prrsv strain jxwn plp can equally efficiently cleave k and k -linkd ub - into dimers and monomer. we also identified critical residues for prrsv plp dub activity. d , an amino acid that is highly conserved among prrsv strains [ ] , was revealed to be a key residue for prrsv plp dub activity (figure ). either a conserved (d n) or non-conserved (d r) substitution led to a significant destruction of plp dub activity ( figure ) . the mutational effect also appears specific, as similar mutations of the nearby residue d (d n and d r) that is also highly conserved did not have an effect (figure ). this result is also in accordance with the bioinformatics prediction ( figure ) . the mutational effect of other residues is contingent on the amino acids used for substitution. for example, the mutations d r and t r largely blocked the plp activity and e r partially blocked the dub activity ( figure ). we suspect that the mutational effect of d r is most likely indirect and could result from electrostatic interaction with e to change the local structure. in a similar manner, the mutation t r can potentially interact locally with e , d , or d , leading to local conformational change of plp , which may have an adverse effect on plp function. this also partially explains why t s, e q, and d e did not have an effect. most interestingly, we found that the t g mutation can selectively block the cis-cleavage activity ( figure c ), without affecting the trans-cleavage activity ( figure a ) and only partially blocking the dub activity in either truncated ( figure b ) or full-length form ( figure b ). this is the first time to report a point mutation that distinguishes cis-cleavage from other activities and paves the way to test the importance of cis-cleavage activity in prrsv infection. it has been viewed that hydrolysis of ubiquitin substrates takes advantage of the trans-activity of viral dubs. surprisingly, we observed an intriguing relationship between prrsv plp dub and cis-activity. that is, several mutations that largely blocked the plp dub activity were found to also ablate its cis-activity, and these include the mutants d n, d r, t r, and d r (figures and c ). in contrast, none of these mutations had an effect on the plp trans-activity cleaving nsp - polyprotein ( figure a ). thus, it seems that the requirement for prrsv plp dub activity is somehow more closely related to cis-activity, suggesting that they may share some intrinsic common mechanistic details in terms of recognition and cleavage of the substrates. to our knowledge, this is the first report revealing such intimate relationship between dub and cis-activity concerning otu cysteine proteases. the molecular basis for these observations is currently not clear, but hopefully, elucidation of the three-dimensional structure of prrsv plp may help resolve the puzzle in the future. a long-asked question is whether the plp dub and cis-cleavage activities are essential for prrsv infection. the inability to differentiate the enzymatic activities by point mutations has limited the evaluation of the contribution of individual enzymatic activities. despite the reports of several studies on the plp dub activities [ , , , ] , none of them established a strong correlation between dub and viral replication, as they did not rule out the mutational effect on both transand cis-cleavage activities. by taking advantage of site-directed mutagenesis, we in the first place provided strong evidence to suggest that the plp cis-activity is not necessary for viral replication ( figure ). this is evidenced by the specific point mutation t g, which selectively blocked the plp cis-cleavage activity but allowed the successful rescue of viable virus. moreover, this mutant exhibited similar growth rate to wt in primary pams ( figure ). in contrast, the mutations (e.g., d n, d r, t r, d r, etc.) that largely disarmed the plp dub activity were lethal to prrsv in marc- cells. although these mutations also crippled the cis-cleavage activity of plp at the same time, the successful rescue of t g mutant strongly argues against an essential contribution of inactive cis-activity to the non-viability phenotype. in addition, the lethal phenotype is less likely due to a potential defect of deisgylation activity of plp , as it has been reported that the hp-prrsv strain jxwn plp used in this study has very limited deisgylation activity [ ] and that isgylation was not observed in prrsv-infected marc- cells [ ] , a cell line that was used for virus rescue. thus, there appears to exist a strong correlation between the lethal phenotype of these mutations and the essential role of plp dub activity in prrsv infection. sun et al. recently characterized the nsp otu domain of a european prrsv strain sd - (type i strain) and reported that plp inhibits nf-κb activation through its dub activity [ ] . they also mutated the residues in the acidic cluster, including the residues d , d , s , d , d , etc., corresponding to the residues d , d , t g and d in this study, respectively [ ] . consistent with our studies, the mutations d a and d a were lethal to prrsv, whereas d a, s a, and d a allowed the successful rescue of the viable virus [ ] . in these studies, the mutant d a did not affect the viral growth, whereas s a and d a crippled the virus in marc- cells [ ] . sun et al. further correlated the growth property with the ability to inhibit nf-κb activation and found that the mutations that were lethal to the virus were these capable of completely inhibiting nf-κb activation, whereas the mutations s a and d a unable to inhibit the activation as wt lead to viable virus [ ] . however, they did not further test the mutational effect of these mutants on cisand trans-cleavage activity and the dub activity. our studies here suggest that the lethal phenotype is not linked to an impairment of cisor trans-cleavage activity, but rather related to an effect on plp dub activity. however, because these are negative results, we could not rule out the possibility that the dub-inactivating mutations may have other unexpected consequences on nsp , or both, which renders the virus non-viable. on the other hand, the mutations e r and d n, which did not affect much the plp dub activity, did not allow the rescue of viable viruses. this result suggests that the mutations may have an adverse effect on other functions of nsp , highlighting the complexity of mutational effect on the function of plp or nsp . another interesting observation is that the partial ablation (t g) of plp dub activity did not affect viral viability whereas a near-complete block (e.g., d n, d r, t r, d r, etc.) was lethal to the virus. these results may suggest that there exists a threshold of dub activity required for prrsv replication, in which the dub activity may be required to remove polyubiquitins from substrates. future study will be directed to investigate detailed mechanisms by which the prrsv plp plays during infection. infections by prrsv often cause de-regulation of inflammatory cytokine production [ ] . for the chinese hp-prrsv, it inhibits inflammation in early infection, but induces inflammatory storm during the late stage in pigs, contributing to lung injury and interstitial pneumonia [ , ] . the regulatory mechanisms of this process have remained poorly understood, but it is known that several nonstructural proteins, including nsp α, nsp β, nsp , and nsp , are capable of inhibiting inflammation response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , whereas the structural proteins n and e promote inflammation [ , ] . prrsv nsp is a type i interferon signaling antagonist [ ] and recently implicated in the modulation of pro-inflammatory cytokine production during infection [ , , ] . liu et al. [ ] reported that mutants of hp-prrsv strain bb carrying deletion of nsp region aa. - or aa. - had the reduced ability to induce the expression of inflammatory cytokines il- β, tnf-α, and il- in pams. however, the corresponding mutants also exhibited reduced growth titer by . to log when compared with the parental virus. moreover, down-regulation is quite subtle (< %). on the other hand, the single-site substitution represents a nice approach by which it can minimize the risk of gross conformational alteration as a result of mutagenesis. at the beginning of this study, we did not mean to study the role of nsp in inflammatory cytokine induction, but with two independent point-mutation mutants (t g and e q) at hand, we unexpectedly established a strong link of nsp to prrsv-induced induction of pro-inflammatory cytokines (figures and ) . the mutants t g and e q retained the similar growth rate to the parental virus jxwn in primary pams, but exhibited significantly reduced ability to induce tnf-α and il- β at both mrna and protein levels (figures and ) . moreover, this reduction rate was by more than % at hpi or later ( figure a ), suggesting a decisive role of nsp in modulating the production of tnf-α and il- β during infection. different from the previous study [ ] , we did not observe a change in expression level of il- . the crippled ability to induce tnf-α and il- β cannot be attributed to an impairment of plp dub activity, as the mutant nsp e q was fully dub active ( figure b ). in addition, because viral dubs often negatively regulate host innate immunity, it is expected that inactivation of dub activity should promote the inflammatory cytokine production as reported by several cases [ , , , , ] . our results, however, showed the opposite, the mutant t g with partially crippled dub activity showed reduced production of tnf-α and il- β (figures and ). thus, with two independent nsp mutants (t g and e q) showing the similar phenotype, our results provide strong evidence to suggest a dub-independent regulatory mechanism by prrsv nsp in inducing tnf-α and il- β production. together, our findings further deepen our understanding of the biological properties and function of prrsv nsp and also provide clues into the pathogenic mechanisms of hp-prrsv. additionally, the results have implications in vaccine development by modifying nsp to reduce cytokine-induced lung injury. in the near future, it will be interesting to address molecular mechanisms of how the point mutations in nsp lead to crippled ability of prrsv to induce pro-inflammatory cytokines. breaking the chains: structure and function of the deubiquitinases atypical ubiquitylation-the unexplored world of polyubiquitin beyond lys and lys linkages the ubiquitin code a ubiquitin replacement strategy in human cells reveals distinct mechanisms of ikk activation by tnfalpha and il- beta a multiubiquitin chain is confined to specific lysine in a targeted short-lived protein k -linked polyubiquitination in cell cycle control revealed by a k linkage-specific antibody the ubiquitin-proteasome pathway is required for processing the nf-kappa-b precursor protein and the activation of nf-kappa-b the role of ubiquitylation in immune defence and pathogen evasion a genomic and functional inventory of deubiquitinating enzymes viral otu deubiquitinases: a structural and functional comparison proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases a deubiquitinating enzyme encoded by hsv- belongs to a family of cysteine proteases that is conserved across the family herpesviridae mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling jupin, i. a viral deubiquitylating enzyme targets viral rna-dependent rna polymerase and affects viral infectivity deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (votu) structure and deubiquitinase activity crimean-congo hemorrhagic fever virus suppresses innate immune responses via a ubiquitin and isg specific protease viral avoidance and exploitation of the ubiquitin system nidovirales: a new order comprising coronaviridae and arteriviridae reorganization and expansion of the nidoviral family arteriviridae isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) experimental reproduction of swine infertility and respiratory syndrome in pregnant sows emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence identification of nonessential regions of the nsp replicase protein of porcine reproductive and respiratory syndrome virus strain vr- for replication in cell culture complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus the porcine reproductive and respiratory syndrome virus nsp cysteine protease domain possesses both trans-and cis-cleavage activities porcine reproductive and respiratory syndrome virus nonstructural protein contributes to nf-kappab activation effect of amino acids residues - and - in nsp of representative porcine reproductive and respiratory syndrome virus strains on inflammatory response in vitro processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes proteolytic processing of the replicase orf a protein of equine arteritis virus the arterivirus nsp protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the votu domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference insights into the porcine reproductive and respiratory syndrome virus viral ovarian tumor domain protease specificity for ubiquitin and interferon stimulated gene product changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis a simple method of estimating fifty per cent endpoints i-tasser server for protein d structure prediction transcriptome analysis reveals dynamic gene expression profiles in porcine alveolar macrophages in response to the chinese highly pathogenic porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nsp β and nsp antagonize the antiviral activity of cholesterol- -hydroxylase via lysosomal degradation evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to lps and lta pathogenesis and control of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome in china regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection immunological features of the non-structural proteins of porcine reproductive and respiratory syndrome virus interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus both nsp beta and nsp are responsible for differential tnf-alpha production induced by porcine reproductive and respiratory syndrome virus strains with different pathogenicity in vitro porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes beta interferon expression by targeting the nf-kappab essential modulator we thank our colleague nianzhi zhang for his help on bioinformatics analysis of plp . the authors declare that they have no conflicts of interest with the contents of this article. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.viruses , , key: cord- -g zhv authors: rowland, raymond r.r; lawson, steven; rossow, kurt; benfield, david a title: lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: g zhv the ability of porcine reproductive and respiratory syndrome virus (prrsv) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to prrsv in utero. in this study, virus replication and prrsv-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at days of gestation with prrsv isolate vr- . eighty-four percent of pigs were born viremic with a mortality of % within days after birth. at approximately days sera from pigs were negative for virus by virus isolation. analysis of virus replication in the tissues of pigs randomly sacrificed between and days showed no evidence of virus in lung and other non-lymphoid organs. however, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. even though replication was at a low level, virus was easily transmitted to sentinel pigs. by days pigs became seronegative and did not transmit virus to sentinel pigs. sacrifice of remaining pigs after days showed no evidence of virus in blood and tissues. this study shows that congenital prrsv-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positivestranded rna virus, prrsv, belonging to the family arteriviridae (wensvoort et al., ; benfield et al., ; nelsen et al., ) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, ) . the arteriviruses, toroviruses, and coronaviruses are members of a single order, nidovirales (cavanagh, ) . arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (snijder and mulenberg, ) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic persistent infection, as well as cause severe and fatal disease (plagemann, ) . the outcome following experimental prrsv infection is dependent on the age and reproductive status of the pig, as well as the isolate used for infection (reviewed in rossow, ) . infection of adult pigs usually produces a nonfatal disease, characterized by mild flu-like symptoms, a transient elevation in temperature and inappetance. the reproductive form of prrs occurs following the infection of pregnant gilts or sows. accordingly, virus-induced reproductive failure can present clinically as increased regular and delayed returns to estrus and not-in-pig sows, as well as abortions, mummified fetuses, stillbirths and weak-born pigs. christianson et al., ; mengeling et al., ; benfield et al., ; rossow et al., ) . surviving neonates exhibit the severest form of respiratory disease with mortality often reaching % within weeks after birth. besides interstitial pneumonia, viral lesions in infected neonates can be found in primary and secondary lymphoid organs, including bone marrow, thymus, lymph node, spleen and tonsil, and non-lymphoid organs, such as brain, heart, kidney, and stomach (rossow et al., (rossow et al., , rossow, ; feng et al., ) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs. another outcome following prrsv infection is persistently infected swine herds, a property that has contributed to the world-wide spread of the disease (albina, ; chung et al., ; duan et al., ; wills et al., ; allende et al., ; bierk et al., ) . it is assumed that persistence is maintained by the ability of prrsv to evade host defenses and establish a long-term asymptomatic infection within individual pigs. even though prrsv-specific antibody appears as early as days post-infection and is followed by serum neutralizing activity and cell-mediated immunity molitor, , ; rowland et al., rowland et al., , persistently infected pigs can continue to shed virus. the mechanistic basis for prrsv persistence is not known. similar to ldv, prrsv may sustain replication in a subpopulation of renewable macrophages, while avoiding host-defenses . another possibility is the restriction of virus replication to immunoprivileged sites, such as the male reproductive tract (christopher-hennings et al., ; sur et al., ) , which is observed during infection with other arteriviruses, such as eav and ldv (holyoak et al., ; anderson et al., ) . in previous work characterizing quasispecies evolution during infection, we proposed that antigenic drift in the ectodomain of gp may lead to the selection of clones that are resistant to neutralizing antibody or change the tropism of the virus for a different population of cells . previous experimental studies designed to understand prrsv persistence have characterized virus replication during prrsv infection of postnatal pigs. these studies indicate that virus is present in the blood for approximately - days after infection; however, in a significant number of animals, virus can be detected in tonsil for up to days and by rt-pcr in semen for days after infection christopher-hennings et al., ; allende et al., ) . more recently wills et al. ( ) reported that six of pigs, infected at days after birth, were positive for the presence of virus at days. however, the ability of these long-term infected pigs to shed virus was not determined. very little is known regarding the course of virus replication in pigs exposed to virus in utero. the purpose of this study was to further understand persistent infection in congenitally infected pigs by characterizing the course of clinical disease, sites of virus replication and the capacity to transmit virus in a group of pigs exposed to prrsv in utero. three pregnant sows at - days gestation were infected with prrsv isolate vr- collins et al., ) passage , which was plaque-purified and propagated on marc- cells, a subclone of the ma- cell line (kim et al., ) . five milliliters of virus, at a concentration of tcid /ml, was divided into two doses and administered into each nare with a cc syringe. the sows were monitored daily for signs of prrsv infection and allowed to farrow naturally. umbilical cord and blood samples were collected from newborn pigs at birth and prior to nursing. blood was then collected from surviving pigs at weekly intervals for the duration of the study. neonates were monitored daily for clinical signs. pigs that succumbed to acute prrsv infection were necropsied and tissues evaluated for pathology and virus replication. at days all surviving pigs were weaned and placed together in a single isolation room in the animal care facility. prrsv-specific antibody in serum was measured using the "old format" herdchek ® prrs elisa (idexx) according to the manufacturer's instructions. this assay was done by personnel at an accredited diagnostic laboratory (animal disease research and diagnostic laboratory at south dakota state university). an s/p ratio greater than . was considered seropositive. prrsv neutralizing activity in serum was measured according to rowland et al. ( ) . serial : dilutions of serum were prepared in culture medium on well plates. an equal volume of the vr- prrsv, at a concentration of tcid /ml, was added to each sample and incubated for h at • c then transferred to a -well plate containing confluent marc- cells. after days the plates were fixed in % acetone and infected cells detected with fitc-labeled anti-nucleocapsid monoclonal antibody, sdow- (nelson et al., ) diluted in pbs with % fbs. neutralizing activity was reported as the log of the highest serum dilution that prevented the replication of prrsv in marc- cells (negative for sdow- staining). virus isolation was performed by incubating dilutions of serum or tissue homogenate on -well plates containing marc- cells. for virus isolation from tissues, approximately g of tissue was homogenized in ml of hanks balanced salt solution and centrifuged at × g for min to remove debris. dilutions ( : and : ) of cleared homogenate or serum were incubated on -well plates of marc- cells for days. plates were then fixed in % acetone and stained with fitc-sdow- . nested rt-pcr amplification of orf , described by christopher-hennings et al. ( ) , was used for the detection of viral rna in serum and tissues. samples were homogenized in an equal volume of lysis buffer ( m guanidinium thiocyanate, mm sodium citrate [ph. ], . % sarkosyl [n-lauryl sarcosine] . m -mercaptoethanol). five hundred microliters of lysate was added to ul of phenol and ul chloroform and mixed. the aqueous phase was removed, the rna precipitated with ethanol and then reconstituted in ul of distilled water. one microliter of rna was reverse transcribed using mulv reverse transcriptase and the outer antisense pcr primer. the outer sense and antisense primers for pcr amplification of a bp product incorporating the entire orf region were -tcgtgttgggtggcagaaaagc- and -gccattcaccacacattcttcc- , respectively. the outer primers were incorporated into a reaction mixture containing cdna, mm mgcl , × pcr buffer and taq polymerase. the product was subjected to a second round of amplification using the nested sense and antisense primers, -ccagatgctggg-taagatcatc- and -cagtgtaacttatcctccctga- , respectively. the second round of amplification incorporated mm mgcl , × pcr buffer and taq polymerase. forty cycles of amplification were performed for each primer pair. all pcr reactions incorporated a • c denaturing step ( s), a • c annealing step ( s), and a • c polymerization step ( s). the reaction products were electrophoresed on an agarose gel and the final bp product detected with ethidium bromide. in situ hybridization for the identification of cells supporting virus replication was done according to lawson et al. ( ) using a modification of the procedure originally described by anderson et al. ( ) . tissues were fixed in % neutral buffered formalin for h at room temperature and embedded in paraffin. thin sections were mounted on denhardt's solution-treated slides and paraffin removed with xylene. after pretreatment, the tissue sections were hybridized with a [ s] dctp labeled random-primed probe complementary to a nucleotide region covering all of orf . slides were hybridized for h at • c, washed, dipped in kodak ntb- autoradiographic emulsion, exposed for approximately days, developed and counterstained with mayer's hematoxylin and eosin y. a positive control included tissues from pigs experimentally infected with prrsv. negative controls included tissues from uninfected pigs and tissues from experimentally infected pigs hybridized with a probe prepared using cdna from ldv orf region. three pregnant sows were infected with vr- at days gestation. clinical signs in sows were minimal, consisting of inappetance in two of the three animals, which began at - days post-inoculation and lasted approximately days. all sows seroconverted and farrowed naturally at days of gestation. outcomes for the piglets from the three infected sows are summarized in table . seven pigs were stillborn and due to the degraded condition of the tissues were not subjected to further study. analysis of virus and antibody in blood and/or umbilical cords from the live neonates showed that at least or % were virus isolation (vi) or rt-pcr positive for prrsv at the time of farrowing indicating that in utero infection was successful. the eight neonates that were identified as prrsv-negative at the time of birth became vi-positive by dpf. neonates as a group exhibited signs consistent with prrsv infection, with pronounced dyspnea being the most common symptom. about half of the pigs exhibited more severe disease signs, including ocular edema and inappetance. microhistology showed prrsv-induced pathological changes in the lungs and other organs of acutely infected symptomatic pigs, including interstitial pneumonia with the infiltration of proteinaceous debris from the interstitial space into the alveoli. lymph nodes were generally enlarged with germinal center hypertrophy, hyperplasia and necrosis. lesions in the brain (encephalitis) and heart (myocarditis) and stomach of some pigs were also observed. there was a % mortality between and dpf. only one pig in the early mortality group was confirmed to have died from a non-prrs-related cause. pigs that survived beyond dpf eventually recovered and showed no external signs of acute infection and there were no prrsv-related deaths after dpf. for the purpose of comparison, we included a litter from a single mock-infected sow. the piglets, born at the same time as the prrsv pigs, appeared normal and showed no mortality within dpf. in the developing fetus, there are two potential sources of virus, which can cause fetal damage. the first source is virus replication within the tissues and organs of the developing fetus. another source is virus replication in the accessory tissues, such as the umbilical cord and placenta. in this study, we were able to obtain umbilical cords of piglets born to infected sows, including umbilical cords from infected newborns and umbilical cords from non-viremic pigs born to infected sows. virus isolation showed all umbilical cords from infected neonates to be positive for prrsv. neonates that were vi or rt-pcr negative for prrsv in serum were also negative for prrsv in umbilical cord tissues. the recovery of virus from umbilical cords presented the possibility that virus was isolated from circulating blood and not from cells supporting virus replication. in situ hybridization, for the detection of cells possessing viral rna, was performed on a single microscopic cross-section of each umbilical cord. we identified prrsv rna-positive cells in of the tissues cross-sections from vi-positive umbilical cords, indicating the presence of infected cells. a representative example of prrsv rna-positive cells in an umbilical cord cross-section is shown in fig. a . positive cells were generally found within the smooth muscle regions of umbilical cord tissues. as a negative control, in situ hybridization was performed on umbilical cord sections from six uninfected pigs. all six were negative for the presence of cells containing viral rna. even though cells supporting virus replication were easily identified in umbilical cords of infected piglets, histology at both gross and microscopic levels revealed no virus-associated lesions. based on the presence of virus and prrsv-specific antibody in pre-suckling serum samples, newborn piglets could be placed into one of the three groups. the first group contained four pigs ( %) that were vi-negative and seronegative. we assume that these neonates were not infected in utero, but were infected immediately prior to farrowing or immediately after farrowing as a result of contact with the dam or infected litter mates. the second group contained ( %) pigs that were vi-positive for prrsv, but seronegative. and finally, the third group was composed of seven ( %) neonates that were both vi-positive and seropositive. each litter contained a mixture of pigs that could be placed in all three groups, indicating that fetuses were infected at different times in the same sow. since pigs are immunocompetent at around days of gestation and there is no passive transfer of antibody from the mother to the fetus (tizard, ) we can conclude that infected ( ) neonates that were born seropositive were infected earlier than those that were vi-positive and seronegative. based on the infection of fetuses at different times in utero, we proposed that the earlier a fetus is infected the greater the probability of early mortality after birth. to test this hypothesis, neonates were divided into two groups. the first group was composed of the piglets that died within dpf and showed symptoms of severe prrs. (one pig succumbed from other than prrsv infection and was eliminated from the analysis.) the second group included the remaining pigs, which recovered and became asymptomatic. in the long-term survivor group only one serum sample was identified as positive for prrsv antibody, as indicated by an elisa s/p ratio greater than . (fig. ) . in pigs that died prior to days, of pigs were seropositive for prrsv. chi-square statistical analysis of these data showed a statistically significant association between the presence of prrsv antibody and mortality prior to dpf (p = . ). it should be noted that in the non-survivor group, of the pigs were vi-negative and seronegative at birth. these two piglets, and two others in the non-survivor group, exhibited clinical features consistent with streptococcus sp. infection, including swollen joints filled with pus, recruitment of pmns into inflammatory lesions, and the presence of streptococci in lung and other tissues. streptococcus suis was isolated from tissues of all four pigs. the presence of virus in the blood was determined by vi, and later when serum samples became vi-negative, by rt-pcr. the analysis of prrsv-specific immune responses included measurements of total anti-prrsv activity by elisa and virus neutralizing activity. these data, collected over a period of days after birth, are graphically summarized in fig. . by dpf, all pigs were vi-positive in serum, with the percentage of vi-positive serum samples declining at about dpf. the decline in the number of vi-positive serum samples was associated with the appearances of peak antibody levels and virus neutralizing activity. the disappearance of virus from the blood also correlated with the recovery of pigs from acute disease. the last vi-and rt-pcr-positive serum samples were obtained at and dpf, respectively. pigs that attained a vi-negative status remained vi-negative throughout the remainder of the study. however, it should be noted that pigs that became pcr negative did not always stay negative. periodically we found serum samples between and dpf, which were pcr-positive, even though the previous and subsequent samples taken from the same animal were negative by pcr. these intermittent pcr-positive results can best be explained by the presence of a small amount of viral rna, but at or near the lower detection limits of the pcr assay (data not shown). there were no positive rt-pcr serum samples after dpf. mean prrsv antibody peaked at approximately dpf with elisa s/p ratios ranging between . and . (n = ), well above the . cut-off that identified a serum sample as seropositive. since pigs were suckling prior to days, we assume that passive antibody from the mother was contributing to the humoral response. after days, antibody gradually declined and by dpf the remaining four pigs had attained a seronegative status (s/p ratio < . ) and remained seronegative throughout the remainder of the study. virus neutralizing activity in serum was first detected in one pig at dpf (neutralizing titer = ). by dpf all pigs showed detectable neutralizing activity. since these measurements were made several weeks after weaning, we presume that virus neutralizing activity was generated by the immune system of the congenitally infected pigs. at their peak, between days and , neutralizing titers ranged between : and : (fig. ) . this relatively low amount of neutralizing activity was not unexpected and is typical of the neutralizing response during infection of older pigs (yoon et al., ; wills et al., ) . interestingly, virus neutralizing activity was detected in sera well beyond days, even though all pigs were seronegative for prrsv by the idexx elisa test (s/p ratios below . ). the source of the virus neutralizing activity in serum was not determined. however, the presence of neutralizing activity in "seronegative" sera can be explained based on the different sensitivities of the assays used to measure antibody and neutralizing activity. for example, we cannot rule out the possibility that pigs possessing an elisa s/p ratio less than . have some prrsv antibody, but at amounts below the confidence limits of the assay. the analysis of virus replication in tissues of congenitally infected pigs focused on three periods of infection, identified by roman numerals ii, iii and iv in fig. . each period was unique in terms of clinical disease signs, viremia, serology and pathology. the first period, indicated by roman numeral ii in fig. , represented the acute, symptomatic stage of infection, which covered the first days after birth. the second period, identified by roman numeral iii focused on a period when pigs were asymptomatic for clinical disease signs and negative in serum for prrsv by vi and largely negative in serum by rt-pcr, but all pigs were still seropositive. and finally, we assessed virus replication in a group of four infected pigs, which had become seronegative (roman numeral iv). during acute infection (roman numeral ii, fig. ) prrsv was easily isolated from all lymphoid and non-lymphoid tissues examined (lung, heart, aorta, kidney, liver, testes, salivary gland, intestine, brain, stomach, thymus, spleen, tonsil, and lymph nodes). in situ hybridization identified cells containing viral rna in the same tissues. fig. b-d shows representative examples of rna-positive cells in lung, salivary gland and kidney. these three tissues represent sources of prrsv shedding by oral-nasal and urinary secretions. also shown are representative examples of virus replication in cells of lymph node, tonsil and thymus (fig. e-g) . in the tonsil, cells supporting virus replication were in close proximity to the tonsilar crypts. these data are consistent with our previous study, identifying multiple sites of virus replication in gnotobiotic piglets at days after experimental infection with vr- (lawson et al., ) . roman numeral iii in fig. shows the next period of infection covered by our study. during this stage, prrsv-specific antibody was declining, but all pigs were seropositive and possessed virus neutralizing activity at a serum dilution of at least : . there were no overt clinical signs of acute prrsv infection and all pigs were vi-negative in serum. virus isolation and rt-pcr were performed on lymphoid and non-lymphoid tissues from nine of the asymptomatic pigs, sacrificed at random between and dpf. vi and rt-pcr results for lung, mandibular lymph node, mesenteric lymph node, tonsil and serum are presented in table . in contrast to the acutely infected pigs, all lung and non-lymphoid tissue samples were negative for virus and viral rna. the absence of detectable amounts of virus in lungs was based on negative results for vi, rt-pcr and in situ hybridization assays of tissue samples from each lung quadrant. virus isolation, rt-pcr and in situ hybridization also failed to identify virus or viral rna in kidney, bladder, heart, aorta, liver, intestines, stomach, testes/ovaries, nasal turbinates, and salivary glands (data not shown). positive vi and rt-pcr results were frequently obtained from homogenates of tonsil (seven of nine pigs) and mandibular lymph nodes (eight of nine pigs; table ). mesenteric, aortic, cervical, tracheal, medial iliac lymph nodes also yielded virus and/or viral rna, but at much lower frequencies (see table for mesenteric lymph node data). virus or viral rna was not detected in spleen or thymus (data not shown). consistent with the vi and rt-pcr results, in situ hybridization identified prrsv rna-positive cells in tonsil and mandibular lymph nodes. prrsv rna-positive cells in a mandibular lymph node at dpf and tonsil at dpf are shown in fig. . only a few rna-positive cells could be identified within a given tissue section. therefore, the locations of rna-positive positive cells were confirmed by performing hybridization of adjacent tissue sections (fig. ) . all pigs sacrificed during this time possessed detectable amounts of virus neutralizing activity. together these data show that prrsv replication during asymptomatic infection is absent from lung and other non-lymphoid tissues and is primarily restricted to tonsil and lymph nodes, even in the presence of humoral immunity. to investigate the significance of this low level of virus replication in asymptomatic pigs, we studied the ability of congenital prrs pigs to transmit virus. age-matched prrsv seronegative sentinel pigs were introduced into the prrsv-infected group at dpf ( sentinels and principals), dpf ( sentinels and principals), dpf ( sentinel, principals) and dpf ( sentinel, principals). sentinel pigs were allowed to commingle with the infected herd for week and then removed to separate isolation rooms for observation and assessment of infection by vi and serology. all sentinel pigs were vi-positive in serum within week after introduction and seroconverted a week later. these results indicate that under natural conditions of normal pig-to-pig contact prrsv is efficiently transmitted to naïve pigs even though virus replication is low. congenital prrs pigs that became seronegative (see roman numeral iv in fig. ) were the subject of the last part of our study. by dpf, all sera from four remaining infected pigs were vi-negative, rt-pcr negative and elisa seronegative for prrsv antibody (s/p ratio less than . ). to determine if prrsv pigs were capable of shedding virus, four age-matched prrsv seronegative sentinel pigs were introduced at dpf and maintained in continual contact with the prrsv pigs for at least days. commingling culminated in the mating of each prrsv pig to an aged-matched sentinel. two litters were obtained from the three prrsv females. the adult female pigs and litters were sacrificed and assessed for prrsv replication. all three female pigs and associated litters were negative for prrsv antibody and viral rna as indicated by negative results for in situ hybridization and by performing rt-pcr in lungs, lymph nodes and tonsils. the single sentinel female pig and litter obtained after mating to the seronegative prrsv boar were also negative for prrsv. since previous work identified long-term virus shedding in semen (christopher-hennings et al., ) , we also performed rt-pcr and in situ hybridization on testes and male accessory reproductive organs of the remaining male pig. there was no evidence of virus replication in any of the male reproductive tissues. the results from this study show that congenital prrsv infection can be divided into distinct stages, unique in terms of serology, virology and clinical disease. the first stage covers the period of in utero exposure to virus. these data demonstrate that not all fetuses are infected at the same time and some fetuses may escape infection altogether. furthermore, the positive association between the presence of antibody at birth with early mortality indicates that the earlier a fetus is infected the more severe the clinical disease (fig. ) . since it takes - weeks to develop antibody, we assume that seropositive fetuses were infected - weeks after infection of the sow or approximately - weeks prior to birth. as a whole, these data show that the placenta is a barrier to prrsv, but this barrier does break down with time. there are at least two sources of virus-related damage that can account for early mortality in the antibody-positive fetuses. the first is the formation of prrsv lesions in fetal organs. however, the target organs in the fetus may be different from those in the postnatally infected pig. for instance, porcine alveolar macrophages, which support virus replication to high levels in postnatal pigs are absent from the fetal lung. in preliminary studies, we found relatively large amounts of virus in the thymus from fetuses obtained - weeks prior to birth (data not shown). feng et al. ( ) observed thymic lesions in congenital prrsv pigs sacrificed at dpf. the possible effect of prrsv infection on fetal/neonatal primary immune organs has obvious implications in the increased susceptibility of congenitally infected pigs to secondary infections. a second source of fetal damage is through the disruption of support tissues, such as placenta and umbilical cord. lager and halbur ( ) reported lesions in umbilical cord, which appeared to be sufficient to cause anoxia in the developing fetus. even though umbilical cord was identified as a sight of virus replication in this study, we were not able to identify prrsv-related lesions. the absence of umbilical cord lesions may reflect the different isolate used in this study. even though immune abnormalities represent a hallmark of prrsv infection, congenital infection did not appear to adversely affect the capacity of surviving pigs to develop an antibody response to prrsv later in life. as a group, the antibody levels in congenital prrs pigs peaked at about days after birth (fig. ) , which is similar to the time-course appearance of antibody in pigs infected several weeks postnatally (yoon et al., ; allende et al., ) . congenital prrs pigs were able to mount a prolonged neutralizing antibody response at levels typically found in experimentally infected older pigs (yoon et al., ) . these results indicate that fetuses exposed to prrsv during late gestation do not become immunotolerant to the virus later in life. a unique aspect of this study was the thorough analysis of virus replication in pig tissues during asymptomatic infection (roman numeral iii, fig. ). this stage of congenital prrsv infection was characterized as the period of time when pigs no longer exhibited clinical signs of acute disease, were vi-negative in serum, but still seropositive. in this study it was represented by a group of nine pigs sacrificed between and days after birth. perhaps, the most interesting finding was the identification of virus in tonsil and mandibular lymph node, but not in lung and other non-lymphoid organs. these results are in agreement with studies identifying tonsil as a source of virus during persistent infection allende et al., ; horter et al., ) , but contradict other studies identifying pulmonary macrophages as the source of virus replication in persistently infected pigs (mengeling et al., ; duan et al., ) . our conclusion that virus is absent from the lungs in persistently infected pigs is based on a thorough analysis of virus replication, including the use of vi, rt-pcr and in situ hybridization. continuous virus replication in regional lymph nodes accounts for the efficient transmission via oral-nasal secretions and in semen during persistent infection (christopher-hennings et al., ) . the basis for an eventual change in organ tropism, from multiple sites of prrsv replication to preferential replication in tonsil and lymph nodes, is not known. in previous work we identified the emergence of a virus sub-population during persistent infection, which possessed a mutation in the ectodomain of orf , the major envelope glycoprotein. this mutation may increase the tropism of prrsv for replication in a macrophage subpopulation that resides in tonsils and lymph nodes. we followed virus and antibody in four congenital prrsv pigs for more than a year. at the time of necropsy these pigs were seronegative for prrsv (s/p elisa ratio less than . ) and showed no evidence of virus replication, as determined by the absence of viral rna in serum and in tissues. in this study we were not able to determine exactly when virus disappeared from these pigs. however, the last rt-pcr positive serum sample was obtained at dpf and sentinel pigs did not become infected when introduced at dpf. the failure of sentinel pigs to become infected after an extended period of intimate contact with the congenital prrsv-infected pigs indicated that prrsv replication was not reactivated. the results from this study show that congenital prrs pigs can shed virus for at least dpf and perhaps for as long at dpf, which support the conclusions of a recent study by wills et al. ( ) . the isolation of virus from lymph nodes at dpf in this study is longer than the isolation of infectious virus at days from pigs infected at month of age (allende et al., ) , but is similar to length of time reported by wills et al. ( ) . because of viral strain differences, it is difficult to assess the impact of congenital infection on virus replication, shedding and persistence by making comparisons with other published studies performed in older pigs. however, in agreement with allende et al. ( ) prrsv replication does not establish a steady-state equilibrium in the manner of other persistent arteriviruses, such as ldv or shfv, but gradually declines over time, with the lymphoid organs as the last vestige of virus replication. the presence of an extended period of ongoing prrsv replication in pigs with substantial anti-prrsv humoral and cell-mediated immune responses suggests that the eventual elimination of the virus from congenital prrs pigs may ultimately reflect the disappearance of prrsv-permissive cells with age. based on our analysis of clinical disease, prrsv-specific antibody and virus replication, congenital prrsv infection can be divided into four distinct stages. stage i covers the period of in utero exposure to virus during which time fetuses are infected at different times and some fetuses remain uninfected. the positive association between the presence of prrsv antibody at birth with early mortality indicates that the earlier a fetus is infected the more severe the clinical disease outcome. stage ii is the period of acute infection. during this time pigs show signs of acute prrsv. in this study virus was isolated from all tissues, and all tissues contained cells supporting virus replication. stage iii is a period when pigs no longer exhibit clinical disease signs, are vi-negative and largely rt-pcr negative in serum and possess peak levels of virus neutralizing activity. even though virus replication is relatively low, persistently infected pigs can efficiently transmit virus to naive pigs. stage iv of congenital prrsv infection is viral clearance. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection lactate dehydrogenase-elevating virus persists in liver, spleen, lymph nodes and testis and results in accumulation of viral rna in germinal centers concomitant with the polyclonal activation of b cells cell-mediated immunity to porcine reproductive and respiratory syndrome virus ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) porcine reproductive and respiratory syndrome diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs nidovirales: a new order comprising coronaviridae and arteriviridae pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid gestation sows and fetuses persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars identification of porcine reproductive and respiratory syndrome virus in semen and tissues from vasectomized and nonvasectomized boars persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs virus quantification and identification of cellular targets in the lungs lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with mac- -positive cells days post-infection temporal characterization of transplacental infection of porcine fetuses with porcine reproductive and respiratory syndrome virus diagnosis of porcine reproductive and respiratory syndrome using infected macrophages from live pigs porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of united states and european isolates of porcine reproductive and respiratory syndrome (prrs) virus using monoclonal antibodies lactate dehydrogenase-elevating virus and related viruses lactate dehydrogenase-elevating virus: an ideal persistent virus? porcine reproductive and respiratory syndrome lymph node lesions in neonatal pigs congenitally exposed to porcine reproductive and respiratory syndrome virus fetal microscopic lesions in porcine reproductive and respiratory syndrome virus-induced abortion porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with aminopurine the molecular biology of arteriviruses porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis immunity in the fetus and newborn mystery swine disease in the netherlands: the isolation of lelystad virus porcine reproductive and respiratory syndrome virus: a persistent infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection this work was supported by the usda national research initiative for competitive grants program grants # - - and # - - , national pork producer council grant # , national science foundation grant # osr- , south dakota agricultural experiment station and the south dakota future fund. we thank curt nelson and scott kistler for their excellent assistance in the care and welfare of the animals. key: cord- -cdas cx authors: morozov, i.; meng, x. -j.; paul, p. s. title: sequence analysis of open reading frames (orfs) to of a u.s. isolate of porcine reproductive and respiratory syndrome virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: cdas cx the sequence of orfs to of a u.s. isolate of porcine reproductive and respiratory syndrome virus (prrsv), atcc vr , was determined by analysis of a cdna λ library. the cdna clones containing prrsv specific sequences were selected using a vr orf specific pcr probe and sequenced. the orfs , and overlapped each other and encoded polypeptides with predicted m(r) of . kda (orf ), . kda (orf ) and . kda (orf ), respectively. no overlap was found between orfs and , and instead there was a bp sequence which separated these two orfs. the nucleic acid homology with corresponding orfs of the european prrsv isolate lelystad virus (lv) was % for orf , % for orf and % for orf . comparison of the orf sequences of vr with that of another u.s. isolate mn- b revealed only % amino acid sequence homology and the presence of deletions in the orf of mn- b. our results further strengthen the observation that there is sequence variation between us and european prrsv isolates. analysis of a cdna )~ library. the cdna clones containing prrsv specific sequences were selected using a vr orf specific pcr probe and sequenced. the orfs , and overlapped each other and encoded polypeptides with predicted m r of . kda (orf ), . kda (orf ) and . kda (orf ), respectively. no overlap was found between orfs and , and instead there was a bp sequence which separated these two orfs. the nucleic acid homology with corresponding orfs of the european prrsv isolate lelystad virus (lv) was % for orf , % for orf and % for orf . comparison of the orf sequences of vr with that of another u.s. isolate mn-lb revealed only % amino acid sequence homology and the presence of deletions in the orf of mn-lb. our results further strengthen the observation that there is sequence variation between us and european prrsv isolates. porcine reproductive and respiratory syndrome virus (prrsv) belongs to the newly proposed virus family arteriviridae, which also includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv) and simian hemorrhagic fever virus (shfv). porcine reproductive and respiratory syndrome (prrs) was first described in the u.s. in [ ] . a similar disease referred to as porcine epidemic abortion and respiratory syndrome (pears) was then reported in europe [ ] . prrsv was first isolated in europe and is believed to be widespread in swine population around the world [ , , ] . all european isolates of prrsv are antigenically and genetically related, whereas there are antigenic variations between us and european isolates as well as among us isolates [ , , ] . the complete nucleotide sequence of the genome of lv has been determined [ ] , but until recently limited information was available about the molecular structure of the genome of north american isolates of prrsv [ ] [ ] [ ] [ ] . we have previously reported the cloning and sequencing of the orfs to ofa u.s. isolate of prrsv vr of high virulence [ ] . the ' end of the genome of the vr and the other u.s. prrsv isolates showed a striking difference when compared to the european isolates [ ] . in this study, we report on the cloning and sequencing of the orfs - of the u.s. isolate vr . for sequencing and characterization of the viral genome of vr a cdna ;~ library was constructed. the crl cells were infected with vr virus at a m.o.i. of . and the total rna from infected cells was isolated at h post infection by using a guanidinium thiocyanate method [ ] . polyadenylated rna was enriched, reverse transcribed and cloned into the )~ zap vector using the uni-zap cdna cloning kit (stratagene, la jolla, ca). a pcr probe generated by orf specific primers dp ( 'gctttgctgtcctccaag ') and dp ( 'gatgcctgacacattgcc ') [ ] were used to screen the library. plaques that hybridized with the probe were isolated and purified. the phagemids containing viral cdna inserts were rescued by in vitro excision using exassist helper phage and e. coli solr cells (stratagene, lajolla, ca). several recombinant phagemids with virus specific cdna inserts with sizes ranging from . to . kb were selected and sequenced by sanger's dideoxynucleotide chain termination method [ ] with an automated dna sequencer (applied biosystems, foster city, ca). universal, reverse and specific internal primers were used to determine the sequence. at least independent clones representing sequence of the orfs to were sequenced. the sequencing data was assembled and analyzed using mac vector (international biotechnologies, inc., ct) and geneworks (intelligenetics, ca) computer programs. the nucleotide sequence reported in this paper has been deposited in the genbank with the accession number u . analysis of the nucleotide sequence identified three partially overlapping orfs. the orf extended from nucleotide to , orf from to , and orf from to . there was an overlap of bp between orfs and , and bp between orfs and . surprisingly, no overlap was found between orfs and . the start codon oforf was located bp downstream of the stop codon of orf . however, the atg start codon of orf and tga stop codon of orf overlapped by only t bp in lv [ , ] . the sequence at the region of the orf and orf junction of lv is atatga. we sequenced the corresponding region of additional independent clones of vr and in all cases the sequence of this region of vr was atttga. the point mutation from a to t in vr and probably some other unidentified changes in this region of vr made the orf atg start codon bp downstream of the stop codon of orf , and a bp non-coding region appeared in the orf and junction of vr . the characteristics oforfs to of vr are summarized in table . the orf encodes a amino acid polypeptide with a predicted size of . kda. the carboxy and amino terminus of the predicted protein are hydrophobic (data not shown) and there are two potential n-glycosylation sites in the orf protein. orf encodes a protein of amino acids and contains potential n-glycosylation sites. the amino terminus of the orf protein is extremely hydrophobic. orf encoded a amino acid protein with a predicted size of . kda. the amino and carboxy termini and regions within the protein are highly hydrophobic. comparison of the nucleotide sequences of vr and lv showed extensive variations. nucleotide sequence identity between vr and lv is % for orf , % for orf and % for orf . alignment of the predicted amino acid sequences of orfs - of vr and lv is presented in fig. . amino acid identity between vr and lv is % for orf , % for orf and % for orf . we also compared the sequence ofvr orf with that of mn-lb, another us isolate of prrsv [ ]. the orf of vr is bp longer and shares an % nucleotide sequence homology with mn-ib. the amino acid homology between the orf of vr and mn-lb is % (fig. lc) . several deletions were found in the orf of mn-lb compared to vr . the orfs and of prrsv are predicted to encode the viral membrane glycoprotein and the viral nucleocapsid protein, respectively [ , ] . analysis of predicted amino acid sequences encoded by orfs - of lv, ldv and eav showed that all of these proteins share features of membrane associated proteins [ , , , ] . the eav orf product was identified as the main envelope glycoprotein ~sequence for vr orfs - is presented in the study, orfs was reported by meng et al. [ ] and lv orfs - was reported by meulenberg et al. [ ] bdistance in nucleotides between proposed junction motif and aug start codon of downstream orf [ ] . our data indicates that the proteins encoded by orfs ~, of vr possess characteristics similar to those of lv and probably are envelope or membrane associated glycoproteins because of their hydrophobicity and presence of potential glycosylation sites. further work is necessary to determine the roles of these proteins. the variability found in the orf sequence between the two u.s. isolates correlate with the findings that orf protein of the mn-lb expressed in e. coli reacted with only % of prrsv positive sera by western blot analysis [ ] . a nested set of subgenomic mrna is formed during replication of prrsv and other members of the arterivirus group [ , , , , ] . all subgenomic mrnas contain a common leader sequence derived from the ' noncoding region of the viral genome. the site of the leader-mrna junction is similar and located upstream of the start codon of each orf. the consensus leader-mrna junction sequence of the six subgenomic mrnas of lv was determined to be (u/a)(c/u/a)(a/ g)acc [ ] . similar sequences were also found as leader-mrna junction regions for ldv [ ] . the potential leader-mrnajunction motifs oforfs to of vr was proposed and compared with those of lv ( table ). the last four nucleotides of the motif for orfs , , , , and in lv are aacc, and for orf is gacc. the aacc motif has been found upstream of orfs and of vr [ ] as well as orfs and . there are two potential junction regions for orf , bp and bp upstream of the orf start codon, respectively (table ) . no aacc motif was found upstream ofvr orfs and . however, the sequences uugacc and cagacc upstream of orf , uugacc and gagacc upstream of orf , may be the leader-mrna junction regions for the mrnas and of vr . multiple potential leader-mrna junction sites suggest that polymorphism of subgenomic mrnas may exist among prrsv isolates. experiments to determine the exact locations ofleader-mrnajunction regions are now in progress. the sequence variations observed in this study between a u.s. and a european prrsv isolate, as well as between two north american prrsv isolates, indicates the heterogenetic nature of prrsv isolates and the need for further characterization of additional prrsv isolates. whether this genetic variation between vr and lv reflects the observed difference in virulence needs to be further studied. comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) sequences of ' end of genome and of ' end of open reading frame la of lactate dehydrogenase-elevating virus and common junction motifs between ' leader and bodies of seven subgenomic mrnas isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group equine arterifis virus is not a togavirus but belongs to the coronavirus-like superfamily structural proteins of equine artefitis virus complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-etevating virus (ldv) cloning, expression, and sequence analysis of the orf gene of porcine reproductive and respiratory syndrome virus mn-lb identification of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus molecular cloning and nucleotide sequencing of the '-terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analysis of the putative m (orf ) and n(orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies porcine reproductive and respiratory syndrome: an overview molecular cloning: a laboratory manual dna sequencing with chain terminating inhibitors experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled antigenic comparison of lelystad virus and swine infertility and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus this work was supported by a grant no. - from the national research initiative competitive grants program of the u.s. department of agriculture, and in part by a grant from the solvay animal health, inc., mendota heights, mn. the authors would like to thank drs. pat halbur and melissa lure for their expert help throughout this project, and dr. harold hills at the nucleic acid facility, iowa state university for his assistance in sequence analysis. received december , key: cord- -t y t v authors: tong, ting; hu, hongwei; zhou, junwei; deng, shuangfei; zhang, xiaotong; tang, wantao; fang, liurong; xiao, shaobo; liang, jiangong title: glycyrrhizic‐acid‐based carbon dots with high antiviral activity by multisite inhibition mechanisms date: - - journal: small doi: . /smll. sha: doc_id: cord_uid: t y t v with the gradual usage of carbon dots (cds) in the area of antiviral research, attempts have been stepped up to develop new antiviral cds with high biocompatibility and antiviral effects. in this study, a kind of highly biocompatible cds (gly‐cds) is synthesized from active ingredient (glycyrrhizic acid) of chinese herbal medicine by a hydrothermal method. using the porcine reproductive and respiratory syndrome virus (prrsv) as a model, it is found that the gly‐cds inhibit prrsv proliferation by up to orders of viral titers. detailed investigations reveal that gly‐cds can inhibit prrsv invasion and replication, stimulate antiviral innate immune responses, and inhibit the accumulation of intracellular reactive oxygen species (ros) caused by prrsv infection. proteomics analysis demonstrates that gly‐cds can stimulate cells to regulate the expression of some host restriction factors, including ddx and nos , which are directly related to prrsv proliferation. moreover, it is found that gly‐cds also remarkably suppress the propagation of other viruses, such as pseudorabies virus (prv) and porcine epidemic diarrhea virus (pedv), suggesting the broad antiviral activity of gly‐cds. the integrated results demonstrate that gly‐cds possess extraordinary antiviral activity with multisite inhibition mechanisms, providing a promising candidate for alternative therapy for prrsv infection. the orcid identification number(s) for the author(s) of this article can be found under https://doi.org/ . /smll. . doi: . /smll. nm. [ ] due to their unique properties in photoluminescence, water solubility, biocompatibility, and resistance to photobleaching as well as cost-effective synthesis and low toxicity, cds have been extensively investigated, [ ] [ ] [ ] and are widely utilized in bio-imaging, bio-sensing, biolabeling, photodynamic therapy, and drug delivery. [ , ] cds have a small size and controllable surface functional groups, which facilitate their high adsorption interactions with different biological surfaces. [ ] recently, functionalized cds synthesized by hydrothermal method have been shown to have antiviral activity. [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, the antiviral mechanism of these cds remains to be further elucidated, and the response of cells to them is still unknown. the most important ingredients of traditional chinese medicines are chinese herbal medicines, herbs and plant preparations, which are used worldwide for their anti-oxidation, anti-inflammatory, antiviral immune regulation, antitumor, and other pharmacological functions. [ ] under the conditions of high cost of antiviral drugs and limited healthcare resources, chinese herbs have been recommended to prevent and treat respiratory disease and herpes simplex in china, especially in poor regions. [ , ] to date, no critically appraised evidence is available on the beneficial effect of chinese herbal medicines on h n influenza, mainly due to lack of both repeated test of intervention and placebo-controlled trial. [ , ] furthermore, chinese herbal poisoning incidents have also been reported. [ , ] as a common chinese herbal medicine and a major component of glycyrrhiza uralensis, glycyrrhizic acid has been characterized with various biological activities, such as antiviral immunoregulation, anti-oxidation, anti-inflammation, and liver protection. [ ] [ ] [ ] [ ] [ ] owing to the ability to inhibit multidrug-resistance-associated proteins, glycyrrhetate and glycyrrhizic acid are used in clinic for cisplatin resistance reversal in hepatocellular carcinoma cells and reduction in liver damage. [ ] [ ] [ ] [ ] however, glycyrrhizic acid also has the disadvantages of poor water solubility and certain cytotoxicity. [ ] porcine reproductive and respiratory syndrome (prrs) is a disease that has been seriously jeopardizing the development of the worldwide pig industry for over two decades. [ ] prrs virus (prrsv), an enveloped single positive-strand rna virus, is the with the gradual usage of carbon dots (cds) in the area of antiviral research, attempts have been stepped up to develop new antiviral cds with high biocompatibility and antiviral effects. in this study, a kind of highly biocompatible cds (gly-cds) is synthesized from active ingredient (glycyrrhizic acid) of chinese herbal medicine by a hydrothermal method. that gly-cds can stimulate cells to regulate the expression of some host restriction factors, including ddx and nos , which are directly related to prrsv proliferation. moreover, it is found that gly-cds also remarkably suppress the propagation of other viruses, such as pseudorabies virus (prv) and porcine epidemic diarrhea virus (pedv), suggesting the broad antiviral activity of gly-cds. the integrated results demonstrate that gly-cds possess extraordinary antiviral activity with multisite inhibition mechanisms, providing a promising candidate for alternative therapy for prrsv infection. carbon dots (cds), a carbon-based zero-dimensional material also known as carbon quantum dots, are a carbon nanomaterial and a kind of spherical carbon particles with a size below etiologic agent of the disease. [ ] to date, prrsv has no effective treatment in the clinic, and the current authorized prevention and treatment strategy involving the use of inactivated and attenuated vaccine cannot block the occurrence and spread of the disease. [ ] previous studies have investigated many microbicides as potential anti-prrsv agents to eliminate prrsv in the body at any stage of prrsv presence. these microbicides include protein, [ ] micrornas, [ , ] antisense oligonucleotides, [ ] [ ] [ ] [ ] chemical compounds, [ , ] and nanobodies. [ ] they are shown to inhibit virus adsorption, invasion, replication, release, and cell-to-cell spread by restricting cell tropism and targeting the prrsv genome. however, they have a relatively slight anti-prrsv effect, with a maximal inhibitory effect of ≈ orders of magnitude. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this study, a kind of highly biocompatible gly-cds was synthesized from active ingredient of chinese herbal medicine, which combines the high biocompatibility of cds with the excellent antiviral properties of glycyrrhizic acid and inhibit the proliferation of prrsv by ≈ orders of magnitude. the size and surface morphology of the gly-cds were characterized by high-resolution transmission electron microscopy (hr-tem). as shown by the hr-tem image in figure a , gly-cds were round shaped and well-dispersed with a uniform size distribution. figure b showed the size distribution of gly-cds measured with a dynamic light scattering (dls) analyzer. consistent with the tem image, gly-cds showed high monodispersity, with a calculated average size of . nm. as shown by the x-ray diffraction (xrd) pattern in figure c , gly-cds possessed the component of amorphous carbon due to the broad peak at around °. [ ] the sharp peak at around . ° may be ascribed to the crystallinity of gly-cds, and the graphitic carbon accounted for only a small amount of gly-cds carbon, so no apparent lattice fringe could be observed in the tem image. [ ] the above results provide evidence that the gly-cds are polymeric cds. the uv-vis absorption and fluorescence (fl) spectra of the gly-cds are presented in figure d . the tiny absorption peak at nm was attributed to π-π* transition. the gly-cds possessed a significant characteristic blue fluorescence at nm, with the maximal excitation wavelength remaining at nm. meanwhile, excitation-dependent fluorescence emission was observed when gly-cds were excited with different wavelengths from to nm (figure e ), due to the normal size distribution of gly-cds. [ ] the uv-vis absorption spectrum and fluorescence spectrum are all consistent with cds reported before. [ , ] figure f shows the gly-cds functional groups measured by the fourier transform infrared spectroscopy (ft-ir). in the spectra of gly-cds, five obvious absorption peaks could be observed at , , , , and cm − , corresponding, respectively, to oh, ch, c  c, c  o, and co, indicating that gly-cds are rich in oh and cooh on the surface. [ ] the results obtained from the preliminary analysis of the ft-ir spectrum proved that the gly-cds conserved part functional groups of glycyrrhizic acid. the elemental constituents and functional groups of gly-cds were further characterized by x-ray photoelectron spectroscopy (xps). figure g shows the full-scan spectrum of gly-cds. the two peaks of c s and o s could be obviously recognized at and ev, respectively. the elemental analysis demonstrated that gly-cds contained . % of carbon and . % of oxygen. the high resolution xps spectra of the c s in gly-cds could be resolved into three peaks (figure h ). the peak at . ev was assigned to c  o of the carboxylic groups, while the other two peaks at . and . ev were ascribed, respectively, to co and cc/c  c. [ , ] the citric-acid-based cds (cit-cds) were used to act as the contrast, because citric acid and ethylenediamine, the raw materials, do not show antiviral activity. we prepared biocompatible cit-cds using the method described in the supporting information. [ ] as can be seen from tem and dls images, the average size of the cit-cds was . nm ( figure s , supporting information). as shown in figure s , supporting information, a characteristic absorption peak could be observed at nm by uv-vis absorption spectrum, the maximum emission of the cit-cds was centered at nm whereas the maximum excitation was recorded at nm in the fl excitation and emission spectra. the results of the ft-ir spectrum suggested that the cit-cds were rich in cooh and nh on the surface while the h nmr spectrum indicating the cit-cds are highly carbonized. figure s , supporting information displays the full scan xps spectrum of cit-cds, and elemental analysis indicated that the cit-cds consisted of . % of carbon, . % of nitrogen, and . % of oxygen. to assess the biocompatibility of gly-cds with the raw materials glycyrrhizic acid in vitro, cytotoxicity experiments were performed. briefly, marc- cells were cultured separately with glycyrrhizic acid or gly-cds at different concentrations for , , , and h. the test results are shown in figure . notably, the viability was more than % for marc- cells when treated with gly-cds at . mg ml − (figure a) , in contrast to the viability of ≈ % when treated with glycyrrhizic acid at . mg ml − (figure b ), which are similar to the cytotoxic concentrations reported in other literature. [ , ] the morphology of the cells treated with the cds and glycyrrhizic acid for h is shown in figure s , supporting information. these experimental results indicated that the biocompatibility of gly-cds was significantly improved when compared with glycyrrhizic acid. finally, the . mg ml − concentration was selected for further assays. cytotoxicity assay results for cit-cds on marc- cells are depicted figure s , supporting information. the anti-prrsv activity of glycyrrhizic acid has been reported previously. [ ] cds have good biocompatibility, but its antiviral effect is specific. here, we focused on the antiviral activity of gly-cds and also explored the effects of traditional cit-cds on prrsv proliferation. plaque assay was used to detect virus infectivity and count the number of viral particles, with the results being expressed in titers. according to the experimental design (scheme s , supporting information), the addition of gly-cds significantly reduced prrsv titers in intracellular and cell supernatants at , , , and h post infection (hpi), with a maximal reduction of ≈ -fold by plaque assay (figure a,b) , demonstrating the strong antiviral effect of gly-cds on prrsv. meanwhile, cit-cds showed no inhibitory effect on the proliferation of prrsv. the influence of gly-cds on prrsv proliferation was further investigated by analyzing the expression levels of small , , the prrsv nucleocapsid (n) protein, one structural protein encoded by prrsv open reading frame (orf ) gene as the viral capsid protein, and the nonstructural protein (nsp ), a protein with a critical role in viral replication. [ ] western blot assay also revealed a remarkable decrease in the expression levels of prrsv n and nsp proteins under the treatment of gly-cds (figure c,d) . meanwhile, indirect immunofluorescence assay (ifa) failed to detect the fluorescence images of prrsv n proteins at all tested time points ( , , , and hpi) under the treatment of gly-cds ( figure e ). nuclei are indicated by the blue fluorescence signal counterstained with -( -amidinophenyl)- -indolecarbamidine dihydrochloride (dapi), and the green fluorescence signal represents prrsv n protein contents stained with a mouse mab specific for prrsv n protein and alexa fluor -conjugated donkey anti-mouse igg. in the above experiments, marc- cells were infected with prrsv (multiplicity of infection, moi = ) for the indicated periods of time. all these observations indicate gly-cds can significantly inhibit prrsv multiplication. the potential mechanism for the antiviral properties of gly-cds were explored by analyzing their effects on the prrsv proliferation at each stage (adsorption, invasion, replication, and release). whether gly-cds can directly inactivate prrsv was first tested. as shown in figure a , gly-cds reduced the number of prrsv by ≈ -fold by plaque assay, indicating that gly-cds can directly inactivate prrsv. in the adsorption process of prrsv, the plaque assay showed no significant difference in the experimental group and control group about their suppression effect on prrsv adsorption (figure b ). in the invasion process of prrsv, the experimental data revealed that the gly-cds treatment decreased the infectious virus titer by about -fold relative to the control group (figure c) , implying that gly-cds may suppress prrsv mainly through inhibiting prrsv invasion. the influence of gly-cds on viral replication was evaluated by real-time quantitative reverse transcription pcr (rt-qpcr) analysis of the negative-sense rna level of prrsv. as shown in figure d , gly-cds reduced the number of prrsv rna copies by nearly -fold, revealing their slight influence on prrsv at the replication stage. moreover, the effect of gly-cds on the release of progeny prrsv was examined. in figure e ,f, no noticeable difference was observed in the virus titers of both intracellular and supernatant prrsv between the experimental group and control group, suggesting that gly-cds had no inhibitory effect on the release of progeny prrsv. taken together, gly-cds suppress prrsv proliferation by targeting the invasion and replication processes, with a certain direct inactivation effect in vitro as indicated by statistical analysis, but without any inhibitory effect on the adsorption and release of progeny virus. the antiviral effect of gly-cds on the other viruses was further explored by using pseudorabies virus (prv) and porcine small , , , and e,f) release processes of prrsv. the mean value was calculated by the t test (mean ± sd, n = ). *p < . , **p < . , ***p < . , ns, non-significant difference, compared with the indicated group. epidemic diarrhea virus (pedv), two causal agents for catastrophic economic losses in the pig industry, as a representative of herpes virus and coronavirus, respectively. the prv is an enveloped, double-stranded dna virus in the family herpesviridae, [ ] while pedv is a large-enveloped rna virus in the genus, alphacoronavirus of the coronavirus family. [ ] in this study, the cytotoxicities of gly-cds on vero cells and pk- cells were evaluated first. as shown in figure a ,c, the viability of either cell line was more than % when treated with gly-cds at . mg ml − for and h. then, the effects of gly-cds on the proliferation of pedv and prv were detected by ifa. in figure b , the blue fluorescence signal stained with dapi represents nuclei, and the green fluorescence signal represents pedv n protein stained with mouse mab specific for pedv n protein and alexa fluor -conjugated donkey antimouse igg. in figure d , the recombinant prv expressing green fluorescent protein (prv-gfp) was transfected into pk- cells. the number of pedv-or prv-infected cells were shown to be obviously reduced by gly-cds versus the control group. these results suggested that gly-cds have significant antiviral activity on different viruses, including rna and dna viruses. interferons (ifn), the major components of natural immunity, play a critical role in antiviral activity. prrsv can interfere with both the induction of host interferons and the function of antiviral interferon-stimulating genes (isgs). [ ] whether gly-cds promote the production of interferons was explored by rt-qpcr analysis of the mrna expression levels of isgs after incubating marc- cells with or without gly-cds at . mg ml − in dmem (containing % fbs) for h. meanwhile, in the positive control group, marc- cells were incubated with the sendai virus (sev) for h to stimulate the host's natural immunity to produce a large amount of type interferon. the group without gly-cds treatment was used as a negative control. as shown in figure , compared with the negative control, gly-cds treatment showed an obvious increase in the mrna expression levels of interferon-stimulated genes, isg- , isg- , isg- , oligoadenylate synthetase (oas), and zinc finger antiviral protein (zap). to date, isg- , isg- , isg- , oas, and zap are the isgs which have been considered to positively regulate the expression of phosphorylated signal transducer and activator of transcription small , , www.small-journal.com /ifn-β/retinoic acid-inducible gene-g to inhibit viral infection and exert various antiviral and pro-inflammatory reactions. [ ] [ ] [ ] [ ] [ ] [ ] [ ] these results imply that gly-cds may inhibit viral infection by regulating the mrna expression levels of interferon-stimulated genes. reactive oxygen species (ros) play an essential role in regulating various physiological diseases, such as brain diseases, inflammatory diseases, cardiovascular diseases, bacterial infection, and so on. [ ] viral infection causes an increase in intracellular ros, thereby blocking the signaling pathways in the body's natural immunity. reducing the ros level by their inhibitors can inhibit virus proliferation. it is reported that the intracellular ros levels can be successfully regulated to suppress viral infection by embedding poly(aniline-co-pyrrole) within an amphiphilic methoxy polyethylene glycol-blockpolyphenylalanine copolymer. [ ] another study has shown that silver nanoparticle-based codelivery of oseltamivir could remarkably inhibit the accumulation of ros and activate the signaling pathways of akt and p phosphorylation to inhibit h n influenza virus infection. [ ] whether gly-cds can regulate the cellular ros level was investigated in this study. briefly, after prrsv infection for - h, the marc- cells were treated separately with gly-cds for h, then an inverted fluorescence microscope was used to detect the cellular ros levels by detecting the fluorescence of ', '-dichlorofluorescein (dcf). in figure , green fluorescence indicates the presence of ros, and the level of prrsv-induced ros was significantly reduced in the gly-cds treatment versus the pprsv treatment (mock). such experimental results suggest that gly-cds can inhibit viral proliferation by suppressing the production of prrsvinduced ros. the antiviral mechanisms of gly-cds were further explored at the intracellular protein level by isobaric tag for relative and absolute quantitation (itraq) quantitative proteomic analysis. briefly, marc- cells were incubated or mock-incubated (dmem containing % serum) with gly-cds at . mg ml − for h. subsequently, a series of experiment was carried out according to the experimental flow chart of scheme s , supporting information, the quality test results of the collected cell samples indicate that the samples meet the quality requirements ( figure s , supporting information) . furthermore, a total of peptides and proteins were identified by searching against the macaca mulatta uniprot proteome database, including proteins that are differentially expressed (total protein data and differentially expressed protein data can be found in the attached excel). a dot-plot was also supplied to present the overview of quantified proteins and differentially expressed cellular proteins ( figure s , supporting information). among them, seven differentially expressed proteins and two non-differentially expressed proteins were detected by western blot assay to validate the liquid chromatography tandem-mass spectrometry (lc-ms/ms) data. in figure a , it was shown that in gly-cds-treated cells, the expressions were downregulated in thioredoxin-like protein (txnl ), nitric oxide synthase (nos ), and olfactomedin (olfm ), small , , upregulated in cathepsin d (ctsd), colony stimulating factor (csf ), and pyridoxal phosphate phosphatase (pdxp), and not significantly changed in kda heat shock protein (hspd ), and mitochondrial and vimentin (vim). the gray value was converted to the quantitative result by imagej software and the ratios (gly-cds/control) identified by western blot assay were consistent with those from itraq-based proteomic analysis. as previously reported, rna helicases are involved in nearly all aspects of rna metabolism, from transcription and translation to mrna decay, and they are enzymes that use atp to bind or remodel rna and rna-protein complexes, with putative atp-dependent rna helicase ddx (ddx ) being a member of this family. [ ] the knockdown of ddx and ddx has been reported to increase sfv infection by small , , affecting the post-penetration stages, but with no effect on vsv infection. ddx is a novel antiviral helicase promoting rig-i-like receptor-mediated signaling. [ ] in ddx -depleted cells, the accumulation of viral rnas and proteins was delayed, leading to an obvious decrease in the production of infectious influenza a virus particles. [ ] in figure b , we can see that the overexpression of ddx in marc- cells showed a certain inhibitory effect on the proliferation of prrsv. nitric oxide (no) regulates the heart by spatial confinement of nitric oxide synthase (nos) isoforms. [ ] nos , a dimeric enzyme, is the most important isoform for no formation in the cardiovascular system, and its expression and activity can be regulated at transcriptional, posttranscriptional, and posttranslational levels. [ ] the deficiency of inducible and endothelial nos was reported to be responsible for delayed union and nonunion development as well as diminished bone formation. [ ] the cardiomyocyte-restricted overexpression of endothelial nos was shown to attenuate β-adrenergic stimulation and reinforce vagal inhibition of cardiac contraction. [ ] as indicated in figure c , downregulation of nos gene can suppress the proliferation of prrsv about % by rt-qpcr assay. the resulting data of the interferon screening were introduced by western blot analysis in figure s , supporting information and the data of the plaque assay confirmed the same results as rt-qpcr assay ( figure s , supporting information). so far, there is no efficient treatment for prrsv in the clinic. as reported, the occurrence and spread of the disease cannot be blocked by the current prevention and treatment strategy that is authorized to use inactivated vaccine and attenuated vaccine. [ ] vaccination could cause hosts to be more susceptible to secondary infection by other viruses or bacteria. [ ] so the development of anti-prrsv drugs have imperative academic research value and clinical application prospects. in recent years, due to their unique properties and biocompatibility, especially non-toxic metabolism, carbon nanomaterials including cds, [ , , , ] nanographene sheets, [ , ] graphene quantum dots, [ ] have been extensively investigated and proved to have outstanding antiviral effects. they are prepared by polyglycerol, -aminophenylboronic acid hydrochloride, benzoxazine, peg-diamine, -ethoxypropylamine, curcumin, and other raw materials. studies have shown that the size, charge, and surface modification of carbon nanomaterials have important influence on their antiviral activity. the mechanisms of inhibiting virus were shown to include blocking the entry of virus, improving expression of isg, changing the structure of surface protein of virus, etc. [ ] [ ] [ ] [ ] [ ] [ ] , ] virus infection to cells is an extremely complex dynamic process, including changes in the structure of the virus, cellular immune stress, and changes in the microecology of the viral parasitic cells. therefore, it is necessary to comprehensively explore the mechanism of gly-cds action from multiple angles. in our previous study, we tested the anti-prrsv activity of glycyrrhizic acid, the results showed that the inhibitory effect of glycyrrhizic acid at µm ( . mg ml − ) was ≈ orders of magnitude. [ ] in this study, . mg ml − gly-cds inhibited prrsv proliferation with ≈ orders of magnitude, indicating that the gly-cds increase the antiviral activity of glycyrrhizic acid. in a recent study, lin et al. reported that the curcumin-carbon quantum dots had higher antiviral effect than curcumin on the proliferation of enterovirus , which is in agreement with our results. [ ] as shown by the infrared spectra in figure f , gly-cds were similar to glycyrrhizic acid in structure, with most functional groups of glycyrrhizic acid being maintained during the formation of gly-cds. however, gly-cds have large surface area and more contact sites as compared with glycyrrhizic acid, leading to polyvalent interactions with the virus and thus higher antiviral activity than glycyrrhizic acid, similar to the enhanced antiviral activity of gold nanoparticles capped with mercaptoethanesulfonate. [ , ] the tem images of prrsv with and without gly-cds ( figure s , supporting information) also proved that gly-cds could interact with prrsv directly, which may result in inhibition of prrsv proliferation. gly-cds were found to inhibit prrsv invasion and replication, which is a more significant physiological process. however, the visual exploration of gly-cds in the virus penetration process is difficult and needs to be improved in further studies. the hydrothermal reaction source, temperature, and time have great effects on the size and functional groups of as-prepared cds, which could further affect the antiviral activity of cds. [ ] in our study, the control (cit-cds) was used mainly to explore the influence of the synthetic materials of cds on their antiviral activity. the results (figure ) showed that cit-cds had no significant inhibitory effect on the proliferation of prrsv, indicating that the source of cds had an important effect on their antiviral activity. what is more, we compared the anti-prrsv activity of gly-cds prepared under hydrothermal reaction for different time periods ( h, h) ( figure s , supporting information). the inhibitory effect of h-gly-cds was shown to be inferior to that of h-gly-cds, probably due to difference in functional groups of cds. this suggests the potential relationship of the functional groups on the surface of gly-cds with antiviral activity, which needs further study. we also systematically explored cellular immune stress caused by viral infection, including changes in ros production and protein expression levels in cells, changes in viral gene levels, protein levels, and infectivity. the induction of isg expression is a classical broad antiviral pathway for elucidating the antiviral mechanism. [ ] gly-cds can induce the upregulation of ifn-stimulating genes, which can be the main reason for its potential use as a broad-spectrum antiviral agent, including pedv and prv. however, gly-cds appear to exhibit stronger antiviral activity against rna viruses (prrsv and pedv in this study) than dna viruses (prv in this study). previous studies demonstrated that cds can induce the production of ifn and ifn-stimulating genes. [ ] however, viruses vary in their susceptibility to interferon, implying the gly-cds may also vary in antiviral activity against different viruses. additionally, we cannot rule out the possibility of other unidentified mechanisms attributed to the broad-spectrum antiviral activity of gly-cds. mass spectrometry-based proteomics can help us to accurately identify and quantify the proteins involved in cellular events, such as cellular responses to nanoparticles underlying www.small-journal.com small , , nano-bio-interactions. proteomics technology can identify the structural functions and expression levels of specific proteins in sample tissues, understand the pathogenic mechanisms, and alter the expression patterns of different proteins. [ ] thus, itraq proteomic analysis was used to evaluate the differential protein expression in cells after treatment with gly-cds as well as the functions of these differential proteins. based on proteomics data, the action mechanism of gly-cds on prrsv proliferation was explored by some cellular responses from marc- cells induced by gly-cds. this means that gly-cds can stimulate cells to regulate the expression of some host restriction factors, including ddx and nos , which have an important role in the subsequent cell infection by prrsv. as an active ingredient of the traditional chinese herbal medicine, glycyrrhizic acid plays an important role in anti-oxidation, anti-inflammation, antiviral immunoregulation, and liver protection. [ ] [ ] [ ] [ ] [ ] however, poor water solubility, relatively high cytotoxicity, and ambiguous action mechanism limit its wide application. additionally, previous studies have reported the side effects of glycyrrhizic acid, such as hypokalemia, edema, and thrombocytopenia. [ ] the gly-cds have higher biocompatibility, and theoretically, they can reduce the side effects of glycyrrhizic acid. however, whether the gly-cds exhibit stronger antiviral activity in vivo and reduce the side effects of glycyrrhizic acid still needs further study in the host (pig) of prrsv. in this paper, gly-cds were synthesized from the traditional chinese herbal medicine by hydrothermal method. the chemical characterization demonstrated that gly-cds have high dispersibility, small size, and other properties. biological experiments indicated that gly-cds have excellent antiviral activity against prrsv (≈ orders of magnitude). the antiviral mechanism of gly-cds was demonstrated from inactivation of prrsv in vitro, inhibition of prrsv invasion and replication, stimulation of cells to produce interferon, inhibition of ros production induced by prrsv infection. proteomics data also indicated that gly-cds can induce upregulation of intracellular antiviral proteins or downregulate pro-viral proteins. synthesis of cds: glycyrrhizic acid ( %) and quinine sulfate ( %) were provided by aladdin chemistry co. ltd. (shanghai, china). naoh (ar), ethylenediamine (ar), and citric acid (ar) were obtained from the sinopharm chemical reagent co. ltd. (shanghai, china). all reagents were directly used without further purification. all solutions were prepared with deionized water from a millipore water purification system. briefly, the gly-cds were synthesized with a modified one-pot hydrothermal method. in a typical synthesis route, . g ( . mmol) glycyrrhizic acid was dissolved in ml deionized water, followed by adding dropwise saturated naoh aqueous solution to adjust the ph to . ± . , transferring the mixture into a ml teflon-lined autoclave and incubation at °c for h (gly-cds). after cooling to room temperature naturally, large precipitate was removed from the mixture by centrifugation at rpm for min, followed by collecting the supernatant and filtering through a . µm filter to further remove small precipitate. finally, the gly-cds were obtained by dialysis against deionized water for h with a cut-off molecular weight kda dialysis bag, with the deionized water being changed every h during dialysis. the gly-cds powders were then collected and freeze-dried for further usage. the quantum yield of the gly-cds was measured to be . %, using quinine sulfate as reference. cells and viruses: porcine kidney (pk- ) cells, african green monkey kidney (vero) cells, and monkey kidney (marc- ) cells were purchased separately from the american type culture collection (atcc) and the china center for type culture collection (cctcc). cells were preserved at °c in % co atmosphere as adherent culture in dulbecco's modified eagle's medium (dmem/high glucose, hyclone) mixed with % fetal bovine serum (fbs, pan, usa). when grown to confluence, the cells were washed with pbs and collected from the culture vessel surface by adding . % trypsin. the hp-prrsv strain wuh (genbank accession no. hm ) (isolated at the end of in china from the pig brains with the syndrome of "high fever") was transfected into marc- cells as previously reported. [ , ] meanwhile, pedv strain aj (genbank accession no. jx ) (isolated in china in from a sucking piglet suffering from acute diarrhea) was transfected into vero cells in dmem containing µg ml − trypsin. [ ] pseudorabies virus (prv) strain ea (genbank accession no. kx ) is a wild virulent strain obtained in china. the recombinant prv expressing gfp (prv-gfp) was transfected into pk- cells. [ ] cytotoxicity assay: when grown to - % confluence, marc- cells were seeded in -well plates, followed by incubation separately with gly-cds, cit-cds, or glycyrrhizic acid at different concentrations in dmem supplemented with % fbs for , , , and h. after replacing the supernatant with µl of fresh dmem (containing % fbs), each well was supplemented with µl of mtt ( -[ , dimethylthiazol- -thiazolyl]- , -diphenyl tetrazolium bromide, sigma) solution ( . mg ml − ). after incubation for h, the supernatant was removed, and the formazan crystals were dissolved in µl per well of dimethyl sulfoxide (dmso) solution. after shaking on rocking shaker for min at rpm, the od values at nm were measured for estimating the percentage of cell relative viability using an enzyme linked immunosorbent assay microplate reader. antiviral assay: briefly, marc- cells were incubated with cds at . mg ml − in dmem (containing % fbs) for h, and rrrsv was preincubated with cds at . mg ml − for h at °c. then the medium containing cds was removed and substituted with the pretreated prrsv at multiplicity of infection (moi) of . after incubation for h, the supernatant was discarded, and cells were washed twice with dmem, followed by incubation separately with cds at . mg ml − for , , , and h. the antiviral effect of gly-cds on prrsv infection was then evaluated by plaque assay, ifa, western blot assay, and rt-qpcr assay. for plaque assay, the supernatant is collected at the indicated time point, then the fresh medium is added to the cells and the cells were stored at − °c; after freeze-thawing three times, the sample was collected as a cell lysate. all collected samples were stored in − °c and the amount of virus in the samples was quantified by plaque assay. the antiviral effect of gly-cds on pedv and prv in vero cells and pk- cells at the concentrations of . mg ml − was detected in a similar way as described above. briefly, pk- cells were infected with gfp-prv (moi = ) for the indicated periods of time. vero cells were infected with pedv (moi = . ) for the indicated periods of time. inactivation assay: prrsv was incubated with . mg ml − gly-cds at °c for h. after pre-cooling at °c for min, marc- cells were infected with the pretreated prrsv at °c for h, followed by plaque assay. adsorption assay: marc- cells were precooled at °c for min, followed by infection with prrsv (moi = . ) for h at °c in the presence of . mg ml − gly-cds in dmem (containing % fbs). after discarding the supernatant, the cells were washed twice with precooled serum-free dmem for plaque assay. invasion assay: after precooling at °c for min, the marc- cells were infected with prrsv (moi = . , . ) at °c for h. after discarding the supernatant and two washes with precooled serum-free www.small-journal.com small , , dmem, the cells were incubated with . mg ml − gly-cds in dmem (containing % fbs) for h at °c. after two washes with serum-free dmem, the plaque assay was performed. replication assay: when marc- cells were cultured to - % confluence in -well plates, the supernatant was discarded and the cells were infected with prrsv (moi = ) at °c for h. next, the virus inoculum was discarded and the cells were washed twice with serum-free dmem to remove non-adsorbed virus particles, followed by culture in dmem ( % fbs) for another h. subsequently, the cells were incubated separately with . mg ml − gly-cds in dmem (containing % fbs) for , , , or h. finally, the total rna was isolated and the negative-sense rna of prrsv was quantified by rt-qpcr assay. release assay: briefly, prrsv (moi = ) was added to the -well plates covered with monolayer marc- cells. after absorption at °c for h, the non-adsorbed virus particles were removed by two washes with serum-free dmem, then the cells were cultured in dmem (containing % fbs) for another h. after discarding the supernatant and two washes with serum-free dmem, the cells were incubated separately with . mg ml − gly-cds in dmem (containing % fbs). at , , , or min, the supernatant was collected and stored at − °c. meanwhile, fresh medium is added to the cells and the cells were stored at − °c; after freeze-thawing three times, the sample was collected as a cell lysate. finally, the virus content in the supernatant and cell lysis were detected by plaque assay. evaluation of ros production: the intracellular generation of ros was evaluated using reactive oxygen species assay kit (beyotime, s ). briefly, after prrsv infection for - h, the marc- cells were treated separately with gly-cds for h, followed by two washes with pbs and staining with ml of µm fluorescence marker ', '-dichlorofluorescein diacetate (dcf-da) for min in the dark at °c in % co . after removing the staining solution and washing twice with pbs, an inverted fluorescence microscope was used to evaluate the relative level of ros production by observing the fluorescence images. for excitation, an argon laser with a wavelength of nm was used and the fluorescent dcf was analyzed at an emission wavelength of nm. itraq quantitative proteomic analysis: briefly, cells were cultured to % confluence, followed by dividing into two groups: treatment with . mg ml − gly-cds or dmem ( % fbs) for h. in each group, there were five biological replicates, with three of them used for itraq quantitative proteomics analysis, and two of them used as parallel experiments to verify the reliability of the proteomics data. for proteomics analysis, the medium was removed at the end of exposure and cells were washed three times with precooled pbs. next, ml of ice-cold pbs was added and cells were harvested using a cell scraper. cell suspensions were transferred to ml falcon tubes, followed by centrifugation at rpm for min, discarding the supernatant and freezing the cell pellet at the liquid nitrogen tank for further analysis. the other two parallel experimental samples were compiled utilizing the observational method of western blot assay. for other experimental methods and procedures regarding total protein extraction, protein examination, itraq mark, fraction separation, and data analysis, refer to the supporting information. supporting information is available from the wiley online library or from the author. proc. natl. acad. sci proc. natl. acad. sci key: cord- - d w xyd authors: jeon, ji hyun; lee, changhee title: cellular cholesterol is required for porcine nidovirus infection date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: d w xyd porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dose-dependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, our data indicate that cell membrane cholesterol is required for porcine nidovirus entry into cells, and pharmacological drugs that hamper cholesterol-dependent virus entry may have antiviral potential against porcine nidoviruses. nidovirales is a large order of enveloped positive-sense, single-stranded rna viruses that consists of the families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, whose members infect a broad range of hosts including humans and other mammals, birds, fish, insects, and crustaceans [ , , , ] . although the genome sizes and virion morphologies of nidoviruses are strikingly different, the genome organization and replication strategy are comparable across the order. the nidovirus genome is composed of two large open reading frames (orfs), a and b, encompassing the ′-proximal two-thirds of the viral genome that encode non-structural proteins (nsps) and the remaining orfs located in the ′-proximal genome part that code for structural proteins [ , ] . the initial translation from replicase orf a and orf b yields large polyprotein (pp) precursors, pp a and pp ab, via a - ribosomal frameshift (rfs), which then undergo autoproteolysis by viral proteases to eventually produce functional nsps, including the viral rna-dependent rna polymerase (rdrp) [ , , ] . the membrane-bound rdrp-containing replication complex engages in viral genomic rna replication and subgenomic (sg) mrna transcription. the latter finally generates a ′ co-terminal nested set of sg mrnas that are used to express nidoviral structural proteins [ , , ] . porcine reproductive and respiratory syndrome virus (prrsv) is a pathogenic macrophage-tropic arterivirus of swine that results in reproductive failure in pregnant sows abstract porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dosedependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, and acute or chronic respiratory illnesses in pigs of all ages. prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months. as a result, prrsv infection suppresses normal macrophage function and immune responses and is often associated with severe disease outcomes, including increased pre-weaning mortality in growing pigs resulting from secondary bacterial or viral infections, thereby affecting the swine production system [ , , ] . porcine epidemic diarrhea virus (pedv) is a pathogenic enterocyte-tropic swine coronavirus that causes acute enteritis with high mortality rates in neonatal piglets. pedv infection is characterized by severe villous atrophy in the small intestine that results in watery diarrhea followed by fatal dehydration and death in newborn piglets [ , ] . although pedv outbreaks have been reported in europe and asia, the most serious epizootics for nearly the past three decades have occurred in asia. however, since the virus first emerged in the united states in [ ] , pedv has become recognized globally as a highly contagious and deadly virus. these two viruses, prrsv and pedv, represent emerging and re-emerging porcine nidoviruses that continue to threaten pork-producing countries around world, leading to huge financial losses to the global swine industry [ , ] . lipid rafts, which are enriched in cholesterol, sphingolipids, and associated proteins, are unique liquidordered microenvironments in the plasma membrane and are involved in a variety of cellular processes as well as in multiple stages of the virus life cycle. cholesterol, a major constituent of lipid rafts, maintains the tight packaging of sphingolipids, and several proteins are partitioned into these microdomains. cholesterol depletion destroys this structural order, leading to disorganization of lipid raft microdomains and dissociation of bound proteins [ , ] . therefore, plasma membrane cholesterol plays important roles in the infection processes of various non-enveloped and enveloped viruses [ , , , ] . in particular, enveloped virus entry requires cholesterol in either the viral or cellular membrane or both [ , , , , , , , , ] . however, there are few reports on the potential relationship between cholesterol and the replication of porcine nidoviruses, although cellular membrane cholesterol has been shown to be a determinant of prrsv entry in african monkey kidney marc- cells [ , ] . in the present study, therefore, we investigated the requirement for cholesterol and its mechanism of action in porcine nidovirus infection. independent depletion of cholesterol from the plasma membrane of target cells by treatment with methylβ-cyclodextrin (mβcd) significantly impaired prrsv and pedv infection. these inhibitory effects on viral replication were partially reversible by replenishment with exogenous cholesterol. in contrast, porcine nidoviruses were shown to be resistant to pharmacological reduction of the cholesterol content of the viral envelope. our data indicate that cholesterol-enriched microdomains are essential for prrsv and pedv in the cellular membrane, but not in the viral membrane. further experiments revealed that pharmacological depletion of cellular cholesterol primarily interferes with virus binding and penetration and subsequently influences post-entry stages of the prrsv and pedv replication cycle, including viral genomic and sg rna synthesis, viral protein expression, and virus production. altogether, our results suggest that cholesterol in the cellular membrane is critical for porcine nidovirus entry and that disruption of the cholesterol-dependent entry process may be an excellent therapeutic option for nidovirus infection in human or veterinary subjects. pam-pcd cells [ ] were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs, invitrogen), antibioticantimycotic solution ( ×, invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ×, invitrogen) in the presence of μg of zeocin (invitrogen) per ml. vero cells were cultured in alpha minimum essential medium (α-mem, invitrogen) with % fbs and antibiotic-antimycotic solution. st-papn cells [ ] were cultured in α-mem with % fbs and antibiotic-antimycotic solution in the presence of μg of g (invitrogen) per ml. the cells were maintained at °c in a humidified % co incubator. prrsv strain vr- was propagated in pam-pcd cells as described previously [ ] . pedv strain sm - was kindly provided by the korean animal and plant quarantine agency and propagated in vero cells as described previously [ , ] . mβcd and water-soluble cholesterol were purchased from sigma (st. louis, mo) and dissolved in ethanol and phosphate-buffered saline (pbs), respectively. these compounds were diluted to the desired concentrations in maintenance medium. prrsv n and pedv n protein-specific monoclonal antibodies (mab) were obtained from choogang vaccine laboratory (cavac; daejeon, south korea). antibodies to porcine cd (pcd ) and β-actin were purchased from abd serotech (raleigh, na) and santa cruz biotechnology (santa cruz, ca), respectively. the polyclonal antibody recognizing porcine aminopeptidase n (papn) obtained from balb/c mice immunized with purified papn (sigma) was a gift from bang-hun hyun (animal and plant quarantine agency, gimcheon, south korea). the cytotoxic effects of reagents on pam-pcd and vero cells were analyzed using a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (sigma) that allows detection of cell viability. briefly, pam-pcd and vero cells were grown at × cells/well in -well tissue culture plates with mβcd or water-soluble cholesterol treatment for h. after days of incubation, μl of mtt solution ( . mg/ml) was added to each well and the samples were incubated for an additional h. the supernatant was then removed from each well, and μl of dmso was added to dissolve the formazan crystals produced by mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd and vero cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with mβcd or ethanol for h and mock infected or infected with prrsv and pedv, respectively, at a multiplicity of infection (moi) of . virus-infected cells were then grown in the presence of mβcd or vehicle for h, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after washing five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with ′, -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on glass microscope slides in mounting buffer ( % glycerol and . % sodium azide in pbs), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). in addition, pam-pcd and st-papn cells stably expressing pcd and papn, respectively, were grown in the presence of mβcd or vehicle for h, fixed, and subsequently subjected to ifa with anti-pcd or anti-papn antibody as described above. cell staining was analyzed using a confocal laser scanning microscope (carl zeiss, gattingen, germany). quantification of virus-infected cells after treatment with mβcd was analyzed by flow cytometry. pam-pcd and vero cells were pretreated with mβcd, infected with virus, and maintained as described above. virus-infected cells were trypsinized at h postinfection (hpi) and centrifuged at × g (hanil centrifuge fleta ) for min. cell pellets were washed with cold washing buffer ( % bsa and . % sodium azide in pbs), and cells were resuspended in % formaldehyde solution in cold wash buffer for fixation at °c in the dark for min, followed by centrifugation and incubation of the pellets in . % triton x- in pbs at °c for min for permeabilization. after centrifugation, the cell pellets were resuspended in a solution of primary anti-n mab, and the mixture was incubated at °c for min. the cells were washed and allowed to react with an alexa fluor -conjugated anti-mouse igg secondary antibody at °c for min in the dark. the stained cells were washed again and analyzed on a facsaria iii flow cytometer (bd biosciences). expression of the viral receptor on the cell surface upon cholesterol depletion was also evaluated by facs analysis as described previously with some modifications [ ] . briefly, pam-pcd and st-papn cells were trypsinized at h post-seeding. the detached cells were fixed and then reacted with the primary antibody or normal mouse igg (santa cruz biotechnology), followed by incubation with secondary antibody as described above. the stained cells were either left untreated or were treated with mβcd at °c for h and analyzed using a flow cytometer. pam-pcd and vero cells were infected with prrsv or pedv and treated with mβcd or vehicle. the culture supernatants were collected at different time points ( , , , , and hpi) and stored at - °c. the prrsv titer was measured by limiting dilution on pam-pcd cells in duplicate by ifa as described above, and the % tissue culture infectious dose (tcid ) per ml was calculated using the spearman-kärber method [ ] . the pedv titer was determined by plaque assay using vero cells as described previously [ ] and was expressed as plaque-forming units (pfu) per ml. viral stocks were treated with mβcd at various concentrations at °c for h followed by ultracentrifugation to remove the mβcd. the mβcd-treated virus supernatants were purified through a % sucrose cushion (wt/vol) prepared in te buffer [ mm tris-hcl (ph . ), mm edta] by centrifugation at , rpm for h at °c in a p at rotor (model cp wx; hitachi, hitachinaka, japan). the virion cholesterol content was determined using fluorescence intensity analysis. briefly, -well plate wells were coated with ng of the purified viruses in mm sodium bicarbonate buffer (ph . ) and incubated at °c overnight. plates were washed three times with washing buffer ( . % tween- in pbs) and blocked with % powdered skim milk (bd biosciences, belford, ma) in pbs at °c for h. after washing, filipin iii (cayman chemical, ann arbor, mi) was added in triplicate for h in the dark. the plates were washed, and fluorescence intensity was measured with a spark m multimode microplate reader (tecan, männedorf, switzerland). in parallel, the purified samples were used to infect pam-pcd or vero cells for h, and the virus-infected cells were independently subjected to facs analysis and virus titration to determine prrsv or pedv infection as described above. pam-pcd and vero cells were first preincubated with vehicle or mβcd at various final concentrations for h and then supplemented with or without μg/ml or μg/ml exogenous cholesterol, respectively, and incubated for h. the cells were then inoculated with prrsv or pedv as described above. the virus inoculum was removed, and the infected cells were maintained in fresh medium containing mβcd and exogenous cholesterol. at h dpi, the virusinfected cells were harvested and subjected to facs analysis to assess the infectivity of prrsv and pedv as described above. in parallel, the cellular cholesterol content was determined using a cholesterol cell-based detection assay kit (cayman chemical) according to the manufacturer's instructions. briefly, virus-infected cells were cultivated in the presence of mβcd and exogenous cholesterol for h, fixed with cell-based assay fixative solution (cayman chemical) for min at rt, and then washed three times for min each with cell-based assay wash buffer (cayman chemical). the cells were incubated with filipin iii for h in the dark. after washing three times in wash buffer, the cells were counterstained with dapi, and filipin staining was visualized using a fluorescent leica dm il led microscope. pam-pcd and vero cells were infected with prrsv and pedv, respectively, at an moi of as described above. at - , , , , , , , , , or hpi, mβcd was added to maintain the indicated final concentration over the remainder of the time course experiment. the virus-infected and inhibitor-treated cells were further maintained and trypsinized at hpi, followed by centrifugation. the harvested cells were subjected to facs analysis to assess the presence of prrsv or pedv infection as described above. binding and internalization assays were performed as described previously with some modifications [ ] . pam-pcd and vero cells grown in -well culture plates were pretreated and infected with prrsv and pedv, respectively, at an moi of at °c for h in the presence of mβcd. unbound viruses were then removed by washing with pbs, and the cells were either incubated at °c (allowing virus binding only) or °c (permitting virus binding and internalization) in the presence of mβcd for h. in the latter case, the cells were further treated with proteinase k ( . mg/ml) at °c for min to remove bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi medium and inoculated onto fresh pam-pcd cell monolayers in -well tissue culture plates. at h post-incubation, bound or internalized viruses were titrated by ifa as described above, and the tcid was determined. for pedv, the serially diluted infected cells were inoculated onto uninfected vero cells, and, after h, viruses were titrated using plaque assay and quantified as pfu per ml. pam-pcd and vero cells were incubated with mβcd for h prior to infection and then inoculated with prrsv or pedv at an moi of for h at °c. the virus inoculum was subsequently removed, and the infected cells were maintained in fresh medium containing mβcd for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and then treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets as described previously [ ] . the rna levels of viral genes were normalized to that of mrna for the β-actin or glyceraldehyde- -phosphate dehydrogenase (gapdh) gene, and relative quantities (rq) of mrna accumulation were determined using the -ΔΔct method. to detect alterations in genomic rna and sg mrna levels in the presence of mβcd during porcine nidovirus infection, the results obtained from drug-treated cells were compared with those from vehicle-treated cells. pam-pcd and vero cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv and pedv, respectively, at an moi of in the presence of mβcd. at the indicated times, cells were harvested in μl of lysis buffer ( . % triton x- , mm β-glycerophosphate, mm ρ-nitrophenyl phosphate, mm mops, mm mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, μg of e per ml, μg of aprotinin per ml, μg of leupeptin per ml, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice and clarified by centrifugation at , × g (eppendorf centrifuge r, hamburg, germany) for min at °c. the protein concentrations of the cell lysates were determined by bca protein assay (pierce, rockford, il). the cell lysates were mixed with × nupage sample buffer (invitrogen) and boiled at °c for min. the proteins were then separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and reacted at °c overnight with primary antibodies against prrsv n, pedv n, or β-actin. the blots were then incubated with secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of : , for h at °c. proteins were visualized using enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify the viral proteins produced, band densities of prrsv n and pedv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/), based on the density value relative to that of the β-actin gene. all statistical analyses were performed using student's t-test, and p-values less than . were considered statistically significant. to investigate whether cholesterol plays a role in viral infection, we used mβcd, which is the most common cholesterol-sequestering agent used for plasma membranes. to examine the effect of mβcd on porcine nidovirus replication, prrsv and pedv were selected because they are economically important viral pathogens in the pork industry. based on mtt assay, none of the doses of mβcd tested in the current study caused detectable levels of pam-pcd or vero cell death (fig. a) . pam-pcd and vero cells were pretreated with mβcd at concentrations of . to mm or with ethanol as a vehicle control for h prior to infection. mβcd or vehicle was present throughout infection. virus production was initially measured by monitoring cytopathic effect (cpe) after infection and then confirmed by immunofluorescence using the respective anti-n protein mab at hpi (fig. b) . in vehicle-treated control cells, visible cpe appeared at hpi (data not shown) and became predominant by hpi, and virus-specific staining was pronounced in many cell clusters, indicating infection and spread of the viruses to neighboring cells. in contrast, mβcd had an obvious inhibitory effect on porcine nidovirus propagation. as shown in fig. b , the cholesterol-sequestering compound dramatically diminished virus-induced cpe (first and fourth panels) and expression of prrsv and pedv genes in a dose-dependent manner. based on the quantification of n protein by flow cytometry, the proportion (%) of virus-infected cells was noticeably reduced after mβcd treatment. a maximum of ~ % inhibition of both viruses was observed in response to . mm mβcd ( fig. c and d) . in addition, the effective doses for inhibiting % (ed ) of the replication of prrsv and pedv were determined to be about μm and μm, respectively. taken together, these data show that cholesterol depletion of target cells efficiently suppresses the replication of porcine nidoviruses. we then investigated the effects of cholesterol depletion on the envelopes of porcine nidoviruses. each viral stock was treated with mβcd up to mm prior to inoculation of the respective target cells. the virion cholesterol content after mβcd treatment was measured using filipin iii as a fluorescent polyene antibiotic that binds to cholesterol. as shown in fig. a , viral cholesterol levels were significantly reduced in mβcd-treated viruses compared to those in vehicle-treated viruses. however, in contrast to depletion of cellular cholesterol, the removal of cholesterol from virions resulted in no significant reduction in the replication of prrsv and pedv, even at the highest concentration used (fig. b) . furthermore, the titers of both prrsv and pedv remained unchanged upon treatment of each virus with mβcd (fig. c) . our results indicate that the viral cholesterol content is irrelevant to prrsv and pedv infection in vitro. to verify the importance of cellular cholesterol in porcine nidovirus infection, we first examined whether replenishment of exogenous cholesterol restored mβcd-induced inhibition of porcine nidovirus infectivity. to accomplish this, cholesterol-depleted cells were treated with μg or μg of exogenous cholesterol per ml, which is the highest noncytotoxic concentration for pam-pcd or vero cells, respectively, before virus inoculation. both mβcd and exogenous cholesterol were supplied throughout the course of infection. the addition of exogenous cholesterol to mβcd-treated and virus-infected cells was found to significantly reverse the antiviral activity of mβcd through depletion of cellular cholesterol. incubation with mβcd alone greatly reduced prrsv production to % and % at . mm and mm, respectively, whereas supplementation with exogenous cholesterol enhanced virus production to % and % at the same concentrations of mβcd (fig. a) . likewise, although pedv infection declined to %, %, and % in the presence of mβcd alone at mm, . mm, and mm, respectively, virus production increased to %, %, and % at the same concentrations of mβcd when exogenous cholesterol was added (fig. b ). to verify these results, we also investigated alterations in cellular cholesterol content in cells treated with mβcd and exogenous cholesterol using a fluorescent filipin iii. cellular cholesterol levels specifically decreased in virus-infected and mβcd-treated cells compared to those in virus-infected and untreated cells. supplementing exogenous cholesterol distinctly elevated the cholesterol level in virus-infected and mβcd-treated cells (fig. ) . altogether, the data reveal that cellular cholesterol content plays a pivotal role in porcine nidovirus infection. to determine the point at which mβcd acts during porcine nidovirus infection, pam-pcd and vero cells were treated with mβcd at various time points postinfection. at hpi, the levels of prrsv or pedv replication were measured indirectly by quantifying the cells that expressed viral n protein using flow cytometry (fig. ). treating cells with mm mβcd at - and hpi resulted in an approximately % and % decrease in prrsv production, respectively, in comparison with control levels (vehicletreated cells). strikingly, the addition of mβcd at hpi and thereafter (post-entry periods) had no significant inhibitory effect on prrsv infectivity compared to the control levels. similarly, treatment of pedv-infected cells with mm mβcd up to hpi suppressed viral production by - %, whereas exposure to the compound at - hpi resulted in no reduction in pedv infectivity. these data showed that mβcd had to be present pre-infection or at an early stage of viral infection to exert its antiviral effect as a cellular cholesterol depletion reagent. therefore, there is an important effect of cholesterol in the pre-entry period during porcine nidovirus infection. next, we sought to pinpoint the step(s) in the replication cycle of porcine nidoviruses that were precisely targeted by pharmacological depletion of cellular cholesterol. to address this, the earliest steps, the two stages of virus entry (virus attachment and penetration), were examined using an internalization assay after treatment with mβcd. pam-pcd and vero cells were inoculated with prrsv and pedv, respectively, at °c for h to allow only virus attachment and were further maintained either at °c or °c to restrict or permit virus internalization, respectively, in the presence of mβcd. the samples incubated at °c were subsequently treated with proteinase k to remove remaining viral particles from the cell surface. serially diluted infected cells were then subjected to an infectious center assay on uninfected pam-pcd and vero cell monolayers, and virus titers were measured days later by ifa or plaque assay. as shown in fig. , the titers of prrsv and pedv were reduced in a dose-dependent manner in cells treated with mβcd maintained at °c to permit virus binding but prevent internalization, indicating that cholesterol depletion has an inhibitory effect on virus attachment to these cells. moreover, production of both viruses was diminished in mβcdtreated cells incubated at °c to allow virus entry to proceed, which suggested that cholesterol sequestration disturbs internalization of prrsv and pedv. in addition, we analyzed the amount of pcd or papn expressed on the cell surface after mβcd treatment and found that the surface expression level of the viral receptor in cells treated with mm mβcd was similar to that of on vehicle-treated cells (fig. ) . taken together, these results indicate that pharmacological depletion of cholesterol hinders virus attachment and its subsequent penetration event without altering viral receptor expression and that cellular membrane cholesterol is indispensable for the porcine nidoviral entry process. like other positive-sense rna viruses, following virus entry, the nidovirus genome is released into the cytoplasm and promptly serves as a template for translation of viral proteins by hijacking the host translational machinery. early nidoviral translation produces the replicase polyproteins that are proteolytically processed into nsps, which subsequently drive de novo synthesis of nidoviral rna. therefore, we focused on the post-entry phases of the viral life cycle to investigate the functional mechanisms of sequestration of cellular cholesterol in pedv infection. because nidoviral infection generates genomic and sg rna species, we first tested whether the removal of cellular cholesterol specifically affected genome replication and sg mrna transcription. for this purpose, relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of mβcd following porcine nidovirus infection. as shown in fig. a , mβcd almost completely inhibited the synthesis of prrsv genomic rna and sg mrna at a concentration of mm when compared to untreated infected cells. furthermore, an analogous effect of mβcd on genome replication and sg mrna transcription of pedv was observed. little pedv genomic rna and sg mrna was detected in cells treated with . mm mβcd (fig. b) . the decreases in viral rna levels caused by mβcd did not reflect nonspecific inhibition of transcription because the internal control (β-actin or gapdh) mrna level remained unchanged in all samples (data not shown). taken together, these results indicated that treatment with mβcd subsequently suppressed synthesis of nidoviral genomic rna and sg mrna. since nidoviral structural proteins are translated from their respective sg mrna transcripts late in the infectious cycle, it is conceivable that suppression of viral protein expression is a consequence of cascade-like inhibition of viral rna synthesis. thus, we examined whether viral protein translation was affected by depleting cholesterol from cell plasma membranes. to accomplish this, pam-pcd and vero cells were exposed to mβcd for h prior to infection, and the compound was allowed to remain in the culture medium during infection and subsequent incubation. the expression levels of prrsv and pedv n proteins in the presence or absence of mβcd were evaluated at hpi by (fig. ). densitometric analysis of the western blots revealed that the intracellular expression of both n proteins was dramatically reduced by mβcd, with a maximum of more than % inhibition at the highest concentration (fig. ) . these data suggested that the sequestration effect of cellular cholesterol on viral protein expression is attributable to its specific preceding actions on viral rna biosynthesis during nidoviral replication. at hpi, cell lysates were prepared, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted using antibodies that recognize the prrsv n protein or pedv n protein. the blot was also reacted with mouse mab against β-actin to verify equal protein loading. viral protein expression was quantitatively estimated by densitometry and expressed as the density value relative to that of the β-actin gene, and mβcd-treated sample results were compared to those of the vehicle control. the values shown are the means from three independent experiments, and the error bars denote standard deviations. **, p < . in addition, virus yields were determined during pharmacological depletion of cellular cholesterol to investigate whether endogenous cholesterol is necessary for production of infectious viral progeny. after infection, viral supernatants were collected at hpi, and viral titers were measured. as illustrated in fig. a , the presence of mβcd suppressed the growth of viral progeny in a dose-dependent manner. the peak viral titer was determined to be . tcid /ml and . pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of mm mβcd reduced titers of prrsv and pedv to . tcid /ml and . pfu/ml, respectively (representing a more than -log reduction compared to control levels). examination of the growth kinetics also indicated that porcine nidovirus replication was markedly delayed when the cells were treated with mβcd at its optimal concentrations for each virus (fig. b) . these findings confirmed that cellular cholesterol content is integral in optimal progeny virus production from host cells. the order nidovirales is a monophyletic group of enveloped, positive-strand rna viruses with human and various animal hosts that produce a ′ co-terminal nested set of sg mrnas during infection. this order unites the four distantly related families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, based on a number of common properties such as genome organization, predicted proteomes, and synthesis of genomic and sg viral rnas, and it also separates them into large-(coronaviruses, toroviruses, and roniviruses), intermediate-(mesoniviruses), and small-genome (arteriviruses) nidoviruses to stress the clear genome size differences [ , , , ] . research on porcine nidoviruses is necessary not only for developing strategies to control these viruses in pig populations but also for understanding the molecular biology of human or veterinary-important nidoviruses. despite extensive attention and research investment, two porcine nidoviruses, prrsv and pedv, continue to plague pig-producing countries, causing a significant economic impact on the swine industry worldwide. this is partially attributable to the lack of efficient vaccines that can confer full protection against nidoviral infections and the lack of antiviral agents to treat these infections. although cholesterol is required for optimal infectivity of diverse non-enveloped and enveloped viruses [ , ] , its contribution to and specific function in porcine nidovirus replication are currently unknown. the present study showed that pharmacological sequestration of cholesterol in the cellular membrane but not the viral envelope exerts an efficient antiviral effect against prrsv and pedv in vitro. this effect could be counteracted by the addition of exogenous cholesterol, indicating the importance of membrane cholesterol for porcine nidovirus infection. depletion of cellular cholesterol using the drug mβcd primarily affected the virus attachment and internalization stages, significantly affecting post-entry steps in the replication of porcine nidovirus. altogether, our data indicate that cellular membrane cholesterol plays a critical role in entry of prrsv and pedv into target cells. because viruses are obligate intracellular parasites, they have evolved elaborate relationships with their host cells and developed the ability to modulate lipid composition, lipid synthesis, and host cell signaling pathways [ ] . in particular, cholesterol-rich microdomains appear to be important in the entry of various viruses. indeed, many viruses have been shown to exploit cholesterol, which is present in either the viral envelope [ , ] , cellular membrane [ , ] , or both [ , ] , for maximal virus entry. furthermore, accumulating evidence indicates that cholesterol is an essential component in the life cycle of several nidoviruses. the depletion of cellular cholesterol inhibits the entry of coronaviruses, including mouse hepatitis virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] , human coronavirus e [ ] , and avian infectious bronchitis virus [ ] as well as arteriviruses, including equine arteritis virus [ ] and prrsv [ , ] . on the other hand, transmissible gastroenteritis virus (tgev) and canine coronavirus require cholesterol both in the target cell membrane and in the viral envelope [ , ] . the current study revealed that prrsv and pedv are sensitive to the depletion of cholesterol only in the cell membrane. although cholesterol depletion has been shown to inhibit prrsv entry into african green monkey kidney-derived marc- cells, whether plasma membrane cholesterol is involved in either virus attachment or penetration or both is unknown [ , ] . in the present study, we used a continuous prrsv-permissive pam cell line that is considered the primary cell target for prrsv in the natural host, making it a good system for studying virushost interactions [ ] . in a previous study, yin et al. [ ] suggested that cholesterol is critical for a post-adsorption step in the entry of tgev, another porcine alphacoronavirus. porcine cd and apn have been shown to confer permissiveness of non-susceptible cell lines to prrsv and pedv, respectively, and have been identified as key molecules in the entry of porcine nidoviruses [ , , ] . a previous report showed that cholesterol depletion did not alter cd expression in marc- cells [ ] . likewise, the present study indicated that pharmacological depletion of cellular cholesterol had no effect on the levels of pcd and papn expression in porcine cells. recent studies have indicated that apn is not a functional cellular receptor for pedv, suggesting that the presence of the authentic virus receptor is essential for viral entry [ , ] . therefore, it is still possible that cellular cholesterol is quantitatively related to a hitherto unidentified receptor for pedv. based on our results, nevertheless, we propose that cellular cholesterol is important in both the binding and internalization stages of prrsv and pedv entry into susceptible cell lines. these findings are striking in that these two viruses are known to use different cell entry mechanisms for the initiation of virus infection: prrsv enters pam cells via receptor-mediated endocytosis followed by ph-dependent fusion between viral and endosomal membranes [ ] , whereas pedv enters target cells via virus-receptor interactions, followed by direct ph-independent fusion of the viral and plasma membranes [ ] . because cholesterol is an essential lipid component of cell membranes, its depletion has the potential to inhibit virus entry via several mechanisms. cholesterol is a critical structural component of lipid rafts, together with sphingolipids. these lipids influence viral infection by regulating viral and/ or cellular membranes and thus can function by preferentially partitioning into specific membrane microdomains [ ] . cholesterol may affect virus entry by modifying interactions between virus particles and host cell membranes, and lipid recognition by certain viral constituents may be essential for virus entry [ ] . in the case of sars-cov, cholesterol in the plasma membrane plays an important role in interactions of the viral spike protein and cellular receptor angiotensinconverting enzyme for optimal infection [ ] . by analogy, it is feasible that cellular cholesterol depletion might disturb binding of prrsv and pedv to specific cellular receptors. secondly, the level of cholesterol is important for maintaining biological membrane fluidity, and its removal could reduce the potential for lateral diffusion in the membrane [ ] . this reduction in membrane fluidity could have an influence on the entry of prrsv and pedv. thirdly, the lipid environment, including the cholesterol level, is known to contribute to the charge of ion channels formed in cellular membranes [ ] . considering this issue, ion channel alterations in response to the lack of cellular cholesterol may affect the entry process of porcine nidoviruses. lastly, cholesterol removal has been shown to result in the inhibition of cellular signaling pathways [ ] . we previously found that prrsv and pedv activate specific intracellular signaling networks such as mitogen-activated protein kinase (mapk) cascade pathways to favor replication of these viruses, but these signaling pathways are irrelevant to virus internalization [ , [ ] [ ] [ ] . based on these previous data, cellular cholesterol does not appear to act through mapk signaling pathways. although our analysis did not provide clear evidence of the mechanism by which cholesterol promotes porcine nidovirus entry, we assume that the mechanism may differ among viruses and that cholesterol-dependent virus entry might be dependent on more than one of the aforementioned pathways simultaneously. in conclusion, our findings indicate that optimal infectivity of porcine nidoviruses requires cholesterol in the plasma membrane and that this is critical for the entry of prrsv and pedv. however, cholesterol depletion resulted in a reduction, but not abolishment, of virus infectivity, indicating that virus entry may still occur with lower levels of cholesterol but that increased cholesterol content makes this process more efficient. future work should address the question of whether cholesterol facilitates prrsv and pedv entry through interactions between viral attachment proteins and cellular receptors and/or by affecting membrane fluidity. the current study indicates that cellular cholesterol is a key player in the early stages of porcine nidovirus infection, including attachment and penetration. impeding porcine nidovirus entry is a viable antiviral strategy because it likely acts on extracellular targets, thereby limiting cell damage, and these viruses might be used as surrogate models for testing antiviral agents against human nidoviruses. although further studies based on in vivo assessments are needed to evaluate the efficacy and safety of the cholesterol-depleting agent mβcd, the results presented here indicate that molecules or drugs that interfere with cholesterol function in virus entry should be considered candidates for antiviral approaches to porcine nidoviral diseases. the fusion peptide of semliki forest virus associates with sterol-rich membrane domains lipid raft disruption by cholesterol depletion enhances influenza a virus budding from mdck cells functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses specific association of glycoprotein b with lipid rafts during herpes simplex virus entry lipids: a key for hepatitis c virus entry and a potential target for antiviral strategies regulation of receptor function by cholesterol suppression of coronavirus replication by inhibition of the mek signaling pathway cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses nidovirales: a new order comprising coronaviridae and arteriviridae lipid-ion channel interactions: increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release plasma membrane cholesterol is required for efficient pseudorabies virus entry the median lethal dose and its estimation duck hepatitis b virus requires cholesterol for endosomal escape during virus entry importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sars-coronavirus with the cellular receptor angiotensin-converting enzyme immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs nidovirales: evolving the largest rna virus genome propagation of the virus of porcine epidemic diarrhea in cell culture assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in marc- cells canine distemper virus infection requires cholesterol in the viral envelope ribavirin efficiently suppresses porcine nidovirus replication extracellular signal-regulated kinase (erk) activation is required for porcine epidemic diarrhea virus replication coronaviridae. in: howley pm mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus jnk and p mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway stress-activated protein kinases are involved in porcine reproductive and respiratory syndrome virus infection and modulate virus-induced cytokine production generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type lipid rafts and hiv pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cells asymmetric requirement for cholesterol in receptor-bearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus interleukin- , interleukin- beta, and interferon-gamma levels are linked to prrs virus clearance a summary of taxonomic changes recently approved by the ictv contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection the role of lipid microdomains in virus biology equine arteritis virus is delivered to an acidic compartment of host cells via clathrindependent endocytosis human coronavirus e binds to cd in rafts and enters the cell through caveolae role of the lipid rafts in the life cycle of canine coronavirus importance of cholesterol for infection of cells by transmissible gastroenteritis virus coronaviruses porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity introduction to nidoviruses how viruses enter animal cells arterivirus molecular biology and pathogenesis family coronaviridae emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences signal transduction via glycosyl phosphatidylinositol-anchored proteins in t cells is inhibited by lowering cellular cholesterol role for influenza virus envelope cholesterol in virus entry and infection cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in marc- cells virus infection and lipid rafts differential effect of cholesterol on type i and ii feline coronavirus infection lipids as modulators of membrane fusion mediated by viral fusion proteins proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp porcine reproductive and respiratory syndrome virus entry into the porcine macrophage cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus critical role of cholesterol in bovine herpesvirus type infection of mdbk cells the authors declare that they have no conflict of interest.ethical approval this article does not contain any studies with animals performed by any of the authors. key: cord- -nbgdmavr authors: kim, youngnam; lee, changhee title: ribavirin efficiently suppresses porcine nidovirus replication date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: nbgdmavr porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. although ribavirin is a well-known antiviral drug against a broad range of both dna and rna viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. the antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic rna synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. investigations into the mechanism of action of ribavirin against prrsv and pedv revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular gtp pool by inhibiting imp dehydrogenase may be essential for ribavirin activity. further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses. the nidovirales are an order of enveloped single-stranded positive-sense rna viruses with animal hosts that include the families arteriviridae, coronaviridae, and roniviridae (cavanagh, ; mayo, ; spaan et al., ) . despite striking differences in genome size and virion morphology, the genome organization and expression strategy of the two groups belonging to the nidovirales order were found to be comparable. the nidovirus genome contains two large orfs, a and b, comprising the two-thirds of the viral genome encoding non-structural proteins (nsps) and the remaining orfs located in the terminal region coding for structural proteins (lai et al., ; snijder and spaan, ) . the initial translation from orf a and orf b yields the a and lab replicase polyproteins, respectively, which are then proteolytically processed into functional nsps including the viral rna-dependent rna polymerase (rdrp) (bautista et al., ; van aken et al., ; ziebuhr et al., ) . the rdrp-containing replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of -coterminal sg mrnas that are individually translated to structural proteins (lai et al., ; snijder and spaan, ) . porcine reproductive and respiratory syndrome virus (prrsv), a pathogenic macrophage-tropic arterivirus of pigs, is the etiological agent of acute respiratory illness in young piglets and reproductive failure in pregnant sows (albina, ) . prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months (albina et al., ; christopher-hennings et al., ; duan et al., ; wills et al., ) . as a result, prrsv infection results in suppression of normal macrophage functions and immune responses, which may render pigs susceptible to secondary bacterial or viral infections, leading to more severe disease than either agent alone (allan et al., ; feng et al., ; harms et al., ; wills et al., ) . porcine epidemic diarrhea virus (pedv), a pathogenic enterocyte-tropic coronavirus of swine, is the etiological agent of acute enteritis, which is characterized by lethal watery diarrhea followed by dehydration leading to death with a high mortality rate in suckling piglets (debouck and pensaert, ) . these two viruses, prrsv and pedv, are devastating porcine nidoviral pathogens that have still continued to plague swineproducing nations, causing tremendous economic losses to the global and asian pork industries (neumann et al., ; pensaert and yeo, ) . ribavirin ( -␤-d-ribofuranosyl- , , -triazole- -carboxamide, also known as virazole) is a synthetic guanosine analog that exhibits broad-spectrum antiviral activity in vitro (sidwell et al., ) . it has been used experimentally against a wide range of both dna and rna viruses, including gb virus b, hantaan virus, hendra virus, respiratory syncytial virus, lassa fever virus, norwalk virus, and west nile virus (chang and george, ; cooper et al., ; day et al., ; mccormick et al., ; lanford et al., ; rockx et al., ; severson et al., ) . most notably, ribavirin is used in combination with interferon-␣ for treatment of chronic hepatitis c virus (hcv) infections (cummings et al., ; davis et al., ) . however, there is still no report regarding an antiviral effect of ribavirin during the replication cycle of porcine nidoviruses. in the present study, therefore, we tried to investigate the antiviral activity of ribavirin and its mechanism of action in target cells upon porcine nidovirus infection. independent treatment of target cells with ribavirin significantly impaired prrsv and pedv infection. further experiments revealed that suppression of ribavirin affects post-entry steps of the replication cycle of prrsv and pedv, including viral genomic and sg rna synthesis, viral protein expression, and virus production. the addition of guanosine to the ribavirin treatment resulted in moderate reversal of the antiviral effects, suggesting that ribavirin activity is involved in the depression of cellular guanosine triphosphate (gtp) levels. sequencing analysis of the prrsv and pedv genomes in the ribavirin-treated and non-treated groups revealed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations during the replication of porcine nidoviruses. altogether, our results suggest that ribavirin may be an excellent therapeutic option for nidovirus infection in a human or veterinary subject. pam-pcd cells were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs, invitrogen), antibiotic-antimycotic solutions ( ×; invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ×; invitrogen) in the presence of g/ml zeocin (invitrogen). vero cells were cultured in alpha minimum essential medium (␣-mem, invitrogen) with % fbs and antibiotic-antimycotic solutions. the cells were maintained at • c in a humidified % co incubator. prrsv strain vr- was propagated in pam-pcd cells as described previously . pedv strain sm - was kindly provided by the korean animal, plant and fisheries quarantine and inspection agency and propagated in vero cells as described previously (hofmann and wyler, ) . ribavirin and mycophenolic acid (mpa) were purchased from sigma (st. louis, mo) and dissolved in distilled water (dw) or dimethyl sulfoxide (dmso), respectively. a monoclonal antibody (mab; sdow ) against the prrsv n protein was purchased from rural technologies (brookings, sd). the pedv spike (s) glycoprotein-specific and n protein-specific monoclonal antibodies (mabs) were kind gifts from sang-geon yeo (kyungpook national university, daegu, south korea). the anti-␤-actin antibody and horseradish peroxidase (hrp)-conjugated secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca). the cytotoxic effects of reagents on pam-pcd and vero cells were analyzed by a -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-pcd and vero cells were grown at × cells/well in a -well tissue culture plate with ribavirin or mpa treatment for h. after one day of incubation, l of mtt solution ( . mg/ml) was added to each well and the samples were incubated for an additional h. the supernatant was then removed from each well, after which l of dmso was added to dissolve the color formazan crystal produced from the mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd and vero cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with ribavirin or mpa for h and mock infected or infected with prrsv and pedv at a multiplicity of infection (moi) of , respectively. the virusinfected cells were further grown in the presence of ribavirin until hpi, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer ( % glycerol and . % sodium azide in pbs) and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-pcd and vero cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv or pedv at an moi of . . at the indicated times, cells were harvested in l of lysis buffer ( . % tritonx- , mm ␤glycerophosphate, mm -nitro phenyl phosphate, mm mops, mm, mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, g/ml e , g/ml aprotinin, g/ml leupeptin, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice, and clarified by centrifugation at , × g (eppendorf centrifuge r, hamburg, germany) for min at • c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with × nupage sample buffer (invitrogen) and boiled at • c for min. the proteins were separated on nupage - % gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk (bd biosciences, belford, ma) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at • c for h, and reacted at • c overnight with the primary antibody against prrsv n, pedv s or ␤-actin. the blots were then incubated with the secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of : for h at • c. proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify viral protein production, band densities of prrsv n and pedv s proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/) based on the density value relative to ␤-actin gene. pam-pcd and vero cells were infected with prrsv or pedv, respectively, at an moi of . as described above. at − , , , , , , , , , or hpi, ribavirin was added to give the indicated final concentration for the remainder of the time course experiment. the virus-infected and ribavirin-treated cells were further maintained and the infection was terminated at hpi by fixing the monolayers with % paraformaldehyde for min at rt. the fixed cells were subjected to ifa to assess the presence of prrsv or pedv infection as described above. an internalization assay was performed as described previously (cai et al., ) . briefly, pam-pcd and vero cells grown in -well culture plates were infected with prrsv and pedv, respectively, at an moi of . at • c for h, respectively. unbound viruses were then washed with pbs and the cells were either incubated at • c or • c in the presence ribavirin for h, followed by treatment with protease k ( . mg/ml) at • c for min to remove the bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added onto fresh pam-pcd cell monolayers in -well tissue culture plates. at h post-incubation, internalized viruses were titrated through ifa as described above and the % tissue culture infectious dose (tcid ) was calculated. for pedv, the serially diluted infected cells were added onto uninfected vero cells and after h, internalized viruses were titrated using a plaque assay as described previously , and the plaques were counted after % crystal violet staining. pam-pcd and vero cells were incubated with ribavirin for h prior to infection and then inoculated with prrsv or pedv at an moi of for h at • c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing ribavirin for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and treated with dnase i (takara) according to the manufacturer's protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets (table ) as described previously (sagong and lee, ) . the rna levels of viral genes were normalized to that of mrna for the ␤-actin or glyceraldehyde- -phosphate dehydrogenase (gapdh) gene and relative quantities (rq) of mrna accumulation were evaluated using the − ct method. to detect alteration of genomic rna and sg mrna levels in the presence of ribavirin during porcine nidovirus infection, the results obtained using ribavirin-treated samples were compared with vehicle-treated results. pam-pcd and vero cells were prrsv or pedv infected with treatment of ribavirin as described above. the culture supernatant was collected at different time points ( , , , , and hpi) and stored at − • c. the titer of prrsv was measured by limiting dilution on pam-pcd cells through ifa as described above and then quantified as the tcid per ml. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. pam-pcd and vero cells were preincubated with m guanosine for h before ribavirin was added to the medium at various final concentrations and then inoculated with prrsv or pedv as described above. the virus inoculum was removed and the infected cells were maintained in fresh medium containing ribavirin and guanosine. at h dpi, the virus-infected cells were fixed and subjected to ifa to evaluate the presence of prrsv or pedv infection as described above. the n gene coding regions for prrsv and pedv were sequenced to monitor mutation frequency. cells were prrsv or pedv infected and treated with ribavirin as described above. at hpi, total rna was extracted by trizol, and rt-pcr was performed to amplify the region encoding the orf to orf genes of prrsv or the n gene of pedv using gene-specific primer sets (table ) . the corresponding pcr product was then cloned into the pgem-t vector (promega, madison, wi). for sequencing of the gene in the recombinant vector, we selected independent bacterial colonies per group and analyzed mutations in the region. a student's t test was used for all statistical analyses and pvalues of less than . were considered statistically significant. virus-specific cpes were observed daily and were photographed at hpi using an inverted microscope at a magnification of × (first panels). for immunostaining, infected cells were fixed at hpi and incubated with mab against the n protein of prrsv or pedv followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescent microscope at × magnification. viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing n proteins through ifa. five fields at × magnification were counted per each condition and the total number of cells per field as determined by dapi staining was similar in all fields. values are representative of the mean of three independent experiments and error bars represent standard deviations. *p = . - . ; † p < . . to examine the effect of ribavirin on porcine nidovirus replication, prrsv and pedv were selected because they are important viral pathogens that economically affect the swine industry. based on mtt assay, none of the doses of ribavirin tested in the present study caused detectable cell death of pam-pcd and vero cells (data not shown). pam-pcd and vero cells were pretreated with ribavirin at concentrations of - m or - m, respectively, or with dw as a vehicle control for h prior to infection. ribavirin was present during the entire period of infection. viral production was initially measured by monitoring the cytopathic effect (cpe) after infection and confirmed by immunofluorescence using n protein-specific mab at hpi (fig. ) . in vehicle-treated control cells, visible cpe appeared at hpi and became predominant by hpi, and n-specific staining was clearly evident in many cell clusters, indicating infection and the spread of the virus to neighboring cells. in contrast, ribavirin had an obvious inhibitory effect on virus propagation. as shown in fig. a and b, ribavirin significantly decreased virus-induced cpe and viral gene expression of prrsv and pedv at concentrations of ∼ m and ∼ m, respectively. n protein staining revealed that the number of cells expressing viral antigen, as quantified by n protein staining results, was also reduced during ribavirin treatment, resulting in a maximum of ∼ % inhibition in response to m and m for prrsv and pedv, respectively ( fig. a and b) . moreover, the effective doses for inhibiting % (ed ) of the replication of prrsv and pedv were determined to be about m and m, respectively. taken together, these data demonstrate that ribavirin efficiently suppresses the replication of porcine nidoviruses. to further determine at which point ribavirin acted during the porcine nidovirus infection period, pam-pcd and vero cells were treated independently with ribavirin at various time points post-infection. at hpi, the levels of prrsv or pedv replication were measured indirectly as viral antigen production by quantifying cells expressing the n protein through ifa (fig. ) . treatment of cells with m ribavirin for up to hpi resulted in more than - % decrease in prrsv production when compared to the untreated control. addition of ribavirin between and hpi led to - % inhibition of prrsv infectivity. similarly, treatment with m ribavirin at − , , and hpi was found to lead to - % suppression of pedv production, while its treatment at - hpi resulted in - % reduction in pedv infectivity. in contrast, no significant impairment of porcine nidovirus propagation was observed when ribavirin was added at hpi. these results demonstrate that the inhibitory effect of ribavirin against the replication of prrsv and pedv was exerted primarily during the initial period of infection, suggesting that its action occurs mainly at early time points after porcine nidovirus infection. to further assess the antiviral activity of ribavirin against porcine nidovirus replication, virus yield was determined during treatment of ribavirin. upon infection, viral supernatants were collected at hpi and viral titers were measured. as fig. a shows, the presence of ribavirin reduced the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be tcid /ml and . pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of m or m ribavirin decreased titers of prrsv and pedv to . tcid /ml and . pfu/ml, respectively (almost log reduction compared with the control). the growth kinetics study further demonstrated that the overall process of porcine nidovirus replication was significantly delayed when cells were treated with ribavirin (fig. b) . consequently, these findings revealed that ribavirin inhibits optimal progeny virus release within the natural host cells. to evaluate which steps of the replication cycle of porcine nidoviruses were targeted by ribavirin, we started focusing on the earliest step, virus entry upon the ribavirin treatment. to address this issue, an internalization assay was performed as described previously (cai et al., ) . pam-pcd and vero cells were inoculated with prrsv and pedv, respectively, at • c for h to allow virus attachment and further maintained either at • c or • c to permit virus internalization in the presence of ribavirin. the samples were then treated with protease k to remove the remaining viruses from the cell surface. the serially diluted infected cells were subsequently subjected to an infectious center assay on uninfected pam-pcd and vero cell monolayers and virus titers were measured days later through ifa or by plaque assay. as shown in fig. c , the titers of prrsv and pedv were virtually the same in cells treated with ribavirin or without ribavirin at • c and determined to be . - . tcid /ml and . × - . × pfu/ml, respectively, indicating no difference between those cells. in contrast, only minimal viral productions of about . tcid /ml (prrsv) and . × pfu/ml (pedv) were observed in cells maintained at • c, which was likely due to inefficient removal of the bound virus. these results indicated that ribavirin has no inhibitory effect on the virus entry process. like all positive-sense rna viruses, following the uncoating process, the nidovirus genome is released into the cytoplasm and immediately serves as a template for viral translation by the same cellular cap-dependent mechanism. early nidovirus translation produces replicase polyproteins that are posttranslationally cleaved into nsps by viral proteases. subsequently, the nonstructural replicase proteins mediate de novo synthesis of viral rna, including genomic rna replication and sg mrna transcription. the nidovirus structural proteins are translated lately from respective sg mrna transcripts. thus, to determine the inhibitory mechanism of ribavirin on the postentry steps of the nidovirus life cycle, we first investigated whether viral protein translation was affected by ribavirin. to accomplish this, pam-pcd and vero cells were treated with ribavirin for h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. the expression levels of the prrsv n and pedv s proteins in the presence or absence of ribavirin were evaluated at hpi by western blot analysis. the production of both proteins productions was drastically diminished during ribavirin treatment (fig. ) . densitometric analysis of the western blots revealed that the intracellular expression of both n and s proteins was reduced by ribavirin, with a maximum of about % inhibition at the concentration of m and m, respectively (fig. ) . this marked reduction was probably not the result of a nonspecific decrease in the translation mechanism, since ponceau s staining results indirectly indicated that the expression levels of the overall cellular proteins remained similar following treatment (data not shown). taken together, these results demonstrated the inhibitory effects of ribavirin specifically against viral translation during porcine nidovirus replication. for positive-strand rna viruses, synthesis of the viral nonstructural proteins is required for viral rna replication and transcription. thus, it is conceivable that impaired viral protein expression in would be caused by inhibition of viral rna synthesis. since nidovirus infection produces two rna entities including genomic versus subgenomic, we therefore determined if ribavirin specifically affected genome replication and sg mrna transcription. to answer this question, the relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of ribavirin upon porcine nidovirus infection. as shown in fig. a , ribavirin exhibited a maximal % and % reduction in the synthesis of prrsv genomic rna and sg after washing with cold pbs, infected cells were maintained in the presence or absence of ribavirin either at • c or • c for an additional hour. bound but uninternalized virus particles were removed by treatment with protease k. the infected cells were then serially diluted and plated onto fresh target cells. at days post-incubation, internalized viruses were titrated by ifa and plaque assay for prrsv (left) and pedv (right), respectively. results are expressed as the mean values from triplicate wells and error bars represent standard deviations. *p = . - . ; † p < . . mrna at a concentration of m, respectively, when compared with untreated infected cells. furthermore, a similar inhibitory effect of ribavirin on genome replication and sg mrna transcription of pedv was observed. the amounts of pedv genomic rna and sg mrna detected in cells treated with m ribavirin were only about % and % of the untreated level (fig. b) . the decreases in viral rna levels after the addition of ribavirin were not due to nonspecific inhibition of transcription, as mrna levels of the internal control (␤-actin or gapdh) remained unchanged in all samples (data not shown). altogether, these results indicated that ribavirin suppresses the synthesis of the nidoviral genomic rna and sg mrna. several mechanisms of action for the antiviral activity of ribavirin have been suggested, including a reduction in cellular gtp pools via inosine monophosphate dehydrogenase (impdh) inhibition and increased mutation frequency on the virus genome leading to error catastrophe (graci and cameron, ) . to assess these known mechanisms, we first examined whether replenishment of guanosine affected the antiviral effect of ribavirin against porcine nidovirus infection. the addition of m guanosine to the ribavirin-treated and virus-infected cells was found to moderately reverse the antiviral activity of ribavirin. the incubation of ribavirin alone reduced prrsv production to % and % at m and m, respectively, whereas supplementation of guanosine to ribavirin inhibited virus production to % and % at the same concentrations (fig. a, left panel) . likewise, while pedv production was declined to %, %, and % in the presence of ribavirin alone at m, m, and m, respectively, it decreased to %, %, and % at the same concentrations when guanosine was added to the ribavirin treatment (fig. a, right panel) . to verify these results, we also tested the effects of mpa, a potent inhibitor of cellular impdh, on the replication of prrsv and pedv. as shown in fig. b , mpa efficiently reduced porcine nidovirus propagation fig. . inhibition of viral protein translation by ribavirin. ribavirin-treated pam-pcd and vero cells were mock-infected or infected with prrsv (a) or pedv (b) for h and were further cultivated in the presence or absence of ribavirin. at hpi, cellular lysates were collected, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted by using the antibody that recognizes the prrsv n protein or the pedv s protein. the blot was also reacted with mouse mab against ␤-actin to verify equal protein loading. each viral protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and ribavirintreated sample results were compared to vehicle-control results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = . - . ; † p < . . inhibition of viral rna transcription by ribavirin. pam-pcd and vero cells pretreated with ribavirin were mock-infected or infected with prrsv (a) or pedv (b) for h and were incubated in the presence of ribavirin. total cellular rna was extracted at hpi, and strand-specific viral genomic rnas (black bars) and sg mrnas (white bars) of prrsv and pedv were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna and sg mrna were normalized to mrna for porcine ␤-actin or monkey gapdh and relative quantities (rq) of mrna accumulation were evaluated. ribavirin-treated sample results were compared with untreated results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = . - . ; † p < . . at concentrations greater than . m and . m in a dosedependent manner for prrsv and pedv, respectively. sequence analysis of the porcine nidovirus genome in the presence or absence of ribavirin was conducted to investigate whether it triggers catastrophic mutations. we analyzed recombinant sequences of the orf to orf -coding region corresponding to nucleotide numbers , - , (prrsv) and the n gene region corresponding to nucleotide numbers , - , (pedv) from each virus grown in the presence of ribavirin. as controls, colonies were also obtained individually from untreated prrsv-and pedv-infected a the prrsv orf -orf coding region ( bp) and the pedv n ( bp) gene were rt-pcr amplified and the amplicon product was then cloned into the pgem-t vector. for sequencing of the gene in the recombinant vector, clones per group were selected and analyzed the number (rate) of mutations. b a total of , nt and , nt for prrsv and pedv were sequenced and a mutation frequency was calculated per nt, respectively. cells. in the ribavirin-treated groups, point mutations occurred in ( %) plasmids for both prrsv and pedv, which corresponded to a mutation frequency of . and . per nt, respectively. interestingly, mutations were also found in ( %) plasmids in the untreated groups for prrsv and pedv, which were calculated as a mutation frequency of . and . per nt, respectively (table ) . these sequencing results indicated that there were no significant differences in the mutation rates of ribavirin-treated and non-treated groups upon porcine nidovirus infection. the nidovirales are an order of enveloped, positive-strand rna viruses with animal hosts, which synthesize a nested set of multiple sg mrnas. this order includes three families arteriviridae, coronaviridae, and roniviridae, which have similar genome organization and replication strategy, but different virion morphology and genome length (mayo, ; lai et al., ; snijder and spaan, ) . porcine nidoviruses are not only devastating viral pathogens to the pig population, but can also be applied as an animal virus model to study human or veterinary-important nidoviruses. among these, despite tremendous efforts to control the diseases, prrsv and pedv have continuously plagued pigproducing countries, leading to significant economic impacts on the global or asian swine industry, respectively. this is partially due to a lack of efficient vaccines capable of conferring full protection against viral infections and antiviral agents for treating porcine nidoviruses. although ribavirin has an antiviral effect on a variety of dna and rna viruses (sidwell et al., ) , its efficacy and mode of action on porcine nidovirus replication are currently unknown. the present study demonstrated that ribavirin also exerts very effective antiviral activity against prrsv and pedv in vitro, as indicated by ed values of approximately m ( . g/ml) and m ( . g/ml), respectively. ribavirin can potentially act via inhibition of various steps of the virus life cycle, including viral translation, genome or transcript capping, rna synthesis, and progeny virus spread (graci and cameron, ) . treatment of cells with ribavirin resulted in significant attenuation of postentry steps during the replication of porcine nidovirus, as determined by lower progeny production, diminished viral protein expression, and decreased synthesis of genomic rna and sg mrna. it was previously reported that the tier of murine norovirus (mnv- ) in the presence of ribavirin dropped about -fold after virus infection, but remained similar after h of virus infection (chang and george, ) . in this study, growth kinetics experiments also indicated that the reduced rate of nidoviral titers in the presence of ribavirin was found to be comparable between and hpi. since ribavirin should be converted to its monophosphate active form (rmp) to exert antiviral activity, it appears that virus infection may disrupt the normal metabolic processes, leading to interference with the conversion of ribavirin (parker, ) . taken together, our data indicate that ribavirin potently elicits antiviral activity against prrsv and pedv in target cells. an important aspect of the antiviral activity of ribavirin may be attributed to the ability to act via multiple mechanisms simultaneously. although the mechanisms of action of ribavirin still remain controversial, five primary mechanisms have been proposed depending on the particular virus. these include reduction in cellular gtp pools by inhibiting impdh, enhanced mutation frequency via its incorporation into the viral genome leading to catastrophic error, modulation of host immune responses, inhibition of mrna capping, and interference with viral rna polymerase. therefore, the mechanisms of action of ribavirin may differ among viruses, and its antiviral activity may be operated predominantly via one or two mechanisms (graci and cameron, ; parker, ) . to elucidate potential mechanisms responsible for the antiviral effect of ribavirin on porcine nidoviruses, we first tested whether the antinidoviral activity of ribavirin is involved in inhibition of impdh and subsequent depression of cellular gtp levels. in previous studies, the addition of guanosine to the culture medium efficiently reversed the antiviral effects of ribavirin against norovirus, yellow fever virus, and human parainfluenza virus (chang and george, ; leyssen et al., ) . the present study so investigated the effects replenishing gtp by adding guanosine to ribavirin-treated cells upon virus infection. consistent with previous reports, the addition of guanosine to the ribavirin treatment significantly reversed the antiviral activity of ribavirin in porcine nidoviruses. furthermore, a noncompetitive impdh inhibitor, mpa, was found to strongly inhibit the replication of prrsv and pedv at concentrations above . m and . m, respectively. in contrast to ribavirin, which has to be converted to the active rmp form to elicit the antiviral activity, mpa does not require metabolic activation to exert its function. thus, the efficient antinidoviral activity of mpa suggests that ribavirin may directly lead to the inhibition of intracellular impdh levels. since rna viruses replicate with high genetic variation, they exist as viral quasispecies that are complex and dynamic mutant distributions that share a dominant nucleotide sequence and a mutant spectrum (ruiz-jarabo et al., ) . therefore, rna viruses reside on the edge of mutation crisis, and the increased average error frequency can cause defective genetic information and reduced viability termed error catastrophe (crotty et al., ; day et al., ) . ribavirin has been shown to trigger catastrophic mutations including lethal mutations in a variety of viruses and these may accumulate as a virus goes through multiple rounds of replication, resulting in extinction of the viral population (crotty et al., ; contreras et al., ; day et al., ; lanford et al., ; severson et al., ) . however, sequence analysis of the conserved n gene regions of prrsv and pedv in the ribavirintreated or untreated groups revealed that the two groups produced similar mutation frequencies ( . / . versus . / . ). these results were not surprising since rna genomes mutate at average rates of − - − per nucleotide copied during replication of rna viruses by the virus-encoded rna-dependent rna polymerase (rdrp) lacking proofreading functions (drake and holland, ) . our data indicated that the antiviral activity of ribavirin against porcine nidoviruses may not be associated with error-prone replication by increasing the probability of ribavirin incorporation into the viral genome. in conclusion, our findings described here demonstrated that ribavirin effectively prevents the replication of porcine nidoviruses in target cells at subcytotoxic doses. additionally, the highest doses of ribavirin used in this study did not increase the frequency of mutations in the nidoviral genome, and instead affected intracellular gtp levels via impdh inhibition. thus, we propose that one of the modes of action of ribavirin to elicit the antiviral effects against porcine nidoviruses occurs via gtp depletion, which may not work in concert with catastrophic mutations. although further studies based on in vivo assessment are needed to evaluate the efficacy and safety of ribavirin, the results presented here indicate that it is a good candidate of choice for antiviral approach against porcine nidoviral diseases. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus suppression of coronavirus replication by inhibition of the mek signaling pathway nidovirales: a new order comprising coronaviridae and arteriviridae interferons and ribavirin effectively inhibit norwalk virus replication in replicon-bearing cells persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars viral rna mutations are region specific and increased by ribavirin in a full-length hepatitis c virus replication system management and prevention strategies for respiratory syncytial virus (rsv) bronchiolitis in infants and young children: a review of evidence-based practice interventions the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen rna virus error catastrophe: direct molecular test by using ribavirin interferon and ribavirin vs interferon alone in the re-treatment of chronic hepatitis c previously nonresponsive to interferon: a meta-analysis of randomized trials interferon alfa- b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis c. international hepatitis interventional therapy group error-prone replication of west nile virus caused by ribavirin experimental infection of pigs with a new porcine enteric coronavirus, cv mutation rates among rna viruses virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii mechanisms of action of ribavirin against distinct viruses experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus propagation of the virus of porcine epidemic diarrhea in cell culture ribavirin induces error-prone replication of gb virus b in primary tamarin hepatocytes coronaviridae porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase a summary of taxonomic changes recently approved by the ictv lassa fever. effective therapy with ribavirin contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states porcine epidemic diarrhea metabolism and antiviral activity of ribavirin a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment memory in viral quasispecies porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-␤ production by inhibiting irf activation in immortalized porcine alveolar macrophages ribavirin causes error catastrophe during hantaan virus replication broad-spectrum antiviral activity of virazole: -beta-d-ribofuranosyl- , , -triazole- -carboxamide arteriviridae family coronaviridae proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus virus-encoded proteinases and proteolytic processing in the nidovirales this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology ( - ). key: cord- -zrsnahi authors: sun, ying; xiao, shaobo; wang, dang; luo, rui; li, bin; chen, huanchun; fang, liurong title: cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in marc- cells date: - - journal: sci china life sci doi: . /s - - - sha: doc_id: cord_uid: zrsnahi cholesterol represents one of the key constituents of small, dynamic, sterol- and sphingolipid-enriched domains on the plasma membrane. it has been reported that many viruses depend on plasma membrane cholesterol for efficient infection. in this study, the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus (prrsv) infection of marc- cells was investigated. pretreatment of marc- cells with methyl-β-cyclodextrin (mβcd), a drug used to deplete cholesterol from cellular membrane, significantly reduced prrsv infection in a dose-dependent manner. this inhibition was partially reversed by supplementing exogenous cholesterol following mβcd treatment, suggesting that the inhibition of prrsv infection was specifically mediated by removal of cellular cholesterol. further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry, especially virus attachment and release. these results indicate that the presence of cholesterol in the cellular membrane is a key component of prrsv infection. porcine reproductive and respiratory syndrome (prrs), characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs, is now considered one of the most economically important diseases in countries with intensive swine industries [  ] . prrs virus (prrsv), the etiology of prrs, is a positive-strand rna virus that belongs to the genus arterivirus, family arteriviridae, along with lactate-dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv) [ ] . it has been demonstrated that prrsv has a strong tropism for cells of the monocyte/macrophage lineage in vivo, and for porcine al-veolar macrophages (pam) and african green monkey kidney cells (marc- ) in vitro [  ] . accumulating evidence has revealed that the replication of prrsv in cultured cells partially depends on the composition of the host cellular plasma membrane [ , ] . cellular surface molecules, such as heparan sulfate and sialoadhesin, play an important role in prrsv attachment and internalization [  ] . moreover, simian vimentin and cd were considered to play a role in prrsv infection of marc- cells [ , ] . however, some links within the detailed mechanism involved in prrsv replication cycle remain poorly understood. the infectivity of enveloped viruses often depends on the lipid composition of the host cell membrane. for instance, infection by semliki forest virus, a species of the alphavirus genus, requires both cholesterol and sphingolipids [ , ] . additionally, virus entry and assembly of certain retroviruses and filoviruses depend on cholesterol-rich membrane microdomains [ , ] . cholesterol is an abundant, key component of the eukaryotic cell membrane that forms liquid-ordered microenvironments in the plasma membrane due to its rigidity and hydrophobicity [ ] . previous studies demonstrated that plasma membrane cholesterol plays an important role in the entry and infection processes of many viruses, especially in some enveloped viruses [ ] , including human immunodeficiency virus type (hiv- ) [ ] , poliovirus [ ] , murine coronavirus [ ] , vaccinia virus [ ] , herpes simplex virus [ ] , foot-and-mouth disease virus [ ] , and severe acute respiratory syndrome-related coronavirus (sars-cov) [ ] . moreover, the plasma membrane cholesterol content also affects the release of influenza a virus from the infected cell [ ] . however, few reports on the potential relationship between cholesterol and the replication of viruses in the arteriviridae exist, although it was recently reported that cholesterol is a determinant of equine arteritis virus entry [ ] . in this study, we investigated whether plasma membrane cholesterol plays a role during prrsv infection and at which step(s) it functions. our study clearly showed that depletion of cholesterol from host cells inhibited virus infection, and primarily affected virus entry and release processes. prrsv strain ch- a, the first field isolate from china, was kindly provided by dr. tong g. z. (shanghai veterinary research institute, shanghai, china). prrsv strain wuh , a highly pathogenic north american-type prrsv, was isolated in [ ] . marc- cells were cultured and maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) at °c in a humidified % co incubator. methyl-β-cyclodextrin (mβcd) and water-soluble cholesterol were purchased from sigma (st louis, usa). to biotinylate prrsv, the virus was first purified following the protocols described by nauwynck et al. [ ] . the titer of the purified virus was approximately × tcid ml  ( % tissue culture infectious doses ml  ), as determined by a cytopathic effect (cpe) assay in marc- cells. the protein concentration of the virus preparation was mg ml  , as determined by the bradford assay with bovine serum albumin (bsa) used as a protein quantitation standard. biotinylation was performed with a protein biotinylation kit (ge healthcare, uppsala, sweden), according to manufacturer's instructions. the biotinylated viruses were collected after purification on a sephadex g- column and diluted in phosphate-buffered saline (pbs; ph . ) at a concentration of . mg ml  and stored at  °c. virus infectivity and the ability to enter cells were not significantly decreased after being biotinylated. to determine the toxicity of mβcd, confluent marc- cells in -well plates were treated with serum-free dmem with or without mβcd. after incubation at °c for h, mβcd-treated cells and mock-treated cells were harvested for the -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay to detect cell viability. to determine if treatment with mβcd reduces the cellular cholesterol content, marc- cells in -well plates were washed three times with cold pbs, and then incubated for h with serum-free dmem (control cells) or various concentrations of mβcd (treated cells). after three additional washes with pbs, cells were harvested and the cholesterol content was measured using the amplex red cholesterol assay kit (molecular probes, oregon, usa), as directed by the manufacturer. to deplete plasma membrane cholesterol, confluent marc- cells in -well plates were washed three times with cold pbs, and then incubated for h with serum-free dmem (control cells) or dmem containing mmol l  mβcd (treated cells). to replenish plasma membrane cholesterol, the protocols established by popik et al. [ ] were used. in brief, confluent marc- cells in -well plates were treated with serum-free dmem (control cells) or dmem containing mmol l  mβcd (treated cells) for h. cells were then washed three times with pbs and incubated with serum-free dmem alone or dmem containing μg ml  water-soluble cholesterol for h. after three washes with pbs, cells were harvested for cholesterol level detection or infected with prrsv. total rna was extracted from cell suspensions or supernatant with rnaprep micro kit (tiangen, beijing, china), according to the manufacturer's instructions. rna was re-versetranscribed into cdna using revertra ace-α (toyobo, osaka, japan) with oligo (dt) . quantitative real-time pcr was performed according to protocols described by egli et al. [ ] . in brief, the generated cdna was amplified by quantitative real-time pcr using primers for the prrsv orf gene, reverse primer ( ′-aaatggggcttctccg-ggtttt- ′) and forward primer ( ′-tcagctgtgcca-aatgctgg- ′), and specific fluorescent probe ( ′-tcc-cggtcccttgcctctgga- ′; sense orientation), which was ′-labeled with -carboxyfluorescein (fam; reporter) and tetramethylrhodamine (tamra) at its ′-end. real-time pcr was performed in a single tube in an abi prism real-time pcr system (applied biosystems, foster city, ca). for each run, four no-template controls containing water instead of dna, as well as five prrsv orf gene standards corresponding to , , , , copies per tube were included. samples of interest were run in triplicate. data from the taqman ® run were analyzed using the sds software (version . ; applied biosystems). confluent marc- cells in -well plates were incubated with μg of biotinylated prrsv in pbs per well at °c for h to achieve the maximal virus-cell attachment. supernatants were removed and the cells were washed three times with cold pbs. cells were next left untreated, or treated with mβcd, or treated with mβcd and cholesterol replenishment, as described above. during the treatment, viruses complete the penetration process. after removal of the supernatant, cells were incubated in dmem containing % fbs for h, and then washed three times with cold pbsa and incubated with : -diluted streptavidin-fluorescein isothiocyanate (fitc) for h on ice. subsequently, cells were washed once with pbs and resuspended in cold pbs containing % paraformaldehyde and fixed for min at room temperature. after removal of paraformaldehyde, cells were washed twice with pbs containing % fbs, and the biotinylated virions were visualized on a confocal laser scanning microscope (lsm meta; carl zeiss, gottingen, germany). student's t-test was used to evaluate differences in results and p< . was considered statistically significant. to investigate the effect of mβcd on the cholesterol level of marc- cells, marc- cells were mock-treated or treated with various concentrations of mβcd. as shown in figure a , mβcd treatment significantly reduced cellular cholesterol levels in a dose-dependent manner. when the concentration of mβcd was up to mmol l  , cellular cholesterol level was reduced by approximately % compared with that in mock-treated cells. as the concentration of mβcd was further increased (> mmol l  ), cellular cholesterol level was no longer reduced, indicating that the remaining % cholesterol represented threshold levels of cholesterol which could not be extracted by higher doses of mβcd. after confirming the effect of mβcd on marc- cellular cholesterol, analyses were performed to exclude the possibility that mβcd causes cell toxicity. to this end, cell viabilities were detected after treatment with various concentration of mβcd by mtt. as shown in figure b , mβcd had little cytotoxicity on marc- cells when the concentration of mβcd was < mmol l  ( figure b) . accordingly, mmol l  mβcd was used for all the subsequent experiments. it is known that the plasma membrane lipid composition plays a key role in the infection by many enveloped viruses [  ]. to investigate the role of plasma membrane cholesterol in the infection of prrsv, marc- cells were mock-treated or treated with various concentrations of mβcd and infected with prrsv strain ch- a at . tcid per well. after incubation for h, cell suspensions were harvested and virus production was determined by tcid assay on marc- cells. as shown in figure a , mβcd treatment inhibited the infection of prrsv strain ch- a in a dose-dependent manner, suggesting that cholesterol is necessary for prrsv replication in marc- cells. moreover, treatment with mmol l  mβcd efficiently reduced virus titer to . tcid ml  compared with that of . tcid ml  in mock-treated cells. to further elucidate the effects of mβcd on the replication cycle of prrsv, mock-treated and mβcd-treated cells were infected with prrsv strain ch- a at . tcid per well. cell suspensions were harvested at various times post-infection (p.i.) and the titers of virus production in this cell suspension were determined. as shown in figure b , virus reproduction was significantly inhibited in mβcdtreated cells, and virus titers at all stages of the replication cycle were notably reduced compared with those in mock-treated cells. specifically, at h p.i., virus titers in mβcd-treated cells were . × -fold lower than that in mock-treated cells. we also investigated the role of plasma membrane cholesterol in the infection of prrsv strain wuh , a highly pathogenic prrsv that has been emerging in china in recent years. significant inhibitory effects were observed ( figure c) , indicating that the inhibitory effects of plasma membrane cholesterol on prrsv replication are not strain-specific. if the inhibition of prrsv replication was specifically due to the cholesterol depletion by mβcd, then cholesterol replenishment in mβcd-treated cells should restore prrsv replication. to clarify this hypothesis, mβcd-treated cells were incubated with dmem containing water-soluble cho- lesterol and then cholesterol levels were measured. as shown in figure d , the cellular cholesterol level of mβcd-cholesterol-treated cells was partially restored to % of mock-treated cell cholesterol levels. subsequently, virus yields from the infected mβcd-cholesterol-treated cells were assayed and compared with that from mβcd-treated cells. the titer of prrsv from the infected mβcd-cholesterol-treated cells was increased to approximately . × -fold of that from mβcd-treated cells ( figure e ). these results definitively demonstrated that decreasing cellular cholesterol levels resulted in the reduced ability of prrsv replication and with the replenishment of cellular cholesterol, virus replication was restored to a certain extent. to further illuminate at which step(s) cholesterol depletion reduces prrsv replication, we first investigated the effect of cholesterol depletion on virus entry. mock-treated, mβcd-treated and mβcd-cholesterol-treated marc- cells in -well plates were infected with prrsv at . tcid per well. after incubation for h, cells were harvested and intracellular prrsv rna levels were measured by real-time rt-pcr. as shown in figure a , compared with the number of viral genomes in mock-treated cells, a significant decrease ( %) in viral genomes in mβcdtreated cells was detected. furthermore, cholesterol replenishment partially restored intracellular viral genomes and there was only a % reduction after adding exogenous cholesterol. these results suggest that entry of prrsv is dependent on the cellular membrane cholesterol. the entry phases of prrsv infection can be divided into two stages: the first stage requires prrsv attachment to cells, and the second stage occurs when prrsv penetrates the cellular membrane. to determine whether mβcd reduces prrsv attachment to marc- cells, mocktreated, mβcd-treated or mβcd-cholesterol-treated marc- cells were infected with prrsv at °c for h. all cells were collected and virus genomes were detected by quantitative real-time pcr assay. as shown in figure b , virus genomes presented in mβcd-treated cells were only . % of that presented in mock-treated cells. furthermore, cholesterol replenishment partially restored virus attachment to approximately % of which presented in mocktreated cells, suggesting that the effect was specifically due to the cholesterol depletion ( figure b ). the effect of cholesterol depletion on virus attachment was defined, but next we determined whether penetration of prrsv into marc- cell was also affected by cholesterol depletion. to this end, marc- cells in -well plates were infected with μg biotinylated-prrsv per well and were mock-treated or treated with mβcd or mβcd-cholesterol. during the treatment, virions attached on these cells completed the penetration process and were detected by confocal microscopy. no significant differences were observed between various groups (data not shown), suggesting that mβcd treatment has little effect on the second stage of virus entry. altogether, these data indicated that cellular membrane cholesterol was required for prrsv entry into marc- cells, and its depletion by mβcd mainly affects virus attachment, rather than penetration. to investigate the effect of mβcd on virus release from marc- cells, cells were infected with prrsv strain ch- a at . tcid per well. at h p.i., cells were left untreated or treated with mβcd or mβcd-cholesterol. at the indicated time points post-treatment, the supernatants were harvested and the virus was detected by real-time rt-pcr. the data indicated that the number of viral genomes present in mβcd-treated cells was notably higher than those in mock-treated cells at each time point, and this increase was partially neutralized by cholesterol replenishment ( figure a ). this suggests that the ability of virus release from marc- is inversely related to cellular cholesterol level. to further determine the effect of cholesterol depletion figure b , the amount of prrsv genomes expressed in cells infected with released virus from mβcd-treated cells was approximately . % of that in cells infected with released virus from mock-treated cells, and cholesterol replenishment partially counteracted this effect. these findings indicate that the infectivity of virus released from mβcd-treated cells was decreased compared with those released from mβcd-cholesterol-treated and mock-treated cells. according to these data, we can conclude that mβcd treatment caused an enhancement of virus particle released from infected cells but the infectivity of released viruses was decreased by this treatment. many studies have reported that cholesterol is involved in the replication of some enveloped virus, such as herpes simplex virus [ ] , vaccinia virus [ ] , murine coronavirus [ ] , sars-cov [ ] , varicella-zoster virus [ ] , and pseudorabies virus [ ] . the present study showed that the inhibition of prrsv infection in vitro occurred as host cell membrane cholesterol decreased due to mβcd treatment. this effect could be counteracted by the addition of exogenous cholesterol and demonstrated the importance of membrane cholesterol for prrsv infection. being an essential component of cell membrane, cholesterol could potentially inhibit virus entry by several different mechanisms. first, cholesterol may affect virus entry by modifying the interaction of the virus particle with host cell membrane. cholesterol level is important for maintaining biological membrane fluidity [ ] , and its removal could reduce lateral diffusion within the membrane. this reduction in fluidity could probably influence entry of prrsv within the membrane. second, the lipid environment, including cholesterol level, is known to affect the charge properties of some ion channels [ ] , and the ion channels formed on cellular membrane require the presence of cholesterol for effective cytoplasmic delivery of the viral genome during virus entry. cholesterol and sphingolipids are components of lipid rafts, and lipid recognition by certain proteins of viruses might be imperative for virus entry [ ] . moreover, there are also other studies indicating that cholesterol removal resulted in an inhibition of cellular signaling pathways [ ] . a previous study demonstrated that the entry of equine arteritis virus, another member of arteriviridae, could also be inhibited by cholesterol depletion, and virus entry requires plasma membrane cholesterol [ ] . in the current study, we examined the effect of plasma membrane cholesterol on prrsv attachment and penetration, the two stages of virus entry. we found that cholesterol mainly affects virus attachment, rather than penetration. it was reported that prrsv enters porcine alveolar macrophages (pams) via receptor-mediated endocytic vesicles and the low ph-dependent endocytic pathway [ ] . thus, it is possible that the effect of plasma membrane cholesterol on prrsv entry into marc- cell is also due to its influence on the endocytosis process. a significant finding of the present study is that cholesterol depletion facilitated the release process of prrsv but also resulted in decreased infectivity of released virus particles. this finding is consistent with reports showing that cholesterol depletion caused an enhancement of influenza a virus particle release from infected cells and a decreased infectivity of virus particles [ ] . in addition, the infectivity of newcastle disease virus could be reduced by cholesterol depletion and possibly due to virus particles released from cholesterol-depleted cells having heterogeneous densities and structural abnormalities [ ] . teissier and pecheur [ ] had demonstrated that the molecular shape of lipids, including cholesterol and sphingolipid, could affect membrane curvature and further influence membrane defor-mation and fluidity. thus, structural abnormalities of virus particles due to cholesterol depletion and the physical function of cholesterol in the plasma membrane may be responsible for the findings reported herein. in conclusion, the current study demonstrated that both efficient entry of prrsv into marc- cells and the release of prrsv are dependent on the presence of plasma membrane cholesterol. these data suggest that further investigation of the prevention and control of prrsv, in particular, prevention of viral entry, is warranted. work was supported by the national natural science foundation of china (grant no. ) and national basic research program of china (grant no porcine reproductive and respiratory syndrome the challenge of prrs immunology lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses characterisation of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at lelystad cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism the porcine reproductive and respiratory syndrome virus requires trafficking through cd -posotive early endosomes, but not late endosomes, for productive infection entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin porcine reproductive and respiratory syndrome virus entry into the porcine macrophage defining the cellular target(s) of porcine reproductive and respiratory syndrome virus blocking monoclonal antibody g role of cd , a tetraspanin, in porcine reproductive and respiratory syndrome virus infection role of cholesterol in fusion of semliki forest virus with membranes membrane fusion of semliki forest virus requires sphingolipids in the target membrane lipid raft microdomains: a gateway for compartmentalized trafficking of ebola and marburg viruses blocking of hiv- infection by targeting cd to nonraft membrane domains functional rafts in cell membranes virus entry, assembly, budding, and membrane rafts role of cholesterol in human immunodeficiency virus type envelope protein-mediated fusion with host cells cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry requirements for ceacams and cholesterol during murine coronavirus cell entry vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts specific association of glycoprotein b with lipid rafts during herpes simplex virus entry productive entry of type c foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle lipid raft disruption by cholesterol depletion enhances influenza a virus budding from mdck cells equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus gp protein encoded by a synthetic orf gene human immunodeficiency virus type uses lipid raft-colocalized cd and chemokine receptors for productive entry into cd + t cells quantitative taqman rt-pcr for the detection and differentiation of european and north american strains of porcine reproductive and respiratory syndrome virus cholesterol dependence of varicella-zoster virion entry into target cells plasma membrane cholesterol is required for efficient pseudorabies virus entry regulation of receptor function by cholesterol attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept lipids as modulators of membrane fusion mediated by viral fusion proteins signal transduction via glycosyl phosphatidylinositol-anchored proteins in t cells is inhibited by lowering cellular cholesterol integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious newcastle disease virus particles open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited key: cord- - vis bwi authors: cui, junru; o’connell, caitlin m.; hagen, connor; sawicki, kim; smyth, joan a.; verardi, paulo h.; van kruiningen, herbert j.; garmendia, antonio e. title: broad protection of pigs against heterologous prrsv strains by a gp -mosaic dna vaccine prime/gp -mosaic rvaccinia (vacv) vaccine boost date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: vis bwi background: porcine reproductive and respiratory syndrome (prrs) viruses are a major cause of disease and economic loss in pigs worldwide. high genetic diversity among prrsv strains is problematic for successful disease control by vaccination. mosaic dna and vaccinia (vacv) vaccines were developed in order to improve protection against heterologous prrsv strains. methods: piglets were primed and boosted with gp -mosaic dna vaccine and recombinant gp -mosaic vacv (rgp -mosaic vacv), respectively. pigs vaccinated with rgp -wt (vr ) dna and rgp -wt vacv, or empty vector dna and empty vacv respectively, served as controls. virus challenge was given to separate groups of vaccinated pigs with vr or mn c. necropsies were performed days after challenge. results: vaccination with the gp -mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both vr and mn c prrsv strains. in contrast, vaccination of animals with the gp -wt vaccines induced responses only to vr . furthermore, vaccination with the gp -mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (pams), and bronchoalveolar lavage (bal) fluids, and less severe lung lesions after challenge with either mn c or vr , which have only % identity. in contrast, significant protection by the gp -wt based vaccines was only achieved against the vr strain. conclusions: gp -mosaic vaccines, using a dna-prime/vacv boost regimen, conferred protection in pigs against heterologous viruses. porcine reproductive and respiratory syndrome (prrs) is a major disease in pigs that causes significant economic losses to industry across the world. in the united states alone, prrs causes over $ million in losses a year [ ] . prrs causes reproductive failure in pregnant sows and respiratory disease in young pigs. the causal virus, prrsv, is a positive-sense, single-stranded rna virus with a lipid envelope, and belongs to the family arteriviridae, order nidovirales [ ] [ ] [ ] . the genome is approximately kb long and encodes at least different viral proteins including fourteen non-structural and eight structural proteins [ ] . prrsv induces both humoral and cellular immune responses in pigs and some of the viral proteins inducing such responses have been identified; however, protection against reinfection is incomplete [ ] [ ] [ ] [ ] . killed virus (kv) vaccines and modified live virus (mlv) vaccines for prrs have been licensed for more than two decades. these vaccines generally reduce the severity of clinical signs and the transmission of virus; however, they do not induce sterilizing immunity. in general, the efficacy of mlv vaccines is superior to that of kv vaccines [ ] [ ] [ ] [ ] . importantly, however, highly variable and generally sub-optimal levels of protection against heterologous prrsv strains have been reported [ , [ ] [ ] [ ] [ ] [ ] . the potential for reversion to the virulence of mlv vaccines and subsequent transmission to susceptible pigs is a concern [ ] [ ] [ ] . the major challenge for the development of efficacious, broadly protective prrs vaccines is the extraordinary genetic variation among disease producing prrsv strains. currently, two major prrsv genotypes are recognized, genotype (european) and genotype (north american), which exhibit nearly % sequence dissimilarity [ , ] . genotype and genotype prrsvs can be further divided into four subgroups and nine different lineages, respectively, based on phylogenetic analysis of orf [ ] . the co-existence of multiple variants within one farm, or even within individual pigs, possibly indicates the occurrence of quasispecies variation of prrsv [ ] . thus, the development of vaccines that can induce protection against different subgroups and lineages is highly desirable. multi-subunit vaccines [ ] , consensus vaccines [ ] , molecular breeding of different viral structural proteins [ ] , mosaic t-cell vaccines [ , ] , a polyvalent vaccine containing five different live-attenuated prrsv strains [ ] and dna prime-mlv boost [ ] are just some of the vaccines that are or have been under evaluation recently. studies on potential vaccines against human immune deficiency virus type (hiv- ), have shown that mosaic sequences designed from naturally occurring hiv sequences could effectively address genetic diversity when used as vaccines [ , ] . mosaic vaccines have been shown to elicit broader immune responses successfully and to confer cross protection in non-human primates and these mosaic hiv vaccines have been trialed in humans [ ] [ ] [ ] [ ] [ ] . we previously reported that a prrsv mosaic vaccine based on gp sequences of the genotype prrsv strain was immunogenic [ ] and induced reactivity to four divergent genotype- prrsv strains that had at least % or more difference in their gp sequence-as shown by the expression of interferon gamma (ifn-γ) by peripheral blood mononuclear cells-and, further, that vaccinated pigs were protected against challenge with vr [ ] . the present study demonstrates further that vaccination of pigs with the gp -mosaic by using a dna prime and rvaccinia boost approach protects them against strains that are more than % divergent in their sequences (vr and mn c). the viruses used in the study included the vr na reference strain (atcc vr- ) and the mn c strain provided kindly by drs. kelly lager and kay faaberg at u.s. department of agriculture-agriculture research service (usda-ars). the viruses were propagated in marc- cells [ ] grown in dulbecco's modified eagle's medium (dmem) containing mm l-glutamine, u penicillin/ml, µg streptomycin/ml and % fetal bovine serum (fbs). the reed-muench formula was used to calculate virus titers [ ] . viruses were purified over continuous cesium chloride gradients, quantified using a nanodrop spectrophotometer (thermo scientific, waltham, ma, usa) and stored at − • c for later use. titrated viruses were used for ex vivo recall immune response assay and challenge experiments. a combination of gene synthesis (dna . , menlo park, ca, usa) and standard sub-cloning was utilized to generate three transfer vectors. each transfer vector contained a cassette with the tetr gene (based on genbank: x ), and either a natural or mosaic version of genotype prrsv orf [ , ] , followed by the emcv ires (based on genbank: nc_ . ) and the egfp gene (based on plasmid pegfp- , genbank: u ). the tetr gene and the orf -ires-egfp genetic segment were placed under back-to-back synthetic vacv early/late promoters [ ] . the teto operator sequence was inserted after the putative vacv d r promoter, as described in [ ] . the cassettes were flanked by bp of the vacv d r gene to the left and bp of the vacv d r gene to the right (based on genbank: nc_ . ). restriction endonuclease analysis was used to confirm plasmid identity. standard homologous recombination was used to generate tetracycline-inducible recombinant vacvs after the transfection of transfer vectors with fugene hd transfection reagent (promega, madison, wi, usa) into bs-c- cells, infected h before with an iptg-inducible vacv strain (based on the wr clone . . . .) in the presence of . mm iptg and µg/ml doxycycline. recombinant egfp-positive tetracycline-inducible vacvs were plaque purified in the absence of isopropyl βd- -thiogalactopyranoside (iptg) and in the presence of µg/ml doxycycline. the elimination of the parental virus was confirmed by pcr analysis of viral dna that was purified using a nucleospin ® blood kit (macherey-nagel, düren, germany). high-titer stocks were generated by infecting hela s cells with recombinant virus at a multiplicity of infection (moi)of . in the presence of µg/ml doxycycline. infected cells were harvested and homogenized days post-infection. homogenates were clarified by centrifugation at × g for min, purified over a sucrose cushion [ ] , and resuspended in x pbs ph . (with no doxycycline). mega (molecular evolutionary genetics analysis) (www.megasoftware.net) (nih, bethesda, md, usa) was utilized to evaluate protein sequence alignments between gp sequences of vr , mn c, mosaic and mosaic and to create a phylogenetic tree. cross-bred three-to four-week-old male and female piglets, which were free of prrsv and porcine circovirus- , were used in this study. the experimental design is summarized in table . gp -mosaic, gp -wt and vector-control vaccines were administered by both intradermal and intramuscular injection. briefly, ml vaccine containing µg dna and µg quil-a ® as adjuvant was injected intradermally ( . ml) on the back of ear and intramuscularly ( . ml) in the neck region at day and again on day . the animals were further boosted at day with a ml suspension containing pfu vacv expressing gp -mosaic, gp -wt or vacv and µg quil-a ® as adjuvant. pigs were challenged at day with vr and at day with mn c to their respective groups. a total of tcid of each virus was administered by intranasal and intramuscular challenge to each animal. blood was collected at days , , , , , the challenge day, and , , , and days after challenge. the pigs were euthanized at days post-challenge. the lungs were evaluated macroscopically, weighed, and bronchoalveolar lavage (bal) was then performed. tissue samples were collected from each lung lobe, tracheobronchial lymph nodes (tbln), spleen, inguinal lymph nodes (iln) and tonsils: portions of each were either fixed in % neutral buffered formalin or kept frozen. fixed tissues were routinely processed to paraffin, and - µm sections were stained with hematoxylin and eosin and evaluated histologically. lung lesion scores were calculated as reported before [ ] . all animal work was performed under a protocol approved by the university of connecticut institutional animal care and use committee, the animal protocol number under which the study was conducted is iacuc a - . peripheral blood mononuclear cells harvested from blood collected at days and on the challenge day, were seeded in -well flat-bottom plates ( × cells/well). they were then stimulated, in duplicate, by adding tcid vr or mn c/well, or mock treated for h at • c in a % co atmosphere. the cells were then collected and total rna was extracted for quantitative real-time pcr analysis. virus neutralization testing was carried out as previously reported [ ] . briefly, each serum was mixed with equal volumes of dmem containing tcid of vr or mn c and the mixtures were incubated at • c for h. the serum-virus mixtures were then added to -well plates containing - % confluent marc- cells and incubated for h at • c in a % co atmosphere (final serum dilution : ). the vr or mn c virus, negative serum, and uninfected cells served as the virus control, negative serum control and cell control, respectively. the neutralizing capacity of serum was quantified by measuring the viral copy numbers by rt-qpcr in supernatants h after the addition of pre-incubated serum-virus mixtures to the cells. trizol ls reagent or trizol reagent (invitrogen, grand island, ny, usa) was used to extract total rna from serum/ culture supernatants or tissues, respectively. the extracted rna was quantified in a nanodrop spectrophotometer (thermo scientific, waltham, ma, usa). the cdna was synthesized from each sample rna using a µl reaction mixture and random primers (invitrogen, grand island, ny, usa) and the following reaction conditions: • c for min, • c for min and • c for min. the cdna was then amplified by sybr green real-time pcr. to this effect, sybr qpcr master mix (bimake, huston, tx, usa), the cdna template and -atg atg rcc tgg cat tct- and -aca cgg tcg ccc taa ttg- were utilized as the forward and reverse primers for orf , respectively. real time pcr was then performed as follows: min at • c, followed by cycles at • c for s and • c for min using bio-rad cfx touch system (bio-rad, hercules, ca, usa). for use in quantification, a standard curve was generated using serial dilutions of viral rna which contained - copies/µl. both positive and negative reference samples were run concurrently with the test samples. viral loads were determined by plotting the ct values against the standard curve. melting curves were analyzed to verify the specificity of the pcr. to test for ifn-γ expression, total rna was extracted by trizol reagent from virus-stimulated or mock-treated pbmcs; this was then used as template for cdna synthesis and this, in turn, was used for real-time pcr following the protocol described above. glyceraldehyde -phosphate dehydrogenase (gapdh) (forward primer: -cgt ccc tga gac acg atg gt- and reverse primer: -ccc gat gcg gcc aaa t- ) was used as internal control to calculate the fold changes in the expression of ifn-γ (forward primer: -tgg tag ctc tgg gaa act gaa tg- and reverse primer: -ggc ttt gcg ctg gat ctg- ) by the delta-delta method [ ] . the scoring of lung lesions was done systematically, as previously described [ ] , by a board-certified veterinary pathologist, blinded to the treatment groups. nine sections, representing all six lung lobes, were scored, with one section from each division of the left cranial lung lobe and two sections from each diaphragmatic lobe. two-way anova or student's t-test was used to evaluate the differences in measurements between the samples within or between groups. the data were analyzed using graphpad prism (version . ) (graphpad software, san diego, ca, usa). the gp amino acid (aa) sequences in mosaic , mosaic , mn c and vr (the prototype of genotype prrsv) were all the same size, with no deletions or insertions. sequence alignments showed aa identity from % to %. to further investigate the antigenic relationship of these strains, a phylogenetic tree was constructed using the neighbor-joining method b. the four sequences clustered into two subgroups ( figure ), with the two most distant being mosaic and mn c. mosaic and mosaic were, relatively, more closely related to vr and mn c, respectively. gp -mosaic-vaccinated pigs had significantly higher levels of neutralizing antibodies in their serum compared to the vector-control animals, both to vr (p < . ) and mn c (p < . ) the virus neutralizing capability of serum from gp -wt-vaccinated animals was greater against vr (p < . ) and against mn c (p > . ) when compared to serum from vector control animals ( figure a) ; vaccination with the gp -mosaic vaccine resulted in broader recall cellular responses than those induced with the gp wt vaccine. thus, a significantly higher (p < . ) relative fold-change in ifnγ mrna expression was detected upon stimulation of pbmcs derived from gp -mosaic-vaccinated pigs, with either vr or mn c strains, when compared to those in equally stimulated pmbcs from vector control pigs ( figure b ). in contrast, the expression of ifn-γ mrna in gp -wtvaccinated pigs was significantly higher only if their pbmcs were stimulated with vr , when compared to those in the equally stimulated pbmcs of the control pigs at the same time points ( figure b ). no changes were detected with the pbmcs of vector-control pigs with any type of stimulation. gp -mosaic-vaccinated pigs had significantly higher levels of neutralizing antibodies in their serum compared to the vector-control animals, both to vr (p < . ) and mn c (p < . ) the virus neutralizing capability of serum from gp -wt-vaccinated animals was greater against vr (p < . ) and against mn c (p > . ) when compared to serum from vector control animals ( figure a) ; vaccination with the gp -mosaic vaccine resulted in broader recall cellular responses than those induced with the gp wt vaccine. thus, a significantly higher (p < . ) relative fold-change in ifn-γ mrna expression was detected upon stimulation of pbmcs derived from gp -mosaic-vaccinated pigs, with either vr or mn c strains, when compared to those in equally stimulated pmbcs from vector control pigs ( figure b ). in contrast, the expression of ifn-γ mrna in gp -wt-vaccinated pigs was significantly higher only if their pbmcs were stimulated with vaccines , , of vr , when compared to those in the equally stimulated pbmcs of the control pigs at the same time points ( figure b ). no changes were detected with the pbmcs of vector-control pigs with any type of stimulation. virus neutralizing capability of serum from gp -wt-vaccinated animals was greater against vr (p < . ) and against mn c (p > . ) when compared to serum from vector control animals ( figure a) ; vaccination with the gp -mosaic vaccine resulted in broader recall cellular responses than those induced with the gp wt vaccine. thus, a significantly higher (p < . ) relative fold-change in ifnγ mrna expression was detected upon stimulation of pbmcs derived from gp -mosaic-vaccinated pigs, with either vr or mn c strains, when compared to those in equally stimulated pmbcs from vector control pigs ( figure b ). in contrast, the expression of ifn-γ mrna in gp -wtvaccinated pigs was significantly higher only if their pbmcs were stimulated with vr , when compared to those in the equally stimulated pbmcs of the control pigs at the same time points ( figure b ). no changes were detected with the pbmcs of vector-control pigs with any type of stimulation. the expression of ifn-γ mrna as fold changes, by either vr or mn c-stimulated pbmcs collected on the challenge day. each dot represents the value of one animal. the variation is expressed as standard error of the means. there were three independent replications. significant differences were calculated by a two-way anova or student's t test (* p < . , *** p < . ). viral loads in serum of both gp -wt and gp -mosaic-vaccinated groups were significantly lower than those in the vector-control group at , and days after challenge with vr (* p < . , ** p < . ) ( figure a) . therefore, the capacity of reducing vr virus loads in serum was relatively similar between the gp -mosaic and the gp -wt vaccines. furthermore, there was a steady decrease in viral loads from days to days after challenge in both gp -wt and gp -mosaic vaccinated groups, while viral loads increased during the course of challenge and reached their peak at days post-challenge (dpc), then declining slightly at dpc in the vector-control group ( figure a ). in contrast, the viral loads in serum of animals receiving the gp -mosaic-vaccine were significantly lower than those of animals receiving the gp -wt vaccine or those of vector-control animals at and dpc with mn c (* p < . ) ( figure b ), which was different compared to vr -challenged groups. viral load levels in gp -wt vaccinated animals, increased during the infection with mn c and reached their peak at dpc, which is comparable to the vector-control group. the viral loads in gp -mosaic vaccinated animals decreased steadily from to dpc and remained unchanged until dpc ( figure b) . furthermore, the viral loads in lung, tbln, spleen, iln and tonsils were significantly lower in gp -mosaic-vaccinated animals than those in the vector-control group (* p < . ) after challenge with mn c, while those of the gp -wt group were similar or slightly lower compared to the vector-control group ( figure c ). viral loads in the lungs, tbln, and tonsil were significantly lower in gp -mosaic-vaccinated animals than those in the vector-control group (* p < . ) after challenge with vr , while those of the gp -wt group were significantly lower in the lungs, spleen and iln compared to the vector-control group (* p < . ). there was no significant difference in vr virus loads between the gp -mosaic and gp -wt groups ( figure d ). control group (*p < . ) after challenge with mn c, while those of the gp -wt group were similar or slightly lower compared to the vector-control group ( figure c ). viral loads in the lungs, tbln, and tonsil were significantly lower in gp -mosaic-vaccinated animals than those in the vector-control group (*p < . ) after challenge with vr , while those of the gp -wt group were significantly lower in the lungs, spleen and iln compared to the vector-control group (*p < . ). there was no significant difference in vr virus loads between the gp -mosaic and gp -wt groups ( figure d ). tests in pams and bal fluids showed a similar pattern in which the viral loads in both the gp -mosaic and gp -wt groups were lower than those in the vector-control group after challenge with vr ( figure a,c) , meanwhile only the viral loads of gp -mosaic-vaccinated animals were significantly lower than those in the vector-control animals after challenge with mn c (*p < . tests in pams and bal fluids showed a similar pattern in which the viral loads in both the gp -mosaic and gp -wt groups were lower than those in the vector-control group after challenge with vr ( figure a,c) , meanwhile only the viral loads of gp -mosaic-vaccinated animals were significantly lower than those in the vector-control animals after challenge with mn c (* p < . and ** p < . , respectively), furthermore there was no apparent difference in mn c viral loads between gp -wt and vector-control animals ( figure b,d) . three independent experiments were performed for each. student's t test was used to calculate significant differences (*p < . , **p < . ). the lung lesion scores after challenge with mn c were significantly lower in gp -mosaicvaccinated animals than those in both gp -wt and vector-control animals when evaluated using nine sections of lung ( figure right panel, p < . ) . in contrast, after vr challenge, lung lesion scores were lower on average in both the gp -mosaic and gp -wt groups than those in vector- bars represent the standard error of the mean. three independent experiments were performed for each. significant differences were calculated by student's t test (* p < . ). (c) the viral copy numbers in pams at necropsy upon challenge with vr . (d) the viral copy numbers in pams at necropsy upon challenge with mn c. each dot represents the mean value of each animal. the bars represent the standard error of the mean. three independent experiments were performed for each. student's t test was used to calculate significant differences (* p < . , ** p < . ). the lung lesion scores after challenge with mn c were significantly lower in gp -mosaic-vaccinated animals than those in both gp -wt and vector-control animals when evaluated using nine sections of lung ( figure right panel, p < . ). in contrast, after vr challenge, lung lesion scores were lower on average in both the gp -mosaic and gp -wt groups than those in vector-control animals. there were no significant differences between the lung lesion scores of gp -wt and gp -mosaic-vaccinated groups after challenge with vr ( figure ). bars represent the standard error of the mean. three independent experiments were performed for each. significant differences were calculated by student's t test (*p < . ). (c) the viral copy numbers in pams at necropsy upon challenge with vr . (d) the viral copy numbers in pams at necropsy upon challenge with mn c. each dot represents the mean value of each animal. the bars represent the standard error of the mean. three independent experiments were performed for each. student's t test was used to calculate significant differences (*p < . , **p < . ). the lung lesion scores after challenge with mn c were significantly lower in gp -mosaicvaccinated animals than those in both gp -wt and vector-control animals when evaluated using nine sections of lung ( figure right panel, p < . ). in contrast, after vr challenge, lung lesion scores were lower on average in both the gp -mosaic and gp -wt groups than those in vectorcontrol animals. there were no significant differences between the lung lesion scores of gp -wt and gp -mosaic-vaccinated groups after challenge with vr ( figure ). there is extraordinary diversity among prrsv strains. the diversity among prrsv strains is as high as, or may even surpass that of, hiv. recently, several vaccine candidates, including a synthetic consensus prrsv strain [ ] , a chimeric prrsv strain containing multiple orfs dna shuffled [ ] [ ] [ ] and an intranasal live virus vaccine with adjuvant [ ] induced various levels of heterologous protection in pigs. we have developed gp -mosaic vaccines that incorporate sequences derived from naturally circulating viruses. the data obtained from our gp -mosaic vaccinated pigs supports the mosaic vaccine approach as an effective strategy to address prrsv diversity. it has also been demonstrated that mosaic t-cell vaccines have great potential for other viruses with extraordinary diversity, such as hiv [ ] [ ] [ ] [ ] [ ] [ ] . in an earlier study, we demonstrated the immunogenicity of gp -mosaic vaccines in swine and their ability to induce cross-reactivity, as shown by their broad recall responses ex-vivo, and conferred the partial protection they provide in pigs [ , ] . the ability of the gp -mosaic vaccines to induce cross-protection in pigs against heterologous prrsv strains was confirmed in the present study. a sequence analysis of two gp -mosaic vaccines and the two genotype prrsv strains utilized in the study, vr and mn c, that belong to lineage and to lineage , respectively, revealed a % difference in the gp aa sequences. the aa sequences were used to generate a phylogenetic tree using the neighbor-joining method [ ] . the gp -mosaic was closer to vr ( % aa identity), while gp -mosaic was closer to mn c ( % aa identity), which suggested that the two gp -mosaic sequences broaden the coverage to strains belonging to different lineages. based on this analysis, a broader protection could be expected with gp -mosaic vaccines. the data of the present study supports clearly the capacity of the gp -mosaic vaccine to cross-protect pigs against vr and mn c that share only % aa identity. vacv provides a very good platform for the development and testing of vaccines, including animal vaccines, human vaccines and cancer immunotherapy [ ] . in our study, a gp -mosaic dna vaccine prime/gp -mosaic rvaccinia (vacv) boost regime was used to immunize pigs. this type of prime-boost regime has previously been demonstrated to increase vaccination efficacy substantially [ , ] . in addition, quil-a ® , used as adjuvant for the priming of the gp -mosaic dna vaccine, was previously shown to further increase the immune responses [ ] [ ] [ ] . therefore, the prime-boost vaccination regime used here for testing gp -mosaic vaccine efficacy, in terms of broad protection, proved appropriate for testing the hypothesis. pbmcs collected on the challenge day from gp -mosaic-vaccinated pigs responded to a broader range of virus strains, as measured by ifn-γ mrna expression, than those from gp -wt vaccinates. thus, pbmc from gp -mosaic-vaccinated pigs expressed higher levels of ifn-γ mrna in response to stimulation with vr or mn c strains, respectively, compared to those of the vector-control pigs. in contrast, pbmc from gp -wt vaccinated pigs expressed higher levels of ifn-γ mrna only when stimulated with vr . these data were consistent with those of our previous study where the gp -mosaic vaccine induced broad recall cellular responses, as measured by significantly higher levels of ifn-γ mrna expression in response to the four diverse genotype prrsv strains tested, including vr , nadc , nadc and sdsu [ ] . the significant induction of ifn-γ is an important asset of a prrsv vaccine, as ifn-γ reportedly plays a critical role in the control of and protection from prrsv infection [ ] . in addition, both the gp -mosaic and the gp -wt vaccines induced relatively high levels of neutralizing antibodies in vr -challenged pigs. these data indicate that the gp -mosaic vaccine preserves virus-neutralizing epitopes that appear to contribute to the overall efficacy of this vaccine. in terms of vaccine-induced protection, both gp -mosaic and gp -wt-vaccinated pigs had significantly lower serum viral loads at , and dpc than the vector-control group after the vr challenge. in contrast, only the gp -mosaic-vaccinated group showed significantly lower viral loads in sera at and dpc than the vector-control group after the mn c challenge. these data demonstrate that the gp -mosaic vaccine is capable of cross-protecting pigs against two divergent prrsv strains ( % difference) while the gp -wt vaccine only provided protection in pigs against the homologous strain. furthermore, a similar pattern was found with respect to viral loads in tissues, where the gp -mosaic-vaccinated pigs had significantly lower viral loads in their lungs, tbln, spleen, iln and tonsils compared to the vector-control group after challenge with mn c, while the gp -wt group only showed slightly lower (numerically, but not significant) viral loads. in the vr challenge experiment, both the gp -mosaic and gp -wt vaccinates had significantly lower viral loads in tissues generally-the viral loads in the gp -mosaic vaccinates were significantly lower in their lungs, tbln and tonsils, while viral loads in the gp -wt group were significantly lower in their lungs, iln and spleen, compared to the vector-control group. similar protective effects were also demonstrated in the gp -mosaic vaccine vaccinates, where lower viral loads were detected in the bal fluids or pams after mn c challenge, further confirming the cross-protective capability of the gp -mosaic vaccine. indeed, this is finding may be particularly relevant since pams are the primary target of prrsv infection. lung lesion scoring is an essential and crucial method to evaluate vaccine efficacy, as infection by prrsv tends to increase susceptibility to coinfection with other virus or bacterial pathogens [ ] [ ] [ ] which may result in more severe lesions and mortality. pigs vaccinated with the gp -mosaic vaccine had significantly lower lung lesion scores than both the gp -wt and vector-control groups after challenge with mn c, while both the gp -mosaic and gp -wt groups had lower lung lesion scores compared to the vector-control group in the vr challenge experiment. taken altogether, our results clearly demonstrate that the gp -mosaic vaccine provided cross-protection against two heterologous prrsv strains, vr , the prototype genotype strain, and mn c, a highly virulent strain, while the gp -wt vaccine provided only homologous protection against vr . in summary, our results demonstrate that our gp -mosaic vaccine is able to induce broader protection against prrs and warrants further research for field applications. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus nidovirales: a new order comprising coronaviridae and arteriviridae porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system proteome-wide screening of the european porcine reproductive and respiratory syndrome virus reveals a broad range of t cell antigen reactivity location of t-cell epitopes in nonstructural proteins and of type-ii porcine reproductive and respiratory syndrome virus antibody response to porcine reproductive and respiratory syndrome virus (prrsv) nonstructural proteins and implications for diagnostic detection and differentiation of prrsv types i and ii characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge. veter microbiol prrsv: comparison of commercial vaccines in their ability to induce protection against current prrsv strains of high virulence failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv. veter rec comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges gucht svan pensaert, m. impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy efficacy of vaccination with porcine reproductive and respiratory syndrome virus following challenges with field isolates in japan vaccination of sows against type porcine reproductive and respiratory syndrome virus (prrsv) before artificial insemination protects against type prrsv challenge but does not protect against type prrsv challenge in late gestation comparison of two commercial type porcine reproductive and respiratory syndrome virus (prrsv) modified live vaccines against heterologous type and type prrsv challenge in growing pigs efficacy of an attenuated european subtype porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs upon challenge with the east european subtype prrsv strain lena appearance of acute prrs-like symptoms in sow herds after vaccination with a modified live prrs vaccine sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents genetic analysis of orf of recent korean porcine reproductive and respiratory syndrome viruses (prrsvs) in viremic sera collected from mlv-vaccinating or non-vaccinating farms phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection dna vaccines co-expressing gp and m proteins of porcine reproductive and respiratory syndrome virus (prrsv) display enhanced immunogenicity a synthetic porcine reproductive and respiratory syndrome virus strain confers unprecedented levels of heterologous protection dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain a gp mosaic t-cell vaccine for porcine reproductive and respiratory syndrome virus is immunogenic and confers partial protection to pigs a prrsv gp -mosaic vaccine: protection of pigs from challenge and ex vivo detection of ifnγ responses against several genotype strains comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductive and respiratory syndrome a dna prime immuno-potentiates a modified live vaccine against the porcine reproductive and respiratory syndrome virus but does not improve heterologous protection polyvalent vaccines for optimal coverage of potential t-cell epitopes in global hiv- variants web-based design and evaluation of t-cell vaccine candidates expanded breadth of the t-cell response to mosaic human immunodeficiency virus type envelope dna vaccination mosaic hiv- vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys mosaic vaccines elicit cd + t lymphocyte responses that confer enhanced immune coverage of diverse hiv strains in monkeys evaluation of a mosaic hiv- vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase / a clinical trial (approach) and in rhesus monkeys (nhp - ) enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line a simple method of estimating fifty percent endpoints compact, synthetic, vaccinia virus early/late promoter for protein expression antibiotic-dependent expression of early transcription factor subunits leads to stringent control of vaccinia virus replication preparation of cell cultures and vaccinia virus stocks analysis of relative gene expression data using real-time quantitative pcr and the −∆∆ct method comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of virus envelope genes from genetically divergent strains broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the gp or m genes chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant a vaccinia virus renaissance. hum. vaccines immunother heterologous prime-boost vaccination with dna and mva vaccines, expressing hiv- subtype c mosaic gag virus-like particles, is highly immunogenic in mice recombinant vaccinia vector-based vaccine (tiantan) boosting a novel hbv subunit vaccine induced more robust and lasting immunity in rhesus macaques immunization of rhesus macaques with echinococcus multilocularis recombinant - - antigen leads to specific antibody response functional analysis of bovine tlr and association with iga responses of cattle following systemic immunisation with h flagella characterization of the immune response and evaluation of the protective capacity of rssna against streptococcus suis infection in pigs increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study. veter microbiol the effect of infection order of porcine circovirus type and porcine reproductive and respiratory syndrome virus on dually infected swine alveolar macrophages the authors thank kay faaberg and kelly lager kindly providing the prrsv strains. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - tkedw authors: lee, changhee; yoo, dongwan title: the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: tkedw the small envelope (e) protein of porcine reproductive and respiratory syndrome virus (prrsv) is a hydrophobic amino acid protein encoded in the internal open reading frame (orf) of the bicistronic mrna . as a first step towards understanding the biological role of e protein during prrsv replication, e gene expression was blocked in a full-length infectious clone by mutating the atg translational initiation to gtg, such that the full-length mutant genomic clone was unable to synthesize the e protein. dna transfection of prrsv-susceptible cells with the e gene knocked-out genomic clone showed the absence of virus infectivity. p -Δe-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic rna, demonstrating that the e protein is essential for prrsv infection but dispensable for virion assembly. electron microscopy suggests that the p -Δe virions assembled in the absence of e had a similar appearance to the wild-type particles. strand-specific rt-pcr demonstrated that the e protein-negative, non-infectious p -Δe virus particles were able to enter cells but further steps of replication were interrupted. the entry of prrsv has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited prrsv replication effectively during the uncoating process. the expression of e protein in escherichia coli-mediated cell growth arrests and increased the membrane permeability. cross-linking experiments in cells infected with prrsv or transfected with e gene showed that the e protein was able to form homo-oligomers. taken together, our data suggest that the prrsv e protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm. porcine reproductive and respiratory syndrome virus (prrsv) has plagued the global swine industry leading to significant economic losses for pig production worldwide (nuemann et al., ) . prrsv is a member of the family arteriviridae, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv) of mice and simian hemorrhagic fever virus (shfv) (meulenberg et al., ; snijder and meulenberg, ) . along with the family coronaviridae, arteriviruses are grouped in the new order of nidovirales (snijder et al., ) . despite the similar virion morphology and genome organization, prrsv is divided into two genotypes, the european genotype (lelystad virus; lv) and the north american genotype, based on their antigenic and genetic characteristics (meng et al., ; nelsen et al., ; nelson et al., ; wootton et al., ) . prrsv is a small, enveloped virus possessing a singlestranded positive-sense rna of ∼ kb in size with a ′ cap and a ′ polyadenylated tail (meulenberg et al., ; sagripanti et al., ; snijder and meulenberg, ; wootton et al., ) . the prrsv genome consists of the ′ untranslated region (utr) , nine open reading frames (orf a, orf b, orf a, orf b and orfs through ) and the ′ utr (meulenberg et al., ; snijder and meulenberg, ; wootton et al., ) . two large orfs la and lb occupy the ′ two-thirds of the genome and encode non-structural proteins, which are suggested to be involved in the genome replication and transcription (bautista et virology ( ) - www.elsevier.com/locate/yviro al., ; van dinten et al., ) . the remaining orfs, a through in the ′ terminal kb region, encode six membraneassociated proteins (gp , e, gp , gp , gp and m) present in the envelope and nucleocapsid (n) protein (meulenberg et al., ; wootton et al., ) . mature virions are spherical, enveloped particles with a diameter of - nm and contain a -to -nm isometric core structure enclosing the genomic rna (benfield et al., ; dea et al., ) . the small envelope (e) protein is a newly identified structural component in arteriviruses. the prrsv e protein, also known as b protein, is translated from the internal orf (orf b) starting from the + nucleotide position in mrna (fig. a) . the e protein is and amino acids for the north american and european type of prrsv, respectively. the e protein is highly hydrophobic but contains a cluster of basic amino acids in the hydrophilic c-terminal region. the e protein is non-glycosylated and intracellular membrane-associated wu et al., ) . in prrsv-infected pigs, the e protein induces specific antibody (wu et al., ) . recent studies with a european prrsv isolate showed that the e protein is incorporated into the virions in association with gp -gp -gp heterotrimers, suggesting a critical role for the heteromultimeric complex in the virus entry process (wissink et al., ) . although the e protein of north american genotype prrsv contains two cysteine residues at positions and , a study has shown that e is unable to form disulfide-linked homodimers . in that study, cysteine residues of the e protein were shown to be non-essential for virus multiplication. the function and significance of e in prrsv replication remain to be determined. in the present study, we investigated the role of e protein during prrsv replication. an infectious cdna clone was used to generate an e gene-knockout mutant prrsv, and we report here that the e protein is essential for virus infectivity but dispensable for virus particle formation. the e protein-negative, non-infectious virus particles were able to enter cells but unable to continue the further steps of replication. furthermore, two ion channel blockers were shown to greatly affect prrsvreplication at early stages of infection, suggesting that ion channel activity was essential for virus uncoating. we also found that expression of the e protein enhanced membrane permeability of hygromycin b in bacterial cells. cross-linking studies revealed that the e protein associated with itself into higher-order structures, including dimers, trimers and tetramers. our study suggests that the prrsv e protein may function as a viroporin in the virion envelope that facilitates uncoating of the virus in order to release the genomic rna into the cytoplasm for subsequent replication. to determine the biological significance of e protein during prrsv infection, an e gene-knockout mutant virus was fig. . (a) the partial genome organization of prrsv. genomic locations of gp and e genes and the e gene-knockout are illustrated. (b) absence of infectivity of the e gene-knockout full-length clone for prrsv, p -Δe. marc- cells were transfected with the full-length cdna genomic clone of p -wt or p -Δe and incubated for days. prrsv-specific cpes were monitored daily and photographed days post-transfection (upper panels). for immunofluorescence, cells were fixed with cold methanol at days post-transfection and incubated with the n-specific mab sdow- (lower panels) (magnification ×). (c) double staining for n (green) and nsp / (red) proteins for p -wt (upper panels) or p -Δe (lower panels). marc- cells transfected with p -wt or p -Δe plasmid dna were fixed at days post-transfection and co-stained with nsp / -specific rabbit antiserum and n-specific mab sdow . yellow indicates merged images where both n and nsp / are co-localized. generated using an infectious cdna clone. the start codon of orf b (e gene) was modified to abolish the e protein expression. with the shuttle plasmid by site-directed mutagenesis, 'atg' for translation initiation of orf b was changed to 'gtg' at genomic positions , to , . this mutation did not alter the amino acid sequence in orf a encoding gp protein. the a g modification was subsequently introduced into the full-length genomic cdna clone by subcloning the eco iii-bsrgi fragment obtained from the shuttle plasmid. transformants were screened by restriction patterns using xmai to determine the ligated clone, followed by nucleotides sequencing to verify the specific modification in the full-length genome. the resulting e gene-knockout genomic clone was designated p -Δe. the infectivity of the e gene-knockout clone p -Δe was examined by transfection of marc- cells. cells were transfected with p -wt or p -Δe, and the appearance of cpe was monitored daily. p -wt induced visible cpe at days post-transfection and showed n-specific staining in clusters of cells, indicating the infection and spread of virus to neighboring cells (fig. b , left panels). in contrast, transfection of p -Δe did not produce any visible cpe for up to at least days post-transfection, suggesting the lack of infectivity. a few single cells exhibited n-specific fluorescence, and these cells represent individually transfected cells with p -Δe (fig. b) . to rule out a possibility that an additional mutation might have been introduced during construction of p -Δe, which may have resulted in the loss of infectivity, six individual mutant clones were independently generated. in these clones, no mutation was identified for the orf gene, and upon transfection of cells, all six p -Δe mutant clones were noninfectious (data not shown). this confirmed the conclusion that p -Δe was non-viable and the absence of infectivity was due to the lack of e protein expression, demonstrating the essential role of e for prrsv replication. the transcription ability of p -Δe was examined by double staining of transfected cells using the alexa greenlabeled sdow mab specific for n and the texas red-labeled rabbit antiserum specific for nsp / non-structural proteins (fig. c) . in cells transfected with p -wt dna, clusters of cells were stained by both antibodies, indicating both n (green) and nsp / (red) expressions (fig. c , upper panels). merging of the two images showed yellow regions where the two proteins colocalized. for p -Δe, dual-staining of n (green) and nsp / (red) was also observed but limited to transfected cells only, and no evidence for the spread of infection was obtained (fig. c , lower panels). the dual staining demonstrated the expression of nsp / and n proteins in transfected cells, indicating the synthesis of both non-structural and structural proteins, which in turn suggests that the prrsv genome replicated and mrna transcription occurred upon transfection of p -Δe dna. the results demonstrate that e protein expression is required for prrsv infectivity, but genome replication and transcription may occur without e protein. it leads us to hypothesize that either virion assembly or virus entry is interrupted in the absence of e protein expression. we next examined whether the infectivity of p -Δe could be restored by provision of the e protein in trans. the e gene was expressed in bhk-t cells and the expression was confirmed by immunofluorescence staining with e-specific antiserum ( fig. a) . bhk-t cells were also suitable for transfection of the full-length cdna clone as n-specific staining was detected in many cells (figs. b and e) . to determine whether p -Δe in the e gene-transfected cells leads to the production of infectious virus particles, bhk-t cells were co-transfected with p -Δe and e gene. the culture supernatant was harvested at h post-transfection from bhk-t cells and transferred to marc- cells. at h postinoculation, marc- cells were fixed, and virus infection was examined by immunofluorescent staining with n-specific mab. marc- cells inoculated with a culture fluid from cells cotransfected with p -Δe and e gene showed bright n-specific fluorescent signal, indicating that the p -Δe replication may be rescued by trans-complementation of the e protein (fig. c ). in contrast, in cells inoculated with the supernatant from co-transfection of p -Δe and n gene (fig. f) , or of p -Δe and an empty plasmid (data not shown), no staining was observed. the staining of marc- cells inoculated with the supernatant from bhk cells co-transfected with p -Δe and n gene was however limited to single cells and no infectivity was produced for up to days post-inoculation (fig. c) , indicating abortive infection of the p -Δe mutant virus in non-complementing marc- cells. attempts to pass the passage- culture supernatant to marc- resulted in no virus replication, confirming that the p -Δe virus particles generated by the provision of e in trans was only capable of a single round of replication. these data demonstrated that the inability of p -Δe to produce infectious virus was due to the absence of e protein in the virions. because p -Δe did not induce infectivity from transfection, it was of interest to examine whether prrsv particles were not produced in the absence of e protein. due to a low transfection efficiency in marc- cells, bhk- cells were used to achieve higher transfection efficiency. when stained with n-specific mab at days post-transfection, numerous cells showed n-specific fluorescence (data not shown), indicating high levels of transfection in bhk- cells, which may be sufficient for the study of particle formation. bhk- cells were transfected with p -wt or p -Δe, and at h post-transfection, the transfected cells were radiolabeled for day with [ s]methionine/cysteine. the cells were harvested and lysed with ripa buffer. the culture supernatants were collected separately and centrifuged through a % (wt/vol) sucrose cushion, and the resulting pellets were dissolved in ripa buffer. the lysates prepared from cells or supernatants were immunoprecipitated with a mixture of individual antibody specific for e, n, or m and resolved by sds-page. as shown in fig. a , intracellular major viral-specific proteins were detectable in all lysates (lower panel), confirming the ability of p -Δe for transcription. similarly, in the culture supernatants, the three major virion proteins gp , m and n were clearly identified (upper panel), showing that, without e protein, virus particles can be produced and released. it was not possible to detect the e protein in the cell lysates or supernatants from p -wt-transfected cells, and this is probably due to the low abundance of e protein in bhk- cells as e is a minor protein (wu et al., ) . this result suggests that the prrsv e protein is not required for particle formation. because the virus particles formed in the absence of e protein were non-infectious, these particles were examined for the presence of viral genome. the culture supernatants, prepared from bhk- cells transfected with p -wt or p -Δe, were first incubated with both rnase a and dnase i to remove any possible contamination of viral rna and the transfected dna from cells. the digestion was carried out in the presence or absence of detergents, followed by proteinase k treatment to inactivate the nucleases. rna was extracted from samples and treated again with dnase i, and the e gene region of prrsv was rt-pcr-amplified and sequenced. in cell culture media mixed with the full-length cdna clone as a negative control, no pcr fragment was amplified in the presence or absence of detergents (fig. b , lanes and ), indicating the appropriateness of dnase i treatment. although no amplification was identified for controls ( fig. b , lanes and ), a -bp product was specifically amplified for both samples of p -wt and p -Δe treated with the nucleases but in the absence of detergents (fig. b , lanes and ). sequencing of the -bp product confirmed the stable incorporation of the 'a g' mutation at the start codon of the e gene in the genome from p -Δe particles. these studies demonstrate that particles formed in the absence of e protein contained the p -Δe genomic rna, which in turn ]methionine/cysteine for h at °c, and the supernatants and cells were separately collected. cell lysates were prepared and subjected to immunoprecipitation with n-specific mab (lower panel). the pellets were prepared from culture supernatants by ultracentrifugation, lysed in ripa buffer and used for immunoprecipitation with a mixture of anti-m, anti-n and anti-e-specific antibodies, followed by sds- % page under reducing conditions (upper panel). lane , mock transfected; lane , p -wt transfected; lane , p -Δe transfected. (b) incorporation of genomic rna in p -Δe virus particles. culture supernatants were pelleted by ultracentrifugation and the pellets were treated with nucleases in the presence (+) or absence (−) of sds and triton x- , followed by proteinase k treatment. rt-pcr was conducted for e gene amplification followed by electrophoresis in . % agarose gel. lane , molecular weight marker; lanes and , culture supernatant spiked with the p -Δe full-length plasmid; lanes and , supernatant from p -wt-transfected cells; lanes and , culture supernatant from p -Δe-transfected cells. (c) electron microscopy of the culture supernatant from p -Δe-transfected cells. particles in the culture supernatants released from bhk- cells transfected with p -wt (upper panel) or p -Δe (lower panel) genomic clones were concentrated by ultracentrifugation through a % (wt/vol) sucrose cushion. pellets were negatively stained by sodium phosphotungstate and visualized by electron microscopy. scale bar, nm. suggests that the lack of infectivity of p -Δe was not due to the improper packaging of the genome. to obtain further evidence for particle formation in the absence of e, electron microscopy (em) was conducted and the microscopic appearance of p -Δe particles was compared to that of p -wt virions. at days post-transfection, the culture supernatant from bhk- cells was harvested and pelleted by ultracentrifugation through a % (wt/vol) sucrose cushion, followed by electron microscopy. prrsv particles were identified in the supernatant of cells transfected with either p -wt (fig. c , upper panel) or p -Δe (fig. c , lower panel). no significant morphologic differences between p -wt and p -Δe particles were noted. each virion for p -wt or p -Δe was a roughly spherical enveloped particle of - nm in diameter, with a densely stained core. the em study confirmed that prrsv particles may be formed in the absence of e protein and also showed that the e proteinnegative particles had similar appearance and size to wild-type prrsv particles. to determine if the lack of infectivity of p -Δe particles was due to a low amount of virus produced from bhk- cells, the supernatants collected from bhk- cells were blindly passaged twice in marc- cells to amplify the infectivity. although extensive cpe was readily evident in marc- cells inoculated with either passage- or passage- of p -wt supernatant, no cpe was detectable with passage- or passage- of p -Δe supernatant, even after days postinoculation (data not shown). to further determine infectivity in marc- cells inoculated with p -Δe virus, time course immunofluorescence was carried out with the n-specific antibody. as shown in fig. a , distinct staining of n was first observed at h post-infection in marc- cells inoculated with p -wt supernatant, and then in many clusters of cells by h post-infection, showing the spread of infectivity to neighboring cells (upper panels). in contrast, cells inoculated with passage- of p -Δe showed no specific staining throughout the experiments, further indicating the lack of infectivity (lower panels). because neither cpe nor n-specific staining was detectable from serial passages of p -Δe virus, rt-pcr was conducted from cells and culture supernatants inoculated with passage- or passage- (fig. b ). to eliminate a possible carry-over contamination of the transfected dna, the rna preparations were treated with rnase-free dnase i prior to the e gene amplification. a -bp product was amplified from both culture media and cells that were inoculated with passage- or passage- p -wt virus (fig. b, lanes , , and ). the -bp product was also obtainable from cells inoculated with passage- p -Δe virus (fig. b, lane ) , but no specific product was amplified from the supernatant (fig. b, lane ) , nor from either supernatants or cells inoculated with passage- p -Δe virus (fig. b, lanes and ) . the amplified fragment was sequenced, and the sequencing results confirmed the 'a g' mutation at the start codon of orf b (data not shown). these results suggest that the e protein-negative, noninfectious p -Δe virus particles entered marc- cells, but further steps beyond entry were interrupted, resulting in the absence of infectivity. to investigate whether the p -Δe virus genome underwent replication following entry, strand-specific rt-pcr was performed from cells inoculated with passage- p -Δe virus. rt-pcr from p -wt-inoculated cells yielded a specific product of the expected size for both positive-and negativestrand genomes and also for positive-and negative-strand mrna (fig. c , lanes in all panels). in contrast, only a minimal amount was amplified for the positive-strand genome from cells inoculated with passage- p -Δe virus (fig. c , panel i, lane ). this product was likely derived from the incoming genomic rna of p -Δe virus. these results indicate that p -Δe virus was unable to replicate, suggesting that the lack of rna replication of p -Δe virus was likely due to an interruption during virus uncoating, a stage prior to genome replication. the e-protein-negative prrsv particles were shown to contain the viral genome and able to enter cells but unable to release the viral rna for initiation of genome replication. this suggests that prrs virions may contain ion channels and the e protein may function as an ion channel protein embedded in the viral envelope. it is postulated that prrsv replication may be suppressed by ion channel blocking agents if ion channel activity is an essential requirement for prrsv infection. this aspect was investigated in marc- cells using amantadine and verapamil. amantadine is known as a proton channel blocker and verapamil is the calcium channel blocking agent. because it has been reported that an acidic environment is required for prrsv infection (kreutz and ackermann, ) , basic lysosomotropic agents including ammonium chloride and chloroquine were also included in the study to examine their inhibitory effects on prrsv replication. strand-specific rt-pcr experiments were carried out and demonstrated that prrsv-infected marc- cells produced reduced levels of positive-sense genomic rna at days post-infection in the presence of the drugs chloroquine, amantadine and verapamil (fig. a, upper panel) . a relatively moderate level of suppression was observed in ammonium chloride-treated cells (lane ). negative-sense genomic rna was not detectable in cells treated with any of the four drugs until days post-infection (middle and lower panels, lanes - ), showing that the initiation of viral rna synthesis was inhibited by the ion channel blockers. furthermore, the number of prrsv-infected cells determined by the n protein staining was significantly reduced by individual drugs in comparison to untreated cells (fig. b , upper panels). prrsv-specific cpe was visible but significantly delayed by the ion channel blockers, indicating the negative effects on virus production (lower panels). the yields of virus production were determined by plaque assays from the supernatants in the presence of each drug. the virus titer in untreated cells reached × pfu/ml by days post-infection (fig. c ). in contrast, fig. . the e protein is essential for prrsv replication. (a) immunofluorescence of n protein in p -Δe inoculated cells. the 'passage- ' virus was prepared as culture supernatant harvested from bhk- cells transfected with p -wt or p -Δe clone. marc- cells were inoculated with 'passage- ' and fixed at the indicated times post-inoculation, followed by staining with n-specific mab. (b) detection of viral rna in culture supernatants and in marc- cells inoculated with 'passage- ' or 'passage- ' virus. total rna was extracted and treated with dnase i followed by rt-pcr for e gene. (c) strand-specific detection of viral rna in cells by rt-pcr. marc- cells were inoculated with 'passage- ' p -wt or 'passage- ' p -Δe, and total cellular rna was extracted at days post-inoculation. the rna was treated by dnase i and rt-pcr was conducted to amplify the region as illustrated in the figure. numbers in parenthesis indicate the ′ most nucleotide position in each primer with respect to the prrsv genome. expected sizes of amplified products are indicated on the right of the gel. there was no detectable virus production at day post-infection in the presence of amantadine or verapamil, and the titers of virus production in the presence of these drugs reached only to a maximum of to × pfu/ml by days post-infection (fig. c) . these results showed that the treatment of infected cells with ion channel blockers greatly reduced the growth rate of prrsv. however, when marc- cells were treated with the drugs at min post-infection, no significant inhibitory effect of virus production was observed (data not shown). therefore, it seems that the ion channel blockers effectively interfered with virus uncoating, a step preceding genome replication and consequently affected prrsv production. it is interesting to note that the inhibitory effect by amantadine are due to the blocking of pores that are necessary for post-internalization during uncoating of the virus (wang et al., ) . because our data suggest that ion channel activity may be involved during uncoating and that the prrsv e protein is likely responsible for this event, the e protein was assumed to function as a viroporin. we examined whether the e protein possessed general features commonly shared by other viroporins (ion channel proteins). viroporins generally contain the properties of membrane permeability alteration and oligomerization (liao et al., ; maldarelli et al., ; paterson et al., ; sakaguchi et al., ; torres et al., ) , and so these two common properties were examined for the e protein. the effect of e expression on bacterial growth was first examined in an inducible protein expression system. when the inner bacterial membrane is intact, intracellular lysozymes cannot reach the cell wall. however, permeabilized membranes allow the lysozymes to gain access to the peptidoglycan, leading to cell lysis. this approach has been shown to be suitable as a permeabilization test of the inner bacterial membrane for viral proteins (bodelón et al., ; ciccaglione et al., ; liao et al., ; madan et al., ) . the prrsv e protein gene was first cloned in the gst-fusion vector and expressed in escherichia coli. the growth rates of transformed cells were analyzed spectrophotometrically. a drastic arrest of cell growth was observed in the e gene-transformed cells upon iptg induction, whereas bacteria carrying the empty plasmid or the prrsv n gene had no effects on their growth during the -min period following induction (fig. a ). this finding suggests that the expression of e affected the cell growth negatively by altering the membrane permeability to intracellular lysozyme. to further determine if the growth rate of cells expressing e was the consequence of permeability modification, a hygromycin b permeability assay was carried out. hygromycin b is normally impermeable to the membrane barrier during a short period of time at low concentration, but it can readily penetrate the permeabilized membrane to cause strong inhibition of intracellular protein synthesis. the hygromycin b permeability assay therefore is widely used to study changes of membrane permeability as well as to identify proteins that can form pores in lipid membranes (aldabe et al., ; arroyo et al., ; bodelón et al., ; chang et al., ; ciccaglione et al., ; de jong et al., ; doedens and kirkegaard, ; han and harty, ; liao et al., ; madan et al., ) . at h induction for e protein expression, hygromycin b was added to the culture media. the cultures were further incubated for min and metabolically labeled with [ s]methionine/cysteine for min, followed by sds-page of cell lysates. the coomassie blue staining showed that equal amounts of proteins were loaded on the gel (fig. b, left panel) . autoradiography indicated that hygromycin b entered cells that expressed the e protein, and protein synthesis was completely blocked in those cells (fig. b, right panel, compare lanes and , lanes and ) . for cells expressing the gst or prrsv n protein, hygromycin b did not inhibit protein synthesis (compare lanes and , lanes and ) . these results indicate that the prrsv e protein enhanced membrane permeability in bacterial cells. a second common property of viroporins is oligomerization. the prrsv e protein contains two well-conserved cysteine residues; however, the e protein was shown not to form a disulfide-linked homodimer . the absence of covalently linked homodimers of e was confirmed using the recombinant e protein expressed in hela cells by vtf - vaccinia virus (fig. a) . although prrsv n-n dimers of kda were readily detected in cells expressing the n protein (lane ), no band corresponding to the predicted dimeric form of e was identified in non-reducing conditions (lane ), concluding that the prrsv e protein does not undergo cysteine-linked homodimerization. because the e protein may form a non-covalent association with itself as shown by gst-pull down assay , the potential of e to form oligomers was further investigated by cross-linking experiments in the presence and absence of other viral constituents. radiolabeled prrsvinfected cells were treated with the membrane-permeable cross-linking reagent dsp, and the cell lysates were immunoprecipitated by n-specific mab or e-specific antiserum followed by sds-page under non-reducing conditions (fig. b) . the prrsv n protein formed a number of higher-order oligomers (lanes , ) , as reported previously (wootton and yoo, ) . when the e protein in virus-infected cells was subjected to cross-linking, numerous multimeric forms of e protein were identified (lane ). the e protein multimerization also was examined in the absence of other prrsv proteins in hela cells expressing e protein by t vaccinia virus, and again higher-order multimeric forms of the e protein were observed (lane ), indicating that oligomerization of e is independent of other viral proteins. altogether, our data indicate that the prrsv e protein exists as non-covalently linked oligomers in virus-infected cells and suggest that the multimerization may be the physical basis for viroporin formation of prrsv e. the present study was conducted to investigate the role of prrsv e protein during virus infection. a reverse genetics approach was used to modify the translation initiation of orf b, so that the modified genome was unable to express the e protein. our experiments show that the absence of e protein expression does not affect genome replication transcription but impairs the production of infectious virus. these data indicate that the prrsv e protein is an essential structural component for infectivity. the e protein however appears not to be an essential component for particle assembly nor genomic encapsidation. the virion particles devoid of e still contained the viral genome and had a similar appearance to that of wildtype virions. for infection, virus particles must proceed through a multiple-step cycle of entry and uncoating, replication and transcription and assembly and release. according to our findings, the e protein does not exert an important function at later events in the infection process such as genome replication, particle assembly and release of virus. we have observed that the e protein-negative, non-infectious virion particles are able to enter cells but subsequent steps of replication are inhibited. this suggests that the particles lacking the e protein most likely remain in the endosome and the viral genome is not released, which is the step between virus entry and genome replication. the uncoating is a critical step for virus infection, during which the lipid envelope is shed and the viral capsid is disassembled to release the genome to initiate a replication cycle in the cytoplasm. for viruses entering cells by receptor-mediated endocytosis, the uncoating process occurs in the endosome where an acidic environment triggers fusion between the viral membrane and the endosomal membrane (reviewed by smith and helenius, ) . prrsv enters cells through receptormediated microfilament-dependent endocytosis in which a low ph is required to trigger fusion for proper uncoating by an unknown mechanism (kreutz and ackermann, ; nauwynck et al., ) . it is possible that an acidic ph in the endosome may lead to conformational changes of viral protein (s) to expose hidden fusogenic domains that facilitate the fusion between viral and endosomal membranes. however, no direct fusion of prrsv with the cell membrane, and no arterivirus membrane proteins possessing the fusogenic property have been reported. it is therefore tempting to speculate that prrsv may contain a viral ion channel protein to promote the uncoating process in the endosome during the early stage of infection. in the present study, we have shown the inhibitory effect of ion channel blockers on prrsv replication, indicating that prrsv may indeed possess virus-coded ion channels whose activity is essential for proper uncoating for infection. virus-coded ion channels, or viroporins, consist of small, highly hydrophobic proteins generally composed of - amino acids. the insertion of these proteins into membranes creates typical hydrophilic pores or selective ion channels with (a) hydrophobic transmembrane domain(s) facing the lipid bilayer, leading to an alteration of membrane permeability to ions or other small molecules (for a review, see fischer and sansom, ; gonzalez and carrasco, ) . expression of these membrane-active proteins may cause cellular membrane leakiness further resulting in the development of cytopathic changes to facilitate particle release late in infection, or may be required to promote virus uncoating at an early stage of infection (reviewed by carrasco, ) . the prrsv e protein structurally resembles a number of viroporins found in other rna viruses, in that it consists of - amino acid residues and contains central hydrophobic sequences with a cluster of basic amino acids at the cterminus. the e protein localizes mainly in perinuclear regions including the er and the golgi complex and associates with intracellular membranes (wu et al., ) . interestingly, coronaviruses, another family member of the order nidovirales, also code for a small hydrophobic membrane, designated e protein, which may play a crucial role during virus morphogenesis (fischer et al., ; liu and inglis, ) . in recent studies, the coronavirus e protein has been shown to modify membrane permeability (liao et al., ; madan et al., ) as well as to form cation-selective ion channels in an artificial membrane (wilson et al., ) . our data in the current study also demonstrate the alteration of membrane permeability by the prrsv e protein and the inhibition of bacterial growth by the increase of hygromycin b penetration into bacterial cells. however, we were not able to show similar results in mammalian cells by infection or e gene transfection, which may be due to the different intracellular localization of e in mammalian cells. in prrsv-infected cells, the e protein appears to remain in the er and golgi complex, where it likely participates in assembly of infectious progeny virus, rather than traveling to the plasma membrane. bacterial cells do not possess such intracellular organelles and the expressed e protein may accumulate at the inner bacterial membrane, leading to membrane perturbation, and thus enhancing membrane permeabilization. failure to observe the direct alteration of membrane permeability by e in mammalian cells implies that the role of e is not linked to membrane disorganization and cell lysis in facilitating virus release. the cross-linking studies show that the e protein can form homo-oligomers, including dimers, trimers and tetramers by non-covalent interactions. all information obtained in the present study support the hypothesis that the prrsv e protein contains a potential for a pore-forming activity and thus may function as an ion channel for virus uncoating. the functional and structural features of the prrsv e protein resemble the influenza a virus m protein that is best characterized as an ion-channel protein. the m protein forms a homo-tetramer in the viral membrane and functions as an ion channel. the m channel allows the translocation of protons from the acidic environment of the endosome to the inner space of virions and alters the ph in the virion. as a consequence, the m protein in the virion is dissociated from the viral ribonucleoprotein complex, which promotes the ribonucleoprotein complex traveling to the nucleus where the influenza replication takes place (pinto et al., ; sakaguchi et al., ) . it is noteworthy that prrsv replication could be inhibited by amantadine, an antiviral drug for influenza virus (kreutz and ackermann, ; fig. ) . in summary, we propose a model for prrsv uncoating based on our findings. in this model, e proteins form pores (ion channels) in the viral envelope. upon internalization by receptor-mediated endocytosis through small clathrin-like coated vesicles, the virion particles are transported to the endosome. there, the e-protein ion channels in the viral membrane undergo conformational changes upon exposure to low ph in the endosome, and allow ions to enter the virion, which triggers the disassembly of inner capsid and the release of viral genome in the cytoplasm, such that further steps of genome replication and infection cycle can proceed. marc- (kim et al., ) , hela and bhk- cells were grown in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs; invitrogen), penicillin ( u/ml) and streptomycin ( μg/ml). bhk-t cells stably expressing the bacteriophage t rna polymerase (generously provided by c.y. kang, university of western ontario, london, ontario, canada) were grown in dmem supplemented with % fbs, u/ml of penicillin, μg/ml of streptomycin and μg/ml of g (geneticin; invitrogen). the cells were maintained at °c with % co . stocks of prrsv (strain pa ) and recombinant vaccinia virus expressing t rna polymerase (vtf - ; fuerst et al., ) were prepared in marc- or hela cells, respectively, as described previously (wootton et al., ) . the n protein-specific monoclonal antibody (mab) sdow , the e protein-specific anti-peptide rabbit antiserum and non-structural proteins and -specific polyclonal rabbit antiserum are described elsewhere nelson et al., ; wootton et al., ) . e. coli strains xl -blue (stratagene) and dh α were used as hosts for site-directed mutagenesis and general cloning, respectively. cdna cloning of the prrsv n or e gene to produce pcite-n; pgex-n or pcite-e; pgex-e, respectively, is described elsewhere (wootton and yoo, ; . to modify the translational initiation codon of prrsv orf b (e), pcr-based site-directed mutagenesis was first conducted to mutate the atg start codon of the e gene to gtg at genomic nucleotide positions , to , using the shuttle vector ptb-shuttle-prrsv- with the following primer pairs; for a g mutation, e-ko-fwd ( ′-gaattgaaatgaagtggggtctatac- ′: nucleotide positions , to , ) and e-ko-rev ( ′-gtata-gaccccacttcatttcaattc- ′: nucleotide positions , to , ), where lowercase letters indicate mutated nucleotides. the a g mutation was translationally silent with respect to orf a encoding the gp protein. pcr-based mutagenesis and screening of mutants were performed as described previously (wootton et al., ) . the shuttle plasmid carrying the a g mutation was digested with eco iii and bsrgi, and a -bp fragment was purified. the wild-type full-length genomic cdna clone was digested with eco iii and bsrgi, and the -bp eco iii-bsrgi fragment was replaced with the corresponding fragment obtained from the shuttle plasmid. the ligated full-length plasmid dna was screened by xmai digestion, and based on the xmai digestion pattern, positive clones were selected. dna manipulation and cloning were performed according to standard procedures (sambrook and russell, ) . the selected clones were sequenced to confirm the presence of the a g mutation in the full-length genomic cdna clone. the resulting plasmid was designated pcmv-s-p -Δe. marc- cells or bhk- cells were seeded on microscope coverslips placed in -mm-diameter dishes and grown overnight to % confluence. the cells were transfected with μg of plasmid dna using lipofectin (invitrogen) according to the manufacturer's instructions. at h post-transfection, cell monolayers were washed twice in pbs and fixed immediately with cold methanol for min at − °c. for time course experiments, marc- cells inoculated with purified passage- viruses prepared in the transfected bhk- cells were fixed at various time points after infection. cells were blocked using % bovine serum albumin (bsa) in pbs for min at room temperature (rt) and then incubated with n-specific mab sdow for h. after washing five times in pbs, the cells were incubated for h at room temperature with goat antimouse secondary antibody conjugated with alexa green dye (molecular probes). for dual immunofluorescence, cells were co-stained with nsp / -specific rabbit antiserum and n-specific mab sdow , followed by incubation with goat anti-rabbit antibody conjugated with texas red (molecular probes) and goat anti-mouse antibody conjugated with alexa green. the coverslips were washed five times in pbs and mounted on microscope glass slides in mounting buffer ( % glycerol and . % sodium azide in pbs). cell staining was visualized using a fluorescent microscope (model ax ; olympus). bhk cells were seeded in mm-diameter dishes and grown to % confluence. cells were transfected for h with μg of the full-length cdna plasmid using lipofectin. the transfected cells were continued for incubation at °c in dmem supplemented with % fbs for h. at h posttransfection, the cells were starved for min in methioninedeficient mem (invitrogen) and metabolically labeled for h with μci/ml of easytag express protein labeling mix ([ s]methionine and [ s]cysteine, specific activity, mbq/ ml) (perkin-elmer). after a -day labeling period, the cells were washed twice with cold pbs and lysed with ripa buffer ( % triton x- , % sodium deoxycholate, mm nacl, mm tris-hcl [ph . ], mm edta, . % sds) containing mm phenylmethylsulfonyl fluoride (pmsf). to prepare radiolabeled viral particle samples, the culture supernatant was harvested, and the cell debris was removed by a low-speed centrifugation at rpm (model ; eppendorf) for min at rt. the virus particles were purified through a % sucrose cushion (wt/vol) prepared in te buffer ( mm tris hcl [ph ], mm edta) at , rpm for h at °c in an sw rotor (model xl- ; beckman). the resulting pellets were resuspended in μl of ripa buffer containing mm pmsf. for immunoprecipitation, the dissolved pellets or cell lysates equivalent to in of a -mm-diameter dish were adjusted with ripa buffer to a final volume of μl and incubated for h at rt with a mixture of e-specific rabbit antiserum, nspecific mab sdow , and m-specific rabbit antiserum. the immune complexes were adsorbed to mg of protein-a sepharose cl- b beads (amersham biosciences) for h at °c. the beads were collected by centrifugation at rpm for min, washed twice with ripa buffer and once with wash buffer ( mm tris-hcl [ph . ], mm nacl). the beads were resuspended in μl of sds-page sample buffer ( mm tris-hcl [ph . ], % glycerol, % sds, . % [wt/vol] bromophenol blue) with % β-mercaptoethanol (βme), boiled for min and analyzed by % sdspolyacrylamide gel electrophoresis (page). gels were dried on filter paper and radiographic images were obtained using a phosphorimager (model phosphorimager si; molecular dynamics). virus preparation from full-length cdna clones bhk- cells were transfected with the full-length cdna plasmid as described above. after washing of cells with dmem at h post-transfection, the transfected cells were further maintained in dmem supplemented with % fbs for days and the culture supernatants were harvested at days posttransfection. the viral particles were purified as described above and the pellets were suspended in dmem. the resulting virus suspension was designated 'passage- '. the passage- virus was used to inoculate fresh marc- cells and the -day harvest was designated 'passage- '. the 'passage- ' virus was prepared in the same way as for passage- . each passage virus was aliquoted and stored at − °c until use. culture supernatants of bhk- cells transfected with the full-length cdna plasmid were harvested at days posttransfection as described above. the culture fluids were centrifuged at rpm (model ; eppendorf) for min to remove cellular debris. the cleared supernatants were purified as described above, and the pellets were suspended in μl of pbs and subsequently stored at °c until use. twenty microliters of the virion suspension was mounted on a formarcoated copper grid. the grids were placed at rt for min, and excess liquid was removed by wicking with filter paper. for negative staining, μl of % sodium phosphotungstate (ph . ) was dropped onto the grids and incubated at rt for s. the samples were viewed with a transmission electron microscope (model h- kev pc-tem; hitachi) operating at kev. viral rna was extracted from either supernatants or lysates of infected cells using the qiaamp viral rna mini-kit (qiagen). to remove any contaminated dna in the rna preparations, samples were treated with u of rq dnase i (promega) at °c for min in mm tris-hcl [ph . ] and mm mgcl . for detection of viral rna in the virions, samples were prepared as previously described (wieringa et al., ) with some modifications. the viral particles in the culture supernatant from bhk- cells transfected with the full-length cdna plasmid were pelleted by as described above. the pellet was resuspended in tnm buffer ( mm tris-hcl [ph . ], mm nacl, mm mgcl ). seventy microliters of each sample suspension were added with μl of rq dnase i ( u/μl; promega) and . μl of rnase a ( mg/ml; sigma), and the mixture was incubated for h at °c in the presence or absence of detergents ( μl of triton x- , and . μl of % sds). after incubation for h at °c, the nucleases were inactivated by the addition of μl of proteinase k ( mg/ml; qiagen) and incubation for min at °c. rna was isolated from each sample by using a qiaamp viral rna mini-kit and subsequently, treated one more time with unit of rq dnase i. after dnase i treatment, rna was re-extracted with an equal volume of phenol:chloroform:isoamyl alcohol ( : : ) mixture and precipitated at − °c for h by adding . volume of . m sodium acetate (ph . ) and volumes of ethanol. the pellets were washed once with % ethanol and dissolved in ultrapure dnase/rnase-free distilled water (invitrogen). the resulting rna samples were used for first-strand cdna synthesis by moloney murine leukemia virus (m-mlv) reverse transcriptase (invitrogen) using the reverse primer orf b-rev ( ′-tcataagatcttctgtaattgctc- ′). the e gene was amplified by taq dna polymerase (invitrogen) using mrna -fwd ( ′-ccgtcattgaac-caacttta- ′) and orf b-rev. for strand-specific rt-pcr, the specific primer pairs (table ) were used to amplify dna fragments representing positive-sense genomic rna, negative-sense genomic rna, positive-sense subgenomic mrna or negative-sense subgenomic mrna . pcr was conducted under the following conditions; initial denaturation at °c for min, cycles of denaturation at °c for s, annealing at °c for s and extension at °c for min, followed by a final extension at °c for min. pcr products were analyzed by . % or . % agarose gel electrophoresis depending on size of the fragment. amplified products were purified using the pcr purification kit (qiagen) and sequenced. stock solutions of ammonium chloride, chloroquine, amantadine and verapamil (sigma) were prepared in water at concentrations of mm, mm, mm and mm, respectively. marc- cells grown in -well plates were preincubated with different concentrations of the reagents for min and subsequently infected with prrsv at a multiplicity of infection (moi) of for h at °c in the presence or absence of the drugs. the virus inoculum was removed and the cells washed three times with mem. the inoculated cells were then incubated in fresh medium in the presence or absence of the reagents and monitored daily for the appearance of cpe. culture supernatants were harvested daily from cells for days and virus titer in the supernatant was determined by plaque assay. at day or days post-infection, marc- cells infected with prrsv in presence or absence of the drugs were fixed with cold methanol and subjected to an immunofluorescence assay as described above. the effect of ion channel blockers on prrsv replication was also determined by strand-specific rt-pcr. total cellular rna was extracted from mock-infected or prrsv-infected cells in presence or absence of the drugs using trizol (invitrogen) and dna fragments representing positivesense or negative-sense viral genomic rna were rt-pcr amplified using orf -specific primers (lee et al., ) or negative-sense-specific primers (table ), respectively, as described above. an inducible e. coli expression system was used to express the prrsv e protein fused with glutathione s-transfererase (gst). plasmids were transformed into e. coli strain dh α to express gst fusion proteins. a single colony was grown in luria bertani (lb) media containing μg/ml ampicillin overnight. one hundred ml of lb media containing μg/ml ampicillin was inoculated with / of overnight culture. when the absorbance of cultures reached at an od of . , mm isopropylthio-β-d-galactoside (iptg) was added to the media to induce protein synthesis. at indicated times after induction, the densities of bacterial cultures were determined by measuring the light scattering at nm. permeability of the plasma membrane of bacterial cells expressing the prrsv e protein to hygromycin b was determined as described previously (liao et al., ) . briefly, bacterial cultures were incubated in the presence or absence of mm of hygromycin b for min at h after iptg induction, and aliquots of l ml were labeled with for min with μci/ml of [ s]methionine/cysteine. the labeled bacterial cells were then harvested and lysed in equal volumes of sds-page sample buffer. proteins were resolved on a % sds-page gel and visualized by coomassie blue staining or autoradiography. the prrsv n or e protein was independently expressed in hela cells using the t -based vaccinia virus vtf - . hela cells seeded on -mm-diameter dishes were grown to % confluence and infected for h at °c with vtf - at an moi of . following infection, fresh medium was added and incubation continued for an additional h. the cells were washed in opti-mem and transfected with . μg of a plasmid for h using the reagent lipofectin. the transfected cells were then starved for min in methionine-deficient mem and incubated for h with μci/ml of [ s]methionine/cysteine. to prepare radiolabeled prrsv-infected cells, marc- cells seeded on a -mm-diameter dish were infected with prrsv containing mm pmsf. the resultant cell lysates were then used in immunoprecipitation with μl of the n-specific mab sdow or μl of the e-specific rabbit antiserum followed by sds- % page analysis and autoradiography as described above. membrane permeabilization by poliovirus proteins b and bc membrane permeabilization by different regions of the human immunodeficiency virus type transmembrane glycoprotein gp functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) modification of late membrane permeability in avian reovirus-infected cells modification of membrane permeability by animal viruses membrane permeabilization by small hydrophobic nonstructural proteins of japanese encephalitis virus hepatitis c virus e protein induces modification of membrane permeability in e. coli cells ultrastructural characteristics and morphogenesis of porcine reproductive and respiratory syndrome virus propagated in the highly permissive marc- cell clone determinants for membrane association and permeabilization of the coxsackievirus b protein and the identification of the golgi complex as the target organelle inhibition of cellular protein secretion by poliovirus proteins b and a analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly viral ion channels: structure and function eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase the ns protein of bluetongue virus exhibits viroporin-like properties enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line porcine reproductive and respiratory syndrome virus enters cells through a low ph dependent endocytic pathway cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication expression of sars-coronavirus envelope protein in escherichia coli alters membrane permeability association of the infectious bronchitis virus c protein with the virion envelope viroporin activity of murine hepatitis virus e protein human immunodeficiency virus type vpu protein is an oligomeric type i integral membrane protein phylogenetic analysis of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the u.s.a. and europe lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav characterization of proteins encoded by orfs to of lelystad virus entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of us and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states influenza b virus bm protein is an oligomeric integral membrane protein expressed at the cell surface influenza virus m protein has ion channel activity the cap structure of simian hemorrhagic fever virion rna the active oligomeric state of the minimalistic influenza virus m ion channel is a tetramer molecular cloning: a laboratory manual how viruses enter animal cells the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses topley and wilson's microbiology and microbial infections: virology volume the transmembrane oligomers of coronavirus protein e proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication ion channel activity of influenza a virus m protein: characterization of the amantadine block structural protein requirements in equine arteritis virus assembly sars coronavirus e protein forms cation-selective ion channels envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b the b protein as a minor structural component of prrsv this study was supported by funding to dy by nserc canada, ontario pork, omaf and the usda nri prrs cap program of the usa. the authors are grateful to pfizer animal health usa for providing the infectious cdna clone for this study. key: cord- -g hhm authors: zhao, ge; zhang, lujie; li, charles; zhao, jianmei; liu, na; li, yuehua; wang, junwei; liu, liheng title: identification of enterobacteria in viscera of pigs afflicted with porcine reproductive and respiratory syndrome and other viral co-infections date: - - journal: microb pathog doi: . /j.micpath. . sha: doc_id: cord_uid: g hhm in order to investigate enterobacteria presence involved in the secondary infections in porcine reproductive and respiratory syndrome (prrs) pigs with different viral co-infections, we identified enterobacteria for guiding clinical treatment. twenty-one diseased pigs were diagnosed with the prrs virus (prrsv) and other virus primers by pcr/rt-pcr in the lung and spleen samples. enterobacteria were isolated using macconkey agar from visceral samples of prrs pigs, and identified by s rdna sequencing. prrsv was positive in % of the lung samples and . % of the spleen samples. seven diseased pigs were diagnosed with only prrsv infection ( . %), pigs with prrsv and or other viruses ( . %) and pigs with prrsv and more than types of other viruses ( . %). prrsv was more inclined to co-infect pigs with porcine group a rotavirus (parv) with the co-infection rate of . % ( / ). approximately types of bacteria were successfully isolated from lung, spleen, liver, kidney and lymph node samples of different prrs pigs. enterobacteria were isolated in % of lung, liver and lymph samples from pigs infected with prrsv alone. however, the isolation rates were significantly decreased in the more than viruses co-infection group. escherichia coli was the most prevalent bacterium, followed by morganella, proteus, shigella, salmonella, klebsiella and aeromonas. most of the isolated enterobacteria were opportunistic pathogens. therefore, timely combination with antimicrobial agents is necessary for effective treatment of prrs-infected pigs. porcine reproductive and respiratory syndrome (prrs) is a swine disease caused by a virus, which poses a significant economic threat to the swine industry worldwide [ ] . the prrs death in prrs infected pigs [ , ] . in practical production, farmers focus more attention on ways to prevent and treat infected pigs. farmers employ vaccine inoculation and isolated rearing to prevent infection [ ] . the viscera, including lung, spleen, liver, kidney and lymph nodes from the pigs with prrs were used for enterobacteria isolation. firstly, the visceral specimen was removed from the plastic bag to a sterile plate under the biosafety cabinets, and sections were cut by using table . through the above examination, we confirmed that the diseased pigs were indeed infected with prrsv, simultaneously. we further determined that some other viruses may infect simultaneously with prrsv. nevertheless, the real causes of death among the nursery pigs with prrs may be the pathogens causing secondary infections [ , ] . therefore, we further investigated the enterobacterial proliferation from the gut to the surrounding viscera, such as lung, spleen, liver, kidney and lymph nodes. from the enterobacterial isolation and identification results (table ) , approximately types of bacteria were successfully isolated from several viscera of different prrs-positive pigs, and no enterobacterium was isolated in healthy samples. if only one type of enterobacterium was isolated, the corresponding organ was supposed to be infiltrated by the gut bacteria. based on this analysis (figure ), we found that the total enterobacterial isolation rate from kidneys ( . %, / ) were significantly lower (p< . ), and the total rates from the other viscera were comparative in levels. we also found that when pigs were co-infected with more types of viruses, the lower bacterial isolation rate was obtained (p< . ). in the prrsv alone infection group, enterobacteria were detected with a % positive rate in lung, liver and lymph samples, whereas in the co-infection group with more than types of virus species, the isolation rates in lung, liver, kidney and lymph were significantly decreased. only the rates from spleens were comparative. in the current study, we first detected other porcine viruses in addition to prrsv in the lung and spleen samples to investigate the most common viruses that tend to co-infect with prrsv. the result showed that prrsv could be detected in % of lung samples, indicating the prrsv infection was really occuring. however, the prrsv detection rate in lung was higher than in the spleen, which confirmed that it is easier for prrsv to invade the respiratory system and then affect other organs, such as the spleen [ ] . for the detected epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark effect of porcine reproductive and respiratory syndrome virus infection on the clearance of haemophilus parasuis by porcine alveolar macrophages secondary infection with streptococcus suis serotype increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs diagnosis and treatment of porcine reproductive and respiratory syndrome a one-step rt-pcr assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr nested reverse transcriptase-polymerase chain reaction for the detection of group a rotaviruses detection of classical swine fever virus in boar semen by reverse transcription-polymerase chain reaction detection of porcine circovirus type in feces of pigs with or without enteric disease by polymerase chain reaction a multiple sybr green i-based real-time pcr system for the simultaneous detection of porcine circovirus type , porcine parvovirus, pseudorabies virus and torque teno sus virus and in pigs a comparison of virus isolation, polymerase chain reaction immunohistochemistry, and in situ hybridization for the detection of porcine circovirus and porcine parvovirus in experimentally and naturally coinfected pigs s/ s rrna sequencing nucleic acid techniques in bacterial systematic pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs co-infection of porcine circovirus type ,porcine reproductive and respiratory disease syndrome virus and porcine parvovirus detection and analysis of porcine circovirus reproductive and respiratory syndrome virus, china animal husbandry & veterinary morganella morganii infections in a general tertiary hospital pathogenesis of proteus mirabilis urinary tract infection emedicine from webmd sherris medical microbiology klebsiella and acute anterior uveitis aeromonas spp. clinical microbiology and disease etiology analysis of the bacterial secondary infection of prrsv in sichuan province from importance of providencia species as a major cause of travellers' diarrhea genetic and biochemical diversity of ureases of proteus morganella species isolated from urinary tract infection sphingomonas paucimobilis bloodstream infections associated with contaminated intravenous fentanyl multidrug resistant acinetobacter baumannii: a descriptive study in a city hospital escherichia fergusonii: an emerging pathogen in south orissa increased incidence of urolithiasis and bacteremia during proteus mirabilis and providencia stuartii coinfection due to synergistic induction of urease activity s: spleen; l: lung; +: positive key: cord- -dzin qb authors: zhang, wei; chen, keren; guo, yang; chen, yaosheng; liu, xiaohong title: involvement of prrsv nsp and nsp in the autophagy process date: - - journal: virol j doi: . /s - - -x sha: doc_id: cord_uid: dzin qb background: autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. methods: lc turnover and the proteins in the endoplasmic reticulum (er) stress pathway were investigated using western blot analysis. the formation and degradation of autophagosomes were detected using immunofluorescence staining. results: autophagy was activated by porcine reproductive and respiratory syndrome virus (prrsv) nsp , nsp and nsp , which are two transmembrane proteins and an rna-dependent rna polymerase, respectively. the formation of autophagosomes was induced by nsp and nsp and developed from the er; the fusion of these autophagosomes with lysosomes was limited. although nsp and nsp are er transmembrane proteins, these proteins did not activate the er stress signaling pathways. in addition, the cytoplasmic domain of nsp plays a pivotal role in activating autophagy. conclusions: the data presented in this study reveal an important relationship between prrsv nsps and autophagy and provide new insights that improve our understanding of the involvement of prrsv nsps in the autophagy process. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome virus (prrsv- ) is a member of the genus arterivirus, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv) [ ] . prrsv infection leads to severe morbidity and mortality worldwide; the main clinical manifestations are reproductive disorders, such as miscarriage, stillbirth, and the delivery of mummified fetuses in pregnant sows, as well as respiratory symptoms in pigs of various ages (particularly piglets) [ , ] . prrsv is an enveloped, single-stranded positive-strand rna virus with a genome of approximately . kb that includes at least open reading frames (orfs); orf a and orf b encode nonstructural proteins (nsps), and orf -orf encode structural proteins [ , ] . these nsps are involved in viral replication and play key roles in regulating genomic transcription [ ] . among these nsps, nsp and nsp are predicted to be transmembrane proteins and nsp possesses viral rna-dependent rna polymerase activity [ ] . prrsv is usually grown in marc- cells, which originated from monkey kidney epithelial cells. autophagy occurs in eukaryotic cells and is a process by which cell homeostasis is maintained through the degradation of proteins and organelles [ , ] . generally, autophagy is triggered in cells stimulated with external factors, such as starvation or viral infection; the marker of autophagy is the formation of autophagosomes [ ] . the integrated autophagy course primarily comprises three processes: first, the formation of autophagosomes; then, the fusion of autophagosomes with lysosomes; and last, degradation of the cargo in lysosomes. the first step of the autophagy process is that a membrane possibly derived from the endoplasmic reticulum (er) elongates, becomes curled, and then surrounds part of the cytoplasm, followed by the degradation of organelles, proteins and other cellular components. in this process, two proteins, namely, atg and atg , form complexes via ubiquitin-like conjugation [ , ] . furthermore, atg interacts with the atg -atg complex to form the atg -atg -atg protein complex, which plays a significant role in forming autophagosomes [ ] . finally, lysosomes fuse with mature autophagosomes to form autophagolysosomes; the contents of the autophagosome are degraded, and the substances inside the autophagolysosomes are delivered to the cytoplasm for transformation into amino acids and energy for cytoplasmic metabolism [ ] . generally, autophagy is accompanied by er stress, which is induced by the accumulation of unfolded or misfolded proteins in the er [ ] . the er is the main site of protein synthesis, lipid production and calcium ion storage in eukaryotic cells. a variety of proteins in the er must be folded, assembled, processed, packaged and transported to the golgi apparatus [ ] . once the cells are stimulated with internal and external factors, the balance between er morphology and function is affected by the changes in molecular biochemistry. protein processing and transport are blocked, and a large number of unfolded or misfolded proteins accumulate in the er, leading to the unfolded protein response (upr) [ ] [ ] [ ] , which operates through three signaling pathways that play significant roles in regulating autophagy during viral infection [ , ] . the er stress response was recently shown to be induced by prrsv infection, leading to the activation of jnk signaling pathways [ ] . in the past decade, a number of studies have reported a connection between autophagy and viral proteins [ , ] . the hcv polymerase ns b was recently reported to colocalize with ns b and interact with atg to initiate autophagosome formation [ ] . in addition, hbx promotes autophagy and prevents the fusion of autophagosomes with lysosomes to maintain the accumulation of solutions in the autophagosomes [ , ] . recent studies examining the relation between autophagy and prrsv revealed that prrsv induced incomplete autophagy and that autophagy enhances the replication of prrsv [ ] [ ] [ ] . additionally, prrsv infection promotes mitochondrial fission and activates apoptosis [ ] [ ] [ ] ; however, few studies have examined the relations between prrsv nsps and autophagy to date. in this study, we detected the involvement of prrsv nsps in autophagy in transfected cells for the first time. prrsv nsp , nsp and nsp induce the formation of autophagosomes. additionally, nsp and nsp are er transmembrane proteins, but these two nsps trigger limited er stress, and the autophagosomes induced by these two nsps are derived from the er and accumulate autophagic cargo to prevent fusion with lysosomes. furthermore, an nsp truncation mutant consisting of only the hydrophobic domains did not induce the formation of autophagosomes. our findings revealed the important functions of prrsv nsps in autophagy. cells of the african green monkey kidney epithelial cell line marc- purchased from atcc were cultivated in dulbecco's modified eagle's medium (dmem, invitrogen) supplemented with % fetal bovine serum (fcs) at °c in a % co atmosphere. hbss (hank's balanced salt solution) was purchased from sigma and can induce autophagy. the prrsv strain ch- a (the first prrsv strain isolated from china) was provided by dr. guihong zhang at the south china agricultural university, china. cellular dna was stained with ′, ′-diamidino- -phenylindole dihydrochloride (dapi), and mitochondria were stained with mitotracker green. rabbit anti-lc , anti-calnexin, and anti-gapdh; mouse anti-atg ; alexa fluor -conjugated anti-rabbit igg; alexa fluor -conjugated anti-mouse igg; and alexa fluor -conjugated anti-mouse igg secondary antibodies were purchased from cell signaling technology (beverly, ma). a mouse anti-mcherry antibody was obtained from abcam (ab ). a prrsv nucleocapsid (n) antibody was purchased from jeno biotech inc. (chuncheon). the mouse monoclonal anti-dsrna antibody was purchased from scicons (hungary). viral rna was isolated with trizol (invitrogen), and cdnas were obtained using a reverse transcription kit (promega). pcr primers were designed to amplify the prrsv nsp transcripts (additional file : table s ) using the prrsv ch- a cdna. the pcr fragments were cloned into a pmcherryn (clontech, , ) expression vector. the truncated prrsv nsp mutant was constructed using a pcr primer pair (additional file : table s ). all multiple cloning digestion sites were selected for digestion with xhoi and bamhi. marc- cells were transfected with μg of the constructed plasmids using lipofectamine (invitrogen). marc- cells were cultured on glass coverslips, followed by transfection with the individual plasmids for h. after three washes with phosphate-buffered saline (pbs), cells were fixed with % paraformaldehyde (pfa) for min at room temperature. after three washes with pbs, the cells were permeabilized with . % triton x- for min and blocked with % bovine serum albumin (bsa) in pbs for one hour at room temperature. next, the coverslips were incubated with primary antibodies (diluted : ) in pbs containing % bsa for h, followed by an incubation with an alexa fluor-conjugated secondary antibody (diluted : ) for h at room temperature in the dark. after three washes with pbs, cellular nuclei were stained with dapi in the dark for min at room temperature. next, the coverslips were washed with pbs, mounted on the microscope slides with antifade mounting medium, and observed under a leica tcs sp confocal microscope. cells were washed with pbs, and total protein was obtained using sds cell lysis buffer ( % sds and mm tris, ph . ) containing mm phenylmethylsulfonyl fluoride (pmsf) and heating for min. a volume of lysate containing μg of protein was separated on % acrylamide gels in a bio-rad system. then, proteins were transferred to a polyvinyl difluoride (pvdf) membrane and blocked with % bsa in tris-buffered saline including tween (tbst) for min. after blocking, the pvdf membrane was incubated with the primary antibody for h, followed by incubation with the hrp-conjugated secondary antibodies. images were captured using an image station mm pro system and image station mm pro software. cells were collected, and total rna was extracted using trizol (invitrogen). two micrograms of rna was obtained with the reverse transcription kit as described above. the synthesized cdnas were amplified with specific primers (additional file : table s ). the final products were visualized on a . % agarose gel that was electrophoresed at v for min. the pearson correlation coefficient per cell was quantified using image-pro plus software. data are presented as means ± standard errors. statistical significance was determined by performing student's t-tests. p values < . were considered statistically significant. the gfp-lc plasmid, which expressed the lc protein tagged at its n terminus with the fluorescent protein gfp, was used to monitor the formation of autophagosomes by indirect immunofluorescence [ ] . gfp-lc was transfected into cells for h, and transfection efficiency was - %. cells were then infected with prrsv ch- a. at h.p.i., the infected cells were fixed, and gfp-lc puncta were observed to assess the formation of autophagosomes. as shown in fig. a and b, compared to the accumulation of gfp-lc puncta in the cytoplasm of mock-infected cells, the accumulation of these puncta in the cytoplasm of hbss-treated and prrsv-infected cells suggested that prrsv induced the formation of autophagosomes. lc conversion is a hallmark of autophagy; therefore, the conversion of lc was assessed by immunoblotting and the levels of lc ii/ lc i were examined to assess the induction of autophagy. marc- cells were infected with prrsv ch- a at h.p.i. or were cultured with hbss for h as a positive control. as shown in fig. c , compared to the lc ii/ lc i ratio in the mock-infected cells, the ratio was increased in the infected marc- cells. we explored whether prrsv dsrna and n proteins were associated with autophagosomes using confocal microscopy to identify whether the autophagosomes induced by prrsv were related to viral replication or assembly. as depicted in fig. d , the majority of the lc protein was colocalized with dsrna and n proteins, indicating that these autophagosomes provide the site for prrsv replication and assembly. prrsv non-structural proteins play an important role in virus replication and assembly and use the substances in the cells to influence cell life activities. because prrsv induced the formation of autophagosomes, we further explored which prrsv nsps played important roles in this process. eukaryotic expression vectors carrying the nsp cdnas with an n-terminal mcherry tag were constructed and transfected into marc- cells (additional file : figure s ). as shown in fig. a and b, the gfp-lc puncta accumulated in nsp -mcherry-, nsp -mcherry-and nsp -mcherry-transfected cells. nsp is an rna-dependent rna polymerase (rdrp) that plays important roles in viral transcription and replication, and nsp and nsp are predicted to be transmembrane proteins; these proteins are anchored on the cytoplasmic membrane and are part of the membrane-bound replication and transcription complex. furthermore, lc levels were detected using immunoblotting to determine the effects of the two transmembrane proteins on autophagy. p /sequestosome- is a protein that can bind to lc as a scaffold protein or a signaling adapter and may be increased at the beginning of autophagy process and degraded gradually. based on the data presented in fig. c , immunoblotting and immunofluorescence assay showed that the expression of the p protein was increased, indicating that nsp and nsp of prrsv induced the formation of autophagosomes. as shown in fig. d , a higher lc ii/ lc i ratio was observed in nsp -mcherry-and nsp -mcherry-transfected cells than in mock-infected cells. we identified some of the effects of these two transmembrane proteins on cell autophagy to determine whether the nsps affected cytoplasmic membrane fusion. the autophagosomes induced by nsp and nsp are derived from the er generally, in the early stage of autophagy, atg complexes with atg and atg l to form the atg -atg -atg l complex [ ] , and this complex attaches to nascent phagophores, which are the precursors of mature autophagosomes that are involved in regulating membrane morphology changes [ ] . therefore, endogenous atg was confirmed at the site of immature autophagosomes. autophagosome membranes have been shown to originate from the mitochondria [ ] or er [ ] . cells transfected with nsp -mcherry and nsp -mcherry were fixed and stained for atg and calnexin, an er marker. the value of overlap of the two different channels was calculated using pearson's correlation coefficient, and a value exceeding . was determined to indicate colocalization. as shown in fig. a , atg puncta were arranged in reticular structures and colocalized with calnexin in the fig. the distribution of autophagy proteins in prrsv-infected marc- cells. a marc- cells were transfected with gfp-lc plasmids and cultured with either dmem or hbss media for h or were infected with prrsv ch- a for h. fixed cells were observed under a fluorescence microscope. nuclei were stained with dapi (blue), and virions were stained with an antibody against the prrsv-n protein (red). scale bars: μm. b statistical analysis of the number of gfp-lc puncta in mock, hbss-treated or prrsv-infected cells; the number represents gfp-lc puncta per cell; data are presented as means ± sd, n = . c lc conversion in marc- cells. marc- cells were mock infected, infected with prrsv for h or cultured in hbss media. cells lysates were subjected to immunoblotting. the ratio of lc ii/lc i reflects the level of autophagy. d marc- cells were infected with prrsv for h, and fixed cells were observed under a fluorescence microscope. nuclei were stained with dapi (blue); dsrna and prrsv-n are labeled in red, and endogenous lc is labeled in green. scale bars: μm cells expressing nsp and nsp . combined with the subsequent findings that prrsv nsp and nsp were localized on the er, these results suggested that the atg puncta were derived from the er. little colocalization of the fluorescently labeled atg proteins with mitotracker green, a mitochondrial marker, was observed in cells transfected with nsp -mcherry and nsp -mcherry (fig. b) . therefore, the autophagosomes induced by prrsv nsp and nsp originate from the er but not from the mitochondria. after mature autophagosomes are formed, they fuse with lysosomes and form autolysosomes to degrade the insoluble substances inside the autophagosomes. fig. the formation of autophagosomes induced by prrsv nsp and nsp . a marc- cells were transfected with gfp-lc and plasmids encoding the individual prrsv nsps; each nsp was cloned into an mcherry-n plasmid, and cells transfected with mcherry-n were used as a negative control. twenty-four hours after transfection, cells were harvested and visualized under a fluorescence microscope. nuclei were stained with dapi, and the number of gfp-lc puncta, which is related to autophagy, was analyzed using graphpad software. scale bars: μm. b statistical analysis of the number of gfp-lc puncta in cells expressing mcherry-n and each nsp; the numbers represent gfp-lc puncta per cell; data are presented as means ± sd, n = . c cells were transfected with mcherry nsp -mcherry and nsp -mcherry for h; the endogenous p protein was stained with a green fluorophore. cell lysates were subjected to immunoblotting. scale bars: μm. d lc protein conversion. marc- cells were mock infected; treated with hbss for h or transfected with mcherry, nsp -mcherry, or nsp -mcherry; and then harvested and lysed. extracts were analyzed by western blotting using an anti-lc antibody. the ratio of lc ii/lc i reflects the level of autophagy we labeled nsp -and nsp -transfected cells with lysosome-associated membrane protein (lamp ), a lysosomal marker, and the mature autophagosome marker lc to investigate the fate of autophagosomes and to obtain a better understanding of the role of these autophagosomes induced by the two nsps. as shown in fig. a , an overlap of lc and lamp was observed in serum-starved marc- cells. as shown in fig. b , in nsp or nsp transfected cells, lamp failed to colocalize with lc , and the pearson correlation coefficient was much lower than that in serum-starved cells, suggesting that lysosomes did not fuse with the autophagosomes induced by the two nsps, as with prrsv-infected cells. these results suggest that the autophagosomes induced by nsp and nsp are derived from the er, but autophagosome-lysosome fusion was limited. nsp and nsp do not induce er stress prrsv nsp and nsp are predicted to be transmembrane proteins, and we studied which organelle membrane is the attachment site of these two integral transmembrane proteins. as shown in fig. a and b, prrsv nsp -mcherry and nsp -mcherry only colocalized with calnexin but not with mitotracker green. thus, prrsv nsp and nsp are integral transmembrane proteins of the er but not the mitochondria. the activation of autophagy is always affected by er stress, which is due to protein processing dysfunctions; the c/ebp homologous protein (chop) is overexpressed [ ] , and the xbp mrna is spliced by phosphorylated ire . prrsv triggers the activation of the ire α and perk branches of the upr, suggesting that chop overexpression is activated by the perk pathway and that the autophagosomes induced by prrsv nsp and nsp are derived from the er; therefore, we examined chop expression using immunoblotting, and we determined the splicing of the xbp mrna using pcr. as shown in fig. c , compared to the expression of chop in mock-infected cells, this protein was noticeably upregulated in prrsv-infected cells; however, the expression of chop in nsp -mcherry-and nsp -mcherry-transfected cells was not significantly fig. autophagosomes originated from the er but not mitochondria. a marc- cells were transfected with nsp -mcherry and nsp -mcherry for h; atg is labeled in green, and nuclei were stained with dapi (blue). scale bars: μm. statistical analysis of the pearson correlation coefficient for calnexin and atg . the pearson colocalization coefficient is presented as the ratio of punctate signals of calnexin that were positive for atg . b nsp -mcherry and nsp -mcherry were expressed in marc- cells. twenty-four hours after transfection, cells were cultured with mitotracker green, a marker of mitochondria, and were then fixed and immunostained with an anti-atg antibody (pink). nuclei were stained with dapi (blue). scale bars: μm. statistical analysis of the pearson correlation coefficient for mitotracker green and atg . the colocalization coefficient is presented as the ratio of fluorescent signals of mitotracker green that were negative for atg changed. thus, the involvement of prrsv nsp and nsp in autophagy was not regulated by an er stress-dependent pathway. the cytoplasmic domain is required for prrsv nsp induced autophagy prrsv nsp and nsp are predicted transmembrane proteins. as shown in fig. a, nsp consists of hydrophobic transmembrane domains and a hydrophilic cytoplasmic domain and nsp consists of hydrophobic transmembrane domains. a deletion mutant in which nsp consisted of only the hydrophobic domains was constructed to detect whether the hydrophilic cytoplasmic domain of nsp , which is out of range of the membrane, affects the formation of autophagosomes. as illustrated in fig. b , the nsp mutant was successfully constructed, and the truncated protein exhibited a lower molecular weight than the normal nsp protein. furthermore, as shown in fig. c , along with the normal nsp protein, the mutant nsp protein was localized on the er but did not induce the formation of autophagosomes. these results reveal that the hydrophilic cytoplasmic domain of nsp plays an important role in inducing autophagy. knowledge about virus-induced autophagy has gradually increased [ ] . however, the relationship between each prrsv protein and autophagy has not been elucidated. as shown in the present study, the induction of autophagy by prrsv nsp and nsp contributed to the formation of autophagosomes derived from the er, and the mature autophagosomes were not degraded by fusion with lysosomes. prrsv nsp and nsp are er transmembrane proteins, but these multiple processes of autophagy were not induced by er stress. moreover, a deletion mutant of nsp revealed that the transmembrane domains are crucial for inducing the formation of autophagosomes. our findings offer new explanations for the activation of autophagy by prrsv nsps. autophagy plays an important role in both the innate and adaptive immune responses, both of which eliminate intracellular microbes [ ] . however, many pathogens that invade cells induce the formation of double-membrane autophagosomes to facilitate their own replication [ ] . almost all viruses induce the formation of autophagosomes, and autophagosomes provide the site for viral replication and assembly [ ] . additionally, individual nonstructural proteins or structural proteins may perform viral functions, such as inducing apoptosis or necrosis. in the present study, prrsv ch- a induced the formation of autophagosomes in the infected cells; these autophagosomes are purported to be the site of viral replication. viral nonstructural proteins play important roles in the replication of the viral rna and in the synthesis of viral particles. in virus-infected cells, the virus interferes with and destroys the normal metabolism of the host cell, causing cell death or apoptosis [ ] . in many studies, viral nonstructural proteins have been shown to play important roles in virus-induced autophagy. in hcv-infected cells, ns / a blocks the rig-i signaling pathway in the early stages of autophagy and modulates the binding of mitochondria-associated immunity-associated gtpase family m (irgm) to promote autophagy [ ] . similarly, jev ns targets irgm to modulate autophagy [ ] , and cbv protein b, whose c-terminal sequence plays a decisive role in autophagy, rearranges the cell membrane [ ] . in our study, gfp-lc puncta accumulated in nsp -mcherry-, nsp -mcherry-and nsp -mcherry-transfected cells and p expression was increased, indicating that nsp , nsp and nsp induced the formation of autophagosomes. furthermore, we detected an increase in the ratio of lc ii to lc i in cells transfected with nsp and nsp , along with an increased expression of p , confirming that nsp and nsp induce autophagy. generally, the process of autophagy includes two steps: the formation of autophagosomes and the degradation of autophagosomes. in the early stage of autophagy, atg interacts with atg and atg to form the atg -atg -atg l complex; this complex is localized in phagophores, which are the precursors of autophagosomes. in addition, the majority of autophagic degradation occurs when autophagosomes envelope cellular substances, which are delivered to the lysosomes for degradation of the contents. in the present study, a marker of phagosomes, which are the precursor of autophagosomes, namely, atg , was colocalized with calnexin, an er marker, but not with mitotracker, suggesting that the autophagosomes induced by nsp and nsp were derived from the er but not from the mitochondria. moreover, autophagy is a complex process. after prrsv infects cells, it incompletely induces autophagy and inhibits the fusion of autophagosomes with lysosomes. this inhibition leads to the accumulation of autophagosomes and enhances prrsv replication. in our study, lc was not colocalized with lamp in nsp -and nsp -transfected cells, suggesting that the fig. a nsp and nsp were located in the er but not the mitochondria. nsp -mcherry and nsp -mcherry were expressed in marc- cells; the er was stained with an antibody against endogenous calnexin (green), and mitochondria were stained with mitotracker green. scale bars: μm. b statistical analysis of the pearson correlation coefficients for calnexin/mitotracker and nsp /nsp . the colocalization coefficient is presented as the ratio of fluorescent signals of calnexin that were positive for nsp /nsp . the colocalization coefficient is the ratio of fluorescent signals of calnexin that were negative for nsp /nsp . c chop expression was examined in mock-infected; prrsv-infected; and mcherry-, nsp -mcherry-, or nsp -mcherry-expressing marc- cells using immunoblotting. the intensities of chop bands were normalized to gapdh. the splicing of the xbp mrna was examined using pcr; xbp u is the unspliced mrna, and xbp s is the spliced mrna mature autophagosomes induced by nsp and nsp were not fused with lysosomes. our findings provide evidence that the membranes of autophagosomes induced by nsp and nsp were derived from the er and inhibited autophagosome fusion with lysosomes. nsp and nsp are two transmembrane proteins that appear to play a role in membrane rearrangement. prrsv nsp and nsp were localized to the er, but not the mitochondria, in the present study, suggesting that the two nsps are transmembrane er proteins but not transmembrane mitochondrial proteins. in a recent study, er stress was activated in prrsv-infected cells. additionally, researchers previously believed that autophagy activation is accompanied by the activation of the er stress pathway. the early er stress response involves the splicing of the xbp mrna and the expression of chop. in the present study, we confirmed that the splicing of the xbp mrna was not noticeably altered and chop expression exhibited a slight increase, indicating that autophagy was not activated by er stress in prrsv nsp -and nsp -transfected cells. because prrsv nsp contains transmembrane domains and a hydrophilic cytoplasmic domain, we deleted the cytoplasmic domain and revealed that the activation of autophagy requires complete nsp protein. in conclusion, prrsv nsp and nsp , which are er transmembrane proteins, induce the formation of autophagosomes. although these autophagosomes are derived from the er and are not degraded by lysosomes, the activation of autophagy by the two nsps does not involve er stress. the hydrophilic cytoplasmic domain of prrsv nsp plays a key role in the activation of autophagy. overall, our findings provide new insights into the connection between autophagy processes and prrsv nsps. the nsp hydrophilic domain is required to activate autophagy. a schematic of the prrsv nsp , nsp and mutant nsp protein structures. b mcherry was detected by immunoblotting in nsp -and mutant nsp -expressing cells c marc- cells were co-transfected with plasmids expressing wildtype/mutant prrsv nsp and gfp-lc for h. fixed cells were detected using fluorescence microscopy. gfp-lc puncta represent mature autophagosomes; nuclei were stained with dapi (blue). scale bars: μm prrsv, the virus porcine reproductive and respiratory syndrome assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states prrsv structure, replication and recombination: origin of phenotype and genotype diversity nidovirales: a new order comprising coronaviridae and arteriviridae the structural biology of prrsv overview: replication of porcine reproductive and respiratory syndrome virus cell biology -autophagy as a regulated pathway of cellular degradation the role of autophagy during the early neonatal starvation period autophagosome formation: core machinery and adaptations molecules and their functions in autophagy the atg -atg conjugate has a novel e -like activity for protein lipidation in autophagy formation of the similar to -kda apg -apg center dot apg multimeric complex, mediated by apg oligomerization, is essential for autophagy in yeast guidelines for the use and interpretation of assays for monitoring autophagy regulation mechanisms and signaling pathways of autophagy endoplasmic reticulum is the sorting core facility in the golgi-lacking protozoan giardia lamblia the unfolded protein response: from stress pathway to homeostatic regulation signal integration in the endoplasmic reticulum unfolded protein response endoplasmic reticulum stress: cell life and death decisions endoplasmic reticulum stress in disease pathogenesis er stress and diseases involvement of unfolded protein response, p and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate coronavirus nsp restricts autophagosome expansion autophagy protein atg interacts transiently with the hepatitis polymerase (ns b) early during infection hepatitis b virus x protein induces autophagy via activating death-associated protein kinase hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication hu hb: autophagy sustains the replication of porcine reproductive and respiratory virus in host cells porcine reproductive and respiratory syndrome virus triggers mitochondrial fission and mitophagy to attenuate apoptosis interplay of autophagy and apoptosis during prrsv infection of marc cell autophagy postpones apoptotic cell death in prrsv infection through bad-beclin interaction apg p is required for the function of the apg p-apg p conjugate in the yeast autophagy pathway the role of atg proteins in autophagosome formation mitochondria supply membranes for autophagosome biogenesis during starvation autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum chop is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum the role of autophagy in host defence against mycobacterium tuberculosis infection an integrated analysis of membrane remodeling during porcine reproductive and respiratory syndrome virus replication and assembly how positive-strand rna viruses benefit from autophagosome maturation the crosstalk between autophagy and apoptosis: where does this lead? hepatitis c virus and autophagy capsid, membrane and ns are the major viral proteins involved in autophagy induced by japanese encephalitis virus protein b of coxsackievirus b induces autophagy relying on its transmembrane hydrophobic sequences the authors wish to acknowledge professor guihong zhang from the south china agricultural university for her donation of experimental materials and her influential guidance to the authors. this study was funded by grants from the natural science foundation of guangdong province ( a ), the national natural science foundation of china ( ) and the guangdong sailing program ( yt h ). additional file : table s . table primers used availability of data and materials not applicable. the major contributions of w.z. were in the writing of the manuscript, designing the experiments and performing the experiments. y.g, k.c, y.c. and x.l. composed the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.received: july accepted: january key: cord- -jrqdyfbg authors: du, yijun; pattnaik, asit k.; song, cheng; yoo, dongwan; li, gang title: glycosyl-phosphatidylinositol (gpi)-anchored membrane association of the porcine reproductive and respiratory syndrome virus gp glycoprotein and its co-localization with cd in lipid rafts date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: jrqdyfbg the porcine reproductive and respiratory syndrome virus (prrsv) glycoprotein (gp ) resembles a typical type i membrane protein in its structure but lacks a hydrophilic tail at the c-terminus, suggesting that gp may be a lipid-anchored membrane protein. using the human decay-accelerating factor (daf; cd ), a known glycosyl-phosphatidylinositol (gpi) lipid-anchored protein, chimeric constructs were made to substitute the gpi-anchor domain of daf with the putative lipid-anchor domain of gp , and their membrane association and lipase cleavage were determined in cells. the daf-gp fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase c (pi-plc), indicating that the c-terminal domain of gp functions as a gpi anchor. mutational studies for residues adjacent to the gpi modification site and characterization of respective mutant viruses generated from infectious cdna clones show that the ability of gp for membrane association corresponded to virus viability and growth characteristics. the residues t (ω − , where ω is the gpi moiety at e ), p (ω − ), and m (ω + ) of gp were determined to be important for virus replication, with m being of particular importance for virus infectivity. the complete removal of the peptide–anchor domain in gp resulted in a complete loss of virus infectivity. the depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in prrsv infection. remarkably, gp was found to co-localize with cd in the lipid rafts on the plasma membrane. since cd has been reported as a cellular receptor for prrsv and gp has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus. porcine reproductive and respiratory syndrome (prrs) is a recently emerged viral disease and causes significant economic losses to the swine industry today. the etiological agent is the prrs virus (prrsv), which is a member of the family arteriviridae (meulenberg et al., (meulenberg et al., , wensvoort et al., ; nelson et al., ) that includes other viruses such as equine arteritis virus (eav), lactate dehydrogenaseelevating virus of mice (ldv), and simian hemorrhagic fever virus (shfv). the family arteriviridae together with the families coronaviridae and roniviridae form the order nidovirales (cavanagh, ; cowley et al., ; gonzález et al., ; smits et al., ) . prrsv contains a single-stranded positive-sense rna genome of approximately kb that encodes two large non-structural polyproteins (pp a and pp a/ b) in the ′-terminal kb region and structural proteins in the ′-terminal kb region: gp (glycoprotein ), e (small envelope), gp , gp , gp , orf a, m (membrane), and n (nucleocapsid) proteins in order (firth et al., ; johnson et al., ; meulenberg et al., ; nelsen et al., ; snijder and meulenberg, ; snijder et al., ; wootton et al., ) . while n protein associates with the genomic rna and makes up the viral capsid, the other proteins are membrane-associated. of these, gp and m form a disulfide-linked heterodimer (mardassi et al., ) that is essential for virus infectivity (faaberg et al., ; snijder et al., ) . the e protein is a myristoylated protein and likely functions as an ion-channel protein embedded in the viral envelope facilitating the uncoating of virus and release of the genome into the cytoplasm (lee and yoo, ) . gp , gp , and gp are minor glycoproteins and form a disulfide-linked heterotrimer essential for viral infectivity (wieringa et al., a (wieringa et al., , b . co-expression of e, gp , gp , and gp results in the transport of these proteins from the endoplasmic reticulum (er) through the golgi, suggesting an important role of the hetero-multimerization for virus assembly and maturation (wissink et al., ) . orf a is a newly identified membrane protein encoded in the internal orf within orf with an unknown function (firth et al., ; johnson et al., ) . gp protein is of and amino acids for the north american (type ii) and european (type i) prrsv, respectively (meulenberg et al., ; murtaugh et al., ) . amino acid sequence analysis of gp reveals two distinct hydrophobic domains, one at the extreme n-terminus at positions - and the other at the c-terminus at - , which likely functions as the signal peptide and a membrane anchor, respectively (meulenberg et al., ) . gp however has a unique structural feature, not commonly seen in the class i-type integral membrane protein since gp does not contain a cytoplasmic tail which normally protrudes into the lumen once it associates with the membrane. the reason(s) for the lack of the cytoplasmic tail in gp is unknown. glycosyl-phosphatidylinositol (gpi) anchor is a c-terminal posttranslational modification found in some eukaryotic proteins residing in the outer leaflet of the cell membrane. genes encoding gpi-anchored proteins specify two signal sequences in the primary translation product: an n-terminal signal sequence for er targeting and a c-terminal hydrophobic sequence that directs its association to the membrane via the lipid (orlean and menon, ) . the process for gpi biosynthesis takes place in the er (takeda and kinoshita, ) , and the proteins subjected to gpi-modification enter the er lumen via the n-terminal er targeting signal which is cleaved off by a signal peptidase in the lumen (gerber et al., ) . in addition to the n-terminal signal sequence, gpi-anchored proteins contain a c-terminal hydrophobic sequence that directs the cleavage of the signal sequence from the protein and the replacement with a preformed gpianchor by action of the transamidase complex (ikezawa, ) . the gpi-linked proteins are then targeted to the membranes. the structural feature of prrsv gp resembles that of a gpi-anchored protein. hundreds of functionally and structurally diverse proteins have been identified as gpi-anchored proteins including the ns protein of dengue virus (jacobs et al., ) , prion proteins for transmissible spongiform encephalopathies (taylor and hooper, ) , adhesion molecules (dustin et al., ) , decay accelerating factor (daf; nickells et al., ) , just to name a few (see a review; ikezawa, ) . gpi modification serves a variety of functions including directing proteins to the cell surface (lisanti et al., ) , association with lipid rafts (taylor and hooper, ) , lymphocytes activation (robinson et al., ) , and signal transduction (cary and cooper, ; jacobs et al., ) . the gpi anchor in particular has the propensity to target proteins to lipid rafts (brown and london, ; metzner et al., ) . lipid rafts are dynamic assemblies of the lipid-ordered phase of microdomains that are highly enriched with cholesterol and sphingolipids in the exoplasmic leaflet of the plasma membrane (ikonen, ; simons and toomre, ) . lipid rafts compartmentalize cellular processes by serving as organizing centers for assembly of signaling molecules, influencing membrane fluidity and protein trafficking, endocytosis, transcytosis as well as for hostpathogen interactions (ikonen, ; pike, ; simons and toomre, ; van der goot and harder, ) . for viruses, lipid rafts play an important role in viral entry (ewers and helenius, ); norkin, ; parton and lindsay, ; vieira et al., , assembly (manié et al., ono and freed, ) and budding (chazal and gerlier, ; scheiffele et al., ) . some viral membrane-associated and also envelope proteins such as the gag protein of human immunodeficiency virus type (hiv- ) (ono and freed, ) , the hemagglutinin of influenza virus (takeda et al., ) , the tegument protein of herpes simplex virus (lee et al., ) , the ns protein of dengue virus (noisakran et al., ) , and the spike protein of severe acute respiratory syndrome coronavirus (sars-cov) (lu et al., ) associate with the lipid rafts. the involvement of lipid rafts in prrsv infection has not been examined. in the present study, we provide evidence that the viral gp protein is a gpi-anchored protein, which co-localizes with the prrsv receptor, cd in the lipid rafts and may be involved in the viral entry process. the topology of gp predicts two hydrophobic domains on the protein; one at residues - and the other at - (http:// mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred; claros and von heijne, ) . the n-terminal hydrophobic domain likely functions as the signal peptide to direct the protein to er membrane and the c-terminal hydrophobic domain to anchor the protein to membrane, but unlike the common structure seen in type i membrane glycoproteins, gp does not possess a hydrophilic cytoplasmic tail following the hydrophobic anchor domain at the c-terminus (fig. ) . the cytoplasmic tail generally protrudes into the lumen when the protein is associated with the er membrane, and the unusual feature of gp resembles the topology of gpi-anchored protein, for example, daf which is a well-characterized gpi-anchored protein (nickells et al., ) . when gp sequences were analyzed using the gpi prediction program (http://gpi.unibe.ch; fankhauser and mäser, ) , a gpi anchor signal was readily detectable. when a large number of gp sequences were examined including the european and north american genotypes, the european-like prrsv circulating in the usa, and the highly virulent prrsv emerged in china (fig. a) , the gpi anchor signal became more prominent and appeared to be highly conserved across the genotypes including ldv and eav. thus, we hypothesized that the prrsv gp protein might be modified by gpi attachment and anchored with the membrane through the gpi. since gp is a minor protein and thus its expression level is low, and because a suitable antibody for gp is not available, a gp -egfp fusion construct was made using a linker of five glycine residues inserted between the egfp and gp sequences to facilitate the detection of gp in cells (fig. b; tsuneki et al., ) . when hela cells were transfected with the pegfp-gp plasmid, the gp -egfp fusion protein of about kda was expressed ( fig. a, lane ) . in addition, a minor band of kda which was slightly larger than egfp alone was identified (fig. a, lane , Δgp ) , suggesting that the c-terminal portion of gp might have been cleaved and the cleaved portion which was fused with egfp was detectable by egfp antibody. such band was absent in cells transfected with pegfp-n which is a fusion construct of the prrsv n protein with egfp ( fig. a, lane ) . this result suggests that gp is possibly a gpi-anchored protein and not all but some of the gp molecules are modified by gpi attachment. a gpi-anchored protein can be distinguished from its un-modified form by its mobility (ikezawa, ) , and thus the migration pattern of gp was examined by sds-page and western blot. hela cells naturally express human daf as a kda protein before gpimodification (karnauchow et al., ) and after glycosylation and gpi-modification, it becomes approximately - kda (nickells et al., ) , which can readily be detectable by daf mab from cell lysates (fig. b, lane ) . to facilitate the detection of gp while maintaining its structural integrity as much as possible, the flag sequence tag of 'ykddddk' was inserted between positions d and e of gp , and pxj -flag-gp was constructed (fig. c) . hela cells transfected with pxj -flag-gp produced the gp protein of approximately kda (fig. b , lane , lower arrow). this form of gp was predominant among two other forms of gp produced in these cells. gp is an integral membrane protein with multiple glycans added onto it (meulenberg et al., ; wissink et al., ) , and the kda protein and two additional bands of smaller sizes likely represent the fully mature and the partially glycosylated forms of gp as previously observed (das et al., ) . a kda band was identified (fig. b, lane , upper arrow) , and this is likely the fully modified gpi-anchored form of gp . to determine the gpi modification of gp , phosphatidylinositolspecific phospholipase c (pi-plc) digestion was conducted. pi-plc is a lipase known to cleave and release a gpi-linked protein from the cellular membrane, and therefore the release of a soluble protein by pi-plc digestion is considered standard biochemical evidence for gpi-anchored proteins (ikezawa, ) . for this experiment, hek- cells were transfected with pxj -flag-gp and at hpt, trypsinized and digested with pi-plc in suspension. the supernatants were then analyzed by western blot using anti-flag ab. in parallel, pi-plc-digested cells were subjected to facs analysis after staining with anti-flag ab. unexpectedly, no protein band equivalent to digested gp was observed from the supernatants, and the intensity of fluorescence was not diminished in pi-plc-digested cells as compared to the untreated cells (data not shown). total lysates of pi-plc-digested cells and untreated cells were also examined by western blot, but no difference was found in their migration patterns. the kda band did not change its migration after pi-plc digestion (data not shown), and we concluded that the gpi modification of gp was resistant to pi-plc digestion. when intact cells are treated with pi-plc, partial or total resistance to digestion may occur due to the inaccessibility of an enzyme to the cleavage site, expression of the protein on the cell surface in both gpi-modified and gpi-unmodified hydrophobic peptide-anchored forms, or tight association of the gpi-modified protein with a plcnon-susceptible protein on the cell surface (rosenberry, ) . the prrsv gp protein may exist as the gpi-modified form and the gpiunmodified hydrophobic peptide-anchored form, which may have caused the inability of pi-plc digestion. thus to further determine if gp was a gpi-modified protein, two chimeric constructs were made such that the known gpi-anchor domain of human daf was substituted with the putative gpi-modification domain of gp protein (figs. a, b) and the c-terminal hydrophobic region of gp protein of prrsv (fig. c ). the residue cross-linked to the lipid moiety is termed ω site, and upstream residues are designated ω-minus while downstream residues are designated ω-plus with respect to their positions from the ω site (ikezawa, ) . the ω site of human daf is serine at position (s ) (nickells et al., ; fig. a) , which is identical to the computer prediction (gpi modification prediction; http:// mendel.imp.ac.at/gpi/gpi_server.html). by using the same program, the potential ω site for gp was predicted to be at e , and based protein of prrsv pa . gp contains two hydrophobic domains (hd), one at the n-terminus and the other at the c-terminus. gp was cloned to express as a fusion protein with egfp using a linker of five glycine ( g) residues. 'ω' at position (e ) indicates the putative gpi modification site, and the arrow between positions and indicates the potential cleavage site specific for pi-plc lipase. numbers indicate amino acid positions. (c) the flag-tag sequence of ykddddkgs was inserted between positions d and e (nucleotide sequence positions and ) of gp gene cloned in pxj , and pxj -flag-gp was constructed. on this prediction the region of - of gp was chosen to replace with the - fragment of human daf to construct pxj -daf/ (fig. b ). the prrsv gp protein was chosen to serve as a gpinegative control and the - fragment of gp was used to replace the - region of daf. this construct was designated pxj -daf/ (fig. c ). using the daf/ fusion construct, gpi modification of gp was reexamined by pi-plc digestion. the endogenous expression of human daf was high in hela cells whereas negligible and undetectable in hek- cells (data not shown). thus, hek- cells were used in this study to express daf/ . daf of orangutan erythrocytes (e or ) migrates as an kda protein, and after digestion with pi-plc, its . for gp , the ω site is predicted at e , and the putative lipase cleavage site is predicted between e and t (arrow). (c) gp is type i integral membrane protein, and the c-terminal amino acids of human daf were replaced with the c-terminal amino acids representing positions and of prrsv gp to generate daf/ . this region of gp contains a hydrophobic trans-membrane sequence plus a short hydrophilic cytoplasmic tail. numbers for - indicate amino acid positions with respect to gp . migration reduces to kda (nickells et al., ) . in our study, the molecular migration of human daf in hela cells decreased from - kda to - kda when digested with pi-plc (fig. a , lanes , ), which was consistent with the orangutan report. after pi-plc digestion, fluorescent cells by daf staining were also reduced by % in hela cells (figs. , ba, ca) , confirming the efficient digestion of daf by pi-plc. in contrast, hek- cells transfected with the empty vector pxj showed no fluorescence for daf (figs. , bb, cb) , and no cleavage product was released by pi-plc digestion (fig. a, lane ) . thus, hek- cells were transfected with pxj -daf, pxj -daf/ , or pxj -daf/ , and analyzed by pi-plc digestion followed by western blot (fig. a) , fluorescence staining (fig. b) , and facs (fig. c) analyses. daf/ , daf, and daf/ were all expressed on the cell surface and detectable by daf-specific mab evr (figs. b, c; panels c, d, e) . after digestion with pi-plc, the fluorescence was lost in daf and daf/ expressing cells, and daf and daf/ proteins cleaved by pi-plc were released to the supernatants, which were then detectable by western blot (fig. a, lanes , ) . in contrast, the intensity of fluorescence and the percentage of fluorescence-positive cells were unaffected for daf/ by pi-plc treatment (fig. b, panel c; fig. c , panel c), and the daf/ protein was not detectable in the supernatant (fig. a, lane ) . these results show that the c-terminal region of gp was modified for pi-plcspecific gpi-attachment, whereas gp protein did not undergo such a modification, which is consistent with previous reports that gp anchors on the cell membrane through the c-terminal hydrophobic transmembrane domain (meulenberg et al., ; wissink et al., ) . mutational studies on the gpi-anchor region since our data showed that prrsv gp was a gpi-modified protein, the importance of individual amino acids surrounding the gpi attachment site (ω site) was examined for pi-plc cleavage. using pxj -daf/ as the parental construct, six mutants were constructed such that the residues adjacent to e (the ω site) were individually changed to valine ( fig. a ; gerber et al., ; furukawa et al., ) . following expression in transfected cells and after pi-plc digestion of wild-type daf/ , the cleaved product released into the supernatant was identified as a - kda band (fig. b, lane ) . mutants daf/ -m v (ω − ) and daf/ -e v (ω) showed similar cleavage properties to those of wild-type daf/ as inferred from the intensity of the cleaved product (fig. b, lanes , ) and the percentage of positive cells (fig. c, panels a, d; fig. d , panels a, d). for daf/ -t v (ω + ), the fluorescence intensity was × as compared to × for daf/ ( these results show that the residues surrounding the ω site of gp contribute to the ability of gpi to anchor to the membrane. these experiments were repeated three times for confirmation, and the representative results are shown in figs. c and d. in summary, all mutations but m * retained the ability of gpi-anchorage to various extents. to determine the significance of residues adjacent to the ω site of gp for virus infectivity, the respective mutations were introduced into the prrsv infectious cdna clone and a total of mutant viral genomic clones were generated. the prrsv infectious cdna clone was modified to place the full-length genomic sequence under the eukaryotic promoter and thus dna transfection of the full-length genomic clones can produce infectious progeny in transfected cells. thus, the wild type and mutant genomic clones were transfected into marc- cells and the cells were incubated for days. cytopathic effects (cpe) were evident for prrsv-gp -wt and some of the mutants. culture supernatants were collected and designated 'passage- '. for mutants that did not produce visible cpe, extracellular and intracellular rnas were examined for the presence of viral genome by rt-pcr for n gene at passages and . the mutant viruses prrsv-gp -m v (ω − ), prrsv-gp -e v (ω), and prrsv-gp -t v (ω + ) grew to titers similar to that of prrsv-gp -wt (fig. a ), and these were the mutants that did not impair the ability for gpianchorage in hek- cells. these mutant viruses grew normally and induced cpe typical for prrsv. mutation at ω − (t v), ω − (p v), and ω + (m v) appeared to be important for virus growth as the titers for prrsv-gp -t v, prrsv-gp -p v, and prrsv-gp -m v decreased in 'passage ' and 'passage ' (fig. a ). their growth kinetics was also slower compared to that of prrsv-gp -wt (fig. b) , and their characteristics were consistent with their fluorescence staining. in particular, prrsv-gp -m v (ω + ) appeared to affect the virus infectivity most notably. prrsv-gp -m v grew slowly and its titer was also low. the truncation mutant prrsv-gp -m * did not exhibit any sign of infectivity (figs. a, b) . furthermore, no viral rna was detectable at passages and and thus it was concluded that the lack of gpi-anchor, thus the lack of membrane association, was lethal for prrsv infectivity. for mouse hepatitis coronavirus, a member virus in the order nidovirales, the cholesterol levels on the cell membrane determine the susceptibility of cells to virus infection, and lipid rafts are required for virus entry and membrane fusion (choi et al., ) . since cholesterol is a major component residing in the lipid rafts of the cell membrane, we examined whether lipid rafts were involved in the entry of prrsv in permissive cells. a cholesterol-depletion experiment was conducted using methyl-β-cyclodextrin (mβcd) in both marc- and pam cells. mβcd is not incorporated into membranes but extracts cholesterol selectively from membranes by binding it in the central non-polar cavity and thus depleting from the plasma membrane (ilangumaran and hoessli, ) . to avoid the possibility that the newly synthesized cholesterol and/or cholesterol from internal compartments may restore the rafts and affect the entry of virus, incubation of mβcdtreated cells with prrsv was limited to h at °c. it was apparent that the mβcd treatments of marc- and pam cells impaired the production of prrsv in a dose-dependent manner (figs. a, b) , suggesting an essential role of cholesterol for prrsv entry. to confirm the involvement of cholesterol in prrsv infectivity, a depletion reversion study was conducted. cells were treated with mβcd for h and then, the depletion was reversed by supplementing with cholesterol at various concentrations, followed by prrsv infection and examination of virus yield from these cells. after replenishment with cholesterol, the virus titer increased dramatically, and at μg/ml of cholesterol supplementation, the production of virus was almost fully restored in both marc- and pam cells (figs. c, d) .these data indicate that the reduction of virus production by mβcd was due to the depletion of cholesterol from the cells and this effect was reversible, suggesting a role of lipid rafts in prrsv infection. the importance of cholesterol for prrsv infection suggests that viral proteins may interact with cellular proteins in the lipid rafts during entry. gpi modification has the propensity to target the gpiproteins to lipid rafts (brown and london, ) and since prrsv gp is a gpi-anchored protein, its localization in the lipid rafts was first examined by dual staining immunofluorescence. daf (a synonym of cd ) is a resident protein in the lipid rafts and thus is frequently used as a marker for lipid rafts (stuart et al., ) . since hela cells constitutively express daf which is readily detectable by daf-specific mab evr (fig. b, lane ; fig. a ), hela cells were used to examine co-localization of gp and daf proteins. the gp protein fused with a flag-tag was detectable on the cell membrane (figs. e, h) and was co-localized with cd (figs. f, i), demonstrating that the prrsv gp protein is a membrane protein localized in the lipid rafts in hela cells. co-localization of gp with cd in the lipid rafts cd has been studied as a cellular receptor for prrsv, and cd renders prrsv non-permissive cells permissive for infection (calvert et al., ; lee et al., ; patton et al., ) . cd has been shown to localize and form high partition in the lipid rafts (wolf et al., ) . since gp co-localizes with daf (fig. ) , colocalization of gp with cd was examined. for this study, cells of the porcine origin were used to co-express both gp and cd . dulac cells are porcine kidney cells but non-permissive for prrsv infection, and thus using these cells, a cell line was generated to stably express porcine cd which was then designated dulac-cd . dulac-cd cells were rt-pcr positive for cd transcription (fig. a, lane ) and the protein was also expressed (figs. b, c). dulac-cd cells became permissive for prrsv infection and the gfp expression was evident when infected with prrsv-p -gfp virus (fig. d) , confirming the expression of porcine cd and infection of prrsv in these cells. when dulac-cd cells were transfected with pxj -flag-gp , cd and gp were found to be co-localized on the plasma membrane (figs. , f, i). these results demonstrate the interaction of gp with cd in the lipid rafts and suggest that this interaction may participate in the initial stage of virus entry into cells. the signal for gpi modification of a protein consists of three parts: a stretch of three amino acids including the residue where gpi attaches (the ω site), a hydrophobic segment of - amino acids, and a hydrophilic spacer segment of usually less than amino acids between them (furukawa et al., ) . for modification, the hydrophobic segment is first cleaved and the lipid is then attached (gerber et al., ) . the gp protein satisfies the above conditions with a hydrophobic segment of residues at the c-terminus and a hydrophilic space of amino acids between the hydrophobic segment and the ω site (fig. a) . in the present study, we provide evidence that the prrsv gp protein can undergo post-translational modification for gpi attachment and anchors to the membrane via the lipid. the gp -egfp fusion protein was cleaved at the ω site and the kda peptide was likely the cleavage product representing the c-terminal portion of gp fused with egfp. gp existed in two forms to anchor to the membrane, and this observation was consistent with other lipid-anchored proteins such as lfa- (lymphocyte functionassociated antigen ) and ncam- (neural cell adhesion molecule ) as they also exist in two forms, a hydrophobic peptide-anchored form and a lipid-anchored form (arai et al., ; cross, ; dustin et al., ) . these alternative forms behave differently. the routes (rothberg et al., ) and rates (keller et al., ) for endocytosis virus at a multiplicity of infection (moi) of , and their supernatants were harvested at indicated times. virus titers were determined by a microtitration infectivity assay and recorded as tissue culture infectious dose (tcid) /ml. experiments were conducted in duplicate and repeated three times. the data are shown as mean titers ± standard error. of the gpi-anchored and hydrophobic peptide-anchored proteins differ. in addition to the transport differences, the lipid-anchored and peptide-anchored forms of a same protein can also have different biological properties. the lymphocyte surface molecules ly- a/e and qa- only activate t cells when present as a gpi-anchored form (robinson et al., ; su et al., ) . the gp protein expressed as the flag-gp fusion protein appeared to be resistant to pi-plc digestion, and this was likely due to the co-existence of lipid-anchored and peptide-anchored forms of gp . in contrast, the daf/ construct became sensitive to pi-plc digestion, and constructs made in the similar way have been shown to be useful for some proteins including cd for the study of hiv. hiv efficiently infected human cells expressing the gpi-anchored cd receptor (diamond et al., ; jasin et al., ) and the cd -gpi fusion protein was efficiently cleaved by pi-plc (anderson et al., ) . replacement of the gpi modification signal by a hydrophobic peptide segment leads to expression of the protein as a peptide-anchored membrane protein (takeda and kinoshita, ) , and this was consistent with our finding that daf/ , in which the gpi modification signal in daf was substituted with the hydrophobic segment of gp , resulted in peptide-anchored membrane association and thus non-digestible by pi-plc. this finding confirms that unlike gp , gp is a type i integral membrane protein (meulenberg et al., ; wissink et al., ) . in contrast to daf/ , daf/ was successfully digested by pi-plc, demonstrating that the c-terminal region of gp indeed possessed the gpimodification signal. m v (ω − ), e v (ω), and t v (ω + ) mutations of gp did not affect the gpi-modification or the growth of mutant viruses, but t v (ω − ), p v (ω − ), and m v (ω + ) were important for both gpi modification and the virus infectivity. the residues at positions ω − , ω − , and ω + were highly conserved among different prrsv isolates of the north american and european genotype, and among those three mutations, m v (ω + ) contributed the most to the gpi modification and virus infectivity. the m * mutation, the c-terminal amino acids truncation mutant, was lethal for infectivity, indicating the essential requirement of this hydrophobic segment for prrsv infection. this is the first report that the gpi-anchor of a viral membrane protein contributes to viral infectivity and growth rates. for viruses to infect target cells, they first bind to a specific receptor on the cell surface. for prrsv, cd is the cellular receptor (calvert et al., ) and has been recently shown to interact with gp and gp proteins (das et al., ) . cd is a macrophagespecific protein in the cysteine-rich scavenger receptor superfamily comprising a large number of cell surface and soluble glycoproteins involved in the recognition of various ligands of proteins, polyribonucleotides, polysaccharides, and lipids (sarrias et al., ) . the localization of a receptor in the lipid rafts is crucial for entry of some viruses (ewers and helenius, ) including sv and murine leukemia virus (lu and silver, ; pelkmans et al., ) . for nidoviruses, cd is the cellular receptor for human coronavirus e and is localized in the lipid rafts (nomura et al., ) , and the importance of lipid rafts for virus replication has also been documented (lorizate and kräusslich, ; lu et al., ; thorp and gallagher, ) . we show in the present study that gp localizes to the lipid rafts of the plasma membrane where it associates with cd , the cellular receptor for prrsv. the lipid-modification of gp contributes to prrsv infectivity, suggesting an important role of gpi for prrsv entry and infection. the daf/ -m * mutant was lethal for infectivity, which is probably due to the inability of gp to anchor to the membrane, and thus unable to associate with the lipid rafts, supporting the notion that the lipid rafts play an important role for prrsv infection. co-localization of gp and cd in the lipid rafts may mediate the entry of prrsv. compared to peptide-anchored gp , lipid-anchored gp is likely to have the priority to locate in lipid rafts where it binds to cd to promote the entry of prrsv. this may explain why certain mutations affecting the ability of lipid-anchor formation of gp on the cell membrane affected the titers and growth of prrsv as seen in other studies (metzner et al., ) . taken together, we have shown here that the prrsv gp protein is a gpi-modified membrane-associated protein. co-localization of gp with cd in the lipid rafts on the fig. . co-localization of prrsv gp and the daf (cd ) protein as a lipid raft marker on the plasma membrane of hela cell. cells were transfected with pxj -flag-gp (d through f) and at h post-transfection, washed with ice-cold pbs. cells were then co-stained with daf-specific mab evr (anti-cd ; a and d) and rabbit anti-flag antibody (b and e), followed by staining with alexa fluor ® conjugated goat anti-mouse igg (h + l) and alexa fluor ® conjugated goat anti-rabbit igg (h +l) secondary antibodies, respectively. images were visualized using a laser-scanning confocal fluorescence microscope (model bx , olympus). panels g through i represent the enlargement of the indicated areas in panels d through f, respectively. cell membrane implicates an important role of the complex for prrsv entry and infection. these findings deepen our understanding on gp and establish the cholesterol and lipid rafts as potential targets in the development of control measures against prrsv infection. marc- cells (a subline of ma- african green monkey kidney cells; kim et al., ) were maintained as described elsewhere (lee et al., ) . porcine alveolar macrophages (pams) were kindly provided by dr. f. zuckermann (university of illinois at urbana-champaign, urbana, il) and grown in rpmi- (invitrogen, carlsbad, ca) containing % fetal bovine serum (fbs; hyclone, logan, ut). hela and hek- cells (nih aids research and reference reagent program, germantown, md) were grown in dulbecco's modified eagle medium (dmem) supplemented with % fbs. dulac porcine kidney cells (obtained from dr. l. babiuk, vaccines and infectious disease organization, saskatoon, canada) were free of porcine circovirus type and grown in modified eagle's medium (mem) supplemented with % fbs. stocks of prrsv (north american genotype strain pa ; wootton et al., ) were prepared in marc- cells. prrsv p -gfp expressing gfp is described elsewhere (pei et al., ) . culture supernatants harvested at days post-transfection with prrsv infectious clones were designated 'passage- '. the 'passage- ' virus was used to inoculate fresh marc- cells, and the -day harvest was designated 'passage- '. the 'passage- ' virus was prepared in the same way as for 'passage- '. each passage virus was aliquoted and stored at − °c until use. virus titers of 'passage- ' and 'passage- ' were determined in marc- cells by a microtitration infectivity assay and recorded as % tissue culture infectious dose per ml (tcid /ml). to determine the growth kinetics of the mutant viruses, marc- cells were infected in duplicate with 'passage- ' virus at a multiplication of infection (moi) of and incubated for indicated times. culture supernatants were collected and titrated by a microtitration infectivity assay and recorded as tcid /ml. monoclonal antibody (mab) against human daf (clone g anti-daf [aa: - ]), anti-flag mab (clone m ), and anti-flag rabbit antibody were from sigma-aldrich (st. louis, mo); anti-gfp rabbit antibody from cell signaling technology (danvers, ma); β-actin mab (ac- ) from santa cruz biotechnology (santa cruz, ca); and anti-porcine cd mab (clone a / ) from abd serotec (raleigh, nc). tissue culture supernatant containing mab evr raised against human daf was kindly provided by k. dimmock (university of ottawa, ottawa, canada) and described elsewhere (karnauchow et al., ) . alexa fluor ®-conjugated goat anti-mouse igg (h + l), alexa fluor ®-conjugated goat anti-rabbit igg (h+ l), and g (neomycin sulfate analog) were purchased from invitrogen (carlsbad, ca). horseradish peroxidase (hrp) conjugated-goat anti-mouse igg and -goat anti-rabbit igg were purchased from jackson immunoresearch laboratories (west grove, pa). phosphatidylinositol-specific phospholipase c (pi-plc) of bacillus cereus, methyl-β-cyclodextrin (mβcd), and water soluble cholesterol were purchased from sigma-aldrich. the eukaryotic expression vector pxj is a derivative of pxj in which the polylinker region was modified (xiao et al., ) . the plasmid pegfp-n contains the prrsv n gene fused to egfp . the prrsv infectious cdna clone pcmv-s-p is described elsewhere (lee et al., ; lee and yoo, ) . to construct pegfp-gp , prrsv gp gene was pcr-amplified from pcmv-s-p using the gp -gfp-fwd and gp -gfp-rev primer pair (table ) and inserted into ecori and bamhi sites in pegfp-n (clontech; mountain view, ca). to generate pxj -flag-gp , the flagtag sequence (ykddddkgs) was inserted between nucleotide positions and (corresponding amino acid positions d and e , respectively) of gp by three-point ligations of pcr products generated using the primer pairs, flag-gp -a and -b, and flag-gp -c and -d (table ) at ecori, bamhi, and hindiii sites (fig. f) . the human daf gene was amplified from plasmid pef /v -daf (provided by k. dimmock, university of ottawa, ottawa, canada) using daf-fwd and daf-rev primers ( table ). the pcr product was digested with ecori and bamhi followed by cloning into pxj to generate pxj -daf. pxj -daf/ was created by overlapping extension pcr (horton et al., ) . briefly, the region representing amino acid positions - of human daf was amplified from pef /v -daf using daf-fwd and daf/ -b primers. the c-terminal region of gp (gp -c, positions - ; fig. ) was amplified from plasmid pcmv-s-p using primers daf/ -c and daf/ -d (table ) . then, overlapping extension pcr was conducted using the pcr products of daf ( - aa) and gp -c ( - aa) as templates and primers daf-fwd and daf/ -d (table ). the daf/ fusion gene ( - aa fragment of daf and gp -c fragment) was inserted at ecori and bamhi sites in pxj to generate pxj -daf/ (fig. ) . plasmid pxj -daf/ was constructed in a similar way using gp -c representing - aa of gp (fig. ) . the primer sequences are listed in table . the topology of gp (http://mobyle.pasteur.fr/cgi-bin/portal.py? #forms::toppred; claros and von heijne, ) and the gpi anchor attachment signal (http://gpi.unibe.ch; fankhauser and mäser, ; eisenhaber et al., ) were predicted using on-line programs available on the bioinformatics resource portal (http://expasy.org/ tool). specific mutations were introduced to plasmids pxj -daf/ and pcmv-s-p using the quikchange xl site-directed mutagenesis kit (stratagene, la jolla, ca) with modifications as described elsewhere . plasmid dna transfection was carried out using lipofectamine™ according to the manufacturer's instruction (invitrogen, carlsbad, ca). hela cells were seeded in -mm diameter dishes and grown to % confluency and transfected with μg of dna diluted in opti-mem (invitrogen, carlsbad, ca). after h incubation, transfection medium was replaced with dmem supplemented with % fbs. at h post-transfection (hpt), cells were washed with pbs and lysed in the buffer ( mm tris-hcl [ph . ], mm nacl, mm edta, mm egta, % tritonx- , % np- and mm pmsf) for western blot analysis. for transfection of pxj -daf, pxj -daf/ , pxj -daf/ , and their mutant derivatives, hek- cells grown in -mm diameter dishes were transfected using μl lipofectamine and μg individual plasmid in ml opti-mem. after h incubation, the transfection mix was replaced with dmem supplemented with % fbs. at hpt, cells were washed once with pbs and trypsinized for pi-plc digestion and facs analysis. for transfection of prrsv infectious clone pcmv-s-p and its derivatives, marc- cells grown in -mm diameter dishes were used. marc- cells were transfected with μg of an infectious clone using lipofectamine as described above. transfected cells were incubated at °c for days in dmem supplemented with % fbs prior to collection of supernatant for virus recovery. hela cells or hek- cells were transfected with pxj -daf, pxj -daf/ , pxj -daf/ or their derivatives and incubated for day. cells were washed with pbs, trypsinized, and collected by centrifugation at rpm for min (eppendorf r). then, the cells were washed twice using pi-plc buffer ( mm tris-hcl [ph . ], mm nacl, . % bovine serum albumin [bsa] ) and resuspended in pi-plc buffer. cells were divided into two equal fractions: one for pi-plc treatment ( . u/ml; sigma-aldrich) and another as a control without treatment. following incubation at °c for min with pi-plc, both fractions of cells were centrifuged at rpm for min (eppendorf r). the supernatants were subjected to western blot analysis whereas the cell pellets were washed twice in ice-cold facs buffer ( . % bsa, . % sodium azide in pbs) followed by fluoresceinactivated cell sorter (facs) analysis. pi-plc-treated or untreated cells were stained with daf-specific mab evr at °c for min. cells were washed twice in ice-cold facs buffer and fixed with % paraformaldehyde in pbs (ph . ) for h at °c. after wash with facs buffer, cells were incubated with alexa fluor ®-conjugated goat anti-mouse igg (h + l) secondary antibody for h, washed twice with facs buffer, and resuspended in % paraformaldehyde in pbs (ph . ) for flow cytometry (bd biosciences lsr ii analyzer). the data were analyzed using the facs express software (ver. research edition, de novo software; los angeles, ca). the supernatants obtained from pi-plc digested cells were centrifuged to remove cell debris at , rpm for min in a microcentrifuge (eppendorf r). the supernatants were collected and boiled in sds-page loading buffer ( mm tris-hcl [ph . ], % sds, . % bromophenol blue, % glycerol, % β-mercapto ethanol) for min, followed by % sds-page and transfer to polyvinylidenedifluoride (pvdf) membranes (millipore). the membranes were blocked with % non-fat dry milk in tbst ( mm tris-hcl [ph . ], mm nacl, . % tween ) for h at room temperature (rt) and incubated with rabbit anti-gfp antibody, anti-flag mab, or anti-daf mab overnight at °c. after washes for min each with tbst, the membranes were incubated with hrp-conjugated goat anti-rabbit or goat antimouse igg for h, washed five times for min each with tbst, and developed using supersignal® west pico chemiluminescent substrate according to the manufacturer's instruction (pierce, rockford, il). cells resuspended in % paraformaldehyde in pbs for facs analysis were spotted on microscope slides and visualized using a fluorescent microscope (olympus, model ax ). dulac-cd cells infected with prrsv p -gfp were also examined directly by fluorescent microscopy. cell cytotoxicity of mβcd was determined for both marc- cells and pams in -well plates at concentrations of , . , . , , , , , and mm. ten millimolar or higher concentration of mβcd was found to be toxic for marc- cells and pams, and thus . - mm concentrations were chosen for subsequent studies. marc- cells and pams were treated with variable concentrations of mβcd for h at °c and infected with prrsv pa at a moi of . after h incubation, cells were washed twice and cultivated in dmem or rpmi- supplemented with % fbs. for cholesterol replenishment, cells were pretreated with mm mβcd for h at °c and then supplemented with variable concentrations of water soluble cholesterol for h at °c (popik et al., ) . after two washes with dmem for marc- cells or rpmi- for pams, the cells were infected with prrsv as above. marc- cells and pams were incubated for days and h, respectively, and the culture supernatants were collected and titrated in marc- cells as described above. dulac cells were transfected with pcdna-cd which contained the porcine cd gene, and selected for neomycin resistance using mg/ml of g in the culture medium. g -resistant cell colonies were picked using cloning cylinders and amplified for screening as described elsewhere (lee et al., ) . the gene expression was confirmed by pcr, rt-pcr, and protein assays by fa and western blot, and the cells expressing porcine cd were designated dulac-cd . hela, dulac, and dulac-cd cells were plated on microscope cover slips and transfected with pxj -flag-gp . at hpt, cells were washed once with ice-cold pbs and incubated with respective primary antibodies at °c for min as follows: daf-specific mab evr and anti-flag rabbit antibody for hela cells transfected with pxj -flag-gp ; cd -specific mab and anti-flag rabbit antibody for dulac-cd cells transfected with pxj -flag-gp . cells were washed twice in ice-cold pbs and fixed with % paraformaldehyde in pbs at °c for h. cells were then washed again three times with ice-cold pbs and incubated with alexa fluor ®-conjugated goat anti-mouse igg (h + l) or alexa fluor ®-conjugated goat anti-rabbit igg (h + l) antibodies. the coverslips were washed five times in pbs, mounted on microscope slides in mounting buffer ( % glycerol and . % sodium azide in pbs), and visualized under a laser-scanning confocal fluorescence microscope (model bx , olympus). intercellular transfer of a glycosylphosphatidylinositol (gpi)-linked protein: release and uptake of cd -gpi from recombinant adeno-associated virus-transduced hela cells association of neural cell adhesion molecule gene polymorphisms with bipolar affective disorder in japanese individuals functions of lipid rafts in biologicalmembranes cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses signal transduction: molecular switches in lipid rafts nidovirales: a new order comprising coronaviridae and arteriviridae virus entry, assembly, budding, and membrane rafts. microbiol murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release toppred ii: an improved software for membrane protein structure predictions gill-associated virus of penaeus monodon prawns: an invertebrate virus with orf a and orf b genes related to arteri-and coronaviruses glycolipid anchoring of plasma membrane proteins the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd human immunodeficiency virus infection is efficiently mediated by a glycolipidanchored form of cd myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth anchoring mechanisms for lfa- cell adhesion glycoproteinat membrane surface sequence properties of gpi-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase lipid-mediated endocytosis disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity identification of gpi anchor attachment signals by a kohonen self-organizing map discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production mutational analysis of the c-terminal signal peptide of bovine liver ′-nucleotidase for gpi anchoring: a study on the significance of the hydrophilic spacer region phosphatidylinositol glycan (pi-g) anchored membrane proteins. amino acid requirements adjacent to the site of cleavage and pi-g attachment in the cooh-terminal signal peptide a comparative sequence analysis to revise the current taxonomy of the family coronaviridae engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension glycosylphosphatidylinositol (gpi)-anchored proteins roles of lipid rafts in membrane transport effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane dengue virus nonstructural protein is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction glycosylphosphatidylinositol-anchored cd /thy- chimeric molecules serve as human immunodeficiency virus receptors in human, but not mouse, cells and are modulated by gangliosides novel structural protein in porcine reproductive and respiratory syndrome virus encoded in an alternative open reading frame present in all arteriviruses the hela cell receptor for enterovirus is decay-accelerating factor (cd ) endocytosis of glycophospholipid-anchored and transmembrane forms of cd by different endocytic pathways enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties a subpopulation of tegument protein vhs localizes to detergent-insoluble lipid rafts in herpes simplex virus-infected cells differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus gp and gp glycoproteins a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line role of lipids in virus replication ecotropic murine leukemia virus receptor is physically associated with caveolin and membrane rafts lipid rafts are involved in sars-cov entry into vero e cells measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus rafts, anchors and viruses-a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus characterization of proteins encoded by orfs to of lelystad virus comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of us and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies characterization of daf- , a high molecular weight form of decay-accelerating factor (daf; cd ), as a covalently cross-linked dimer of daf- association of dengue virus ns protein with lipid rafts human coronavirus e binds to cd in rafts and enters the cell through caveolae simian virus infection via mhc class i molecules and caveolae plasma membrane rafts play a critical role in hiv- assembly and release gpi anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids exploitation of major histocompatibility complex class i molecules and caveolae by simian virus modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages porcine reproductive and respiratory syndrome virus as a vector: immunogenicity of green fluorescent protein and porcine circovirus type capsid expressed from dedicated subgenomic rnas caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er the challenge of lipid rafts human immunodeficiency virus type uses lipid raft-colocalized cd and chemokine receptors for productive entry into cd (+) t cells a glycophospholipid anchor is required for qa- -mediated t cell activation a chemical modification that makes glycoinositol phospholipids resistant to phospholipase c cleavage: fatty acid acylation of inositol the glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus the scavenger receptor cysteine-rich (srcr) domain: an ancient and highly conserved protein module of the innate immune system influenza viruses select ordered lipid domains during budding from the plasma membrane lipid rafts and signal transduction phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses heterodimerization of the two major envelope proteins is essential for arterivirus infectivity a novel cell entry pathway for a daf-using human enterovirus is dependent on lipid rafts the glycosyl phosphatidylinositol anchor is critical for ly- a/e-mediated t cell activation gpi-anchor biosynthesis influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion the prion protein and lipid rafts requirements for ceacams and cholesterol during murine coronavirus cell entry novel g s mutation of human alpha nicotinic receptor promotes agonist-induced desensitization by a protein kinase c-dependent mechanism raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack host-cell lipid rafts: a safe door for micro-organisms? mystery swine disease in the netherlands: the isolation of lelystad virus intra-and intermolecular disulfide bonds of the gp b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity formation of disulfide-linked complexes between the three minor envelope glycoproteins (gp b, gp , and gp ) of equine arteritis virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate cloning, expression, and transcriptional properties of the human enhancer factor tef- the authors thank aimee bachand for construction of daf/ and daf/ and federico zuckermann for providing z-mac cells. this study was supported by the national research initiatives of the us department of agriculture cooperative state research education and extension service, grant number - - awarded to dy. key: cord- - oltss authors: patel, deendayal; opriessnig, tanja; stein, david a.; halbur, patrick g.; meng, xiang-jin; iversen, patrick l.; zhang, yan-jin title: peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: oltss porcine reproductive and respiratory syndrome (prrs) has been devastating the global swine industry for more than a decade, and current strategies to control prrs are inadequate. in this study we characterized the inhibition of prrs virus (prrsv) replication by antisense phosphorodiamidate morpholino oligomers (pmo). of peptide-conjugated pmo (ppmo), four were found to be highly effective at inhibiting prrsv replication in cell culture in a dose-dependant and sequence-specific manner. ppmo up and hp are complementary to sequence in the ′ end of the prrsv genome, and p and p to sequence in the translation initiation regions of orf and orf , respectively. treatment of cells with up or hp caused a . log( ) reduction in prrsv yield, compared to a control ppmo. combination of p and p led to higher level reduction than p or p alone. up , hp, and a combination of p and p inhibited prrsv replication in porcine alveolar macrophages and protected the cells from prrsv-induced cytopathic effect. northern blot and real-time rt-pcr results demonstrated that the effective ppmo led to a reduction of prrsv rna level. up and hp inhibited virus replication of other strains of prrsv. results from this study suggest potential applications of ppmo for prrs control. porcine reproductive and respiratory syndrome (prrs) causes an estimated us$ million in losses per year to the swine industry in the usa (neumann et al., ) . the clinical manifestations of prrs includes severe reproductive failure, post-weaning pneumonia, growth reduction, and increased mortality (keffaber, ; loula, ) . the causative agent of this disease is prrsv, an enveloped, single-stranded and positivesense rna virus (j.j. . prrsv is a member of the family arteriviridae, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus for years; however, there is evidence of reversion to virulence of at least one of the current vaccine strains following use in pigs (opriessnig et al., ) . therefore, alternative strategies are needed for effective prrs control. this study explored the inhibition of prrsv propagation in cell cultures by peptideconjugated phosphorodiamidate morpholino oligomers (ppmo) specific for prrsv sequences. pmo are structurally similar to single-stranded dna in that each subunit includes a purine or pyramidine base. in pmo each base is joined to a novel backbone consisting of one morpholine ring and phosphorodiamidate linkage per subunit (summerton, ; summerton and weller, ) . pmo are uncharged, water-soluble, and highly resistant to nuclease degradation (hudziak et al., ) . pmo bind to target mrna by watson-crick base pairing and exert an antisense effect by preventing access to critical segments of rna sequence, such as a translation initiation site, through steric blockade. this is a distinctly different process from the rnase h-dependent mechanism induced by the often-used antisense structural type phosphorothioate dna (summerton, ) . a number of studies have shown that pmo can effectively and specifically block translation of target mrna in vitro and in vivo via intravenous, intraperitoneal, subcutaneous, transdermal or oral administration (arora et al., a,b; brent and drapeau, ; hudziak et al., ; qin et al., ; stein et al., ; taylor et al., ) . it has been shown that pmo conjugated to short arginine-rich cell penetrating peptides have a significantly higher efficiency of delivery into cultured cells than do non-conjugated pmo (moulton et al., ; deas et al., ) . ppmo were found to be fairly stable in cells and human serum for at least h (youngblood et al., ) . sequence-specific antiviral efficacy of ppmo in cell cultures has been documented against sars coronavirus (neumann et al., ) , eav (van den born et al., ) , flaviviruses (deas et al., ; kinney et al., ) , influenza a virus (ge et al., ) , and kaposi's sarcomaassociated herpesvirus (zhang et al., ) . recently ppmo were also applied in vivo and protected animals from challenge with ebola virus (enterlein et al., ) , coxsackievirus b (yuan et al., ) , and murine coronaviruses (burrer et al., ) . prrsv has an rna genome approximately -kb in size, which consists of untranslated region (utr), nine open reading frames (orf a, orf b, orf a, orf b, and orfs through ) and utr (meng et al., ; j.j. meulenberg et al., ) . two large orfs, la and lb, together occupy over kb and encode non-structural proteins, which are likely involved in genome replication and transcription (j.j. . the remaining orfs, a through encode six membraneassociated proteins (gp , e, gp , gp , gp , and m) and nucleocapsid (n) protein (meulenberg et al., ) . in prrsvinfected cells, a set of six or seven nested viral subgenomic rna is formed (conzelmann et al., ; j.j.m. . all of the subgenomic rna have an identical -leader sequence derived from the end of genomic rna, as well as an identical terminal sequence preceding poly-(a) tails of variable length. the generation of co-terminal mrna species is believed to be accomplished through a process known as dis-continuous subgenomic mrna synthesis (sawicki and sawicki, ; van marle et al., ) . the end of each subgenomic mrna of north american strains contains a common leader sequence of bases (nelsen et al., ) , whereas in lelystad virus, the prototype strain of the european prrsv genotype, the leader is bases (j.j. . the utr of north american and european prrsv strains are and bases, respectively, excluding the poly-(a) tail, and is common to all of their subgenomic mrna. prrsv subgenomic rnas are polycistronic in structure, but it is believed that only the first open reading frame (orf) of each subgenomic rna is translated into a viral protein (meng et al., b) . in a previous study (zhang et al., ) , we tested six ppmo and found one of them ( up ), designed to target the terminal region of the prrsv genome, to be a highly effective inhibitor of prrsv replication in a sequence-specific and dose-dependent manner. considering the lengthy genome and sequence heterogeneity of prrsv strains, we sought to evaluate ppmo that target other sites of the genome and that have broad reactivity against different strains of prrsv, and also to further refine our evaluation of efficacy and ppmo mechanism of action, in the hopes of defining a potential strategy for addressing prrsv with ppmo technology. in this study, we evaluated a dozen ppmo targeting a variety of different sites in prrsv genomic, subgenomic and negativesense rna. the peptide component of the ppmo used in this study was recently developed, and possesses improved characteristics compared to the peptide used in our previous ppmo study. we found that it was possible to obtain a greater-thanadditive effect by using a combination of two ppmo. the effects of ppmo on prrsv rna synthesis were also assessed in an attempt to gain insight into the mechanism of ppmo-mediated inhibition of prrsv replication. ppmo are also shown in this study to inhibit prrsv replication in pam cultures, the primary target cells for prrsv infection in pigs. cell line atcc crl (meng et al., a) was maintained in dmem medium supplemented with % fetal bovine serum (fbs). prrsv strains atcc vr (meng et al., a) , lelystad (j.j. , and nvsl - (nvsl, ames, ia) were used to inoculate crl cells at . multiplicity of infection (moi) for ppmo testing. other prrsv strains used in this study include fl- , , , b, , , , and (kindly provided by dr. fernando osorio, university of nebraska-lincoln), and ingelvac mlv (kindly provided by dr. kay s. faaberg, university of minnesota). for virus titration, a series of fold dilutions of virus were added to monolayer crl cells in a -well plate. the degree of cytopathic effect (cpe), characterized by cell rounding, clumping and detachment, was assessed microscopically h after prrsv inoculation, in comparison with mock-infected cells. tissue culture infectious dose (tcid ) per milliliter was calculated based on cpe develop-ment according to the method of reed and muench, as described previously (zhang et al., ) . for pam culture, broncho-alveolar lavage was collected from to weeks old prrsv-negative piglets, as described previously (shibata et al., ) . broncho-alveolar lavage fluid was centrifuged at × g for min, and cell pellets resuspended in rpmi- medium supplemented with % fbs, mm lglutamine, u/ml penicillin, g/ml streptomysin sulfate and g/ml gentamycin. the cells were plated at a density of × cells/well in -well cell culture plates. pam were incubated for h at • c in a humidified % co incubator and washed once with plain rpmi- media before virus inoculation. ifa was carried out as reported previously (zhang et al., ) with an n-specific monoclonal antibody sdow (nvsl, ames, ia) (nelson et al., ) . specific reactions between sdow and n protein were detected with goat anti-mouse igg-fluorescein isothiocyanate (fitc) conjugate (sigma, st louis, mo) and observed with fluorescence microscopy. for pam cells, ifa was performed by fixation of pam cells with % paraformaldehyde and permeabilization with . % triton x- , followed by incubation with antibody sdow . pmo were synthesized at avi biopharma inc. (corvallis, or) by methods previously described (summerton and weller, ) . the peptide nh -(rxr) xb-cooh (where r = arginine, x = -aminohexanoic acid, and b = ␤-alanine) was covalently conjugated at the end of each pmo (fig. a) . the conjugation, purification, and analysis of ppmo were similar to the methods described elsewhere (abes et al., ; moulton et al., ) . a random sequence ppmo (named 'cp ') having little agreement with prrsv or primate mrna sequences was used as control for non-sequence-specific activity of the ppmo chemistry. ppmo were dissolved in water at a concentration of mm and stored at • c. for experiments, ppmo were diluted to desired concentrations in dmem. crl cells were seeded in -well plates at × cells per well and grown overnight to near confluency. treatment of crl cells was conducted as previously described (zhang et al., ) . briefly, cells were inoculated with virus at . moi for h at • c. after inoculum removal, ppmo diluted in dmem was added to the cells and incubated for h at • c. dmem medium without ppmo was included as a mock treatment control and ppmo cp as a negative control. after removal of ppmo, maintenance medium (dmem supplemented with % fbs) was added. the cells were then incubated for h at • c, after which both supernatant and cells were harvested for further analysis. for experiments using ppmo combination treatments, each of the two constituent ppmo were present at equal molar concentration. for the cross strain inhibitory assay, similar procedures as described above were used, except that different prrsv strains were used. macrophages were incubated in -well cell culture plates for h prior to treatment. cells were inoculated with prrsv strain vr at . moi and incubated for h. the cells were washed once with plain rpmi medium before addition of ppmo. the ppmo were diluted in plain rpmi medium, added to the cells and incubated for h at • c. the ppmo solution was removed and culture medium (rpmi supplemented with % fbs) was added. the cells were cultured and observed for cpe development. total rna was isolated from prrsv-infected crl cells by trizol ® reagent (invitrogen, carlsbad, ca). ppmotreated crl cells (same procedure as above) were grown in -well cell culture plates and harvested at h p.i. by direct lysis in trizol ® reagent after removal of culture supernatant. cells treated with cp or mock-treated were included as controls. rna was quantified by quant tm universal microplate spectrophotometer (biotek instruments, winooski, vermont) and stored at − • c for further analysis. rna samples ( . g) were denatured at • c for min and immediately placed on ice for min. the denatured rna was separated at v for h in % agarose gel containing formaldehyde. the separated rna were transferred onto nylon membrane and hybridized with a dna probe derived from prrsv orf sequence with the pcr primers sb f , -gcgga tccca aataa caccg gcaag c- and sb r , -cgtct agatg ccagc ccatc atgct gag- . the probe was labeled with dig- -dutp by pcr dig probe synthesis kit (roche diagnostics, indianapolis, in). the blotted membrane was incubated with anti-digoxigenin-alkaline phosphates for min, cspd chemiluminescent substrate (roche) for min, and then exposed on x-ray film. for quantitative rt-pcr analysis, rna was first treated with rnase-free dnase (promega, madison, wi) to remove carryover dna from the rna isolation procedure. reverse transcription was carried out using superscript tm iii first-strand synthesis system and random hexamers (invitrogen). real-time pcr primers were the same as reported previously (zhang et al., ) . a fragment of bases from the end of the prrsv genome was cloned into pcdna vector, and used as template to generate standard curves for the real-time pcr, which was conducted in chromo tm four-color real-time system (bio-rad laboratories, hercules, ca) with iq sybr green supermix (bio-rad). transcripts of ␤-actin were also amplified from the same samples in order to assure normalized quantitative rt-pcr detection of prrsv rna from the cells. pairs of dna oligonucleotides corresponding to ppmo target sequence in prrsv were duplexed and subcloned upstream of luciferase coding sequence in a t promoter-containing reporter plasmid, pcineoluc, as described previously (zhang et al., ) . dna sequencing was conducted to confirm the presence of desired sequences in the resulting plasmids. cp was used as a negative control in the reporter assay. each plasmid dna was linearized downstream of the luciferase sequence. in vitro transcription was conducted with the t ribomax tm express large scale rna production system (promega). in vitro translation was carried out with rabbit reticulocyte lysate translation system (promega). bright-glo tm luciferase assay system (promega) was used to detect luciferase yield. luminescence signals were measured with victor tm multilabel counter (perkin-elmer life and analytical sciences, wellesley, ma). the viability of crl and pam cells after ppmo treatment was determined with celltiter-glo ® luminescent cell viability assay (promega). briefly, cells were treated with ppmo under conditions similar to those described above in "ppmo treatment of crl cells". mock-treated cells were included for comparison. celltiter-glo reagent was added and incubated for min at room temperature. the luminescence signal was measured with victor tm multilabel counter. relative percentages of luminescence intensity were calculated by comparison to mock-treated controls. the student's t-test was used to assess the significance of differences of viral yield or rna level between the groups of ppmo-treated cells. a two-tailed p-value of less than . was considered significant. prrsv genomic sequences were retrieved from the gen-bank database. parallel sequence alignments of prrsv strains from each of the two major genotypes show that the and utrs, and the translation initiation regions of all orfs, within each genotype, are highly conserved. these regions likely participate in essential events of the viral life cycle (tan et al., ; verheije et al., ) , making them rational sites for antisense ppmo targeting. for this study, ppmo were designed against prrsv vr , a virulent strain of the north american genotype. ppmo sequences and target site locations in the prrsv genome are specified in table and depicted schematically in fig. b . the terminal region has proven to be a productive ppmo target region in a number of positive-strand rna viruses (burrer et al., ; deas et al., ; van den born et al., ; zhang et al., ) . ppmo up and hp were designed to basepair to the end region of prrsv rna genome, in an attempt to interfere with translation of viral rna replicases. hp is complementary to the hairpin loop region, which was predicted from secondary structure analysis ( ). ppmo complementary to negative-sense eav rna inhibited eav replication in cell culture (van den born et al., ) . nsp is complementary to the end of negative-sense prrsv rna, and was designed to interfere with the production of positivesense viral rna. nsp is complementary to negative-sense prrsv rna in the transcription regulatory sequence (trs) region, in an attempt to interfere with synthesis and/or transla- tion of the viral subgenomic rna. up is complementary to the end of prrsv rna genome, in an attempt to interfere with negative-strand rna synthesis. ppmo bp , p , p , p , p , p , and p are complementary to the translation initiation regions of orfs b, , , , , , and , respectively, to attempt blocking the translation of these orfs. ppmo up , which is complementary to nucleotide - of prrsv ' terminal sequence and was previously shown to be highly effective (zhang et al., ) and cp were included as positive and negative controls, respectively. a virulent prrsv strain, vr , was used to evaluate the ability of the ppmo to inhibit prrsv replication in crl cells. at . moi, vr reached peak replication in these cells at approximately h post-inoculation. in initial experiments, ppmo were applied to monolayer cells at a final concentration of m in dmem and incubated for h immediately after the virus inoculation period. the cells were then observed daily for cpe development and supernatants were collected at days p.i. for virus titration in crl cells to determine prrsv yield. of the ppmo tested, up and hp were found to be highly effective at inhibiting prrsv cpe development compared to cp -or mock-treatment. the cells treated with p and p also showed less severe cpe than the controls. the other ppmo had no effect on preventing cpe development. titration of prrsv in cell culture supernatants showed that treatment of crl cells with ppmo up or hp caused a . log tcid reduction in prrsv yield in comparison to cp ( fig. a) . treatment of the cells with p and p led to a reduction in prrsv yield of and log tcid , respectively, compared to cp . none of the other ppmo reduced prrsv yield significantly. ppmo up , hp, p , and p were selected for further characterization in this study. confirmation of the cpe observations and prrsv yield titration was obtained by ifa on crl cells after virus inoculation and ppmo treatment (fig. b ). ifa using a monoclonal antibody against the prrsv n-protein showed that treatment of cells with up led to an absence of prrsv-positive cells. in cells treated with hp, a few fluorescent-positive cells were observed. in cells treated with p and p , some prrsv-positive cells were observed, but far fewer than those treated with cp and up . these results indicate that the presence of up , hp, p , and p inhibited prrsv replication in vr -inoculated cells. ppmo up , hp, p , p , and cp were further tested at doses between and m for inhibition of vr replication in crl cells (fig. ) . cp had little effect on virus replication in comparison with the mock treatment control. each of the other four antisense ppmo reduced virus yield in a dose dependent manner (fig. ) . treatment of cells with up at m or hp at m concentration led to . log tcid reduction in prrsv yield compared to cp . ppmo p or p at m concentration led to reduction in prrsv yield to below the detection level of this assay. to assess the cytotoxicity of the ppmo used in this study, cell viability assays were conducted on crl and pam cells treated with ppmo at m. the experiment was repeated twice, each with three replicates. in comparison with mock treatment control, the average relative percentages of cell viability for crl cells after treatment was % ( up ), % ( hp), % ( p ), % ( p ), and % (cp ). the average percentages of viable pam cells after ppmo treatment were % (cp ), % ( up ), % ( hp), and % ( p + p ). these results virus titers are shown as tcid (log /ml). "mock" samples is from cells receiving virus inoculation but no ppmo treatment. statistical significance of difference in viral yields between ppmo and mock treatments: * p < . ; ** p < . . cells that were treated with m up or hp had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph. the experiment was repeated three times and error bars are shown. (b) immunofluorescence assay with sdow monoclonal antibody. specific fluorescence is clearly visible at h post prrsv (vr ) infection, while no fluorescence is observed in uninfected (−) control. treatment with ppmo up , hp, p , and p resulted in reduction of virus replication, while up and cp did not appear to have any effect under identical conditions. the images below the green fluorescence images were taken with a dapi filter to show the total number of living cells. demonstrated that ppmo generated little cytotoxicity in both crl and pam cells, further indicating that the suppression of virus replication observed in the antiviral experiments above was due to sequence-specific effects. in an attempt to derive a "selectivity index" (si) (the ratio of the concentration of drug causing % cytotoxicity [cc ] divided by the concentration of drug causing a % inhibition of viral production) relevant to the various conditions of this study, we sought to determine the cc for ppmo up , hp, p , and p . crl cells were treated for h with concentrations of each ppmo from to m. cell viability was determined at h after the treatment period. the cc for up , hp, p , and p are , , , and m, respectively. based on a % inhibition of viral production of approximately , . , fig. . dose-dependent inhibition of prrsv replication. virus yield in cell culture is reduced concomitantly with increasing concentrations of up , hp, p , and p . cells receiving some of the treatments had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the sample presence in the graph. the dose-dependent inhibition of prrsv replication by up , hp, p , and p is significantly different from cp (p < . ). the experiment was repeated three times and error bars are shown. . , and m, respectively, for these four ppmo (fig. ) , we conclude that the si for these antisense ppmo is , , , and under these conditions. to investigate the effect of treatment with ppmo combinations on prrsv replication, ppmo were paired and tested at equal molar concentrations, for example, a combination at m final concentration consisted of each ppmo being present at m. the ppmo up was not included in this test since it was already established as effective at m. the ppmo that target regions in prrsv thought to be involved in transcription events were tested in pairs: hp + p , hp + p , up + nsp , up + nsp . other pairs of ppmo targeting the translation initiation regions of orfs to were also tested: p + p , p + p , p + p , and p + p . the results of virus titration showed that only the combination of p fig. . combinatory inhibition assay of ppmo. two ppmo were added at equal molar concentration to cells after prrsv inoculation. the combination treatment of p and p led to significant lower virus yield (p = . ) than did either ppmo alone. cells that were treated with or m p + p had virus yields not detectable (nd) in this assay, and a bar is arbitrarily drawn to show the presence of the samples in the graph. and p ( p + p ) caused significant reduction (p = . ) in prrsv yield in comparison with a constituent individual ppmo (fig. ) . none of the other ppmo combinations showed significantly higher inhibitory activity than that of a constituent individual ppmo. to determine ppmo effect on prrsv rna synthesis, crl cells were inoculated with vr , treated with ppmo and harvested at h p.i. for rna isolation. ppmo up , hp, nsp , nsp , and up were included in this experiment as they were designed to block prrsv rna synthesis. the combination of ppmo p + p was also used to see if it had any effect on prrsv rna synthesis. ppmo p , p , and cp were included, for comparison. in northern blot analysis, at least seven distinct rna species were detected in the mocktreated sample, consistent with what would be expected from using a probe that detects orf , and indicating that all prrsv genomic and subgenomic rna containing orf sequence was no detectable rna is seen after treatment with up or hp. rna molecular weight markers are on the left. prrsv rna transcript designations are indicated on the right. as a loading control, the transcript of ␤-actin was detected and is shown in the lower panel. (b) quantitation of prrsv rna by real-time rt-pcr with primers designed to amplify a region of orf a. treatment of the cells with ppmo up , hp, p + p , up , nsp and nsp at m led to significant reduction of prrsv genomic rna level (p < . ). the cells were harvested at h p.i. and total rna was isolated for real-time rt-pcr. cells treated with up had no prrsv rna detectable (nd) in this assay and a bar is arbitrarily drawn to show the sample in the graph. the prrsv rna copy numbers in each sample was calculated in comparison with a standard curve, after normalization with ␤-actin transcript levels (see section . ). detected. prrsv rna in samples treated with up or hp was below the detection level (fig. a) . ppmo p or p had no effect on viral rna level, but the combination of p and p led to significant reduction of viral rna level (fig. a) . ppmo up , nsp , and nsp also apparently led to a modest reduction of viral transcription in comparison with cp . the northern blot shows that the, control ppmo, cp , produced a small yet noticeable effect on prrsv rna level compared to mock-treatment. however, the slight non-specific inhibition caused by cp was far less than the high level of inhibition produced by several of the antisense ppmo. real-time rt-pcr with primers specific for prrsv orf a was also used to evaluate rna levels. only prrsv genomic rna could be detected with the primers. in cells treated with up , prrsv rna was not detectable (fig. b) . in cells treated with hp or p + p , prrsv genomic rna was reduced by -and -fold, respectively, compared to cp -treated cells. the presence of up , nsp , and nsp also led to -, -, and -fold reduction of prrsv genomic rna in comparison to cp -treated cells. treatment with either p or p did not reduce prrsv rna. these results are consistent with the northern blot analysis above. to further characterize the sequence-specificity of the pmomediated inhibition of prrsv replication, we conducted a cell-free luciferase reporter assay. the target sequences of ppmo up , hp, p , and p were subcloned upstream of luciferase in a reporter expression plasmid. in this assay, ppmo binding to its rna target typically results in inhibition of translation of the downstream luciferase coding sequence. effects of ppmo treatment on target rna translation were assessed by measurement of luciferase production. incremental concentrations of ppmo were added to in vitro translation reactions to determine ppmo inhibitory effect on translation. as shown in fig. , nm or higher concentration of up , p , hp, and p reduced luciferase yield by more than %, while cp had no or little effect on translation of corresponding rna. ppmo p was slightly less effective than the other three ppmo in this test. the results of this assay indicate that the antisense ppmo specifically bound complementary rna and thereby inhibited the translation of luciferase. the ppmo in this study were designed against the vr strain of prrsv. to determine the efficacy of the ppmo against eleven other prrsv strains, nvsl - , fl- , b, , , , , , , mlv, and lelystad, cross strain inhibition assays were conducted. the lelystad strain is a prototype of the european genotype. the rest are strains of the north american prrsv genotype. ppmo up , hp, and the combination of p + p were tested against fig. . inhibition of target rna translation by ppmo in a cell-free luciferase reporter assay. relative percentages of luciferase level were calculated in comparison with signal of control ppmo sample at nm. the level of luciferase production decreased in response to increasing concentrations of ppmo up , hp, p , and p , while cp has little effect on luciferase rna translation. each of the prrsv strains. virus inoculation and ppmo treatment were conducted in the same manner as described above for vr . virus titration results showed that up and hp effectively suppressed replication in all strains except lelystad (fig. a) . the p + p combination led to a noticeable reduction in virus titer of a majority of the north american isolates, including nvsl, b, , , , and mlv. replication of lelystad was not inhibited by any of the ppmo tested. sequence alignment of the ppmo target sites in the prrsv strains showed that lelystad had low sequence identity with the other strains (fig. b ). prrsv nvsl, , , and b have one nucleotide mismatch with vr in the end of the up target site. in the hp target site, the only mismatch found was in nvsl, which has a single nucleotide difference in the middle of the ppmo target region. in the p target site, nvsl and have two mismatches, lelystad has five mismatches and two deletions, and all others have one mismatch. in the p target site, one mismatch occurs in all strains except lelystad, which has five mismatches. the sequence alignment results indicate that nucleotide mismatches in the prrsv target sites of p and p are likely responsible for the low level of inhibition produced by these two ppmo. alveolar macrophages are the primary target cells for prrsv infection in pigs (yoon et al., ) . evaluation of ppmo effect on virus replication in pam has relevance to the biology of a natural infection. to facilitate adaptation to laboratory culturing, pam cells were pre-incubated for h before their inoculation with prrsv. after virus inoculation, pam were treated with ppmo up , hp, p + p , or cp , and then observed at h p.i. for cpe development. the pam that were treated with fig. . cross strain inhibition assay. (a) virus yield titration shows inhibition of ten north american prrsv strains by ppmo up and hp. lelystad is a prototype of european prrsv genotype. all other strains are within the north american prrsv genotype. "mock" denotes virus infected cells receiving no ppmo. treatment of cells with ppmo up or hp led to suppression of prrsv replication of all north american strains, producing virus yields not detectable (nd) in this assay. bars are arbitrarily drawn for those samples with no detectable virus ("nd") to show the presence of samples in the graph. (b) sequence analysis identifies nucleotide mismatches between ppmo and their complementary target sites in prrsv rna. prrsv strain names are listed in the first column. the sequence of strain vr is used as the reference sequence, as the ppmo were designed against it. ppmo names are listed above the blocks of prrsv sequences. "lely" stands for lelystad strain, which has little similarity in the utr to other strains, as indicated by symbol "-" in the alignment of up and hp target sequences. for all other sequences, only nucleotides differing from reference sequence are shown, and identical nucleotides are indicated with ".". missing nucleotides are indicated with "-". the initiation codons atg of orfs and are underlined. genbank accession numbers for prrsv strains in the alignment are listed in parenthesis: ppmo up , hp, or combination of p + p showed no or little cpe development, while the cp -or mock-treated control cells suffered severe cpe (fig. a ). this result indicates that ppmo treatment protected the cells from prrsv-induced cpe development, and also that prrsv replicates rapidly in pam. to investigate the ppmo-mediated protection of pam from cpe development, we treated of pam with up twice, h apart. no cpe was observed in the cells at days p.i. (data not shown), indicating that ppmo treatment protected pam from cpe for at least week after prrsv inoculation. ifa was conducted with pam cells after prrsv inoculation and ppmo treatment. the cells were fixed at h p.i. to mini-mize cell death in mock-treated control. results showed that the approximate percentages of prrsv-positive cells were % with up treatment, % with hp, % with p + p , and % in mock-treated control (fig. b) . the results demonstrated that treatment with antisense ppmo reduced prrsv infection in pam cells. prrsv yield in pam culture was titrated in crl cells. treatment of pam with ppmo up , hp, and p + p led to -, -, and -fold reduction in virus yield, respectively, compared to mock-treated control (fig. c) . the reduction in virus titer in pam was considerably less than had been observed in crl cells after similar ppmo treatment. employing morpholino oligomers to inhibit rna virus replication has become an appealing prospect after reports of the successful knockdown of cellular proteins via inhibition of pre-mrna splicing and/or translation of mrnas (heasman, ) . in rna virus genomes, there are a variety of regions for pmo targeting. in our previous study, six ppmo, designed to target the and utrs, the trs-region of genomic rna, and the end of negative-sense prrsv rna, were evaluated (zhang et al., ) . only one of those ppmo, up , targeting the terminal region of the genome, was found to be effective at inhibiting prrsv replication. in the present study, ppmo were designed to target a wider variety of sites within the prrsv genome, including the and utrs, translation initiation regions of orfs through , and negative-sense prrsv rna. of the ppmo tested here, up was found to be the most highly effective ppmo, followed by hp, p , and p . in this study, ppmo up appeared more effective than up did in our previous study (zhang et al., ) . the target sites for up and up are located in the terminal region of the prrsv rna genome and overlap by nucleotides. we note, however, that comparing results between the two studies is complicated by the fact that the first study employed ppmo in which the peptide component was r f r (p ), while in the present study we utilized the more recently developed (rxr) (p ) peptide. ppmo hp complements a hairpin sequence in the utr, and was also quite effective, reducing prrsv yield by . log ( fig. a) . a ppmo targeting the corresponding region in eav was likewise found to be highly effective (van den born et al., ) . it has previously been reported that a combination of pmo compounds can outperform a single agent. this was shown in vivo with non-conjugated pmo against ebola virus , and the authors of that study suggested that slowing virus replication may allow sufficient time for development of antiviral immune responses and viral clearance. likewise ge et al. showed that a combination of ppmo produced greater efficacy than either ppmo alone against influenza a virus in cell culture (ge et al., ) . our results showed that treatment with a combination of p and p led to significantly higher prrsv inhibition than did either of the two ppmo alone. however, many other ppmo combinations in this study failed to outperform their constituent single agents. to elucidate the mechanism of pmo-mediated inhibition, we conducted northern blot and rt-pcr analyses (fig. ) . treatment of cells with up or hp reduced prrsv rna to the level of below detection, indicating inhibition of prrsv rna synthesis, likely accomplished through blocking translation of the replicase encoded by orf a/b. treatment with a combination of p and p also produced a reduction in prrsv rna level, probably due to the blockage of the translation of the transcripts for orfs and . based on their target locations in the end of prrsv genome, we speculate that the combination of p and p may have reduced prrsv genome replication by interfering with assembly or progression of the replicase complex. our results clearly show a reduction of prrsv rna level in cells treated with up or hp. a ppmo-associated reduction in prrsv rna production was also observed by rt-pcr in our previous study (zhang et al., ) . conversely, similar experiments against eav showed no significant change in the level of any viral rna transcripts, as assessed by gel hybridization analysis (van den born et al., ) . this seeming discrepancy may have resulted from differing experimental conditions. for rna isolation, cells were harvested at h p.i. in the prrsv, but h in the eav experiments (van den born et al., ) . there likely are some differences in the replication mechanics between prrsv and eav, since ppmo designed to target the respective trs-regions were reported to be effective against eav (van den born et al., ) , yet ineffective against prrsv (zhang et al., ) . among the ppmo tested in this study, only were found to be significantly efficacious. the reasons for the ineffectiveness of the other ppmo are unclear, but could include inaccessibility of prrsv target sequence, or, that successful ppmo/target rna duplexing did not affect prrsv replication. ppmo up , hp, and combination of p + p effectively inhibited prrsv-induced cpe development in primary pam, the major target cell type of prrsv infection in host animals. ifa confirmed the ppmo inhibition of virus replication. these results indicate that ppmo may indeed have potential to control, or reduce the severity of, prrsv infection in pigs. it was unexpected that ppmo-mediated reduction of virus yield in pam was less than that observed in crl cells. this difference but may be because prrsv has a higher replication rate in pam than it does in crl cells, and the ppmo were unable to significantly interfere with the more rapid viral replication. alternatively, differential uptake of ppmo by crl and pam cultures may have had an effect. however, virus replication in pam after treatment with up did not result in cytopathic effect, and the cells were healthy for at least week after virus inoculation. further study is being undertaken to further investigate ppmo-mediated protection of primary cells. ppmo up and hp inhibited replication of ten north american prrsv strains in our cross-strain inhibition assay. sequence alignment (fig. b) showed that the region targeted by these ppmo contains highly conserved sequences. in addition, analysis of prrsv sequences from the genbank indicates that the complementary sequence of up and hp are highly conserved across north american prrsv strains. thus, these two antisense ppmo may be suitable for application against most north american prrsv strains. the broad inhibition by up and hp also further confirms that the utr of prrsv is a highly productive target region for ppmo, and that this region likely plays an important role in arterivirus replication. a one base mismatch between p or p ppmo and their respective target sites in ten north american prrsv strains may explain the low activity observed. (fig. a and b) . it is noteworthy that all strains had identical sequences in both the p and p target sites. thus, a minor sequence modification of ppmo targeting these regions may lead to improved efficacy. a cell viability assay of ppmo-treated crl cells and pam detected no cytotoxicity, indicating that the suppression of prrsv replication observed in the antiviral assays was due to ppmo-specific inhibition of prrsv molecular events. the absence of ppmo-induced cytotoxicity at effective antiviral concentrations is an important attribute of these compounds, when considering potential in vivo applications. the prevalence of prrsv and financial losses associated with prrsv infection in swine herds is high, and current strategies to control prrs, including the use of commercial and autogenous vaccines, are inconsistent and generally less than adequate (meng, ; opriessnig et al., ) . specific anti-prrsv drugs would be useful to complement other strategies of prrsv prevention and control. under the conditions of this study, four ppmo showed potent and specific anti-prrsv activity in cell culture, and can be considered potential drug candidates for use in prrsv control. further investigation into the pharmacokinetic, toxicological and antiviral properties of these ppmo in vivo is warranted. vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents phosphorodiamidate morpholino antisense oligomers inhibit expression of human cytochrome p a and alter selected drug metabolism bioavailability and efficacy of antisense morpholino oligomers targeted to c-myc and cytochrome p- a following oral administration in rats comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) targeted 'knockdown' of channel expression in vivo with an antisense morpholino oligonucleotide antiviral effects of antisense morpholino oligomers in murine coronavirus infection models molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers morpholino oligos: making sense of antisense? resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers mystery pig disease: an update for the practitioner heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development molecular cloning and nucleotide sequencing of the terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the usa and europe characterization of a high-virulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav characterization of proteins encoded by orfs to of lelystad virus subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence cellular uptake of antisense morpholino oligomers conjugated to argininerich peptides porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv in vivo evaluation of a morpholino antisense oligomer directed against tumor necrosis factoralpha coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands in vivo replication of porcine reproductive and respiratory syndrome virus in swine alveolar macrophages and change in the cell population in bronchoalveolar lavage fluid after infection inhibition of vesivirus infections in mammalian tissue culture with antisense morpholino oligomers morpholino antisense oligomers: the case for an rnase h-independent structural type morpholino antisense oligomers: design, preparation, and properties comparison of the leader sequences of north american isolates of reference and field strains of porcine reproductive and respiratory syndrome virus (prrsv) antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences kissing interaction between noncoding and coding sequences is essential for porcine arterivirus rna replication gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers mystery swine disease in the netherlands: the isolation of lelystad virus isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells inhibition of coxsackievirus b in cell-cultures and in mice by peptide-conjugated morpholino oligomers targeting the ires monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers inhibition of replication and transcription activator and latency-associated nuclear antigen of kaposi's sarcoma-associated herpesvirus by morpholino oligomers we are grateful to the chemistry department of avi bio-pharma inc. for the synthesis and quality control of all ppmo used in this study, to dr. fernando osorio at university of nebraska-lincoln for his gifts of prrsv isolates fl- , , , b, , , , and , and to dr. kay s. faaberg at university of minnesota for her gift of prrsv ingelvac mlv used in cross strain inhibition assay in this study. this project was supported by a grant from the national pork board. key: cord- -ov fy b authors: nazki, salik; khatun, amina; jeong, chang-gi; mattoo, sameer ul salam; gu, suna; lee, sim-in; kim, seung-chai; park, ji-hyo; yang, myoun-sik; kim, bumseok; park, choi-kyu; lee, sang-myeong; kim, won-il title: evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ov fy b the host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (prrsv) from infected pigs is currently poorly understood. to better understand the dynamics of host–pathogen interactions, seventy-five of pigs infected with prrsv-ja and control pigs were euthanized at , , , and days post-challenge (dpc). blood, lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected to evaluate the cellular immune responses. the humoral responses were evaluated by measuring the levels of anti-prrsv igg and serum virus-neutralizing (svn) antibodies. consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between and dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by dpc. at peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived dc/macrophage and conventional dc frequencies were increased, and these effects coincided with the early induction of local t-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, bal, and bln as early as dpc. conversely, the systemic t-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between and dpc. taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining prrsv through the early induction of t-cell responses at the sites of virus replication. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded positive-sense rna virus with an approximate . -kb genome, belongs to the genus betaarterivirus of the family arteriviridae (ictv ). in pigs, prrsv causes porcine reproductive and respiratory syndrome (prrs), which is characterized by reproductive failure in breeding sows and severe respiratory distress in young and growing pigs [ ] . prrs results in colossal economic losses in the swine industry worldwide, and these losses are still observed three decades after its emergence in the united states and europe. after the exposure of pigs to prrsv, the virus replicates in alveolar macrophages (am) and further spreads rapidly throughout the body via a lymphohematic route. this viral spread results in acute infection characterized by viremia that lasts for approximately month [ ] , and a few studies have reported a nonviremic persistent infection of secondary lymphoid tissues lasting for approximately days or longer [ ] . in general, the viremia peaks at approximately - days post-infection (dpi) and is almost cleared by dpi depending on the viral strain and age of the pigs [ , ] . additionally, the immune response against prrsv depends on the strain, but the virus usually has immunosuppressive properties [ , ] , which leads to the increased susceptibility of pigs to secondary microbial infections [ ] . the interactions between prrsv and host immune responses have been widely studied, but most studies investigated systemic immune responses using pbmc and/or serum [ ] . previous studies have shown that interstitial pneumonia constitutes the major lung lesions in prrsv-infected pigs and that significantly decreased numbers of alveolar macrophages are found in bronchoalveolar lavage (bal) and lung parenchyma samples from prrsv-infected pigs [ ] . however, to the best of our knowledge, the kinetics of local immune responses in the lungs or lymph nodes during the course of infection compared with those of peripheral immune responses have not been previously studied. this information would provide a more in-depth understanding of the sequential activation of both immune compartments and the correlation between local or peripheral immune responses and virus clearance in infected pigs. as a result, achieving a comprehensive understanding of the immune responses against prrsv infection remains an important goal in prrsv research. during prrsv infection, the pig immune system is capable of escalating an immune response to ultimately clear the virus from the body [ ] . for clearance, proper stimulation of the pig innate immune system is required to direct the development of protective adaptive immunity against prrsv. interestingly, the preferential sites for prrsv replication are alveolar macrophages present in the lungs, which form the major component of the respiratory dc/macrophage network. this network is predominantly involved in sensing foreign antigens, controlling inflammation, and initiating the adaptive immune response [ ] . different dc subsets with specific functional specializations exist in the respiratory dc/macrophage network in the lungs and are reportedly resistant to prrsv infection [ , ] . however, upon activation, these dc travel to lymphoid tissues to present antigen to t lymphocytes and thereby serve as the link between innate and adaptive immunity [ , ] . t cells, in turn, play a critical role in the development of anti-prrsv immunity due to their cytotoxic effector functions in clearing infected cells from the body and developing and regulating antigen-specific immune responses [ ] . however, whether the peripheral virusspecific immune response is appropriately correlated with the local immune response during infection, which could result in the precise use of the peripheral response as a surrogate for scrutinizing the local immune response during viral clearance, remains unclear. therefore, discerning the local and peripheral immune responses during prrsv infection is important for understanding the basic mechanism of viral clearance from the host. cytokines secreted by immune cells act on their targets in an autocrine, paracrine, and/or endocrine manner to prompt local and/or systemic immune responses. in porcine respiratory diseases, proinflammatory cytokines play a key role in activating and synchronizing the adaptive immune responses to clear the virus from the body [ ] . however, the tissue damage caused by excessive production of these cytokines is controlled by the secretion of anti-inflammatory cytokines, which results in the maintenance of homeostasis in the body [ ] . moreover, effective instigation of the local inflammatory response in the lungs accompanied by significant changes in the proinflammatory cytokine levels in serum has been observed in pigs with respiratory diseases [ , ] . nevertheless, whether the local or the systemic cytokine/ chemokine response plays the primary role in the clearance of prrsv from pigs during the acute phase of infection remains unclear. in this context, the present study aimed to investigate the trend of host immune responses against prrsv infection during disease progression and to elucidate the innate and adaptive immunological mediators modulated by the prrsv-ja strain both systemically in peripheral blood and locally in the bronchoalveolar lavage, lung parenchyma and bronchial lymph nodes (bln) of infected pigs. marc- cells, an african green monkey kidney cell line that is highly permissive to prrsv [ ] , were used for virus propagation and assays. these cells were maintained in rpmi- medium (gibco ® rpmi- , life technologies, carlsbad, ca, usa) supplemented with % heat-inactivated foetal bovine serum (fbs, life technologies), mm l-glutamine, and an antibiotic-antimycotic cocktail (anti-anti, life technologies) containing iu/ml penicillin, µg/ml streptomycin, and . µg/ml fungizone ® [amphotericin b] in a humidified chamber with % co at °c. in this manuscript, this medium is designated rpmi growth medium. the north american prrsv- strain ja (genbank: ay . ) was used in the present study. one hundred -week-old piglets purchased from a prrsv-seronegative farm were randomly assigned to two groups and housed in separate animal rooms. after days of acclimatization, the pigs in the infected group (n = ) were intramuscularly inoculated with ml of the prrsv-ja strain ( × tcid /ml) diluted in sterile pbs. the control pigs (n = ) remained uninfected. feed and water were provided ad libitum to all the pigs. five pigs from the control group and , , , and pigs from the infected group were humanely euthanized on days , , , and post-challenge (dpc), respectively. euthanasia was performed by electrocution after the intramuscular injection of ml of azaperone ( mg/ml, stressguard ® , dong bang inc., seoul, south korea). three infected pigs died during the course of the experiment due to high fever and reduced body growth. an overview of the animal study is presented in figure . after euthanasia, the lungs, trachea and bronchi were aseptically extracted and lavaged with ml of sterile pbs. the collected lavage fluid was centrifuged at × g and room temperature for min to separate the bronchoalveolar lavage fluid (balf) and cells (bal), and these samples, along with the lung parenchyma and bln, were also used for immune cell analysis. these tissues and balf were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at − °c for rna extraction and cytokine analysis, respectively. for histopathology, the lung tissues were also collected in % neutral-buffered formalin. blood was collected from the euthanized pigs at , , , and dpc, and the serum and pbmc were separated. in addition, blood samples from pigs, including uninfected and infected pigs that were going to be euthanized on and dpc, were also collected at , , , , , and dpc, and the serum and pbmc were separated. the body weights of all the pigs were measured at dpc, and the body weight gains of the euthanized pigs were measured at , , , and dpc. serum viremia was measured at , , , , , , and dpc, and the viral load in the lungs was quantified in the euthanized pigs at , , , and dpc. viral rna was extracted from serum using a magmax viral rna isolation kit (ambion; applied biosystems, life technologies, inc.) according to the manufacturer's instructions. real-time reverse transcription-polymerase chain reaction (rt-qpcr) employing the prime-q pcv prrsv detection kit (genet bio, daejeon, south korea) was performed for the quantification of serum viremia. one-step rt-qpcr was performed in accordance with the manufacturer's instructions. pcr amplification was performed using a model fast real-time pcr system (applied biosystems, foster city, ca, usa). the cycling conditions were as follows: (i) cdna synthesis for min at °c; (ii) -min predenaturation step at °c; and (iii) cycles of denaturation for s at °c figure overview of the animal study. prrsv-negative piglets (n = ) were purchased and acclimatized for days. seventy-five pigs were then infected, and pigs were used as a negative control (nc). pigs belonging to both the nc and infected (i) groups were euthanized at , , , and days post-challenge (dpc), and lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected. blood samples for sera and peripheral blood mononuclear cells (pbmc) were collected weekly post-challenge. the body weight of the pigs was also monitored from the day of purchase to the end of the experiment. and annealing/extension for s at °c. to calculate the amount of prrsv in each sample, the cq values were converted to virus titres (tcid /ml) by generating a standard curve through the titration of prrsv- strain ja . in addition, marc- cells were used to quantify the virus titres in lung tissues using a microtitration infectivity assay [ ] . briefly, tissue homogenates ( % [weight/ volume]) of finely chopped lung pieces were prepared in dulbecco modified eagle medium (dmem) with antibiotics, and these mixtures were vortexed vigorously for - min and then centrifuged at ~ × g and °c for h. the collected supernatant was filtered through a . -μm sterile syringe filter and used as an inoculum for the measurement of virus titres. the virus titres were calculated at to days post-inoculation based on the cytopathic effect (cpe) and are expressed as tcid /ml [ ] . the lungs of the necropsied pigs in both groups were subjected to pathological evaluation on each day of necropsy. the microscopic lung lesions were given a score on a scale from to to reflect no lesion, mild interstitial pneumonia, moderate multifocal interstitial pneumonia, and severe interstitial pneumonia, respectively. the microscopic lesions were examined from five different lobes of the lungs, and the average value was ultimately utilized for scoring purposes. the serum samples from uninfected and challenged pigs were tested for anti-prrsv antibody (igg) using a commercially available elisa kit (prrs ab elisa . ; bionote inc., hwaseong-si, republic of korea) according to the manufacturer's instructions. samples with an s/p ratio (the ratio of the net optical density of the test samples to the net optical density of the positive controls) ≥ . were considered to be positive for the prrsv antibody. the serum virus-neutralizing (svn) antibodies were measured through a fluorescent focus neutralization (ffn)-based svn assay with marc- cells as described previously [ ] , with some modifications. after heat inactivation at °c for h, the serum samples were serially (twofold) diluted using rpmi- growth medium. two-hundred-microlitre mixtures were prepared by mixing each diluted serum sample with fluorescent focus-forming units per ml (ffu/ml) of prrsv-ja at a ratio of : and were then incubated for h at °c in a humidified atmosphere with % co . each mixture was transferred onto a monolayer of marc- cells in -well plates and incubated for another h at °c. the medium was replaced with µl of fresh rpmi growth medium per well and further incubated for h at °c. the cells were later fixed using ice-cold % (v/v) acetone, air-dried, and stained with mouse anti-prrs nc mab a (median diagnostic, gangwondo, korea) and fitc-conjugated goat anti-mouse igg (h + l) (bethyl laboratories, tx, usa). subsequently, the plates were washed at least three times with pbs and observed under a fluorescence microscope to examine the prrsv-specific ffu. the svn titre is expressed as the reciprocal of the highest dilution at which a % or higher reduction in the number of ffu was observed. pbmc were isolated from the blood samples ( ml) by the density gradient method using leucosep ™ centrifuge tubes (greiner bio-one north america inc., nc, usa) and leucoprep ™ lymphocyte separation media (greiner bio-one north america inc.) according to the manufacturers' instructions. the blood samples were briefly stratified on leucoprep ™ solution at a ratio of : (blood:leucoprep) and centrifuged at × g for min. the purified pbmc were collected, washed twice with sterile pbs (ph . ) and resuspended in . ml of sterile pbs supplemented with % heat-inactivated fbs (gibco, carlsbad, ca, usa). contaminating red blood cells (rbc) were removed by treatment with rbc lysis buffer (ebioscience, ca, usa). for pathological evaluation, the right-sided lobes were clamped to collect specimens for rna extraction and histopathology, and the left lobes of the lungs were used for bal collection according to a previous study [ ] . the lungs were lavaged with - ml of pbs containing µg/ml ampicillin (usb corporation cleveland, oh, usa) and an antibiotic-antimycotic cocktail (anti-anti, life technologies), and the harvested fluid was centrifuged for min at × g. the resulting supernatant was collected as bal fluid (balf), whereas the cell pellet (bal cells) was washed three times with pbs after rbc lysis. the cells were resuspended in facs buffer ( % fbs in phosphate-buffered saline and . % sodium azide). the bln were passed through a -μm cell strainer (spl life sciences, pocheon, korea) in pbs and then washed with facs buffer according to a previous study [ ] . the single cell suspension obtained was used for flow cytometric analysis. mononuclear cells from lung parenchyma were prepared based on a previous study [ ] , with few modifications. briefly, lung tissue was collected, washed in sterile ice-cold pbs and suspended in serum-free rpmi media containing dnase i ( u/ml, sigma, st. louis, mo, usa) and collagenase d ( mg/ml, roche diagnostics, mannheim, germany). single-cell suspensions were prepared using the gentlemacs octo dissociator (miltenyi biotec, san diego, ca, usa) and incubated at °c for min. subsequently, the cells were passed through a -μm cell strainer, washed, and resuspended in facs buffer for flow cytometry analysis after rbc lysis. finally, the cells were counted with a countess ™ automated cell counter (invitrogen, carlsbad, ca, usa), and their viability was tested by trypan blue (sigma-aldrich, st. louis, mo, usa) exclusion [ ] . for cell surface staining, single-cell suspensions were incubated on ice for min with specific antibodies as listed in additional file , and the cells were then washed three times with facs buffer. when necessary, secondary antibodies conjugated with fluorochrome were used. natural killer (nk) cells, dc and macrophages required only cell surface staining, whereas the different subsets of t cells required intranuclear and intracellular staining. two subsets of nk cells have been phenotypically defined based on nkp marker expression: nkp + and nkp − nk cells [ , ] . following pbmc staining, a similar gating hierarchy was followed by excluding the unstained cells, doublets and cd + cells, and the cd − lymphocytes were further analysed for cd α and nkp expression. among cd − cells, two populations were found in the pbmc, namely, nkp + and nkp − nk cells, and both of these cells were cd α + (additional file a). bal cells were subjected to cell surface staining for the cd surface marker, and the viability of these cells was analysed using propidium iodide (pi) staining (additional file b). additionally, dc and macrophages were segregated from the bal cell population based on staining and gating strategies outlined previously (additional file c) [ , ] . from the mhc-ii + cell population, five phenotypically and functionally defined subpopulations were distinguished using the cd and cd a (sirpα) surface markers. among the mhc-ii + cells, cd a + / cd high cells were defined as am, whereas cd a + / cd int , cd a + /cd low , cd a + /cd − and cd a − /cd − cells were defined as monocytederived macrophages (momɸs), monocyte-derived dendritic cells (modc), conventional dendritic cells (cdc ) and conventional dendritic cells (cdc ), respectively, based on a previous study [ ] . regulatory t cells (tregs), which require intranuclear staining of foxp after cell surface staining, were fixed with cold fixation/permeabilization buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min and were then stained for foxp at °c for min. based on a previously described staining and gating strategy [ ] , tregs (cd + foxp + cells) were apparent among the cd + cd − population (additional file d). the t-cell subsets subjected to intracellular staining were stained according to previous studies [ , ] , with few modifications. briefly, single-cell suspensions were treated with a mixture of × cell stimulation cocktail (ebioscience, thermo fisher scientific, seoul, korea) and × brefeldin a (ebioscience, thermo fisher scientific, seoul, korea) in rpmi growth media and incubated at °c in a humidified chamber with % co for - h. the cells were then stained with antibodies for various cell surface markers in cold facs buffer for min at °c, properly washed twice with cold facs buffer, and fixed with intracellular (ic) fixation buffer (ebioscience, thermo fisher scientific, seoul, korea) at °c for min. for intracellular staining, the cells were washed twice with permeabilization buffer ( μl/well) and stained with cytokine-specific antibodies in cold permeabilization buffer at °c for min. subsequently, the cells were washed twice with permeabilization buffer. the gating strategy employed for obtaining various t-cell phenotypes after the gating of singlet lymphocytes is demonstrated in additional file e. a -μl suspension of the stained cell populations in facs buffer was run on an accuri c flow cytometer (bd accuri ™ c plus, bd biosciences, md, usa). bd accuri ™ c plus software version . . . (bd biosciences, md, usa) was used to analyse the data after setting compensation settings according to monocolour and isotype control stains. the data are presented as percentages of all the cell subsets. the cytokine levels in the sera and balf of the uninfected and infected pigs at , and dpc were measured using a porcine-specific procartaplex ™ multiplex immunoassay (thermofisher scientific, vienna- , austria) according to the manufacturer's instructions. magnetic microsphere technology based on porcine cytokine/chemokine antibody-immobilized magnetic beads was employed in the immunoassay for cytokine quantification [ ] . the concentration of each cytokine was measured by running the samples on the luminex ® ™ system (luminex corporation, austin, tx, usa). appropriate standards provided in the kit were utilized to determine the concentration of each cytokine. the machine was verified and calibrated using a luminex ® / ™ verification kit and a luminex ® / ™ calibration kit (luminex corporation, austin, tx, usa) prior to use. graphical presentations of the data were prepared using graphpad prism . (graphpad, san diego, ca, usa), and the data were statistically analysed using spss advanced statistics . software (spss, inc., chicago, il, usa). a nonparametric t-test (mann-whitney u test) was used to compare the viral loads in the lung tissues, the average daily weight gain (adwg), the phenotypes of various cell subsets and the cytokine responses between two groups. the normalized dead cd + cells were analysed by repeated anova (tukey post hoc test) to determine the overall difference, and pairwise comparisons were also performed at different days post-challenge. spearman rank correlation and linear regression were used to determine the associations between two parameters. differences were considered statistically significant if p < . and are indicated by asterisks and different letters over the bars. the highest viremia and lung viral loads in prrsv-ja challenged pigs were detected between and dpc (figures a, b) . the mean peak virus titre in serum at dpc was recorded as . tcid /ml, whereas the mean peak live viral load in the lungs at dpc, which was measured through the microtitration infectivity assay, was . tcid /ml. the virus was gradually cleared by dpc, at which point, the serum and lung viral loads had decreased to mean values of . and . tcid / ml, respectively. as expected, the control group maintained an uninfected state throughout the experiment. the effect of viremia on body weight gain was observed in the infected pigs by calculating the adwg of the pigs ( figure c ). the adwg in the prrsv-ja -challenged pigs was significantly lower than that in the control pigs at , and dpc. the adwg per control pig was recorded as . kg, whereas the adwg of the infected pigs was reduced to . kg. as expected, the growth rate of the pigs was negatively affected by viremia following infection, and this negative correlation was significant (r = − . ; p ≤ . ) ( figure d ). moderate to severe interstitial pneumonia with alveolar wall thickening due to type pneumocyte proliferation and inflammatory cell infiltration was detected in the infected pigs during the period of peak viremia. thus, the highest microscopic lung lesion score in the infected pigs was recorded at dpc, and the scores of the lung lesions in these pigs decreased at later time points but remained at significantly higher levels compared with those in control pigs (figures a, b) . an elisa based on the nucleocapsid (n) protein was employed to measure the prrsv-specific antibody (igg) response in the infected and noninfected pigs ( figure a ). at dpc, prrsv-ja -specific igg were detected in the serum of the infected pigs. additionally, the sample-to-positive (sp) value gradually increased in the infected group from to dpc, whereas in the control group, the pigs did not produce any prrsv-specific igg at any time point. a low svn titre was observed in the challenged pigs at and dpc, when most of the virus was already cleared from the body ( figure b ). in general, the svn antibody responses predominantly appear at the later stages of infection, which is the phase at which most of the virus is cleared from the body, and might play a minor role in the clearance of virus. nk cells, which are a specialized subpopulation of lymphocytes, are the innate immune cells critically responsible for directly killing virus-infected cells, which ultimately leads to viral clearance in the host [ ] . to observe the effect of prrsv infection on nk cells in pigs, the frequencies of two different nk cell subsets in the pbmc population were analysed. the percentages of nkp − nk cells (cd + nkp − in cd − ) in the pbmc populations of the uninfected and infected pigs were higher than those of nkp + nk cells (cd + nkp + in cd − ) (additional file a). moreover, compared with the control pigs, the infected pigs exhibited a significantly (p ≤ . ) increased frequency of nkp + nk cells at the early time point of dpc, whereas the frequency of nkp − nk cells was slightly increased at dpc and significantly increased at dpc. the frequency of nkp + nk cells returned to normal values at dpc, but the frequency of nkp − nk cells in the infected pigs remained at significantly higher values up to dpc ( figures a, b) . the associations between nk cells in pbmc and serum viremia were evaluated post-infection ( figures c, d) . the nkp + nk cell population revealed a significant (r = . ; p < . ) positive correlation with viremia; however, no statistically significant correlation between the levels of nkp − nk cells and viremia was observed. the receptors of host cells determine the cell tropism of prrsv. among others, cd , a cysteine-rich scavenger receptor (srcr), acts as the determinant receptor for prrsv entry and infection [ , ] . the bal cells were subjected to cd staining, and the results show that although the virus did not affect the cd + cells until dpc, the infected pigs exhibited a significant (p ≤ . ) reduction in the cd + cell percentage at dpc, showing a gradual recovery with the decline in the viral loads at dpc ( figure a ). the reduction in the cd + cell population was attributed to the death of cd + cells, which was confirmed by observing the viability of these cells through pi staining. the mean frequencies of dead cd + cells in the infected pigs, which were normalized to those in the uninfected pigs, was significantly (p ≤ . ) higher at and dpc ( . % and . %, respectively) ( figure b ). the class ii major histocompatibility complex (mhc-ii) is essential for the presentation of antigens to t cells and is constitutively expressed on macrophages and dc [ , ] . the mhc-ii + cells were analysed to observe the changes in the dc/macrophage network in the lungs. the mhc-ii + cells, such as cd + cells, show significant (p ≤ . ) decreases at and dpc ( figure c) . interestingly, the percentage of am (cd a + /cd high /mhc-ii + cells), which constituted the majority of mhc-ii + cells, decreased significantly at dpc after infection (p ≤ . ) (figure d ). in contrast, the cd a + /cd int /mhc-ii + cell (momɸ) and cd a + /cd low /mhc-ii + cell (modc) frequencies were significantly (p ≤ . ) reduced in the infected pigs early during the infection process ( dpc) ( figures e, f) . however, the cd a + / cd int /mhc-ii + cell percentages in the infected pigs were significantly (p ≤ . ) increased at and dpc, and a higher (p ≤ . ) cd a + /cd low /mhc-ii + cell frequency was also detected at , , and dpc in the infected pigs compared with the control pigs. cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells were not frequently detected among bal cells and accounted for less than % of the five subsets ( figures g, h) . at dpc, a significantly higher (p ≤ . ) percentage of these cell populations was observed in the infected pigs, and this value decreased with the reduction in the viral load and the recovery of am. the associations between the lung viral loads and different subpopulations of macrophages and dc in bal were evaluated after pooling the results of infected pigs at each time point (figure ) . during the infection, mhc-ii + cells and am displayed a significant negative correlation (p < . ) with lung viral loads; however, other subsets exhibited a significant (p < . ) positive correlation. during infection, prrsv-ja alters the dynamics of the respiratory dc/macrophage network by destroying am while increasing the populations of antigen-presenting cells (apc), which bridge the gap between innate and adaptive immune systems by presenting the foreign antigen to t cells. the peripheral immune response was measured by analysing the t-cell populations in the pbmc of uninfected and infected pigs at , , , and dpc (additional file a). the th (ifn-γ + in cd + cd − ) response in the infected pigs was significantly higher (p ≤ . ) than that in the control pigs at dpc, and these responses continued to increase until dpc. the cytotoxic t lymphocyte (ctl) (ifn-γ + in cd − cd + ) response in the infected pigs started to increase at dpc and was significantly higher (p ≤ . ) compared with that in the control pigs at dpc ( figure a ). at dpc, the th (il + in cd + cd − ) cell response was significantly higher (p ≤ . ) in the infected pigs compared with the control pigs, and the response was further escalated at later time points. the il- -producing cd − cd + cell population was significantly higher (p ≤ . ) in the infected pigs compared with uninfected pigs at dpc. the delay in the induction of effector t cells was mainly perceived peripherally in blood. to observe the activation of the local adaptive immune responses by innate immune cells at the sites of replication and the persistence of prrsv, the t-cell phenotypes in the bln, bal and lung parenchyma of the euthanized control and infected pigs were analysed at , , and dpc. the percentages of various immune cells in the lungs, bal and bln of uninfected and prrsv-ja infected pigs at different stages of infection are summarized in additional files b, c, and d. overall, various t-cell responses were significantly induced in the lungs as early as dpc, and significant responses in bal and bln were first detected at dpc and were maintained until dpc. the th cell (ifn-γ + in cd + cd − ) frequency in all the local tissues was significantly (p ≤ . ) induced in the challenged pigs as early as dpc, and the frequency in bal cells tended to be higher. the ctl (ifn-γ + in cd − cd + ) frequency was significantly (p ≤ . ) higher in the lungs of the infected pigs compared with that of the control pigs at dpc, whereas the frequency in bln and bal cells was higher at the early time point and significantly higher at dpc. the prrsv-ja -infected pigs also displayed a higher induction (p ≤ . ) of th cells (il + in cd + cd − ) in the lymph nodes and lung tissues at dpc, whereas a slight increase in these cells was observed in bal cells. the il- -producing cd − cd + population was significantly (p ≤ . ) induced in the lungs and bal cells of the infected pigs at dpc, whereas in bln, this cell population showed a slight increase at dpc and a significant increase (p ≤ . ) at dpc (figures b-d) . therefore, compared with the weak and delayed peripheral responses, early and effective cellular immune responses were triggered in local tissues by prrsv-ja infection. tregs are well known for their immunosuppressive activities, and previous studies have demonstrated that prrsv infection induces treg responses that might be responsible for ineffective adaptive immune responses [ ] [ ] [ ] . unlike previous studies, no upregulation of tregs (cd + foxp + in cd + cd − ) was detected in pbmc isolated from prrsv-infected pigs. moreover, tregs were significantly (p ≤ . ) reduced in the bal throughout the course of infection, but no such decline was observed in the lungs (figure ). however, the treg frequencies in bln of the infected pigs initially exhibited a significant decline at dpc but then increased significantly at dpc. the levels of seven different innate and adaptive cytokine/chemokine proteins in the sera and balf at , and dpc were compared between the uninfected and infected pigs ( figure ) . the results reveal that interferon-α (ifn-α) was significantly (p ≤ . ) induced in the sera and balf of the infected pigs at dpc. however, at peak viremia ( dpc), the ifn-α level was reduced peripherally in the sera of the infected pigs, whereas the cytokine level was elevated locally in the balf. nevertheless, significant increases in the level of this cytokine (p ≤ . ) were maintained both locally and systemically in the infected pigs compared with the uninfected pigs. furthermore, proinflammatory cytokines/chemokines, such as tumour necrosis factor-α (tnf-α), interleukin- β (il- β), il- , il- and il- , show a similar pattern of early induction at dpc in the sera of the infected pigs followed by decreases as viremia increased. in the sera, significant (p ≤ . ) changes in il- β were only observed in the infected pigs at and dpc. however, significant (p ≤ . ) induction of il- β and il- locally in the balf was observed in the infected pigs at dpc. in addition, elevations in all the proinflammatory cytokine/chemokine levels were observed in the infected pigs as the viral load increased, and significant increases in the levels of il- β, il- , il- and il- were observed in the infected pigs compared with the uninfected pigs at dpc. moreover, il- , an anti-inflammatory cytokine, was significantly induced (p ≤ . ) at and dpc in the balf of the infected pigs. overall, the clearance of the virus from the pigs also coincided with the increased secretion of anti-viral and proinflammatory figure local and systemic levels of cytokines/chemokines in the pigs. the concentrations of cytokines/chemokines in the a sera and b balf of pigs belonging to both groups at , , and dpc were measured using a multiplex luminex-based cytokine immunoassay. the bars in the graphs represent the mean ± sem of the cytokine levels, and the asterisks (*) indicate a statistically significant difference between the averages found for the uninfected (nc) pigs and those obtained for the infected pigs at each time point (* indicates p ≤ . and ** indicates p ≤ . ). cytokines locally in the balf of prrsv-infected pigs, although no considerable shifts were detected in the serum. despite exhaustive research, the understanding of the protective immune responses against prrsv in pigs remains limited. previous studies have mainly focused on certain aspects of immune responses and/or have used a narrow time window to study the immune responses during prrsv infection; therefore, the available data offer a limited picture of the host defence system. to the best of our knowledge, the present study constitutes the first investigation of various aspects of immune responses, such as local vs. systemic and innate vs adaptive, during the course of prrsv infection to obtain a broader picture of the host defence against prrsv. here, we demonstrate the critical role of local immune responses in the clearance of prrsv from pigs due to the early induction of dc/macrophage subsets, the activation of protective t cells in local lymphoid and lung tissues, and the stimulation of proinflammatory cytokines locally in the lungs. in pigs that were intramuscularly inoculated with the biologically characterized prrsv-ja strain [ ] , the viral titre reached its peak value in the serum and lungs between and dpc. in addition, moderate to severe interstitial pneumonia and a significant decrease in the adwg, which are characteristics of prrsv infection [ ] , were detected in the challenged pigs by dpc. mild histopathological lesions (with a lesion score lower than ) characterized by alveolar wall thickening due to type pneumocyte proliferation were also observed in some of the uninfected healthy pigs that were free of other respiratory pathogens. these observations were likely obtained due to the stress induced by environmental factors, such as housing conditions, weaning, individual space, and ambient temperature variations, as previously reported [ , ] . similar to previous reports [ ] , the prrsv-ja strain induced delayed (≥ dpc) and weak (≤ ) svn titres in infected pigs. an svn antibody titre of offers sterilizing immunity, whereas a titre greater than is considered protective against prrsv infection [ ] . the anti-prrsv antibody response induced by prrsv-ja in the infected pigs crossed the threshold of . (s/p ratio) between and dpc, and this response was in accordance with the response produced by other prrsv strains [ ] , but the role of these antibodies in protection against prrsv infection is unknown [ ] . nk cells, an important component of the innate host defence system, play a critical role in the resolution of viral infections [ ] . in general, the potential roles of nk cells in relation to prrsv immunity are poorly understood [ , ] . in the current study, two defined nk cell subpopulations were distinguished in pbmc based on an approach similar to one previously described [ ] . similar to previous observations, the nkp + nk cell frequency in pbmc collected from both control and infected pigs was lower than that of nkp − cells. nkp + and the nkp − nk cells execute analogous cytolytic activities but produce different levels of ifnγ, with nkp + nk cells producing higher amounts of ifn-γ. moreover, nkp can be expressed in nkp − nk cells after stimulation with interleukins (il)- , il- and il- [ ] . in the current study, early increases in the frequencies of nkp + and nkp − nk cells in prrsv-ja -challenged pigs were clearly detected. previous studies have shown similar increases in the cd − cd − cd + cell population after infection with different strains of prrsv [ , ] . induced proliferation of nk cells has also been perceived after influenza infection, suggesting that this subset of lymphocytes is capable of antigen-specific clonal expansion [ ] . prrsv, however, significantly suppresses nk cell-mediated cytotoxicity to evade the host immune response [ ] , but these previous studies did not consider the subsets of nk cells. the specific action of different subsets of nk cells on prrsv has not yet been explored, and further studies are needed to explain the role of nk cells in the containment of the virus during infection. the main targets of prrsv are cells belonging to the monocyte and macrophage lineages, particularly am. however, dc are also reportedly vulnerable to prrsv infection [ ] . the dc/macrophage network, which senses the foreign antigen and initiates the immune response, constitutes one of the main components of the respiratory immune system [ ] . it is plausible to expect that the viral infection of these cells alters this network and thus affects downstream immune responses. therefore, dissecting how these immune cells respond to prrsv infection is important to better understand the nature of prrs. until now, limited studies have investigated the alterations in the respiratory dc/macrophage network in pigs during disease progression [ , ] , and the association of the alteration in this immune network with the t-cell response has not been reported. here, we attempted to explore the dynamics of this important host defence mechanism in relation to prrsv infection. during infection, prrsv replicates in the cd + cells in the lungs and induces apoptosis at the early stages of infection [ ] . in the current study, a flow cytometric analysis of bal cells revealed that prrsv-ja decreased the cd + cell population in the infected pigs at the time when peak viral loads were detected. after the viral load decreased, the cd + cell population recovered to the normal levels. pi staining of the cd + bal cells revealed that the decrease in the cd + cell population was due to cell death. a similar reduction in the macrophage population after prrsv- infection was previously reported [ ] . following the strategy described previously [ ] , different subsets of dc and macrophages were identified in bal cells, and the changes in the populations of these subsets during disease progression were studied. previous reports verify that prrsv infection impairs dc function directly by downregulating mhc-ii expression [ , ] . intriguingly, among the five cell subsets in mhc-ii + cells, only the cd a + /cd high / mhc-ii + (am) cell population decreased significantly at peak viremia, revealing a significant negative correlation between the population of these cells and the viral loads in the lungs, whereas significant positive correlations were found for the other subset populations. the increases in the cd a + /cd int /mhc-ii + and cd a + /cd low /mhc-ii + cell populations can be attributed to the higher influx of monocytes and their differentiation in the lungs during inflammation. moreover, cd a + /cd − /mhc-ii + and cd a − /cd − / mhc-ii + cells exhibit strong migration and antigenpresenting capabilities [ ] , which can be credited to the increased influx of these cells into the lungs during the course of infection. our results were in agreement with those obtained in previous studies, which found that the populations of these cells are increased in the bal after prrsv- infection [ ] . interestingly, cdc s activate allogenic naïve t cells and aid induction of the th response, whereas cdc s induce a th response in pigs [ ] . after sensing antigen, the mature migrating dc recruit nk cells to drain ln, thus providing an early source of ifn-γ and thereby promoting th polarization [ ] . thus, dc work together with nk cells to regulate innate immunity and further dictate the direction and intensity of the adaptive immune response [ ] . the effective immune response to counter viral infections is governed by the proper activation of t lymphocytes by apc [ ] . in the current study, we analysed the dynamics of t lymphocytes in pbmc, lung parenchyma, bal, and bln to detect the central cause of the clearance of prrsv from the body. delayed induction of th , th and ctl responses was observed in the pbmc of the infected pigs after dpc, when most of the virus had been cleared from the blood, whereas in bln, the virus persists for a longer period, which leads to early and sustained (> dpc) induction of t cell responses. previous studies also found a delayed induction of effector t cells in the peripheral blood of pigs infected with prrsv [ , ] . intriguingly, significant increases in the frequencies of these t lymphocyte subpopulations were detected in the lung parenchyma and lymphoid tissues of the infected pigs at early stages of infection ( dpc), when the virus levels were at their peak in the body. in agreement with our findings, a higher frequency of t-helper cells and ctl has been observed in local tissues, such as the bal, lymph nodes and lung parenchyma, of pigs after swine influenza infection, and this induction has been linked to viral clearance [ ] . the local cell-mediated immune responses are considered crucial for the clearance of the influenza virus [ ] . moreover, during human respiratory syncytial virus infection, increased populations of cd + and cd + t cells exhibiting effector functions have been observed in the bal of infected patients [ ] . a recent study investigating t-cell proliferation in prrsv- -infected pbmc at dpi and the expression of lymph node-homing receptors revealed that the t-helper cell response plays a main role in viral clearance and that the ctl response is strongest at the site of infection [ ] . in addition, the induction of th cells in local tissues has also been found to be essential for the resolution of respiratory infections [ ] . in the present study, the cell responses in bln and bal were found to be significantly increased from or dpc and were maintained until dpc, when the virus was completely cleared. therefore, it can be concluded that local t-cell responses in the lungs, bln and bal are induced markedly faster than systemic responses and are maintained at significantly high levels, even after virus clearance, substantiating the critical role of local immune responses in the clearance of prrsv from pigs. the treg lineage is responsible for the maintenance of homeostasis in the immune system by suppressing the activation of various immune cells, including other t cells, nk cells and dc [ ] . treg play a vital role in the pathogenesis of some viral infections that result in severe inflammatory lesions, such as influenza [ ] . however, the treg response in pigs after prrsv infection is controversial. although some studies have revealed increased frequencies of tregs after prrsv infection [ , , ] , other studies revealed that the induction of tregs was not changed and even suppressed after infection [ , ] . these conflicting results could be due to the strainspecific response of the pigs to the virus after challenge or to the method used to evaluate the changes in the treg response. several studies have revealed that tregs are induced due to prrsv infection, but most of these studies were performed using in vitro or ex vivo assays in which pbmc and mononuclear cells isolated from the lungs and lymph nodes of pigs were infected or reinfected with prrsv and subsequently observed to monitor the proliferation of various t-cell subsets [ , , ] . however, some ex vivo assays have also demonstrated the inability of certain strains of prrsv to induce tregs [ ] . in the current study, the treg frequencies were directly evaluated in the peripheral blood, bal, lung and lymphoid tissues collected from prrsv-ja -infected pigs. during the acute phase of infection, the treg frequencies largely remained unchanged, but suppression was observed in the bal throughout the infection period and in bln at dpc. comparable results were obtained in a previous in vivo study, revealing that the treg numbers remained unchanged in the pbmc, lymph nodes and tonsils of the infected pigs up to dpc [ ] . similar results were observed in another study, which showed no significant upregulation of tregs in the lymph nodes and pbmc at the acute phase of prrsv infection [ ] . furthermore, the decrease in treg frequencies in ja infected pigs could be attributed to phenotypic plasticity, which resulted in most of the naïve cd + t cells differentiating into effector cells, such as th and th , after being antigenically stimulated during the acute infection [ ] . a previous study revealed that the influenza virusinfected pigs showed a similar suppression of tregs in the bal and lymph nodes and an increased frequency of t-helper cells during the acute phase of infection [ ] . moreover, in some acute infections of mice with lymphocytic choriomeningitis virus (lcmv) and influenza virus, little or no effect on the immune response was observed after the removal of tregs from mice [ , ] . based on the findings from these studies, it can be deduced that tregs play only a minor role during many acute infections, but further studies are needed to understand the ultimate cause of this decline in the treg populations, specifically in bal cells during prrsv infection. cytokine secretion by immune cells plays a major role in protection against invading pathogens or in the induction of pathology [ ] . the levels of proinflammatory cytokines have often been associated with the severity of the disease. however, lower mrna and protein expression levels of proinflammatory cytokines have been associated with protection against viruses in pigs infected with prrsv strains that typically induce mild to moderate clinical signs [ ] . prrsv has been shown to reduce or suppress ifn-α production during infection [ ] . similarly, in the current study, ifn-α was peripherally suppressed in the infected pigs with increasing viral replication; however, in the balf, the levels of ifn-α were maintained, even at peak viremia, possibly playing a role in local viral clearance. similar to our observations, the induction of ifn-α with increasing viremia has also been observed locally in lymphoid tissues and the balf in prrsv-infected pigs [ ] . moreover, local induction of the proinflammatory cytokines il- β and il- in lymphoid tissues is reportedly linked to the clearance of prrsv from infected pigs [ ] . similar induction of the proinflammatory cytokines il- α, il- , il- , and tnf-α figure chronological order of the immune responses elicited in prrsv-infected pigs. this figure shows an overall representation of the immune responses elicited in prrsv-infected pigs during the acute phase of infection, which is the stage when the virus reached its maximum titre. most of the virus is cleared from blood and tissues by dpc due to the pig immune responses. the delayed induction of serum virus-neutralizing antibodies and the t-cell response in peripheral blood at days post-exposure, when most of the virus was cleared from the body, indicate that, in addition to the other factors, the early induced local t-cell response plays a key role in the clearance of the virus. has been observed in local tissues in previous studies [ ] . in the current study, the induction of proinflammatory cytokines locally in the balf at peak viremia could thus be linked to their contribution to the clearance of the virus from the pigs. in contrast, the induction of il- in local tissues during early infection was observed in the present study. the production of il- , which has been previously observed, reportedly contributes to the persistence of prrsv by suppressing the immune response in pigs [ ] . however, il- production also protects pigs from the tissue damage caused by proinflammatory cytokine overexpression [ ] . tregs are thought to produce il- , but in the current study, the treg frequencies did not change significantly. however, in addition to tregs, monocytes, th cells, mast cells and b cells are also known sources of il- [ ] . consequently, measuring the systemic cytokine response might not provide a full picture of the protective events orchestrated by cytokines locally in the lungs of prrsv-infected pigs. in conclusion, the early stimulation of the dc/macrophage frequencies coincided with the induction of a protective t-cell response in local lymphoid tissues and lung parenchyma, which are known sites of prrsv replication. moreover, the local proinflammatory cytokine responses at peak viremia augmented the clearance of the virus from the infected pigs. a temporal sequence of immunobiological events after prrsv infection in pigs is proposed in figure , and the observed schematic suggests that local t lymphocytes might play an important role in the clearance of the virus from pigs during the acute phase of infection. in addition, early nk cell induction and delayed t-cell responses in peripheral blood were also perceived, and these might play a complementary role in viral clearance. future studies are needed to further elucidate and refine the understanding of the anti-prrsv immune response in pigs, and these future studies should primarily focus on obtaining a better understanding of and deciphering the local immune responses with the aim of developing an effective strategy to restrain the virus. characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr infected pigs porcine reproductive and respiratory syndrome virus type . lena triggers conventional dendritic cells activation and t helper immune response without infecting dendritic cells infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs the respiratory dc/ macrophage network at steady-state and upon influenza infection in the swine biomedical model broncho alveolar dendritic cells and macrophages are highly similar to their interstitial counterparts innate and adaptive immunity against porcine reproductive and respiratory syndrome virus in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding role of interleukin transcriptional regulation in inflammation and autoimmune disease inflammatory cytokine expression in swine experimentally infected with actinobacillus pleuropneumoniae cytokines and the acute phase response to influenza virus in mice enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line the use of a microtitration technique for the routine assay of african swine fever virus a simple method of estimating fifty per cent endpoints different biological characteristics of wild-type porcine reproductive and respiratory syndrome viruses and vaccine viruses and identification of the corresponding genetic determinants porcine alveolar macrophages: poor accessory or effective suppressor cells for t-lymphocytes effect of polymorphisms in porcine guanylate-binding proteins on host resistance to prrsv infection in experimentally challenged pigs swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus nkp expression discriminates porcine nk cells with different functional properties porcine cd alphadim/-nkp high nk cells are in a highly activated state porcine reproductive and respiratory syndrome virus induces cd + cd + cd + foxp + regulatory t cells (tregs) quantification of th and th cells with intracellular staining following pma/ionomycin stimulation in vitro immune responses of porcine alveolar macrophages reflect host immune responses against porcine reproductive and respiratory syndrome viruses significance analysis of xmap cytokine bead arrays cell mediated innate responses of cattle and swine are diverse during foot-and-mouth disease virus (fmdv) infection: a unique landscape of innate immunity a brief review of cd and its role in prrsv infection sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus t-cell antigen receptor genes and t-cell recognition the detailed distribution of mhc class ii antigens in normal human organs induction of inducible cd + cd + foxp + regulatory t lymphocytes by porcine reproductive and respiratory syndrome virus (prrsv) induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus phenotypic and functional characterisation of porcine cd (+)cd (high) regulatory t cells pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs porcine reproductive and respiratory syndrome: neb- prrsv infection did not potentiate bacterial pathogens comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr ), atcc vr , and two recent field isolates of prrsv convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs porcine cd (+) nkp (+) lymphocytes have nk-cell characteristics and are present in increased frequencies in the lungs of influenza-infected animals response of the cdc and cdc subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype prrsv strain characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells induced recruitment of nk cells to lymph nodes provides ifn-gamma for t(h) priming dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity t cell responses to viral infections-opportunities for peptide vaccination resistance to and recovery from lethal influenza virus infection in b lymphocyte-deficient mice cd + t cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections the t-cell response to type porcine reproductive and respiratory syndrome virus (prrsv) th lymphocytes in respiratory syncytial virus infection the development and function of memory regulatory t cells after acute viral infections induction of porcine reproductive and respiratory syndrome virus (prrsv)-specific regulatory t lymphocytes (treg) in the lungs and tracheobronchial lymph nodes of prrsv-infected pigs european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells harnessing the plasticity of cd (+) t cells to treat immune-mediated disease partial depletion of natural cd (+) cd (+) regulatory t cells with anti-cd antibody does not alter the course of acute influenza a virus infection role of regulatory t cells during virus infection proinflammatory cytokines and viral respiratory disease in pigs differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells immunohistochemical expression of il- , il- , ifn-alpha and ifn-gamma in lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs interleukin- , interleukin- beta, and interferon-gamma levels are linked to prrs virus clearance porcine reproductive and respiratory syndrome virus infection activates il- production through nf-kappab and p mapk pathways in porcine alveolar macrophages interleukin- and the interleukin- receptor publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are pleased to acknowledge the undergraduates at the college of veterinary medicine and the laboratory technicians at the veterinary diagnostic center of jeonbuk national university (jbnu) for their constant assistance throughout the study. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . key: cord- -qnn hp e authors: cong, yingying; verlhac, pauline; reggiori, fulvio title: the interaction between nidovirales and autophagy components date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: qnn hp e autophagy is a conserved intracellular catabolic pathway that allows cells to maintain homeostasis through the degradation of deleterious components via specialized double-membrane vesicles called autophagosomes. during the past decades, it has been revealed that numerous pathogens, including viruses, usurp autophagy in order to promote their propagation. nidovirales are an order of enveloped viruses with large single-stranded positive rna genomes. four virus families (arterividae, coronaviridae, mesoniviridae, and roniviridae) are part of this order, which comprises several human and animal pathogens of medical and veterinary importance. in host cells, nidovirales induce membrane rearrangements including autophagosome formation. the relevance and putative mechanism of autophagy usurpation, however, remain largely elusive. here, we review the current knowledge about the possible interplay between nidovirales and autophagy. nidoviruses rank among the most complex rna viruses and their molecular genetics clearly discriminates them from other rna virus orders [ ] . still, our knowledge about their life cycle, mostly unveiled with studies on coronaviruses (covs), is very limited [ , , , ] . to enter cells, nidoviruses bind to cell surface receptors, an event that precedes the fusion of the viral and cellular membranes ( figure , step ), which is presumably mediated by one of the surface glycoproteins [ , ] . the fusion takes place either at the plasma membrane or in the endosomes and releases the nucleocapsid into the host cell cytoplasm [ ] (figure , step ). after genomic rna uncoating from the nucleocapsid, two large replicase open reading frames (orfs), orf a and orf b, are translated by host ribosomes to yield two large polyprotein precursors that undergo autoproteolytic processing to eventually produce the non-structural (nsp) proteins. the nsp proteins interfere with the host defenses but also induce the formation of double-membrane vesicles (dmvs) and convoluted membranes, on which they collectively form the replication-transcription complexes (rtcs) [ , ] (figure , steps , , and ). these complexes mediate the synthesis of the genomic rna and a nested set of subgenomic rnas that directs the translation of the structural proteins (the nucleocapsid n protein, the membrane m protein, the envelope e protein and the spike s protein) and some accessory proteins, like the hemagglutinin esterase in the case of severe acute respiratory syndrome (sars)-cov or mouse hepatitis virus (mhv) [ ] [ ] [ ] (figure , steps and ). newly synthesized genomic rnas associate with the cytoplasmic nucleocapsid proteins to generate the so-called ribonucleoprotein complexes [ , ] . the viral structural envelope proteins are inserted into endoplasmic reticulum (er) and targeted to the site of virus assembly, the er, or the golgi, where they interact with the ribonucleoprotein complex to initiate the budding of virus particles into the lumen of the membrane compartment [ , , ] (figure , steps , and ) . newly formed virions then egress the host cell through secretion via the exocytic pathway [ , ] (figure , step ). viruses , , of they interact with the ribonucleoprotein complex to initiate the budding of virus particles into the lumen of the membrane compartment [ , , ] (figure , steps , and ). newly formed virions then egress the host cell through secretion via the exocytic pathway [ , ] (figure , step ). generalization of nidovirales life cycle, based on the information acquired studying arteriviruses and coronaviruses. infection starts with the binding of the viral particle to a cell surface receptor and subsequent cell entry through membrane fusion in endosomes upon endocytosis (step ). translation of the released genomic rna (grna) yields replicase polyproteins (step ), i.e., polyprotein a (pp a) and polyprotein ab (pp ab), which undergo autoproteolytic processing to generate nonstructural proteins that assemble into replication-transcription complexes (rtcs). the rtcs are part of a complex membranous network composed of double membrane vesicles (dmvs) and convoluted membranes (step ). the rtcs first engage in minus-strand rna synthesis to produce both single strand full-length and subgenomic (sg) minus-strand rnas (step ). subsequently, they use sg mrnas as templates for the production of the grna and plus-strand sg mrnas required to express the structural protein genes (step ). newly synthesized s, e, and m structural proteins are inserted in the endoplasmic reticulum (er) (steps and ), whereas the n nucleocapsides are translated and oligomerize in the cytosol, where they interact with rtcs and associate with the grna to form the ribonucleoprotein complexes (step ). virion assembly takes place in the er and/or golgi (step ), and involves the inward budding of the limiting membrane of these compartments, which is triggered by the interaction between the structural proteins and the ribonucleoprotein complexes. mature virions are released extracellularly by exocytosis (step ). within the term autophagy are grouped all those catabolic pathways mediating the delivery of cytoplasmic material into the mammalian or plant/yeast vacuole for degradation. there are three major types of autophagy, i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy [ ] . macroautophagy (hereafter referred to as autophagy) is conserved among eukaryotes that allows the turnover of excess or damaged cellular components, including long-lived arteriviruses and coronaviruses. infection starts with the binding of the viral particle to a cell surface receptor and subsequent cell entry through membrane fusion in endosomes upon endocytosis (step ). translation of the released genomic rna (grna) yields replicase polyproteins (step ), i.e., polyprotein a (pp a) and polyprotein ab (pp ab), which undergo autoproteolytic processing to generate nonstructural proteins that assemble into replication-transcription complexes (rtcs). the rtcs are part of a complex membranous network composed of double membrane vesicles (dmvs) and convoluted membranes (step ). the rtcs first engage in minus-strand rna synthesis to produce both single strand full-length and subgenomic (sg) minus-strand rnas (step ). subsequently, they use sg mrnas as templates for the production of the grna and plus-strand sg mrnas required to express the structural protein genes (step ). newly synthesized s, e, and m structural proteins are inserted in the endoplasmic reticulum (er) (steps and ), whereas the n nucleocapsides are translated and oligomerize in the cytosol, where they interact with rtcs and associate with the grna to form the ribonucleoprotein complexes (step ). virion assembly takes place in the er and/or golgi (step ), and involves the inward budding of the limiting membrane of these compartments, which is triggered by the interaction between the structural proteins and the ribonucleoprotein complexes. mature virions are released extracellularly by exocytosis (step ). within the term autophagy are grouped all those catabolic pathways mediating the delivery of cytoplasmic material into the mammalian or plant/yeast vacuole for degradation. there are three major types of autophagy, i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy [ ] . macroautophagy (hereafter referred to as autophagy) is conserved among eukaryotes that allows the turnover of excess or damaged cellular components, including long-lived proteins and organelles, to maintain cellular energy levels and ensure survival [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this process is characterized by double-membrane vesicles called autophagosomes, which sequester the cytoplasmic components targeted to destruction and deliver them into lysosomes for degradation [ ] [ ] [ ] [ ] [ ] (figure ). the process can be divided into three different steps: the initiation step, when the phagophore or isolation membrane is formed, the elongation step, during which the phagophore expands and close to generate an autophagosome, and the maturation step, where the autophagosome fuses with the lysosome (figure ). autophagy is regulated by several signaling cascades, including the one centered on the mammalian target of rapamycin (mtor) [ , , ] . autophagosomes are formed through the concerted action of the autophagy-related (atg) genes [ , ] . the proteins encoded by the atg genes have been classified in five functional groups. the unc- like autophagy activating kinase (ulk) kinase complex, the class iii hvps phosphatidylinositol -kinase complex, and the atg cycling system, on one hand, play a key role in the initiation of autophagy and phagophore formation. the atg and microtubule-associated protein light chain (lc ) conjugation systems, on the other hand, mediate the elongation and closure of the phagophore. the first of these atg protein complexes responds to upstream regulatory signals, such as the inactivation of mtor, and key in initiating the formation of an autophagosome, is the ulk kinase complex, which is composed of ulk or ulk , atg , fak family kinase-interacting protein of kda (fip ), and atg [ , ] . the class iii hvps phosphatidylinositol -kinase, which is part of a complex including hvps , atg l , and beclin , generates the pool of phosphatidylinositol -phosphate (ptdins p) on autophagosomal membranes that facilitates the recruitment of ptdins p effectors such as double fyve-containing protein (dfcp ) and the human wd-repeat protein interacting with phosphoinositides (wipi) proteins [ ] [ ] [ ] [ ] [ ] [ ] . the hvps -containing complex as well as the transmembrane protein atg , are two other important factors during the early stage of autophagosome biogenesis [ , ] . subsequently, two ubiquitination-like systems, which ultimately recruit members of the lc /atg protein family onto autophagosomal membranes through their conjugation to phosphatidylethanolamine, are essential for the completion of the forming autophagosomes [ ] . finally, the fusion of autophagosomes first with endosomes and then with lysosomes, leads to the formation of amphisomes and autolysosomes, respectively, where the degradation of the autophagy cargoes take place [ ] . proteins and organelles, to maintain cellular energy levels and ensure survival [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this process is characterized by double-membrane vesicles called autophagosomes, which sequester the cytoplasmic components targeted to destruction and deliver them into lysosomes for degradation [ ] [ ] [ ] [ ] [ ] (figure ). the process can be divided into three different steps: the initiation step, when the phagophore or isolation membrane is formed, the elongation step, during which the phagophore expands and close to generate an autophagosome, and the maturation step, where the autophagosome fuses with the lysosome (figure ). autophagy is regulated by several signaling cascades, including the one centered on the mammalian target of rapamycin (mtor) [ , , ] . autophagosomes are formed through the concerted action of the autophagy-related (atg) genes [ , ] . the proteins encoded by the atg genes have been classified in five functional groups. the unc- like autophagy activating kinase (ulk) kinase complex, the class iii hvps phosphatidylinositol -kinase complex, and the atg cycling system, on one hand, play a key role in the initiation of autophagy and phagophore formation. the atg and microtubule-associated protein light chain (lc ) conjugation systems, on the other hand, mediate the elongation and closure of the phagophore. the first of these atg protein complexes responds to upstream regulatory signals, such as the inactivation of mtor, and key in initiating the formation of an autophagosome, is the ulk kinase complex, which is composed of ulk or ulk , atg , fak family kinaseinteracting protein of kda (fip ), and atg [ , ] . the class iii hvps phosphatidylinositol -kinase, which is part of a complex including hvps , atg l , and beclin , generates the pool of phosphatidylinositol -phosphate (ptdins p) on autophagosomal membranes that facilitates the recruitment of ptdins p effectors such as double fyve-containing protein (dfcp ) and the human wd-repeat protein interacting with phosphoinositides (wipi) proteins [ ] [ ] [ ] [ ] [ ] [ ] . the hvps -containing complex as well as the transmembrane protein atg , are two other important factors during the early stage of autophagosome biogenesis [ , ] . subsequently, two ubiquitination-like systems, which ultimately recruit members of the lc /atg protein family onto autophagosomal membranes through their conjugation to phosphatidylethanolamine, are essential for the completion of the forming autophagosomes [ ] . finally, the fusion of autophagosomes first with endosomes and then with lysosomes, leads to the formation of amphisomes and autolysosomes, respectively, where the degradation of the autophagy cargoes take place [ ] . it has long been believed that the atg proteome is exclusively involved in autophagy, but recent findings have revealed that single atg genes or functional clusters of atg genes fulfill important cellular functions outside the context of their role in autophagy [ ] [ ] [ ] . some of these functions have it has long been believed that the atg proteome is exclusively involved in autophagy, but recent findings have revealed that single atg genes or functional clusters of atg genes fulfill important cellular functions outside the context of their role in autophagy [ ] [ ] [ ] . some of these functions have been discovered by studying host-pathogen interactions [ , , ] . for example, atg but no other atg proteins play a unique role in the defense against mycobacterium infection [ ] . more recently, it has been shown that fip and atg participate in the controlling of picornaviral infections outside the context of autophagy [ ] . here, we will review the current knowledge on the interplay between nidovirales and autophagy. there are currently no data available for several viruses and few nidovirales families. thus, this compendium will focus on the documented viruses in the arterivirus and coronavirus families (summarized in table ). green shading indicates a pro-viral role for autophagy, grey shading indicates conflictual reports and blue shading highlights an unconventional role of autophagy-related (atg) proteins. the table also provides the information about how autophagy has been experimentally manipulated in the studies addressing the role of this pathway with the indicated virus, the used cell types, and the references. -ma: -methyladenine; bafa : bafilomycin a , lc : microtubule-associated protein light chain , mefs: mouse embryonic fibroblasts. the two most studied arteriviridae are prrsv and the equine arteritis virus (eav). the prrsv strain, which was historically first characterized and is commonly referred to as atypical (i.e., ap prrsv), causes the abortions in - % of the sows, and fever and anorexia leading to the death of - % of them [ ] . however, in , the emergence of a novel virulent highly pathogenic prrsv (hp prrsv) strain, carrying mutations in nsp β, nsp , and orf genes, caused higher morbidity ( %) and mortality ( %) rates in piglets and sows [ ] . the equine arteritis virus, in contrast, infects horses and donkeys, and can cause abortions in pregnant females and mortality in neonates [ ] . the first study on the role of autophagy in arterivirus life cycle was carried out with the hp prrsv strain [ ] . infected cells displayed the presence of a higher number of autophagosome-like double-membrane vesicles, an accumulation of green fluorescent protein (gfp)-lc -positive puncta and higher levels of lipidated lc , indicating an induction of autophagy [ ] . inhibition of autophagy with either -methyladenine ( -ma), a non-specific hvps inhibitor, or depletion of atg or beclin , led to a significant reduction in both expression of prrsv nsp and prrsv titer. conversely, induction of bulk autophagy using rapamycin (mtor inhibitor) resulted in an enhancement of viral replication [ ] , a result that later was confirmed by others [ ] [ ] [ ] [ ] . in one of these subsequent studies, pujhari et al. showed that while the virus titer in the supernatants of infected cells treated with rapamycin was higher than in the control, intracellular levels of prrsv n protein or nsp assessed by flow cytometry were lower [ ] . this latter result is the opposite of the ones obtained by the other studies where rapamycin treatment led to an up-regulation of nsp expression [ ] [ ] [ ] [ ] . two subsequent works reached the same conclusion of autophagy having a beneficial role in prrsv life cycle [ , ] . liu et al. confirmed autophagy induction by both ap and hp prrsv strains [ ] . interestingly, they also observed a decrease in the virus titer in cells treated with bafilomycin a (bafa ), a drug inhibiting either autophagosome-lysosome fusion or lysosomal degradation, which suggested that autolysosomes might serve as replication platforms for prrsv [ ] . in contrast, sun et al. showed, using confocal microscopy, that the hp prrsv infection inhibits the fusion between autophagosomes and lysosomes [ ] . this result indicates that prrsv might trigger an incomplete autophagy and implicates that this could be beneficial for the virus life cycle. to gain insights into a possible molecular connection between autophagy and prrsv, they also transfected cells with either nsp or nsp , which encode two transmembrane components of arterivirus rtcs that play a central role in dmvs formation [ ] . interestingly, they found the co-localization between endogenous lc and ectopically expressed nsp , but not nsp , by confocal microscopy and fractionation on continuous density gradients, suggesting that the accumulated autophagosomes during prrsv could represent the replicative platforms [ ] . thus, it still remains to be firmly demonstrated whether the results obtained with the ectopic expression of nsp recapitulates a situation occurring in cells exposed to prrsv. recently, three additional research articles provided evidences that autophagy but also apoptosis are induced by prrsv in host cells [ , , ] . wang and collaborators investigated apoptosis and autophagy activation in the thymus of piglets infected with the hp prrsv strain because they previously showed that this virus leads to thymic atrophy and thymocyte apoptosis [ ] . their investigation concluded that the hp prrsv could induce apoptosis in bystander cells and autophagy in both infected and bystander cells in the thymus of infected piglets [ ] . in a follow-up study, another laboratory found that hp prrsv replication was attenuated in autophagy deficient marc- cells and potentiated by inhibiting apoptosis using z-vad-fmk, a caspase pan-inhibitor [ ] . interestingly, hp prrsv replication could be restored in the autophagy deficient cells by blocking apoptosis, suggesting a functional interplay between autophagy and apoptosis during prrsv replication. subsequently, zhou et al. confirmed the activation of autophagy and a subsequent induction of apoptosis over the course of a prrsv infection in marc- cells [ ] . in their study, inhibition of autophagy by -ma caused a significant increase in prrsv-induced apoptosis, also unveiling a potential connection between both mechanisms. in line with this conclusion, they also observed an increase in the expression of bcl- -associated death promoter (bad), a pro-apoptosis protein, and beclin , an autophagy regulator. interestingly, co-immunoprecipitation and confocal microscopy experiments revealed the formation of a bad-beclin complex in infected cells [ ] . beclin knockdown significantly decreased viral replication and prrsv-induced autophagy as expected, while knocking down of bad resulted in an induction of autophagy and enhanced viral replication [ ] . the authors concluded that the enhancement of autophagy could promote prrsv replication by postponing apoptosis through the formation of the bad-beclin complex [ ] . in a study exploring a potential connection between eav and autophagy, monastyrska et al. found that the dmvs induced by this virus are decorated with lc but the eav life cycle proceeded unaffected in cells lacking atg [ ] . although autophagy was not required, depletion of lc markedly reduced eav replication and it could be fully restored by expression of a non-lipidable form of lc [ ] . similar to mhv, eav-induced dmvs were also positive for edem (er degradation enhancing alpha-mannosidase like protein ) leading to the conclusion that eav might also hijack the er-derived membranes of edemosomes to ensure its replication [ , ] . it still needs to be investigated, however, whether other atg proteins are dispensable for eav life cycle. furthermore, it is unclear whether both autophagy and apoptosis are induced over the course of an eav infection as observed for prrsv. the most studied alphacoronaviruses (alpha-covs) are the transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv), which both infect suckling piglets and lead to a high mortality rate [ , ] . recurrent outbreaks of pedv have occurred across asia and the usa, causing significant economic losses [ ] . alpha-cov also includes human pathogens such as hcov- e and hcov-nl , which are associated with respiratory tract infections such as the common cold to bronchiolitis [ , ] . despite their medical and veterinary relevance, however, the exact mechanisms of alpha-cov replication and pathogenesis are not well characterized yet. sun et al. recently performed a high throughput mass spectrometry analysis in pedv-infected vero cells [ ] . their goal was to identify which cellular proteins are differentially expressed during viral infection to better understand the impact of pedv on host cells. interestingly they found that autophagy might be among the altered pathways as sequestrosome (sqtsm /p ) and lc expression levels were upregulated. a subsequent study thus focused on the potential interplay between autophagy and pedv [ ] . the authors found that pedv infection induces autophagy in vero cells using different assays such as lc -positive puncta formation, transmission electron microscopy (tem) and western blot assessment of both lc lipidation and sqstm /p turnover. in line with these observations, -ma treatment or the ablation of either beclin or atg , reduced the production of infectious viral particles. treatment of the infected cells with rapamycin, however, did not change the viral titer, probably because of the multiple effects of this compound on the cell physiology. altogether, these data showed that autophagy induction during pedv infection could be beneficial for the virus. two more recent studies have addressed the link between autophagy and tgev replication but they have reached opposite conclusions [ , ] . they both demonstrated that autophagy is induced in tgev-infected cells using methods such as tem, lc puncta formation and western blot analysis of both lc lipidation and sqstm /p degradation. in their article zhu et al. also showed that the selective degradation of mitochondria by autophagy, i.e. mitophagy, might be induced by tgev as they observed in infected ipec-j cells a reduced mitochondrial mass, a light oxidative stress, and mitochondria in autophagosome-like vesicles [ ] . in support of this notion, the authors also found that tgev n protein and gfp-lc localize to mitochondria. interestingly, induction of mitophagy by mitochondria depolarization using carbonyl cyanide m-chlorophenyl hydrazone (cccp) increased the viral titer, suggesting that this pathway might be beneficial for viral replication. similarly, induction of bulk autophagy using rapamycin also led to more production of progeny virus [ ] . conversely, incubation with -ma or atg depletion inhibited viral replication assessed by n protein expression and viral titers. zhu et al. thus concluded that autophagy, and mitophagy in particular, plays an important role in tgev life cycle [ ] . this conclusion, however, differs from the one reached in a parallel study. guo and collaborators found that both hvps and lysosomal inhibitors increased both the number of cells infected by tgev and the viral titer, while rapamycin had an opposite effect [ ] . moreover, silencing lc , atg , or atg expression in st cells promoted tgev replication, showing that autophagy has an antiviral function [ ] . the apparent discrepancies between these two studies could be explained by the use of different tgev strains (shxb versus h ) and/or cell lines (ipec-j versus st). further investigations are thus needed to conclusively determine whether autophagy plays a role in tgev life cycle. it will be particularly important to establish this in tissues that are normally infected by pedv. the first investigations on the interplay between cov and autophagy focused on mhv, a betacoronavirus (beta-cov) that is often used as a model virus to study the mechanism of cov infections. as a result, there is a relatively large amount of data about various aspects of mhv life cycle. importantly, the genus beta-cov also includes the highly pathogenic human viruses sars-cov and mers-cov, two viruses that cause acute respiratory symptoms and they are on the who list of viruses likely to cause future epidemics (figure ). like other cov, mhv replication takes place on interconnected structures formed by convoluted membranes and dmvs, with the latter being reminiscent of autophagosomes [ ] . this structural similarity prompted the investigation of a possible interplay between autophagy and cov replication. interestingly, the first two studies on the importance of atg proteins during mhv replication reached conflicting conclusions. prentice et al. argued that components of the autophagy machinery are required for mhv replication while zhao et al. demonstrated that autophagy was dispensable for the same process [ , ] . in particular, both teams monitored viral replication in murine embryonic fibroblasts (mefs) knocked out for atg . the first group found that both mhv replication and dmv formation was impaired in atg −/− knockout cells, while the second did not observe any effect on mhv life cycle in absence of atg [ , ] . the fact that mhv infection does not require intact atg machinery was also later confirmed by another group using atg −/− mefs [ ] . data from both laboratories, however, established that the viral rtcs are co-localizing with endogenous lc , which on one hand was in agreement with observations gained from sars-cov, but on the other hand was conflicting with results obtained with mhv by a third group [ , ] . these apparent discrepancies were subsequently explained by showing that endogenous lc but not ectopically expressed gfp-lc co-localizes with cov rtcs [ ] . data from different groups strongly support an er involvement in convoluted membranes and dmvs biogenesis, as those structures can be found connected to the er and transmembrane nsp proteins can be glycosylated and localize to the er when individually expressed [ , ] . the rtcs and dmvs, however, do not co-localize with marker proteins of the er, ergic, and the golgi [ , ] and disruption of the secretory pathway has no major effect on cov replication [ ] . this indicates that dmvs' biogenesis might not involve the secretory pathway. in contrast, the er-associated degradation (erad) tuning pathway, a vesicular transport route out of the er, has been shown to be important for cov infection [ ] . erad allows for the degradation of misfolded er proteins and it is negatively regulated during normal growing conditions, in absence of stress. this tuning down of the erad activity is mediated at least in part by small vesicles called edemosomes, which specifically capture key positive erad regulators such as edem and os- , and degrade them in compartments of the endolysosomal system [ ] . interestingly, edemosomes are decorated with non-lipidated from of lc (also called lc -i) and their formation might require selected components of the atg machinery, such as atg [ , ] . reggiori et al. eventually revealed that dmvs were associated with lc -i and positive for both edem and os- , suggesting that mhv might actually highjack part of the membranes of the erad tuning pathway [ ] . although expression of a non-lipidable lc impaired dmvs biogenesis and viral replication, absence of edem and os- had no effect. thus, the authors hypothesized that one or more nsp proteins might associate with components of the machinery of edemosomes, such as a cargo receptor or a coat protein, to subvert these vesicles and generate the dmvs. lc -i could be such a candidate but no interaction between lc -i and mhv nsp proteins was detected using the yeast two-hybrid assay [ ] . how mhv highjacks edemosomes and what the exact role of lc -i is in this process are questions that remain unanswered. overall, beta-cov interactions with autophagy and atg proteins appear to be complex. although mhv hijacking of lc -positive edemosomes for its replication appears to be clearly established, this finding has not yet been extended to other beta-cov or to other cov in general. co-localization between sars-cov rtcs and endogenous lc , however, has been reported [ ] . beta-cov do not require canonical autophagy for their life cycle [ ] [ ] [ ] but it cannot be excluded a priori that they could need a non-canonical form of autophagy, independent from atg and atg [ ] . furthermore, while autophagy might be induced during infection or transient expression of single viral proteins [ , ] , there is currently no evidence that this is directly regulated by beta-cov. indeed, autophagy stimulation could be part of a cellular response caused by either the presence of toxic exogenous proteins or er stress induced by the massive production of viral proteins [ ] [ ] [ ] . a recent study concluded that expression of nsp fragments derived from several cov, which comprise the papain-like protease domain and one transmembrane segment, induce autophagy through direct binding to lc and beclin [ ] . this conclusion, however, has to be carefully considered since the authors of this study observed gfp-lc puncta formation in cells ectopically expressing the nsp fragment but they did not examine whether these puncta are indeed autophagosomes. moreover, the relevance of the use of a truncated form of nsp in absence of a cov infection remains to be determined. additional studies are thus needed to address the questions if and, eventually, how beta-cov trigger autophagy. gammacoronaviruses (gamma-covs) are viruses that mainly infect poultry but are also transmissible to humans. they replicate in the respiratory tract and thus cause respiratory defects. the infectious bronchitis virus, which causes major loses in the poultry industry, is the model virus for gamma-cov. similarly to other cov, the ibv genome encodes several nsp proteins that help with replication and interfere with host cell functions. cottam et al. have reported that infections with ibv trigger the formation of endogenous lc -positive puncta in host cells [ , ] . interestingly, they noted that a fraction of these puncta partially co-localized with double stranded viral rna. by screening several ibv nsp proteins, they found that ectopically expressed nsp localized with the er and was able to autonomously induce the formation of gfp-lc puncta. this raised the question whether the gfp-lc puncta induced by nsp were edemosomes [ ] . in contrast to edemosomes, however, formation of these gfp-lc -positive vesicles required atg and lc lipidation, suggesting that they are canonical autophagosomes [ , , ] . interestingly, nsp from mhv and sars-cov also induced gfp-lc puncta formation indicating that the nsp -dependent mechanism for autophagy induction might be conserved among covs. cottam et al. also argued that nsp might reduce the expansion of the autophagosomes as well, while maturation into autolysosomes is still possible [ ] . these results have been obtained using ectopic expression of nsp and thus the relevance of nsp -mediated induction of autophagy during cov infection remains to be explored. the investigation of the potential interplay between nidovirales and autophagy is still at its beginning. nonetheless, it can already be firmly concluded that nidovirales infections trigger autophagy in host cells. several viral families and virus species such as torovirinae, mesoviridae, and roniviridae, have yet to be investigated while for others, such as prssv and tgev, opposite conclusions have been reached regarding whether autophagy induction is beneficial or detrimental for the viral life cycle. the apparent contradictory results could be due to the use of different cell lines and tissues, and/or virus strains. some of these discrepancies could also be due to potential noncanonical functions of atg proteins as was shown for mhv. further investigations are therefore needed to reconcile these results. another drawback of several of the studies cited in this review is the extensive use of drugs to modulate autophagy during viral infection. as none of the employed compounds inhibits autophagy specifically, they can have adverse effects on cellular or viral biology, making the interpretation of the results difficult. the genetic ablation of atg proteins is a better option but it must be kept in mind that these factors are also part of other pathways [ , ] . as a result, it is crucial to compare the results obtained by depleting more than one atg protein. moreover, the few studies that have depleted atg proteins have blocked the initial steps of the autophagic pathway ( figure and table ) without analyzing the steps following the completion of an autophagosome. this is relevant since some viruses such as influenza a or epstein-barr virus, have been shown to manipulate this part of the pathway and therefore it is critical to investigate whether it could also be the case for nidovirales [ , ] . it also remains unclear which step of the virus life cycle is impacted, as most studies relied on assays quantifying general parameters such as the viral protein levels, the number of infected cells, and /or the number of produced infectious viral particles. results that were obtained by studying viruses from other orders have revealed that autophagy and atg proteins can practically play a key role in every step of a virus life cycle, from entry to assembly and egression [ ] . while it is indisputable that large part of the investigated nidovirales induces autophagy in host cells, it still remains unclear whether this is due to a subversion of autophagy by the virus or whether it is a physiological response to the cellular stress caused by either the infection or the transfection of single viral proteins. future research should therefore also focus on the identification of a potential direct molecular link between viral and atg proteins. such studies could also pave the way to the development of novel antiviral therapies targeting the virus-autophagy interaction. evolving the largest rna virus genome the molecular biology of coronaviruses virus taxonomy: ninth report of the international committee on taxonomy of viruses nidovirus transcription: how to make sense the coronaviridae retrospective testing and case series study of porcine delta coronavirus in us swine herds coronavirus entry and release in polarized epithelial cells: a review the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range identification and characterization of genetically divergent members of the newly established family mesoniviridae an insect nidovirus emerging from a primary tropical rainforest complete genome sequence of an isolate of a novel genotype of yellow head virus from fenneropenaeus chinensis indigenous in china genetic engineering alveolar macrophages for host resistance to prrsv assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc nidovirus entry into cells heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses nidovirus ribonucleases: structures and functions in viral replication are nidoviruses hijacking the autophagy machinery? the nidoviruses: (coronaviruses and arteriviruses) autophagy: renovation of cells and tissues autophagy and the endo/exosomal pathways in health and disease autophagy-from molecular mechanisms to clinical relevance autophagy in leukocytes and other cells: mechanisms, subsystem organization, selectivity, and links to innate immunity autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity advances in autophagy regulatory mechanisms. cells autophagy and mammalian viruses: roles in immune response, viral replication, and beyond autophagy: from phenomenology to molecular understanding in less than a decade autophagy as a regulated pathway of cellular degradation guidelines for the use and interpretation of assays for monitoring autophagy the autophagosome is overrated! autophagy autophagy: molecular machinery for self-eating signaling the beginning from the end the ulk complex: sensing nutrient signals for autophagy activation autophagosome formation-the role of ulk and beclin -pi kc complexes in setting the stage autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum the puzzling origin of the autophagosomal membrane dynamic association of the ulk complex with omegasomes during autophagy induction temporal analysis of recruitment of mammalian atg proteins to the autophagosome formation site mammalian atg (wipi ) localizes to omegasome-anchored phagophores and positively regulates lc lipidation dynamic and transient interactions of atg with autophagosomes, but not membrane integration, are required for autophagy mammalian autophagy: core molecular machinery and signaling regulation hidden behind autophagy: the unconventional roles of atg proteins non-autophagic roles of autophagy-related proteins atg proteins: are we always looking at autophagy? autophagy using microbes as a key tool to unravel the mechanism of autophagy and the functions of the atg proteins unique role for atg in neutrophil-mediated immunopathology during m. tuberculosis infection an sirna screen for atg protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication segregation and rapid turnover of edem by an autophagy-like mechanism modulates standard erad and folding activities basal autophagy is involved in the degradation of the erad component edem edem reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the copii exit sites induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication role of phosphatidylinositol- -kinase (pi k) and the mammalian target of rapamycin (mtor) signalling pathways in porcine reproductive and respiratory syndrome virus (prrsv) replication autophagy sustains the replication of porcine reproductive and respiratory virus in host cells porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication highly pathogenic porcine reproductive and respiratory syndrome virus infection induced apoptosis and autophagy in thymi of infected piglets interplay of autophagy and apoptosis during prrsv infection of marc cell open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex autophagy postpones apoptotic cell death in prrsv infection through bad-beclin interaction coronavirus replication complex formation utilizes components of cellular autophagy coronavirus replication does not require the autophagy gene atg coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication an autophagy-independent role for lc in equine arteritis virus replication clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus equine arteritis virus: a review of clinical features and management aspects production and characterization of a monoclonal antibody against spike protein of transmissible gastroenteritis virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines human coronavirus nl , a new respiratory virus understanding human coronavirus hcov-nl analysis of protein expression changes of the vero e cells infected with classic pedv strain cv by using quantitative proteomic technique porcine epidemic diarrhea virus induces autophagy to benefit its replication mitophagy in tgev infection counteracts oxidative stress and apoptosis rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum localization and membrane topology of coronavirus nonstructural protein : involvement of the early secretory pathway in replication integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus rna synthesis and virus-induced remodeling of endoplasmic reticulum membranes disposal of cargo and of erad regulators from the mammalian er discovery of atg /atg -independent alternative macroautophagy coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate endoplasmic reticulum stress triggers autophagy the coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mrna concentrations severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity coronavirus nsp restricts autophagosome expansion lc -associated phagocytosis (lap): connections with host autophagy. cells matrix protein of influenza a virus blocks autophagosome fusion with lysosomes epstein-barr virus blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication manipulation or capitulation: virus interactions with autophagy. microbes infect author contributions: y.c., p.v. and f.r. organized all related references and wrote the manuscripts. all authors read and approved this manuscript. the authors declare no conflict of interest. key: cord- -u bbgn k authors: yun, sang-im; lee, young-min title: overview: replication of porcine reproductive and respiratory syndrome virus date: - - journal: j microbiol doi: . /s - - -z sha: doc_id: cord_uid: u bbgn k porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. the genome replication of prrsv is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. replication of the viral rna genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic rna template; this replication complex is embedded within particular virus-induced membrane vesicles. prrsv rna replication is directed by at least replicase proteins that have both common enzymatic activities, including viral rna polymerase, and also unusual and poorly understood rna-processing functions. in this review, we summarize our current understanding of prrsv replication, which is important for developing a successful strategy for the prevention and control of this pathogen. porcine reproductive and respiratory syndrome virus (prrsv) is the etiologic agent of prrs wensvoort et al., ; benfield et al., ; collins et al., ) , an economically devastating, pandemic disease of swine that is typically characterized by reproductive failure in breeding herds and respiratory problems and growth retardation in growing pigs (done and paton, ; botner, ; van reeth, ; zimmerman et al., ; rossow, ; suarez, ; rowland, ) . two prrs outbreaks were first reported in the late s in north america (keffaber, ; hill, ) and central europe (paton et al., ) . the disease is now found in most pig-producing countries and affects the swine industry and food safety worldwide (albina, ; blaha, ; lunney et al., ; shi et al., a) , causing enormous economic losses each year (brouwer et al., ; garner et al., ; zimmerman et al., ; nieuwenhuis et al., ) . in the us, the annual loss due to prrs is estimated to exceed $ million (neumann et al., ) . in particular, the emergence of highly pathogenic prrsvs in china and vietnam in (li et al., ; tian et al., ; feng et al., ; zhou et al., ; and their rapid spread to several neighboring asian countries (an et al., ) have raised a growing concern that new pathogenic prrsvs can spread throughout the world, posing a significant threat to the global agricultural community (normile, ; lunney and chen, ; murtaugh et al., ; zhou and yang, ) . because of the current burden of prrs and the emergence of highly pathogenic forms of prrsv on a global level, control of this virus remains a research priority in all pig-producing countries. prrsv belongs to the family arteriviridae in the order nidovirales, which also includes two other families, the coronaviridae and roniviridae (gorbalenya et al., ; de groot et al., ) . within the arteriviridae family, prrsv forms a single genus arterivirus, together with equine arteritis virus (eav), lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus (plagemann and moennig, ; cavanagh, ; de vries et al., ; faaberg et al., ) . like other arteriviruses, prrsv is an enveloped virus (dokland, ) containing a non-isometric nucleocapsid core (spilman et al., ) that encapsidates a plus-strand genomic rna of ~ kb in length (meulenberg et al., ) . this genomic rna consists of a -untranslated region (utr), open reading frames (orfs), and a -utr (snijder and spaan, ; britton and cavanagh, ; firth et al., ; johnson et al., ) (fig. ) . based on its genetic diversity and geographic distribution, prrsv is divided into two major genotypes (murtaugh et al., the ~ -kb plus-strand genomic rna of prrsv is shown on top. two long -proximal orfs (orf a and orf b) are translated into two large polyprotein precursors, pp a and pp ab; the latter is synthesized by a - ribosomal frameshift. the two polyproteins are cleaved into at least nsps: encoded in orf a (nsp α, nsp β, nsp to nsp , nsp α, nsp β, and nsp ) and encoded in orf b (nsp to nsp ). this proteolysis is regulated by four viral proteases, namely nsp α, nsp β, nsp , and nsp . an additional protein designated nsp tf is translated by a - ribosomal frameshift in the nsp -coding region. (b) synthesis of the viral structural proteins from the six subgenomic mrnas. eight short -proximal orfs are translated from a nested set of six major subgenomic mrnas: orf a (gp / a), orf b (e, envelope), orf (gp ), orf (gp ), orf (gp ), orf (m, membrane), orf (n, nucleocapsid), and a newly discovered protein encoded in orf a that overlaps with the end of orf . ; shi et al., a) : type , represented by the european prototype lelystad strain ; and type , exemplified by the north american prototype vr- strain (benfield et al., ; collins et al., ) . interestingly, despite their concurrent emergence and similar clinical symptoms (halbur et al., ) , the two genotypes show ~ % genetic divergence (mardassi et al., ; kapur et al., ; allende et al., ; nelsen et al., ; meng, ; oleksiewicz et al., ; forsberg et al., ) , with a high degree of antigenic variation (wensvoort et al., ; nelson et al., ; drew et al., ; wootton et al., ) . over the last decade, this genetic/antigenic diversity has expanded continuously and rapidly (murtaugh et al., ; stadejek et al., ; mateu et al., ; pesch et al., ; han et al., ; stadejek et al., stadejek et al., , balka et al., ; li et al., li et al., , shi et al., b) , highlighting the dynamic nature of prrsv evolution and epidemiology. at present, a larger number of genetically heterogeneous prrsvs are widely co-circulating throughout the world than ever before (dewey et al., ; goldberg et al., ; ropp et al., ; thanawongnuwech et al., ; fang et al., ) , posing a significant challenge for the diagnosis, prevention, and control of prrsv infection. prrsv is transmitted both horizontally (pig-to-pig infection) and vertically (transplacental infection) to fetuses during mid-to-late gestation (christianson et al., (christianson et al., , yaeger et al., ) ; horizontal transmission occurs through both direct and indirect contact (cho and dee, ; zimmerman et al., ) . direct contact is the most efficient route of prrsv transmission, via a variety of porcine secretions from infected animals in which the virus has been detected: e.g., saliva (wills et al., a; prickett et al., ) , milk (wagstrom et al., ) , nasal fluids (rossow et al., ) , and se-men (swenson et al., ; christopher-hennings et al., ) . although its mechanism(s) remains elusive (mateu and diaz, ; lunney and chen, ; yoo et al., ; murtaugh and genzow, ) , prrsv persistence in pigs plays an important role in viral transmission because the virus is present at low levels in the infected animals (wills et al., b (wills et al., , allende et al., ; bierk et al., ; batista et al., batista et al., , horter et al., ) . in addition to these direct routes of prrsv transmission, indirect routes of a particular concern include contaminated fomites (dee et al., (dee et al., , otake et al., b) , needles (otake et al., c ), transport vehicles (dee et al., , aerosols (torremorell et al., ; brockmeier and lager, ; mortensen et al., ; otake et al., a otake et al., , kristensen et al., ; trincado et al., ; fano et al., ; dee et al., ; pitkin et al., ) , and insects as a mechanical vector (otake et al., d (otake et al., , a (otake et al., , b schurrer et al., schurrer et al., , . prrsv infection is initiated by the attachment of the virions to the highly sulfated, negatively charged glycosaminoglycans on the surface of susceptible cells (jusa et al., ; vanderheijden et al., ; delputte et al., ) , followed by binding to cd (duan et al., a , b vanderheijden et al., ; delputte and nauwynck, ; delputte et al., delputte et al., , van breedam et al., b) , which triggers receptor-mediated clathrin-dependent endocytosis (kreutz and ackermann, ; nauwynck et al., ; vanderheijden et al., ) . at the early endosomes, the viral genome is released into the cytoplasm through a reaction mediated by cd (calvert et al., ; van gorp et al., das et al., ; van gorp et al., ) and presumably other cellular factors (misinzo et al., ) . once the genome enters the cytoplasm, orf a and orf b, located in the -proximal three-quarters of the viral genome, are translated to produce two large polyproteins, pp a and pp ab (snijder and meulenberg, ; snijder and spaan, ) , with the expression of pp ab controlled by a - ribosomal frameshift (brierley et al., ; den boon et al., ) (fig. a) . autocatalytic processing of these precursors generates at least nonstructural proteins (nsps) (ziebuhr et al., ; fang and snijder, ) : encoded in orf a (nsp α, nsp β, nsp to nsp , nsp α, nsp β, and nsp ) and encoded in orf b (nsp to nsp ) (snijder et al., (snijder et al., , den boon et al., ; van dinten et al., ; wassenaar et al., ; chen et al., a; li et al., ) . this proteolytic processing is mediated by four viral proteases residing in nsp α, nsp β, nsp , and nsp (den boon et al., ; snijder et al., ; van aken et al., b) . also, an additional viral protein is synthesized by a - ribosomal frameshift in the nsp -coding region, yielding a transframe fusion nsp tf with the nterminal two-thirds of nsp (fang et al., ) . most, if not all, of the nsps assemble into a replication and transcription complex (rtc) that accumulates at the virus-induced er-derived double-membrane vesicles (van der meer et al., ; pedersen et al., ; kroese et al., ) . the rtc directs both genome amplification ("replication") and subgenomic mrna synthesis ("transcription") (fang and snijder, ) ; the latter, a hallmark of all nidoviruses (pasternak et al., ; sawicki et al., ; snijder and spaan, ) , produces a nested set of six major subgenomic mrnas that are both -and -coterminal with the genomic rna and thus consist of nucleotide sequences that are non-contiguous in the genomic rna (de vries et al., ) . through the six subgenomic mrnas, eight mostly overlapping orfs situated in the -proximal region of the viral genome are expressed, presumably by utilizing each subgenomic mrna for the translation of one or two of its most -proximal orfs (conzelmann et al., ; meng et al., ) (fig. b) . these orfs encode eight structural proteins that constitute the infectious virion (snijder and meulenberg, ; snijder and spaan, ; dokland, ) : i.e., four minor components encoded in orf a (gp / a), orf b (e, envelope), orf (gp ), and orf (gp ); three major components encoded in orf (gp ), orf (m, membrane), and orf (n, nucleocapsid) (meulenberg et al., ; meulenberg and petersen-den besten, ; van nieuwstadt et al., ; snijder et al., ; wu et al., ) ; and a recently identified protein encoded in orf a that overlaps with the -end of orf (firth et al., ; johnson et al., ) . at the late stage of viral replication, multiple copies of the n proteins bind to the newly synthesized genomic rna to form a nucleocapsid complex (tijms et al., ) , which buds into the lumen of the smooth er and/or golgi complex (wood et al., ; stueckemann et al., ; dea et al., ; weiland et al., ; pol et al., ) and acquires the six viral envelope proteins, i.e., e, m, and gp to gp (snijder et al., b; wieringa et al., ; zevenhoven-dobbe et al., ; wissink et al., ) . in this event, the role of the protein product of orf a is unclear. finally, the progeny virions accumulated in the intracellular membrane compartments are released into the extracellular space through exocytosis (dea et al., ) . although considerable research has been focused on prrsv, little is known about the proteolytic processing pathway and the structure and function of most of the prrsv nsps. the initial functional assignments of the prrsv nsps have primarily been based on the experimental data of eav, the arterivirus prototype (fig. a ). nsp α and nsp β: prrsv nsp α and nsp β each contain a cysteine protease domain responsible for autocatalytic processing at the nsp α/ β (den boon et al., ; sun et al., ; chen et al., a) and nsp β/ (den boon et al., ; chen et al., a) junctions, respectively. the atomic structure of prrsv nsp α reveals three domains (sun et al., ) : (i) a n-terminal zinc-finger domain, (ii) a papainlike cysteine protease (pcpα) domain with a zinc ion bound at the active site that is required for its proteolytic activity, and (iii) a c-terminal extension bound to the substrate binding site of the pcpα domain. similarly, the crystal structure of prrsv nsp β reveals four domains (xue et al., ) : (i) an n-terminal metal-dependent nuclease domain, (ii) a linker domain, (iii) a papain-like cysteine protease (pcpβ) domain, and (iv) a c-terminal extension bound to the substrate binding site of the pcpβ domain, as observed for prrsv nsp α. in the case of both nsp α and nsp β, their c-terminal extensions occupy the protease active site after their release from the polyprotein, suggesting that they function in cis (sun et al., ; xue et al., ) . in prrsv, inactivation of the pcpα activity in nsp α blocks subgenomic mrna synthesis without altering genome replication, whereas when pcpβ activity is eliminated in nsp β, no sign of viral rna synthesis is seen; therefore, both pcp protease activities are apparently required for productive viral rna synthesis (kroese et al., ) . similarly, mutagenesis studies have shown that eav nsp (which contains a tandem of the pcpα and pcpβ domains, with pcpα having lost its enzymatic activity) is involved in regulating the accumulation of minusstrand templates to control the relative abundance of viral mrnas, thereby coordinating genome replication, subgenomic mrna synthesis, and virus production (tijms et al., (tijms et al., , nedialkova et al., ) . both prrsv nsp α/ β (chen et al., a) and eav nsp (tijms et al., ) are translocated to the nucleus in infected cells, but no consensus nuclear localization signal has yet been found. the interaction of eav nsp with the cellular transcription co-factor p suggests that it might be important for viral and/or cellular transcription (tijms and snijder, ) . nsp and nsp : prrsv nsp is predicted to have four domains: (i) an n-terminal cysteine protease domain, (ii) a large hypervariable domain, (iii) a transmembrane domain, and (iv) a c-terminal tail (han et al., ) . the cysteine protease belongs to the mammalian ovarian tumor domain (otu)-containing protein superfamily (makarova et al., ; han et al., ) ; it cleaves at the nsp / junction that functions both in cis and in trans (snijder et al., ; han et al., ) and is crucial for the viral replication cycle (han et al., ) . in eav-infected cells, nsp is localized to the perinuclear membranes, which are presumably derived from the er and are involved in the formation of the membrane-bound rtc, where viral rna synthesis occurs (van der meer et al., ; pedersen et al., ) . in the ab-sence of eav replication, the co-expression of eav nsp and nsp is both necessary and sufficient to modify host cell membranes during the formation of the rtc . also, eav nsp interacts with nsp (snijder et al., ) , and nsp has been implicated in the process of remodeling intracellular membranes (posthuma et al., ) . biochemical and morphologic studies of eav replication have shown that the nsps containing transmembrane domains (e.g., nsp , nsp , and nsp ) are part of the membrane-bound rtc, suggesting that they play an important role in recruiting other viral components of the rtc that lack the membrane-spanning domains (van der meer et al., ) . in vitro, the eav rtcs isolated from infected cells require a cytosolic host factor for viral rna synthesis, which reproduces the synthesis of both viral genome and subgenomic mrnas . interestingly, prrsv nsp contains a cluster of linear b-cell epitopes that are dispensable for virus replication (oleksiewicz et al., ; chen et al., b) but capable of modulating the host immune response (chen et al., b; li et al., ) . nsp : prrsv nsp contains the main protease ( c-like serine proteinase) responsible for all nsp processing, except for the nsp α/ β, nsp β/ , and nsp / junctions (van dinten et al., ; ziebuhr et al., ) . cleavages at the nsp / , nsp / , and nsp / junctions have been confirmed experimentally by the use of recombinant prrsv nsp (tian et al., ) . the crystal structure of both prrsv and eav nsp s reveals a chymotrypsin-like fold with a canonical catalytic triad (s-h-d), as well as a novel α/β c-terminal extension (barrette-ng et al., ; tian et al., ) that may be involved in regulating viral polyprotein processing (van aken et al., a) . nsp : arterivirus nsp includes the viral rna-dependent rna polymerase (rdrp) (den boon et al., ) . in prrsv, the rdrp domain is located in the c-terminal region, which contains an upstream n-terminal region of unknown function (gorbalenya et al., ; fang and snijder, ) . enzymatically active eav rdrp can be purified from e. coli and initiates rna synthesis by a de novo mechanism on homopolymeric templates in a template-dependent fashion (beerens et al., ) . nsp : prrsv nsp is predicted to have three domains (gorbalenya et al., ; fang and snijder, ) : (i) an n-terminal zinc-binding domain, (ii) a linker domain, and (iii) a nucleotide triphosphate binding or helicase domain (den boon et al., ) . bacterially expressed prrsv and eav nsp s possess atpase activity and can unwind dsrna and dsdna in a -to- direction (bautista et al., ; seybert et al., ) . the zinc-binding domain of eav nsp is also critical for this activity (seybert et al., ) . in eav, the zinc-binding domain contains a set of conserved cys and his residues and is critical for viral rna synthesis (van dinten et al., ) . the linker domain (s p) has been implicated in subgenomic mrna synthesis (van dinten et al., ; van marle et al., b) . nsp : arterivirus nsp contains the uridylate-specific endoribonuclease (nendou) domain, which is a major genetic marker unique to nidoviruses (snijder et al., a; gorbalenya et al., ; fang and snijder, ) . bacterially expressed nsp has been used to show that the endoribo-nuclease activity of both prrsv and eav nendous exhibits broad substrate specificity in vitro, but its function in infected cells is elusive (nedialkova et al., ) . viruses with mutations in the eav nendou active site are viable but have a defect in subgenomic mrna synthesis (posthuma et al., ) . recently, ifn-mediated host innate immunity has been shown to be modulated by a panel of prrsv nsps (i.e., nsp α, nsp β, nsp , nsp , and nsp ) with different intensities (beura et al., ; chen et al., a; li et al., ) . in the case of prrsv nsp , the otu domain-containing cysteine protease has been shown to possess deubiquitinating and interferon antagonism activity, thereby evading ubiquitin-and isg -dependent innate immunity (frias-staheli et al., ; sun et al., ) . other nsps: to date, no specific functions have been demonstrated for the other prrsv nsps (nsp , nsp , nsp α, nsp β, nsp , and nsp ). also, it should be noted that during the proteolytic processing of eav nsps, many cleavage intermediates of unknown function have been observed (snijder et al., ; van dinten et al., ) , and alternative major and minor processing pathways have also been characterized . based on the "discontinuous rna transcription" model (sawicki and sawicki, ) , the plus-strand genomic rna of prrsv is thought to serve as a template for either (i) continuous minus-strand rna synthesis, which produces the genome-length minus-strand template for genome replication; or (ii) discontinuous minus-strand rna synthesis, which generates a nested set of six major subgenome-length minus-strand templates, one for each subgenomic mrna synthesis. all the subgenomic mrnas are both -and coterminal with the genomic rna, with a common short "leader" sequence corresponding to the -proximal region of the genome joined to different "body" segments that are co-linear with its -proximal region (pasternak et al., ; sawicki et al., ) . this leader-body joining is guided by regulatory transcription-regulating sequences (trss); in the genomic rna, these rna motifs are located at the -end of the leader sequence (leader trs) and upstream of each structural protein-coding region (body trs) ( van marle et al., a; pasternak et al., ; van den born et al., ) . in prrsv, the -proximal one or two orfs of each subgenomic mrna are translated to produce eight viral structural proteins that constitute an infectious virion (meulenberg et al., ; meulenberg and petersen-den besten, ; van nieuwstadt et al., ; snijder et al., ; dea et al., ; molenkamp et al., ; wu et al., ; johnson et al., ) : gp (gp a), e, gp , gp , gp , m, n, and a protein product of orf a (fig. b) . the viral envelope contains the two major (gp and m) and four minor (e, gp , gp , and gp ) membrane proteins that are all required for the production and infectivity of infectious virions; however, the four minor proteins are dispensable for virus assembly (wieringa et al., ; wissink et al., ) . e protein has an ion channel protein-like property and is embedded in the viral membrane, presumably promoting uncoating of the virion and release of the viral genome into the cytoplasm (lee and yoo, ) . gp is heavily glycosylated (dea et al., ; das et al., ) , and its glycans on the viral surface prevent the recognition of epitopes by neutralizing antibodies (vu et al., ) ; a subset of the gp proteins is secreted from the cells as a nonvirion-associated soluble form (mardassi et al., ) . gp has a neutralizing epitope in the hypervariable region (meulenberg et al., ) that might be associated with the e, gp , and gp proteins through non-covalent interactions (wieringa et al., ; wissink et al., ; das et al., ) . gp is a triple membrane-spanning protein with a short ectodomain (~ aa) and a long cytoplasmic tail (~ - aa) (meulenberg et al., ; mardassi et al., ) , which contains major neutralizing epitopes (wissink et al., ; ansari et al., ) . m is the most conserved membrane protein and has a membrane topology similar to that of gp (dea et al., ) . n is a serine phosphoprotein that forms a dimer and is distributed in the cytoplasm and the nucleus (rowland and yoo, ; you et al., ) . in the viral membrane, the gp and m proteins are embedded as disulfidelinked heterodimers, whereas the e, gp , gp , and gp proteins are associated with each other through non-covalent interactions (meulenberg et al., (meulenberg et al., , mardassi et al., mardassi et al., , meulenberg and petersen-den besten, ; van nieuwstadt et al., ; wu et al., ; wissink et al., ) . prrsv has a very restricted cell tropism. in vivo, it targets specific subsets of porcine macrophages, primarily alveolar macrophages (lawson et al., ; duan et al., a duan et al., , b teifke et al., ) ; in vitro, it can also infect monocyte-or bone marrow-derived porcine dendritic cells when stimulated with gm-csf/ il- (loving et al., ; wang et al., ; chang et al., ; flores-mendoza et al., ; silva-campa et al., ), but not lung dendritic cells (loving et al., ) . prrsv entry into porcine macrophages is the first step in a highly coordinated process of virus-host interactions. based on recent findings (welch and calvert, ; van breedam et al., a) , highly sulfated, negatively charged glycosaminoglycans such as heparan sulfates can be used as low-affinity attachment factors that concentrate virus particles on the cell surface (jusa et al., ; vanderheijden et al., ; delputte et al., delputte et al., , . once this interaction has taken place, the viral gp /m complex binds to the nterminal portion of cd (also called sialoadhesin or siglec- ) (duan et al., a (duan et al., , b vanderheijden et al., ; delputte et al., ; van gorp et al., ; van breedam et al., b) . this interaction is directed by the sialic acid-binding domain at the n-terminus of cd and sialic acids on the virion surface (delputte and nauwynck, ; delputte et al., ; van breedam et al., b) , which trigger receptor-mediated, clathrin-dependent endocytosis (kreutz and ackermann, ; nauwynck et al., ; vanderheijden et al., ) . once internalized, the particles are transported to early endosomes, where the viral genome is released into the cytoplasm in a reaction that depends on both the acidic environment and scavenger receptor cd (nauwynck et al., ; calvert et al., ; van gorp et al., ). the role of cd is mediated through its cysteine-rich domain (van gorp et al., ) and by interaction with gp and gp (das et al., ) . the protease cathepsin e and an additional serine protease are also implicated in this process (misinzo et al., ) . other host factors, such as simian vimentin (kim et al., ) and cd (shanmukhappa et al., ) , have been identified in marc- cells, a cell line susceptible to prrsv infection (kreutz, ) . prrs is controlled by several different strategies, including management (e.g., herd depopulation/repopulation, herd closure, and regional elimination), biosecurity, and vaccination (corzo et al., ; thanawongnuwech and suradhat, ) . of these strategies, vaccination is the most cost-effective for controlling prrs, but it does not completely prevent prrsv infection. two types of prrsv vaccines are commercially available: modified-live virus (mlv) and killed virus (kv) vaccines (yoo et al., ; charerntantanakul, ; kimman et al., ; cruz et al., ; huang and meng, ) . the mlv vaccine confers effective protection against genetically homologous prrsvs but only partial protection against genetically heterologous prrsvs (meng, ; murtaugh et al., ; labarque et al., ; okuda et al., ) ; it is of particular concern that the live vaccine viruses have the potential to spontaneously revert to virulence and spread the disease (botner et al., ; madsen et al., ; mengeling et al., ; storgaard et al., ; wesley et al., ; nielsen et al., ; opriessnig et al., ; amonsin et al., ; grosse beilage et al., ; li et al., ) . the kv vaccine, on the other hand, is safe but offers limited protection at best against either homologous or heterologous prrsvs (scortti et al., ; zuckermann et al., ; vanhee et al., ) . thus, the current vaccines fail to provide sustainable disease control and prevention, particularly against the genetically heterologous prrsvs (cano et al., a (cano et al., , b , making it difficult to achieve global eradication. although significant progress has been made in understanding the routes of prrsv transmission and in developing and implementing control measures for prrsv infection, there is clearly an urgent need for novel strategies that may be applicable to the development of a safer, more effective vaccine against prrsv. despite the clinical importance of prrsv in animal health, only limited information is available to date regarding the biological functions of the viral nonstructural and structural proteins in replication and pathogenesis. in particular, the molecular characterization of the replicase proteins and their roles in prrsv rna synthesis have represented a major challenge in prrsv biology. we and others have established a reverse genetics system for prrsv by constructing a full-length infectious cdna that allows genetic manipulation of the viral genome and from which molecularly cloned viruses can be rescued. this system offers a unique opportunity to address some of the key questions in prrsv biology. new information will give us a better understanding of the molecular and genetic basis of prrsv replication and pathogenesis, a prerequisite for the development of new and promising strategies to nauwynck, h.j., duan, x., favoreel, h.w., van oostveldt, p., and evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under controlled field conditions evaluation of mosquitoes, aedes vexans, as biological vectors of porcine reproductive and respiratory syndrome virus transmission of porcine reproductive and respiratory syndrome virus by fomites (boots and coveralls) transmission of porcine reproductive and respiratory syndrome virus by needles mechanical transmission of porcine reproductive and respiratory syndrome virus by mosquitoes, aedes vexans (meigen) transmission of porcine reproductive and respiratory syndrome virus by houseflies (musca domestica) nidovirus transcription: how to make sense sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis blue ear' disease of pigs open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex new insights into the genetic diversity of european porcine reproductive and respiratory syndrome virus (prrsv) use of a production region model to assess the airborne spread of porcine reproductive and respiratory syndrome virus lactate dehydrogenaseelevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses comparative morphogenesis of three prrs virus strains site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle formation of the arterivirus replication/transcription complex: a key role for nonstructural protein in the remodeling of intracellular membranes oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type infections characterization of emerging european-like porcine reproductive and respiratory syndrome virus isolates in the united states porcine reproductive and respiratory syndrome experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs the interaction between prrsv and the late gestation pig fetus nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands a contemporary view of coronavirus transcription retention of ingested porcine reproductive and respiratory syndrome virus in houseflies spatial dispersal of porcine reproductive and respiratory syndrome virus-contaminated flies after contact with experimentally infected pigs failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv a complex zinc finger controls the enzymatic activities of nidovirus helicases role of cd , a tetraspanin, in porcine reproductive and respiratory syndrome virus infection molecular epidemiology of prrsv: a phylogenetic perspective phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage heterodimerization of the two major envelope proteins is essential for arterivirus infectivity the molecular biology of arteriviruses arteriviruses identification of a novel structural protein of arteriviruses non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex the end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease proteolytic processing of the replicase orf a protein of equine arteritis virus the arterivirus nsp protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the arterivirus nsp protease is the prototype of a novel group of chymotrypsin-like enzymes, the c-like serine proteases cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses examination of the selective pressures on a live prrs vaccine virus replication of lactate dehydrogenase-elevating virus in macrophages. . mechanism of persistent infection in mice and cell culture ultrastructural pathogenesis of the prrs virus crystal structure of porcine reproductive and respiratory syndrome virus leader protease nsp alpha the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars detection of european porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridizationimmunohistochemistry double labelling experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled genetics and geographical variation of porcine reproductive and respiratory syndrome virus (prrsv) in thailand taming prrsv: revisiting the control strategies and vaccine design emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark structure and cleavage specificity of the chymotrypsin-like serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp autoprotease equine arteritis virus non-structural protein , an essential factor for viral subgenomic mrna synthesis, interacts with the cellular transcription co-factor p nuclear localization of non-structural protein and nucleocapsid protein of equine arteritis virus a zinc finger-containing papain-like protease couples subgenomic mrna synthesis to genome translation in a positivestranded rna virus airborne transmission of actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus in nursery pigs attempts to transmit porcine reproductive and respiratory syndrome virus by aerosols under controlled field conditions mutagenesis analysis of the nsp main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp porcine reproductive and respiratory syndrome virus entry into the porcine macrophage the m/gp( ) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner secondary structure and function of the -proximal region of the equine arteritis virus rna genome orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis processing of the equine arteritis virus replicase orf b protein: identification of cleavage products containing the putative viral polymerase and helicase domains the porcine reproductive and respiratory syndrome virus requires trafficking through cd -positive early endosomes, but not late endosomes, for productive infection sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor the arterivirus replicase arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection effects of heparin on the entry of porcine reproductive and respiratory syndrome virus into alveolar macrophages involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein shedding of porcine reproductive and respiratory syndrome virus in mammary gland secretions of sows porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease electron microscopic studies on the morphogenesis of prrsv in infected cells-comparative studies a brief review of cd and its role in prrsv infection antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mystery swine disease in the netherlands: the isolation of lelystad virus evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus structural protein requirements in equine arteritis virus assembly duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus: routes of excretion porcine reproductive and respiratory syndrome virus: a persistent infection envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus the major envelope protein, gp , of a european porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its n-terminal ectodomain electron microscopic study of tissue cultures infected with simian haemorrhagic fever virus antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b the crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein nsp beta reveals a novel metal-dependent nuclease evidence for the transmission of porcine reproductive and respiratory syndrome (prrs) virus in boar semen modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus infectious cdna clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging rescue of disabled infectious single-cycle (disc) equine arteritis virus by using complementing cell lines that express minor structural glycoproteins porcine reproductive and respiratory syndrome in china highly virulent porcine reproductive and respiratory syndrome virus emerged in china virus-encoded proteinases and proteolytic processing in the nidovirales porcine reproductive and respiratory syndrome virus (porcine arterivirus) general overview of prrsv: a perspective from the united states assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge this work was supported by a utah agricultural experiment station grant (uta ), utah science technology and research funds, and a korean national research foundation grant . this research was approved as journal paper number uaes # . s.i.y was supported by the basic science research program ( - ) from the national research foundation funded by the korean ministry of education, science, and technology. we thank dr. deborah mcclellan for reading the manuscript. key: cord- -smlq y authors: dhakal, santosh; renukaradhya, gourapura j. title: nanoparticle-based vaccine development and evaluation against viral infections in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: smlq y virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. the economic burden caused by virus infections such as porcine reproductive and respiratory syndrome virus, swine influenza virus, porcine epidemic diarrhea virus, porcine circovirus , foot and mouth disease virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. pigs can also have a role in zoonotic transmission of some viral infections to humans. inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. thus, improvements over existing vaccines are necessary to: ( ) increase the breadth of protection against evolving viral strains and subtypes; ( ) control of emerging and re-emerging viruses; ( ) eradicate viruses localized in different geographic areas; and ( ) differentiate infected from vaccinated animals to improve disease control programs. nanoparticles (nps) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. nps help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. some of the nps-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (m) cells and dendritic cells (dcs). in conclusion, nps-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. this review highlights the recent developments in nps-based vaccines against porcine viral pathogens and how the nps-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy. viruses are the obligate intracellular nano-sized particles, which depend on host cell machinery for propagation and survival. they carry deoxyribonucleic acid (dna) or ribonucleic acid (rna) as their genomic material. there are several viruses from both dna and rna virus families that infect and produce disease in pigs [ ] . there are many economically important swine viral infections which cause considerable morbidity and mortality, and responsible for significant economic losses to the pork industry (table ). depending on their cellular and tissue tropisms, viruses cause pathological changes and clinical signs associated with respiratory system, reproductive and gastrointestinal tracts, nervous system, skin and extremities, alone or in combinations [ , ] . porcine reproductive and respiratory syndrome virus (prrsv), an enveloped and positive-stranded rna virus of arteriviridae family, causes porcine reproductive and respiratory syndrome (prrs) [ ] . prrs is responsible for over one billion dollar loss per year through direct and indirect costs in the us swine industry [ ] . two entirely distinct genotypes of prrsv circulate in european (genotype /prrsv ) and north american countries (genotype /prrsv ) and cause tremendous economic loss. prrsv is transmitted through oral-nasal secretions and semen. the clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. it is the most common pathogen associated with porcine respiratory disease complex (prdc) [ ] . swine influenza (flu) constitutes another persistent health challenge to the global pig industry. flu infection is caused by influenza a virus of orthomyxoviridae family which has negative-sense, single-stranded, segmented rna genome. influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets [ ] . the clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses [ ] . influenza of swine origin occasionally infect humans and can even lead to pandemics as of [ ] . porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) are enteric pathogens of young pigs [ ] . these viruses belong to coronaviridae family and have positive-sense, single-stranded rna genome. tgev did serious economic damage to the swine industry in s but with the advent of vaccines it has been largely controlled [ ] . pedv still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. in / , pedv outbreak in the us led to over a billion-dollar loss [ ] . rotaviruses are double-stranded rna viruses of reoviridae family, cause enteric infections in pigs. rotavirus of groups a, b, c, e and h are involved in porcine enteric infections. some of these porcine rotaviruses also have zoonotic potential [ ] . foot and mouth disease (fmd) is another highly contagious, acute viral disease in pigs. the etiologic agent, fmd virus (fmdv), is a positive-sense, single-stranded rna virus of picornaviridae family [ ] . fmdv is transmitted through direct contact with infected animals or contaminated sources. clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. fmdv causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions [ ] . classical swine fever (csf) or hog cholera can result in high morbidity and mortality in pigs. it is caused by csf virus (csfv), an enveloped, positive-sense, singlestranded virus of flaviviridae family. transmission of csfv occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets [ ] . clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. csf is a notifiable disease to world organization for animal health (oie). the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . for example, the / outbreak of csfv in the netherland resulted in death of million pigs and economic losses of . billion dollars [ ] . united states is free of csfv; however, this virus is endemic in many parts of the world including central and south america, africa and asia. porcine circovirus (pcv ), a single-stranded dna virus of circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (pcvad). pcv is transmitted horizontally as well as vertically. direct contact is the most efficient way of horizontal transmission of this virus. the clinical signs of pcv infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures [ ] . five genotypes of pcv (pcv a to pcv e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide [ ] . porcine parvovirus (ppv) is the common cause of reproductive failure in swine herds. this single-stranded dna virus of parvoviridae family is transmitted through oral-nasal routes. stillbirths, mummification, embryonic death, and infertility (smedi syndrome) are linked to ppv infection. conventionally, ppv was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate [ ] . aujeszky's disease or pseudorabies in pigs is caused by suid herpesvirus , a double stranded dna virus belonging to herpesviridae family. the causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [ , ] . african swine fever (asf) causes hemorrhagic infection with high morbidity and mortality. the etiologic agent, asf virus (asfv), is a double stranded dna virus of asfarviridae family [ ] . virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus ornithodoros. clinical disease may range from asymptomatic infection to death with no signs. acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death [ ] . asf is oie notifiable disease. united states is free of asfv, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the us and other nonendemic regions through animal trades [ ] . the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [ ] . besides the rna and dna viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis e virus, porcine endogenous retrovirus, porcine sapovirus, japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [ , , ]. different types of vaccines that are available against economically important swine viruses are listed in table . vaccines against prrsv are being used in the us since s [ ] . both inactivated and modified-live virus vaccines are available and used globally. these vaccines are effective in reducing clinical disease and viremia mainly against homologous but not against heterologous infections [ ] . therefore, different strategies are ongoing to develop live, inactivated, subunit and mucosal prrsv vaccines to induce better immunity and broader protection [ , [ ] [ ] [ ] . swine influenza vaccines are also most effective when the vaccine strains closely match with the circulating strains [ , ] . to increase the immunity and protection, vaccines containing multiple strains of influenza a virus (iav) and autogenous vaccines are widely used [ , ] . cocirculation of multiple lineages of iav and frequent antigenic drift are responsible for reduced field efficacy of current swine influenza vaccines. moreover, the most commonly used whole inactivated iav vaccines administered via intramuscular route do not induce adequate mucosal antibody and cellular immune responses, suffer maternal antibody interference in young piglets and even can cause enhanced respiratory disease [ , ] . the emergence of highly virulent strains of pedv in recent years has highlighted the need of safe and effective vaccines against porcine enteric coronaviruses that prevents clinical disease, mortality and virus shedding in neonates [ ] . modified live vaccines against rotavirus are available for use in pigs against rotavirus a but their efficacy under field conditions is questionable indicating the need of alternatives for porcine rotavirus management [ ] . the available inactivated vaccines provided great help in prevention and control of fmd outbreaks in many countries. however, the development of these vaccines needs high level biocontainment facilities. further, the fmdv serotypes undergo continuous antigenic drift and escape the vaccine-induced immunity [ ] . thus, fmd vaccines with less stringent regulatory procedures and multi-serotype protective efficacy are needed in the future. safe and highly efficacious live-attenuated vaccines are available against csfv but differentiation of infected from vaccinated animals (diva) is not possible with these vaccines, which limit their use during outbreak control or disease eradication programs [ ] . inactivated whole virus or subunit vaccines based on pcv a are highly adopted in pig farms and are efficacious in reducing clinical signs and improving the production parameters. however, infections are still widespread in vaccinated farms [ , ] . further, the replacement of pcv a to pcv b and recently to pcv d is in part contributed by the selection pressure exhibited by pcv a-based vaccines [ ] which highlights the need of vaccines that protect against multiple genotypes. the currently used inactivated vaccines of porcine parvo virus protect against old ppv strains but not against the newly emerging strains demanding for more efficacious vaccines [ , ] . fortunately, pseudorabies has been eradicated in many countries including the us by using inactivated and attenuated vaccines together with stringent biosecurity measures. however, it is still a problematic disease in many countries including china and is also maintained in feral swine populations in other countries [ , ] . the frequent emergence of virulent strains even in the vaccinated herds demands updated vaccine technology to achieve efficient control and ultimate global eradication of pseudorabies [ , ] . vaccine is not available so far against asfv, and the control measures depend entirely on early identification and culling of infected herds and adoption of strict sanitary measures [ ] . vaccine development is hindered by the antigenic diversity and multitude of immune-evasion strategies used by the virus. an effective vaccine will definitely help in control and eradication of asfv from endemic countries and prevent its geographical expansion [ ] . [ , ] porcine epidemic diarrhea (ped) rna particle, inactivated and live-attenuated virus (in asia) protective immune response in sows better mucosal immunity [ , ] foot and mouth disease (fmd) inactivated virus less stringent requirements in vaccine production protection against multiple serotypes [ ] classical swine fever (csf) live-attenuated virus diva potential [ ] porcine circovirus associated disease (pcvad) inactivated, recombinant subunit multi-genotype protection [ , ] porcine parvovirus infection inactivated virus protection against novel strains [ , ] pseudorabies inactivated, live-attenuated virus protection against novel emerging strains [ , ] african swine fever (asf) none novel cross-protective vaccine [ ] importance of nanoparticle-based vaccine delivery platforms development of vaccines has made significant impact on reducing the viral infectious disease burden in both humans and animals. however, there are still many diseases for which either we do not have vaccines or need substantial improvements over existing ones [ , ] . in the past few decades, nanoparticles (nps)-based technologies have elicited significant interests in the development of novel vaccine candidates as they offer multiple benefits over inactivated virus or subunit soluble antigens. nps-based vaccines (nanovaccines) are prepared either by encapsulating vaccine components within the nps or by decorating the particle surface with viral antigens. nps protect antigens from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release. all of these properties help in induction of better immune responses compared to soluble antigen vaccines [ ] . the different mechanisms used by various nps to facilitate immune modulation of antigen presenting cells (apcs) are depicted graphically in figure . briefly, nps can enhance antigen adsorption and uptake by apcs; they can also facilitate antigen processing mechanisms; nps can induce maturation of dcs and promote antigen cross-presentation through major histocompatibility complex (mhc) class i to cd + t cells; and induce production of different innate cytokines that regulate humoral and cellular immune responses. nps-loaded antigens are readily phagocytosed by apcs; soluble antigens are not [ ] . moreover, dendritic cells (dcs), the key player involved in bridging innate and adaptive immunity, preferentially internalize nps compared to microparticles (> nm). for example, when poly(lactic-co-glycolic acid) (plga) particles of size nm to µm encapsulating ovalbumin were tested on mouse bone-marrow derived dendritic cells, nm sized particles were taken up efficiently compared to larger ones [ ] . the nm sized plga nps resulted in greater activation of dcs and stronger antigen-specific t cells responses in immunized mice compared to soluble antigens and larger particles [ ] . besides controlled delivery of antigens, nps also provide adjuvant-like functions. vaccine adjuvants either work as antigen delivery systems facilitating antigen uptake and presentation by apcs or they activate innate immune receptors for cytokine production and maturation/migration of dcs [ ] . adjuvant-induced innate immune responses determine the type of adaptive immune responses generated such as t helper (th ) versus t helper (th )-biased immunity [ ] . alum, the most widely used adjuvant in humans, is safe and inexpensive. its compatibility has been proved favorable with different vaccine antigens. however, despite inducing potent antibody responses, alum is a weak-inducer of cell-mediated immunity. adverse reactions are observed at injection site with alum-based adjuvants [ , ] . in veterinary vaccines, oil-in-water emulsions or saponins are the most common adjuvants. these can also cause adverse reactions at the injection sites [ , ] . while number of adjuvants are available for parenteral vaccinations, very limited options are available for intranasal (in) or other alternative routes of immunization [ , , ] . nps can serve as an alternative adjuvant for human and animal use as they act both as antigen delivery system and activate the innate immune responses [ ] [ ] [ ] . further, the modern vaccination approach has shifted from traditional whole pathogen-based antigens to small fraction (subunit) of the pathogen. however, purified whole inactivated pathogen and subunit or recombinant antigens by themselves are poorly immunogenic and require a potent immunostimulatory platform to augment the immune response. this can be achieved through nps-based technologies [ , ] . nps-based platforms can be used to deliver multiple antigens or antigen/adjuvant combinations, which improves antigen uptake and concurrent activation of apcs leading to innate immune programming [ , ] . co-delivery of cpg oligodeoxynucleotide and tetanus toxoid in nanospheres induced significantly greater t cell proliferative response and to times greater igg antibody isotypes in mice after subcutaneous immunization compared with the group that received tetanus toxoid and cpg oligodeoxynucleotide in soluble form [ ] . likewise, co-delivery of melanoma antigen and toll-like receptor (tlr) agonist in plga nps induced therapeutic anti-tumor effects that are mediated through potent cd + t cell activation [ ] . nps can be surface modified to target microfold (m) cells, macrophages or dcs, and could be used for mucosal vaccination through oral, nasal or other mucosal routes of immunization. in mice, surface coating of plga nps encapsulating hepatitis b virus vaccine antigens with lectin resulted in efficient targeting of oral delivered nps to mucosal m cells and induced secretary iga antibody response in mucosal surfaces [ ] . likewise, dcs targeted chitosan nps loading plasmid dna encoding nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) induced better nucleocapsid protein-specific mucosal iga antibody response compared to soluble unentrapped antigens after nasal immunization in mice [ ] . a targeted t-cell mediated immune response is critical in protection against intracellular pathogens such as viruses. beneficially, nps-delivered antigens are useful in antigen cross-presentation to cytotoxic t lymphocytes (ctls) and development of robust cell-mediated immune response [ , ] . plga-based particulate vaccines are shown to induce efficient t-cell immunity in mice and pigs [ ] [ ] [ ] [ ] . similarly, rodent and pig studies have shown that polyanhydride nps-based vaccines also enhance cellular immunity [ , ] . thus, immunogenic properties of different polymer-based nps could be exploited to improve the efficacy of vaccines for use against porcine viral infections. in this review, only studies conducted in pigs related to the development and evaluation of nps-based vaccine candidates by using virus-like particles (vlps), biodegradable polymers, polysaccharides and liposomes against porcine viral infections are included (table ) . vlps are constructed using viral structural proteins, which can self-assemble but are non-infectious as they lack the viral genomic material. vlps mimic the virion and can effectively induce innate and adaptive immune responses [ ] . vlps are produced using different bacterial, insect, yeast or mammalian expression systems [ ] . due to their smaller size and particulate nature, vlps-based vaccines are processed and presented not only through mhc class ii but also through mhc class i pathway leading to the generation of antibodies as well as ctl responses [ , ] . the potential use of vlps in porcine viral vaccine development is evident through the success in commercialization of human papilloma virus (hpv), hepatitis b virus and malaria vaccines by adapting this technology [ ] . in one study, prrsv vlps containing five (gp , gp , gp , gp a and m) and two (gp and m) viral surface proteins were generated using the baculovirus expression system. prrsv vlps vaccine was mixed at : ratio with mycobacterium tuberculosis whole cell lysate (m. tuberculosis wcl) adjuvant and administered into pigs. vlps-vaccinated pigs were partially protected with -log reduction of virus titers in lungs. vlps-vaccinated pigs also had enhanced ifn-γ response compared to mock challenge pigs [ ] . however, in another study, when pigs were vaccinated in with prrsv vlps expressing n, m, gp and e proteins, enhanced viremia accompanied with higher level of ifn-α cytokine response was observed [ ] . the contrasting results in prrsv vlps study suggest the need for further research to fully evaluate the potential of vlps-based prrsv vaccines for swine. influenza-associated vlps expressing ha, na and m proteins of pandemic (h n ) virus were inoculated twice intramuscularly with or without emulsigen (mvp lab, usa) adjuvant to pigs. this vaccine induced robust serum igg, mucosal iga and virus neutralizing antibody responses in pigs. after homologous virus challenge, vlps-vaccinated pigs had significantly reduced pneumonic lesions and virus titers were substantially lowered in upper and lower respiratory tracts compared to mock vaccinated animals [ ] . many studies have been conducted with the goal to develop vlps-based fmdv vaccine using various expression systems encoding different viral antigens. rabbit hemorrhagic disease virus (rhdv) vlps expressing t-cell epitope of a protein of fmdv (rhdv- a-vlps) was generated. this vlps vaccine induced maturation of bone marrow derived dendritic cells in vitro [ ] . pigs immunized im with rhdv- a-vlps together with montanide isa adjuvant (seppic, france) induced higher serum igg and iga antibody responses. this vaccine also increased number of ifn-γ secreting cells and lymphoproliferative responses in pbmcs compared to vaccine delivered without adjuvant and in rhdv- a-vlps inoculated pigs; however, challenge experiments were not performed [ ] . guo et al. constructed fmdv vlps expressing capsid proteins vp , vp and vp and immunized pigs by im route [ ] . vlps-vaccinated pigs produced virus-specific neutralizing antibodies and ifn-γ response in peripheral blood mononuclear cells (pbmcs) as good as the inactivated fmdv vaccine control. after challenge with homologous virus, vlps-vaccinated pigs did not show specific clinical signs [ ] . in another study, vp epitope peptides (ep - ) of fmdv were inserted into the coat protein genes of male-specific coliphage (ms ) (cp-ep - vlps) and injected im to pigs. this formulation resulted in induction of virus neutralizing antibodies and protected % of the immunized pigs compared to only % protection in peptide alone vaccinated animals. however, the protection was lower than inactivated vaccine ( %) indicating the need : of further improvement in this vlps either by using longer sequence of epitope or addition of other adjuvants [ ] . vlps generated by insertion of vp epitopes of fmdv into porcine parvovirus vp were administered im to pigs. this vlps-vaccine induced higher virus neutralizing antibodies compared to synthetic peptide vaccine and resulted in better protection to challenge fmdv infection [ ] . vlps have also been developed and tested against porcine neurotropic viruses [ , ] . porcine encephalomyocarditis virus (emcv) vlps containing structural protein p , nonstructural protein a and protease c were generated. after im administration together with montanide ims n vg adjuvant (seppic), vlps-vaccine induced sustained production of virus neutralizing antibodies comparable to commercial vaccine control. there was absence of any severe injection site reactions in vlps-vaccinated pigs [ ] . this suggests the potential of developing vlps-based vaccine against emcv disease in pigs. likewise, in a recent study, japanese encephalitis virus genotype i (gi) vlps encoding premembrane (prm) and envelope (e) proteins were constructed. after subcutaneous immunization, this vaccine formulation induced robust neutralizing antibody response and protection against both homologous gi and heterologous giii jevs viruses. this finding indicates the cross-protection potential of vlps-based jev vaccine in pigs [ ] . early study on pcv vlps used full length cap protein in escherichia coli expression system [ ] . pigs vaccinated against pcv using cap vlps and isa adjuvant (seppic) by im route induced cap-specific igg antibodies. vaccinated animals were apparently healthy with normal body weight gain and absence of any clinical signs of disease [ ] . li et al. [ ] showed induction of cap-specific igg antibodies in pigs vaccinated by subcutaneous (sc) injection of cap vlps. vaccinated pigs demonstrated reduced fever, viremia and mild pathological changes in lungs and lymph nodes compared to unvaccinated challenge animals. in another study, vlps co-expressed with cap protein and porcine gm-csf were administered im to pigs. this vaccine formulation induced significantly higher virus neutralizing antibodies in pigs. after virus challenge, vlps-vaccinated pigs had normal body weight gain compared to cap protein alone and commercial pcv vaccine groups. virus clearance, however, was observed in equally in vlps as well as other control vaccine groups [ ] . only a single vlps-based vaccine study for porcine parvovirus was found [ ] . ppv-vlps expressing major structural protein vp were administered im with double oil emulsion (doe) mineral oil adjuvant to weaned pigs. there was an induction of significantly higher neutralizing antibodies in vlps-vaccinated animals compared to inactivated vaccine group. further, when gilts immunized with this formulation were challenged with virulent ppv, virus was not detected in any of the fetuses. thus, ppv-vlps can be a potential vaccine candidate to prevent ppv-induced reproductive failure [ ] . in summary, vlps of various origin can be used to develop more efficient vaccines against porcine viral infections. further studies are needed to evaluate their immunogenicity and protective efficacy under field conditions. plga is a co-polymer of lactic acid and glycolic acid. it is the most widely explored synthetic polymer in vaccine studies. it is a safe and non-toxic compound, and its hydrolysis products are readily assimilated into existing metabolic pathways [ ] . plga nanoparticles are prepared either by oil in water emulsification or nanoprecipitation methods [ , ] . plga nps bear a net negative charge. they enter apcs through pinocytosis and endocytosis, undergo reversal of charge and endolysosomal escape of entrapped vaccine cargo leading to antigen processing in cytoplasm, resulting in cross-presentation of antigen to cd + t cells through mhc class i pathway [ , ] . plga nps are involved in maturation of dcs of mice and human origin, and controlled release of entrapped antigens leading to efficient expansion and differentiation of memory t-cells [ , ] . in rodent studies, induction of robust t-cell immunity is observed with plga nps-based vaccines containing various vaccine antigens [ , ] . further, plga is approved for drug deliveries in humans by the us food and drug administration (fda) and european medicine agency (ema) [ ] . plga nps enhance antigen uptake and induce maturation of porcine apcs [ , , ] . single dose of in immunization with plga nps-encapsulated inactivated/ killed prrsv antigen (nps-kag) induced activation of innate natural killer (nk) cells, γδ t-cells and secretion of innate cytokine ifnα [ ] . nps-kag vaccine also induced greater frequency of cd + t cells; increased secretion of ifn-γ; lowered frequency of t-regulatory cells; and reduced secretion of inflammatory cytokines compared to control kag-vaccinated animals [ , ] . in a subsequent study, when nps-kag was co-administered in with m. tuberculosis wcl adjuvant, a balanced th / th immune response and augmentation of mucosal iga antibody response was observed. after heterologous prrsv challenge, pigs that received nps-based vaccine showed no clinical signs and also had significant reduction in lung virus load [ , ] . plga nps were also used to encapsulate highly conserved influenza peptides and evaluated for efficacy in pigs after in administration. plga nps-based subunit vaccine resulted in induction of epitope-specific t-cell response but not the antibody response [ ] . the t-cell biased immune response was also observed in pigs after in immunization with plga nps-encapsulated inactivated/killed influenza virus (plga-kag) vaccine in pigs [ ] . in plga-kag vaccine administered animals observed reduced fever; lowered pneumonic lesions; and increased virus clearance from lungs after heterologous virus challenge compared to kag vaccine controls [ ] . in another study, pedv kag was encapsulated in plga nps and used to immunize pregnant sows by in route. this nanovaccine induced higher virus-specific igg and neutralizing antibodies in serum and greater igg, iga and neutralizing antibody responses in colostrum. it also induced greater cell proliferation and ifn-γ responses in restimulated pbmcs compared to kag vaccine controls. importantly, piglets born to nps-vaccinated sows had higher virus neutralizing antibodies and were better protected against homologous virus challenge than kag controls [ ] . these studies suggest that plga nps can be used as an efficient means of enhancing virus-specific cell-mediated immune responses in pigs. polyanhydrides are another type of synthetic polymer widely studied for vaccine deliveries [ ] . polyanhydride nps are synthesized by polycondensation or emulsification processes and are biodegradable, biocompatible and safe for vaccine delivery [ , ] . they activate innate immune responses in a manner similar to lipopolysaccharides (lps) [ ] . the surface-eroding nature of polyanhydride nps provides safe microenvironment for the encapsulated antigens and facilitates slow and sustained antigen release [ , ] . induction of better antibody and cell-mediated immune responses by polyanhydride npsbased vaccines has been reported against viral, bacterial and parasitic infections [ , ] . inoculation of polyanhydride nps-based siv kag vaccine (kag-nanovaccine) by in route enhanced cell-mediated but not the antibody responses in pigs [ ] . after heterologous virus challenge, kag-nanovaccine group had six to eightfold reduction of nasal virus shedding compared to kag vaccine controls [ ] . in a subsequent study, when kag-nanovaccine formulation was supplemented with cpg-odn adjuvant, both cell-mediated as well as mucosal iga antibody responses were improved [ ] . after heterologous virus challenge, cpg-odn-adjuvanted kag-nanovaccine provided better protection through a significant reduction in influenza-induced fever, -fold reduction of nasal virus shedding and -fold reduction in lung virus titers compared to pigs immunized with five-times greater quantity of soluble killed antigen (kag) vaccine [ ] . this study also indicates the dose-sparing ability of polyanhydride nps. thus, polyanhydride nps can also be used to induce better cellular as well as humoral immune responses in pigs. chitosan, alginate and other polysaccharides have also attracted attention as materials for nps formulation and drug delivery studies. chitosan is a natural polymer derived from deacetylation of chitin and is composed of glucosamine and n-acetylglucosamine residues [ ] . due to the availability of amino and carboxyl groups in an acidic microenvironment, chitosan nps have net positive surface charge which makes them highly mucoadhesive and increases their half-time of antigen retention on mucosal surfaces [ , ] . further, chitosan nps can reversibly open the epithelial cell tight junctions thereby improving paracellular and intracellular antigen transport across mucosal epithelial surfaces [ , ] . chitosan nps also enhance antigen uptake by apcs, induce apc maturation and active secretion of innate cytokines [ , ] . thus, chitosan nps form an attractive mucosal vaccine delivery vehicle. chitosan-based nps are used in pigs to deliver adjuvants such as bee venom and plasmid encoding porcine il- and il- /il- genes, which improved induction of better virus-specific immune responses of respective vaccines against prrsv and pcv [ , ] . chitosan nps enhance antigen uptake by porcine apcs and activate them to produce innate cytokines including ifn-alpha, tnf-alpha and il- β [ ] . chitosan nps encapsulated siv kag (cnps-kag) vaccine administered twice through in route without any additional adjuvant in pigs induced the cross-reactive mucosal iga antibodies. chitosan nps-based vaccine also induced ifn-γ response in pbmcs and tracheobronchial lymph nodes (tbln) better than kag vaccine controls. this vaccine formulation substantially reduced the challenge heterologous virus titers by up to -fold in both the upper and lower respiratory tracts compared to soluble kag vaccine. this finding emphasizes the potential benefits of using chitosan nps in future development of mucosal swine influenza vaccine for pigs [ ] . recently, dendrimer-like-alpha-d-glucan (nano- ) nps derived from sweet corn variety sugary- was examined as an alternative, safe, cost-effective and potent adjuvant [ , ] . nano- are positively charged nps which efficiently adsorb negatively charged antigens through electrostatic interactions. rodent studies have shown that nano- nps enhance antigen uptake by dcs, induce their maturation, activate them to produce pro-inflammatory cytokines and help in induction of antigen-specific antibodies [ , ] . in a recent study, we observed that nano- nps with or without addition of siv killed antigen (kag) can stimulate porcine apcs and produce cytokines such as ifn-α, tnf-α and il- β [ ] . pigs immunized via in route with nano- nps adsorbed siv kag at two-to-one ratio (nano- + kag) resulted in cross-reactive mucosal iga responses better than kag controls. moreover, pigs immunized im with nano- adsorbed ovalbumin (nano- + ova) had significantly greater igg and igg antibodies in serum compared with pigs vaccinated with ova alone [ ] . these findings highlight the possibility of using cornderived nano- nps as a potential adjuvant in porcine viral vaccine development. liposomes can encapsulate both hydrophilic and hydrophobic molecules in aqueous and non-aqueous phases of their vesicles [ ] . liposome vesicles protect antigens from enzymatic degradation, enhance antigen internalization by apcs and maintain controlled release of antigens [ ] . liposome-encapsulated antigens can enhance both cellular and humoral immune responses [ , ] . in a pig study, liposome nps were used as an im adjuvant for a pcv dna vaccine [ ] . liposome nps-adjuvant induced higher neutralizing antibodies and ifnγ response in pigs and reduced viremia of a challenge virus compared to alum-adjuvanted vaccine, providing the evidence that liposome nps can be a potent adjuvant in pigs [ ] . in our recent study, we used liposome nps to encapsulate ten highly conserved peptides of different influenza viruses of human and pig origin and immunized pigs through in route co-administered with monosodium urate (msu) crystal adjuvant [ ] . the liposome-adjuvant based vaccine enhanced the mucosal iga antibody response and induced peptide and virusspecific t-helper/memory cells and ifnγ responses resulting in reduced fever and modest reduction in virus titers in the respiratory tract of pigs [ ] . these studies highlight the fact that liposome-based nps can be used as an attractive vaccine delivery platform against porcine viral infections. virus infections have significant impact on pig industry worldwide. use of available vaccines have definitely helped in achieving strong control over some of the porcine viral infections such as food and mouth disease, transmissible gastroenteritis, classical swine fever and pseudorabies. vaccination also helped in reducing the clinical signs and increasing the production parameters in pcv -associated disease. however, for many other porcine viruses, further improvements in existing vaccine platforms and development of novel vaccine delivery systems are necessary to: ( ) induce better mucosal and cell-mediated immunity; ( ) protect against emerging and re-emerging strains; ( ) enhance the breadth (heterologous, cross-genotype and heterosubtypic) of immunity; and ( ) differentiate between infected and vaccinated animals. nps-based vaccine delivery platforms such as vlps, biodegradable polymers and liposomes have great potential as they-( ) protect vaccine antigens from degradation; ( ) facilitate antigen uptake and processing by apcs; ( ) impart adjuvant potential; ( ) can be used in mucosal and other alternate routes of immunizations; and ( ) induce effective mucosal and cellular cross-protective (broader) immunity. research efforts are ongoing to develop porcine viral vaccines using nps-based technologies. however, more collaboration(s) and in-depth studies are warranted to make this innovative vaccine antigen delivery technology successful and practical for application in food animal industry. to date, almost all of the immunomodulatory mechanisms of nps-based vaccine delivery platforms have been studied in rodent disease models, which may or may not reflect the situation in pigs or other domestic animal species [ ] . likewise, proper understanding of effect of size, charge and other physicochemical properties of nps after delivery through different routes of immunization in pigs is necessary to make efficient translation of this robust nps-based vaccine technology. similarly, studies should also focus on nps stability at different storage conditions and immunogenicity over a long period of time as they will directly associate with commercial aspect of the vaccine product. recent advances in nps-based adjuvant and vaccine delivery platforms in pigs demonstrate great promise to yield better candidate vaccines against many porcine viral infections with enhanced efficacy in the field. these nanovaccine technologies can also be adopted to develop effective vaccines against viral infections in other animal species, and knowledge gained could be exploited for improving the efficacy of existing human viral vaccines. the important viral infections of pigs emerging and re-emerging swine viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system assessment of the economic impact porcine reproductive and respiratory syndrome virus on united states pork producers optimal use of vaccines for control of influenza a virus in swine assessing production parameters and economic impact of swine influenza, prrs and mycoplasma hyopneimoniae on finishing pigs in a large production system the role of swine as "mixing vessel" for interspecies transmission of the influenza a subtype h n : a simultaneous bayesian inference of phylogeny and ancestral hosts vaccines for porcine epidemic diarrhea virus and other swine coronaviruses lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts porcine rotaviruses: epidemiology, immune responses and control strategies the pathogenesis of foot-and-mouth disease in pigs a review of classical swine fever virus and routes of introduction into the united states and the potential for virus establishment african and classical swine fever: similarities, differences and epidemiological consequences the classical swine fever epidemic - in the netherlands: descriptive epidemiology epidemiology and transmission of porcine circovirus type (pcv ) porcine circovirus type (pcv ) vaccines in the context of current molecular epidemiology molecular epidemiology and evolution of porcine parvoviruses pseudorabies virus in wild swine: a global perspective vaccines against pseudorabies virus (prv) a review of african swine fever and the potential for introduction into the united states and the possibility of subsequent establishment in feral swine and native ticks immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig genotype i of japanese encephalitis virus virus-like particles elicit sterilizing immunity against genotype i and iii viral challenge in swine improved vaccine against prrsv: current progress and future perspective challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective influenza a virus vaccines for swine foot-and-mouth disease vaccines classical swine fever vaccines-state-of-the-art ten years of pcv vaccines and vaccination: is eradication a possibility? pcv d- is the predominant type of pcv dna in pig samples collected in the u.s. during biology of porcine parvovirus (ungulate parvovirus ) control of swine pseudorabies in china: opportunities and limitations african swine fever: a re-emerging viral disease threatening the global pig industry vaccination greatly reduces disease, disability, death and inequity worldwide veterinary vaccines and their importance to animal health and public health role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine biodegradable particles as vaccine delivery systems: size matters modes of action for mucosal vaccine adjuvants vaccine adjuvants: putting innate immunity to work alum adjuvant: some of the tricks of the oldest adjuvant mechanism of immunopotentiation and safety of aluminum adjuvants adjuvants in veterinary vaccines: modes of action and adverse effects adjuvants for veterinary vaccines-types and modes of action recent progress in mucosal vaccine development: potential and limitations respiratory nanoparticle-based vaccines and challenges associated with animal models and translation chitosan nanoparticles act as an adjuvant to promote both th and th immune responses induced by ovalbumin in mice poly(anhydride) nanoparticles act as active th adjuvants through toll-like receptor exploitation liposome-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators vaccine delivery using nanoparticles potent antigen-specific immune responses stimulated by codelivery of cpg odn and antigens in degradable microparticles enhancement of immune responses by co-delivery of a cpg oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres co-delivery of cancer-associated antigen and toll-like receptor ligand in plga nanoparticles induces potent cd + t cell-mediated anti-tumor immunity m-cell targeted biodegradable plga nanoparticles for oral immunization against hepatitis b dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery enhanced and prolonged crosspresentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles cytotoxic t cell vaccination with plga microspheres interferes with influenza a virus replication in the lung and suppresses the infectious disease entrapment of h n influenza virus derived conserved peptides in plga nanoparticles enhances t cell response and vaccine efficacy in pigs biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs induction of potent antigen-specific cytotoxic t cell response by plga-nanoparticles containing antigen and tlr agonist polyanhydride nanovaccine against swine influenza virus in pigs virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development virus-like particle engineering: from rational design to versatile applications phagocytic processing of exogenous particulate antigens by macrophages for presentation by class i mhc molecules efficient major histocompatibility complex class i presentation of exogenous antigen upon phagocytosis by macrophages major findings and recent advances in virus-like particle (vlp)-based vaccines development of a porcine reproductive and respiratory syndrome virus-like-particlebased vaccine and evaluation of its immunogenicity in pigs intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus ′, ′-cgamp vaccigrade adjuvant exacerbates viremia after virus challenge pandemic h n influenza virus-like particles are immunogenic and provide protective immunity to pigs chimeric calicivirus-like particles elicit specific immune responses in pigs foot-and-mouth disease virus-like particles produced by a sumo fusion protein system in escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle promising ms mediated virus-like particle vaccine against foot-and-mouth disease immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying b and t cell epitopes of foot-and-mouth disease characterization of porcine circovirus type (pcv ) capsid particle assembly and its application to virus-like particle vaccine development construction and immunogenicity of recombinant porcine circovirus-like particles displaying somatostatin a novel subunit vaccine co-expressing gm-csf and pcv b cap protein enhances protective immunity against porcine circovirus type in piglets a novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure poly lactic-co-glycolic acid (plga) as biodegradable controlled drug delivery carrier plga-based nanoparticles: an overview of biomedical applications the preparation of sub- nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery delivery of a peptide via poly(d, l-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity duration of antigen availability influences the expansion and memory differentiation of t cells biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination poly (d, l-lactide-co-glycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets recent advances in polyanhydride based biomaterials amphiphilic polyanhydrides for protein stabilization and release activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants structural and antigenic stability of h n hemagglutinin trimer upon release from polyanhydride nanoparticles evaluation of cpg-odn-adjuvanted polyanhydride-based intranasal influenza nanovaccine in pigs chitosan-based gastrointestinal delivery systems strong adhesion and cohesion of chitosan in aqueous solutions contact timeand ph-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces effect of chitosan on the permeability of monolayers of intestinal epithelial cells (caco- ) effect of chitosan on epithelial permeability and structure the effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells immunogenic properties of a bcg adjuvanted chitosan nanoparticle-based dengue vaccine in human dendritic cells nasal delivery of chitosan/alginate nanoparticle encapsulated bee (apis mellifera) venom promotes antibody production and viral clearance during porcine reproductive and respiratory syndrome virus infection by modulating t cell related responses enhancement of immune response of piglets to pcv- vaccine by porcine il- and fusion il- / gene entrapped in chitosan nanoparticles mucosal immunity and protective efficacy of intranasal inactivated influenza vaccine is improved by chitosan nanoparticle delivery in pigs alpha-d-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants corn-derived alphad-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs liposomes as vaccine delivery systems: a review of the recent advances mucosal vaccine development based on liposome technology liposomeencapsulated antigens are processed in lysosomes, recycled, and presented to t cells development of porcine circovirus (pcv ) open reading frame dna vaccine with different adjuvants and comparison with commercial pcv subunit vaccine in an experimental challenge liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs intranasal delivery of influenza antigen by nanoparticles, but not nkt-cell adjuvant differentially induces the expression of b-cell activation factors in mice and swine porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the research reviewed in this article was supported by agriculture and food research initiative competitive grant no. - - from the usda-nifa and nanovaccine institute ( - ), iowa state university to rgj. salaries and research supports were provided by the state and federal funds appropriated to oardc. we thank dr. steven krakowka for scientific editing of the manuscript. authors' contributions sd wrote the article; gjr edited and revised the article. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - p ma authors: duan, x.; nauwynck, h. j.; pensaert, m. b. title: effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: p ma in this study, the susceptibility of porcine peripheral blood monocytes (bmo), peritoneal macrophages (pmφ) and alveolar macrophages (amφ) to prrsv was examined. to test the effect of differentiation and activation on their susceptibility, amφ and bmo were aged, cultivated in either adhesion or suspension and treated with bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma). it was found that freshly isolated pmφ and bmo were non-permissive to prrsv. pmφ remained refractory but a few bmo became susceptible after day cultivation. amφ were permissive with a significant increase of their susceptibility after one day cultivation. in a binding assay, it was demonstrated that the attachment of biotinylated prrsv to amf is much more efficient than to pmφ and bmo. two monoclonal antibodies (mabs) d and d which block prrsv infection of amφ and are directed against a candidate receptor for prrsv only reacted with the cell membrane of amφ. pma treatment of amφ blocked prrsv replication in the cells in a dose-dependent manner. the blocking effect of pma decreased after h continuous pre-treatment and diminished after h continuous pre-treatment. pma treatment did not affect the binding of prrsv and mab d and d to amφ. direct or indirect treatment of amφ and bmo with lps or cultivation in suspension did not significantly affect their susceptibility. these results provide clear evidence that prrsv has a strongly restricted tropism for only some sub-populations of porcine monocytes/macrophages and that some specific states of differentiation and activation of monocytes/macrophages considerably affect their susceptibility. porcine reproductive and respiratory syndrome virus (prrsv) resembles lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), three other members of the arterivirus group with regard to morphology, genetic organisation and structural proteins [ , ] . one peculiar common characteristic of these viruses is that they have a strong tropism for monocytes/macrophages. of many procine cell systems tested, only porcine alveolar macrophages (amf) support replication of prrsv [ , , , ] . replication of prrsv in some cultivated porcine peripheral blood monocytes (bmo) has also been reported [ ] . unexpectedly it was also found that prrsv replicate in two non-porcine cell lines: an established cell line from monkey kidney, marc- [ ] , and a proprietary cell line, cl [ ] . similarly, the replication of ldv and shfv is also highly restricted to primary cultures of host macrophages in vitro. eav forms an exception as it replicates in many different cell types [ ] . prrsv also shows a strict cell speci®city``in vivo''. cells of the macrophage lineage have previously been identi®ed as the predominately and consistently infected cell type in prrsv infected pigs [ , , , ] . furthermore, several observations indicated that prrsv only replicates in some sub-populations of monocytes/macrophages. it was found that amf even during the period of the highest virus titres in the lungs after natural or experimental infection, only a low percentage of levaged amf carries prrsv antigens [ , ] . also, immature macrophages and macrophages progenitor stem cells are not or less susceptible to a prrsv infection. this was particularly evident for bone marrow cells, where replication of prrsv was not detected in experimentally prrsv infected pigs [ ] . similar evidence comes from``in vitro'' experiments in which different sub-populations of alveolar macrophages fractionated by density gradient centrifugation were shown to have different susceptibilities to a prrsv infection [ ] . such heterogeneity may be a re¯ection of the sate of differentiation and/or activation of the macrophages [ ] . a number of factors such as ageing, some cytokines and a few chemical products have been found to be able to differentiate and activate mononuclear phagocytes [ ] . bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma) are two frequently used stimulants. both lps and pma have a strong, rapid and easily reproducible activating capacity. their structure and sites of activation in the cells have been extensively studied [ , ] . lps is a structural component of the outer membrane of gram-negative bacteria. it activates monocytes and macrophages and stimulates them to produce certain factors, including tnf-a, il- , and prostaglandin e [ , ] . studying the effect of lps treatment on the susceptibility of amf to prrsv may give new insights in the pathogenesis of dual infection with prrsv and bacteria. pma has effects on monocyte/macrophages, including dramatic changes in cell shape, spreading, endocytosis, and release of lytic mediators and regulators [ , , ] . pma also stimulate premonocytic cells of some continuous cell lines to differentiate into a more mature monocyte/macrophage phenotype [ ] . because of those remarkable properties, pma has been used to investigate the relationship between cell activation/differentiation and viral replication with some viruses [ , ] . in this study, it was examined if porcine monocyte/macrophage lineage cells isolated from peritoneal cavity, lungs and peripheral blood are susceptible to prrsv and if their susceptibility was related to the degree of virus attachment. furthermore, the effect of differentiation and activation of monocytes/macrophages by ageing, adhesion and treatment with lps and pma on their susceptibility to prrsv was evaluated. two prrsv isolates were used: the lelystad strain of prrsv (kindly provided by dr. wensvoort) and a belgian isolate of prrsv designated v . a lelystad virus stock of the thirteenth passage grown in porcine amf with a titre of . tcid /ml was used in this study. the v was adapted to marc- cells, puri®ed and biotinylated as earlier described [ ] . brie¯y, a ®fth passage of v was ®rst clari®ed by centrifugation at  g for min, then precipitated at  g for h in a beckman t rotor at c. the pellets were resuspended in / of original volume in tne buffer ( mm nacl, mm edta, mm tris-hcl, ph . ) and centrifuged on a . to . m discontinuous sucrose gradient in a sw rotor at  g for h. after centrifugation, the virus band was harvested and its purity was determined with a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the titre of the resulting virus preparation was to  tcid /mg as determined on marc- cells. the number of virus particles, as determined by negative staining electron microscopy, was to  /mg virus protein. puri®ed prrsv was labelled with biotin by using a protein biotinylation kit (amersham, buckinghamshire, uk). the virus was pelleted and re-suspended in biotinylation buffer ( mm na co , ph . ) at a protein concentration of mg/ml. after a brief sonication ml biotin reagent was added per mg of viral protein. the mixture was shaken for h at c and the reaction was terminated by addition of tris-hcl (ph . ) to a ®nal concentration of mm. biotinylated virus was collected after purifying on a sephadex g- column and diluted in pbs at a concentration of . mg/ml. after biotinylation, the titre of prrsv was reduced by %. biotinylated virions were stored at À c. a swine myeloid cell speci®c monoclonal antibody (mab), . . , was used to determine the percentage of porcine monocyte/macrophage cells [ ] . mabs against prrsv nucleocapsid protein, wbe and wbe ± were used for immuno¯uorescenece [ ] . two monoclonal antibodies (mab), d and d , which have been raised against amf and which are able to block prrsv infection of amf [ ] , were used for membrane immuno¯uorescence staining to test their reactivity with various cells. isotype matched irrelevant mabs e and g directed against suid herpesvirus type [ ] , were used as negative controls. porcine alveolar macrophages (amf) amf were obtained from -to -weeks old conventional belgian landrace pigs from a prrsv negative herd according to the method previously described by wensvoort et al. [ ] . brie¯y, the lungs were lavaged with ml cold phosphate buffered saline solution without calcium and magnesium (pbs). the lavaged cells were collected by centrifugation at  g for min at c. after two washings with cold pbs, a cell smear was made and stained with hemacolar reagents (diagnostica merck, darmstadt, germany) and the percentage of neutrophils was determined. the cells were stained with . . by membrane immuno¯uorescence. the percentage of cells from the monocytes/macrophages lineage was estimated by subtracting the percentage of neutrophils from the . . positive cells. by doing so, more than % of lung lavage cells were found to be monocyte/ macrophage lineage cells. pmf were isolated by lavaging the peritoneal cavity of four -to -weeks old conventional belgian landrace pigs originating from a prrsv negative herd with ml cold pbs. after centrifugation at  g for min at c and two washings with pbs, the cells were collected and the percentage of monocyte/macrophage lineage cells was determined with the technique described above. more than % of lavaged peritoneal cells were characterised as monocytes/macrophages. bmo were separated and cultivated as previously described [ ] . brie¯y, peripheral blood was obtained from four to weeks old conventional belgian landrace pigs originating from a prrsv negative herd and peripheral blood mononuclear cells (pbmc) were isolated from blood by ficoll-paque (pharmacia, uppsala, sweden) density gradient centrifugation according to the method recommended by the manufacturer.  pbmc were layered on a polystyrene cell culture dish (corning glass works, corning, ny, usa) which was coated with ml autologous plasma. after h incubation at c, non-adherent cells were removed by three washings with pbs. adherent cells representing enriched monocytes were harvested by gently¯ushing, the number of cells were counted and the percentage of monocytes was estimated with mab . . . more than % of cells were found to be monocyte/macrophage lineage cells. the viability of all cells used was b as assessed by % nigrosin staining. amf, pmf and bmo were brought in -well cell culture plates (nalge nunc international, roskilde, denmark) at a concentration of cells per well. the medium used for cultivation was rpmi supplemented with % of foetal calf serum. amf, bmo and pmf were inoculated by replacing the medium with ml stock solution containing . tcid prrsv. after incubation at c for h, three washings with pbs were performed. then, the cells were refed with medium and further incubated at c with % co . to evaluate the effect of maturation of monocytes/macrophages on their susceptibility to prrsv, freshly isolated amf, pmf and bmo from ®ve donors were seeded in -well tissue culture plates at a concentration of cells/ml/well and further incubated in rpmi medium plus % of foetal bovine serum at c with % co . after and h incubation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. to test if the adhesion of bmo and amf affect their susceptibility to prrsv, freshly isolated amf and bmo from ®ve donors were cultivated either in suspension in te¯on inserts (poly labo, strasbourg, france) or attached to polystyrene in -well cell culture plates at a concentration of cells/well in rpmi medium plus % of foetal bovine serum at c with % co . after h cultivation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and hours after inoculation. the effect of pma treatment on prrsv replication in bmo was examined by treating one day cultivated bmo with ng/ml phorbol -myristate -acetate (pma) (sigma-aldrich vertriebs gmbh, deisenbofen, germany) for h before prrsv inoculation. after pma treatment, the cells were washed three times, then inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. in order to examine the effect of duration of pma pre-treatment of amf on prrsv replication, amf were treated with ng/ml pma for different time periods prior to prrsv inoculation. after pma treatment, the cells were washed three times then inoculated with prrsv. intracellular virus titre was determined at h after inoculation. to test the effect of pma treatment during virus replication in amf, the amf were treated with ng/ml pma for h at different times after prrsv inoculation. intracellular virus titre was determined at h after inoculation. to estimate the dose dependent effect of pma treatment of amf on the prrsv replication, one day cultivated amf grown in -well-plates were treated with different concentrations of pma for h after h prrsv inoculation. the prrsv positive wells were determined by ®xing and staining the plate using an immunoperoxidase monolayer assay (impa) as earlier described [ ] after h inoculation. direct and indirect lps treatments were performed to evaluate the effect induced directly by lps or indirectly by lps-induced cytokines. for direct treatment, bacto lipopolysaccharide (lps) of e. coli :b (difco laboratories, detroit, michigen, usa) was used at a concentration of mg/ml medium. for indirect treatment, % of the supernatant from h lps treated porcine amf was used. after and h treatment, the cells were washed three times with pbs and inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. pma and lps had no negative effect on the viability of the treated cells as assessed by nigrosin staining after each treatment. a cytotoxicity bioassay with pk cells for detecting tnf [ ] and a proliferation assay on d (n )m cells for determining il- concentrations [ ] were performed to asses the ef®cacy of the lps treatment. for determining the titre of extracellular virus, medium from prrsv infected cells was collected and centrifuged at  g for min. ml of tenfold dilutions of the supernatants were inoculated in -well microtiter plates (nalge nunc, roskilde, denmark) previously seeded with amf. for determining the titre of intracellular virus, amf, pmf and bmo were harvested by¯ushing, washing, washed twice with pbs and lysed by freeze-thawing. ml of tenfold dilutions were inoculated in -well microtiter plates previously seeded with cultivated amf. inoculated -well plates were ®xed h after inoculation and stained using an immunoperoxidase monolayer assay (ipma) as earlier described [ ] . macrophages and monocytes were detached by thorough¯ushing of the bottom of polystyrene dishes and washed once with pbs. cell smears were made using a cytocentrifuge at  g for min. the smears were ®xed in acetone for min at À c. a streptavidin-biotin immuno¯uorescence technique was performed. brie¯y, the smears were pre-incubated with : diluted sheep serum to block non-speci®c staining. the smears were ®rst incubated with a mixture of mabs wbe and wbe ± , then with biotinylated sheep-anti-mouse immunoglobulin and ®nally with streptavidin-¯uorescein isothiocyanate (fitc)-conjugate (amersham, buckinghamshire, uk). the smears were washed three times with pbs between each incubation. to con®rm the speci®city of the staining, two mouse ascites¯uids containing irrelevant mabs against suid herpesvirus type of the same isotype as wbe and wbe ± were used as negative controls [ ] . positive cells were counted using a leica dm rbe¯uorescence microscope. the membrane reactivity of biotinylated prrsv, mabs d and d to pbmc, bmo, pmf and pma-treated ( ng/ml pma for h) and -untreated amf were evaluated by ā ow cytometer facscalibur (becton dickinson)¯ow cytometer.  cells were washed three times with cold pbs and incubated with ml of % paraformaldehyde at room temperature for min. after washing once with cold pbs, the ®xed cells were ®rst incubated with biotinylated prrsv ( mg) or mabs ( ng/ml of d , d or e ), then : diluted streptavidin-¯uorescein isothiocyanate (fitc)-conjugate or goat antimouse igg fitc (amersham, buckinghamshire, uk) for h on ice. the cells were washed three times after each incubation. the mean¯uorescence intensity of each sample was measured on the¯ow cytometer. relative mean¯uorescence measurements were corrected for auto¯uorescence of control cells. all data were statistically analysed by the student's t-test. prrsv productively replicated in amf. table shows the virus titres and percentage of prrsv positive cells in freshly isolated and one day cultivated amf at , and h after prrsv inoculation. the virus titres and percentage of viral antigen positive cells in freshly isolated porcine amf at and h after inoculation were respectively to log tcid and to times lower than those of one day cultivated ones, which is signi®cantly different (p` . ). a productive replication of prrsv was not detected in freshly isolated bmo obtained from four pigs. however, when the bmo were inoculated after one day of cultivation, x to . tcid per cells virus and . to . % viral antigen positive cells were detected at hours and hours post inoculation, respectively ( table ) . productive replication of prrsv was not found in freshly isolated pmf obtained from ®ve pigs. after one day cultivation, pmf remained refractory to prrsv infection. as shown in table , no statistically signi®cant differences in both viral antigen expression and virus titres were observed between amf and bmo cultivated in suspension in te¯on inserts and those attached to polystyrene dishes. the ef®cacy of activation after lps treatment was shown by the presence of tnf-a (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) and il- (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) in the supernatant of amf and bmo after h treatment with lps. however, no signi®cant differences in both viral antigen expression and virus titres were found between untreated and the directly or indirectly lps treated amf (table ) . no virus replication was detected in the bmo directly or indirectly treated with lps (data not shown). no virus replication was detected in the bmo treated with pma. when amf were pre-treated with pma for a short time ( to h), they became resistant to prrsv infection (fig. ) . after h continuous exposure to pma, amf gradually regained their susceptibility to prrsv and after h continuous pre-treatment, virus production reached the level similar to that of untreated cultures. the effect of a h treatment of amf with pma at different time points after a prrsv inoculation on the virus replication is shown in fig. . simultaneous virus inoculation and pma treatment for h reduced the virus production to a level of . tcid per cells. replication of prrsv was completely blocked when amf had been inoculated for h before pma treatment. the longer the interval between inoculation and treatment, the higher the virus titre. the blocking effect was concentration dependent as shown in fig. . concentrations as low as  À mg/ml pma completely inhibited prrsv infection. the membrane reactivity of bmo, amf and pmf to biotinylated prrsv and anti-amf monoclonal antibodies d and d as shown in fig. , the binding of biotinylated prrsv to bmo, amf and pmf was demonstrated by¯ow cytometry with a signi®cantly higher uorescence intensity in amf. the binding of biotinylated prrsv to the amf was not affected by pma treatment. after staining with anti-porcine amf monoclonal antibodies d and d , the membrane¯uorescence was detected only on amf and not on pbmc, bmo, and pmf (fig. ) . the results of this study show that only some subsets of cells from the porcine monocyte/macrophage lineage are susceptible to prrsv and that the speci®c differentiation and activation state may considerably affect their susceptibility to prrsv infection in vitro. cells of the porcine monocyte/macrophage lineage from different anatomic locations are clearly heterogeneous in their permissiveness for prrsv infection. prrsv replication was detected in porcine amf, while freshly isolated bmo and pmf were completely resistant. these results are in agreement with earlier``in vivo'' experiments, in which the replication of prrsv was found in alveolar macrophages but not in peripheral blood mononuclear cells [ ] . in the present study, very low virus titres and few infected cells were detected in cultivated bmo. this is in contrast with the observations of voicu et al. [ ] , who found much higher virus titres in cultivated bmo. this variation may be due to differences in the genotype/phenotype of the donor animals, differences in prrsv isolates and differences in techniques for the isolation of bmo. genetic, antigenic and pathogenic variations among prrsv isolates in the usa and europe have been reported [ , ] . even with amf and cl , two commonly used cell systems for prrsv isolation, nearly one third of american prrsv isolates grown in one cell type failed to grow in the other one [ ] . the experiments also revealed that amf show some restriction to a prrsv infection when they were freshly isolated, even though they are relatively the most sensitive cell type for prrsv isolation [ , ] . the susceptibility clearly increases after one day cultivation. a similar increase of permissiveness to prrsv infection was observed in a very small number of cultivated bmo. these results suggest that the state of monocyte/macrophage differentiation plays an important role in determining their susceptibilty to prrsv. similar enhancing effects of differentiation on virus replication have been observed with other viruses such as the respiratory syncytial virus (rsv), parain¯uenza virus- , african swine fever virus, cytomegalovirus or herpes simplex virus (hsv), human immunode®ciency virus type (hiv- ), visna-maedi virus and pseudorabies virus in monocytes/macrophages [ , ] . since bmo are precursors of tissue macrophages and readily mature into macrophages when cultivated in vitro [ ] , it is logical to predict that some bmo may mature into susceptible macrophages similar to alveolar macrophages by cultivation in vitro. resident pmf, which represent another population of well-differentiated tissue macrophages from a restricted lineage, were completely resistant to a prrsv infection. in this study, treatment of amf with pma was associated with a clear reduction of the replication of prrsv and the blocking effect of pma was transitory. when amf were continuously pre-treated for to h, the virus replication was almost completely blocked. the cells regained full susceptibility after h of continuous treatment. this rather peculiar ®nding can be explained by the fact that, while the activating capacity of monocytes and macrophages by pma reaches a maximum after h exposure, a prolonged exposure to pma for more than h causes macrophages and monocytes to become refractory to pma stimulation [ ] . when monocyte/macrophage lineage cells are treated with pma, both enhanced or decreased viral replication has been observed with other viruses. for example, an increased virus replication with herpes simplex virus has been noticed upon cell differentiation [ ] whereas a down-regulation of viral replication was observed with feline immunode®ciency virus [ ] . the mechanism through which pma inhibits prrsv infection of amf is unclear. in contrast to the pma treatment, lps treatment of porcine amf and bmo did not in¯uence the susceptibility to prrsv infection. the biological properties of pma are generally considered to be due to the activation of protein kinase c, an enzyme involved in many cellular responses. although the activation of protein kinase c is also an essential process for lps induced activation of the macrophage, the signal pathways used by pma and lps in macrophages are quite different [ ] . therefore, it seems that pma modulated inhibition of prrsv infection might be mediated by a speci®c pma-signalling pathway in porcine amf. recent observations suggest that prrsv enters amf through a process of receptor-mediated endocytosis (unpubl. data). pma treatment does not change the binding activity of prrsv and monoclonal antibody against the putative prrsv receptor(s) to amf. therefore, the blocking effect of pma does not act through down-regulating the expression of the virus receptor(s) on the cells. mononuclear phagocyte differentiation is a process of normal cellular development and homeostasis and re¯ects a permanently altered expression of a cell's genetic potential, while macrophage activation is the process that causes reversible changes in macrophage phenotype and functions [ ] . the differentiation may induce some factors that are essential for virus replication, such as receptors or transcription factors, while activation may up-or downregulate the expression of these factors in cells. inef®cient binding of biotinylated prrsv to pmf, bmo and pbmc suggests that these cells lack the more speci®c binding sites that are expressed on the membrane of amf. different binding activity of mab d and d , which block prrsv infection of amf and recognise a candidate receptor on the cell, to different monocytes/macrophages provide further evidence that the restriction of prrsv replication in some subsets of monocytes/macrophages is due to a reduced expression of virus receptor(s). comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterisation of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: pk( ) infection of peritoneal macrophages in vitro and in vivo with feline immunode®ciency virus lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes heterogeneity of porcine alveolar macrophages sub-population: immune functions and susceptibility to pears virus two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus virus quanti®cation and identi®cation of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus infection of alveolar macrophages can be blocked by monoclonal antibodies against cell surface surface antigens macrophages in viral infections immunohistochemical identi®cation of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrumdeprived pigs simple, sensitive and speci®c bioassay of interleukin- enhanced replication of porcine reproductive syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the u.s.a. and europe diagnosis of porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav in vitro induction of cytologic functional differentiation of the immature human monocyte cell line u- with phorbol myristate acetate replication of virulent aujeszky's disease virus (adv) in different blood mononuclear cell subpopulations virus production and viral antigen expression in porcine blood monocytes inoculated with pseudorabies virus effect of speci®c antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types preparation and characterization of monoclonal antibodies reactive with porcine pbl lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses pathological, ultrastructural, and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs mechanism generating functionally heterogeneous macrophages: chaos revisited lps induces selective translocation of protein kinase c-beta in lps-responsive mouse macrophages, but not in lps-nonresponsive mouse macrophages interaction of porcine reproductive and respiratory syndrome virus with swine monocytes mystery swine disease in the netherlands: the isolation of lelystad virus antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mechanism of intrinsic macrophage-virus interaction``in vitro isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome we thank c. bracke for technical assistance. we are grateful to dr. g. wensvoort for the supply of the lelystad isolate of prrsv and dr. t. w. drew for the gift of monoclonal antibodies wbe and wbe ± . received january , key: cord- -fi vo gx authors: shi, xibao; chang, yongzhe; zhang, xiaozhuan; wang, li; li, chunxi; jiang, kai; chen, jing; wang, chao; deng, ruiguang; fan, jianming; zhang, gaiping title: small interfering rna targeting nonstructural protein α (nsp α) of porcine reproductive and respiratory syndrome virus (prrsv) can reduce the replication of prrsv in marc- cells date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: fi vo gx abstract porcine reproductive and respiratory syndrome virus (prrsv) is one of the most economically devastating and pandemic diseases of swine, which is poorly controlled by current methods. the inhibition of specific genes by small interfering rna (sirna) has been proven to be a potential therapeutic strategy against viral infection. previous studies have indicated that the nonstructural protein α (nsp α) of prrsv may take an important role in virulence of prrsv. the present work was involved to explore the effect of sirna targeting nsp α on the replication of prrsv in marc- cells, and the results showed that over-expression of nsp α enhanced the replication of prrsv and that sirnas specifically targeting nsp α significantly inhibited the replication of prrsv in marc- cells. in conclusion, this work indicated that nsp α may be a viral pathogenicity factor of prrsv and that sirnas specifically targeting nsp α may be a new strategy to control prrsv in the future. small interfering rna targeting nonstructural protein α (nsp α) of porcine reproductive and respiratory syndrome virus (prrsv) can reduce the replication of prrsv in marc- cells a b s t r a c t porcine reproductive and respiratory syndrome virus (prrsv) is one of the most economically devastating and pandemic diseases of swine, which is poorly controlled by current methods. the inhibition of specific genes by small interfering rna (sirna) has been proven to be a potential therapeutic strategy against viral infection. previous studies have indicated that the nonstructural protein α (nsp α) of prrsv may take an important role in virulence of prrsv. the present work was involved to explore the effect of sirna targeting nsp α on the replication of prrsv in marc- cells, and the results showed that over-expression of nsp α enhanced the replication of prrsv and that sirnas specifically targeting nsp α significantly inhibited the replication of prrsv in marc- cells. in conclusion, this work indicated that nsp α may be a viral pathogenicity factor of prrsv and that sirnas specifically targeting nsp α may be a new strategy to control prrsv in the future. © elsevier ltd. all rights reserved. prrsv, a positive-stranded rna virus, is a member of family arteriviridae (snijder et al., ) . prrsv is one of the most economically important diseases of swine which is characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs (rossow, ) . infection with prrsv also predisposes pigs to secondary infection by bacterial and viral pathogens (mateu and diaz, ) . however, to date, there is no efficient antiviral agent or method available against prrsv, and unfortunately, both traditional-control strategies and conventional vaccines are insufficient to provide sustainable control of prrsv (darwich et al., ) , so it is very important and urgent to develop therapeutic strategies to control prrsv effectively. rna interference (rnai) is sequence-specific gene silencing which is mediated by -to -nt rna duplexes (sirnas) and can be as an exciting method to silence viral genes, especially for the single strand rna genomes (meister and tuschl, ) . to date, rnai has been used against several viruses including hepatitis b virus and dengue virus kahana et al., ) . the prrsv genome has ten open reading frames (orfs) and could produce nonstructural proteins (termed nsp α, nsp β, etc.) (snijder et al., ) . previous studies have indicated that the nsp α was essential for the synthesis of prrsv subgenomic mrna and it may play an important role in the virulence of prrsv (kroese et al., ; nedialkova et al., ) . however, whether the prrsv nsp α facilitates the replication of the virus and sirnas targeting the nsp α can influence the replication of prrsv was not clear. in this work, we firstly constructed an expression plasmid encoding a gfp-nsp α fusion polypeptide (pcdna . -gfp-nsp α)(gfp-nsp α) by subcloning from the plasmid pcdna . -flag-nsp α (shi et al., ) to pcdna . -gfp (shi et al., ) using the restriction endonuclease hind iii and ecori, and the results of the western-blot experiment in fig. a confirmed the successful expression of gfp-nsp α. secondly, in order to investigate the effect of nsp α on the replication of prrsv, the marc- cells were grown in -well plates overnight, and then the cells were transfected with expression plasmid gfp-nsp α or the control plasmid pcdna . -gfp. six hours after transfection, the cells were infected with bj- prrsv at a moi of . , and hours later, the cells were lysed by freezing and thawing repeatedly. the supernatants were obtained and the virus yields were measured by % tissue culture infected dose (tcid ) using the reed-muench method in marc- cells. the result in fig. b showed that the viral titer in the marc- cells transfected with pcdna . -gfp-nsp α has been increased to . times as compared with that in the marc- cells transfected with control plasmid. having demonstrated that overexpression of nsp α enhanced prrsv titers in marc- cells, it is reasonable to design the sirna of nsp α and study its effect on the replication of prrsv. we designed three sirnas ′-cagtcttgaagg ctctaca- ′, ′-ctgaacttccaacaaagaa- ′ and ′-ccagtgg aaacctgaactt- ′ that specially targeted nucleic acid sequence of nsp α, and a control sirna ′-cctacgccaccaatttcgt- ′ (a random sequence not found in the virus and host genome). marc- cells grown in -well plates were co-transfected in triplicate with the nsp α sirna ( nm/well) and the plasmid gfp-nsp α ( ng/well). twenty four hours later, the cells in five random fields were analyzed by fluorescence microscopy ( ×) and only one of them were shown in fig. . the results in fig. a showed that all of the three sirnas targeting nsp α could inhibit the expression of gfp-nsp α and did not influence the expression of gfp. similar results were obtained when hek t cells were in place of marc- cells (data not shown). finally, two sirnas targeting nucleic acid sequence of nsp α were selected to determine whether they could reduce the replication of prrsv in marc- cell. marc- cells grown in -well plates were transfected in triplicate with the nsp α sirna ( nm), nsp α sirna ( nm) or control sirna ( nm). six hours later after the transfection, the cells were infected with prrsv at a moi of . or mock infected, and hours later, the cells were lysed and then viral titers were measured by tcid . the results in fig. b showed that sirnas targeting nucleic acid sequence of nsp α could significantly reduce the replication of prrsv. nsp α was a multi-functional protein, and both our and other previous studies have shown that prrsv nsp α was an interferon antagonist and found that mutation to the nucleic acid sequence of nsp α influenced the synthesis of prrsv subgenomic mrna (kroese et al., ; shi et al., ) . our present study showed that over-expression of prrsv nsp α can enhance the replication of prrsv in marc- cells (fig. ) , so the present work gave direct evidence that nsp α may be a viral pathogenicity factor for prrsv. identification and targeting of viral pathogenicity factor are critical for understanding and controlling the virus. targeting pathogenicity factors is an attractive strategy for vaccine development such as live attenuated influenza virus vaccines encoding altered ns proteins (richt et al., ) and highly efficient coronavirus vaccines (zust et al., ) . in this work, we also explore whether the sirna, which targeted the nucleic acid sequence of nsp α, influenced the replication of prrsv, and the results showed that sirna targeting nsp α significantly reduced the replication of prrsv (fig. ) . the inhibition of specific genes by sirna in recent years has been proven to be a potential therapeutic strategy against viral infection (mahmoodur et al., ) , especially for positive single stranded rna viruses since their genomes function as both the mrna and the replication template , and additionally, a recent improved live prrsv vaccine has indicated that orf a and orf b were virulence determinants in prrsv (wang et al., ) , therefore, it is reasonable to propose that our present results provided a basis for generating the new prrsv vaccine by targeting prrsv nsp α. in conclusion, our present work indicated that nsp α may be a pathogenicity factor of prrsv and a promising target for controlling prrsv in future. dation china (grant no. ). marc- cells grown in -well plates were co-transfected with pcdna . -gfp-nsp α ( ng/well) or pcdna . -gfp ( ng/ well) and nsp α sirna ( nm), nsp α sirna ( nm), nsp α sirna ( nm) or control sirna( nm). twenty-four hours later, the cells in five random fields were analyzed by fluorescence microscopy ( ×) and only one of them was shown. (b) marc- cells grown in -well plates were transfected with nsp α sirna ( nm), nsp α sirna ( nm) or control sirna ( nm). six hours later, the cells were infected with prrsv at moi of . or mock infected, and hours later, the cells were lysed and then viral titers were measured by tcid . the results shown were from one of three independent experiments with similar observations. error bars represented the standard deviations. *p < . compared with the results in control. certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology rnai: antiviral therapy against dengue virus rnai: antiviral therapy against dengue virus inhibition of foot-and-mouth disease virus replication by small interfering rna the nsp alpha and nsp papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis rna interference: the story of gene silencing in plants and humans the challenge of prrs immunology mechanisms of gene silencing by double-stranded rna arterivirus nsp modulates the accumulation of minus-strand templates to control the relative abundance of viral mrnas vaccination of pigs against swine influenza viruses by using an ns -truncated modified live-virus vaccine porcine reproductive and respiratory syndrome the nonstructural protein papain-like cysteine protease was necessary for porcine reproductive and respiratory syndrome virus nonstructural protein to inhibit interferon-beta induction the zincfinger domain was essential for porcine reproductive and respiratory syndrome virus nonstructural protein- alpha to inhibit the production of interferon-beta arterivirus molecular biology and pathogenesis attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines this work was supported by the national natural science foundation of china (grant no. ), a program (no. cb ), the doctoral starting up foundation of henan normal university ( ), the key project of national natural science fund (no. ) and another national foun- key: cord- -emboxdzu authors: gómez-laguna, jaime; salguero, francisco j.; pallarés, francisco j.; carrasco, librado title: immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: emboxdzu porcine reproductive and respiratory syndrome (prrs) virus (prrsv) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages and inducing apoptosis of immune cells. an imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin- , in prrs may impair the immune response of the lung. pulmonary macrophage subpopulations have a range of susceptibilities to different prrsv strains and different capacities to express cytokines. infection with prrsv decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. prrsv infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (cd ) and induce the synthesis of anti-inflammatory mediators. the balance between pro- and anti-inflammatory cytokines modulates the expression of cd , which may affect the pathogenicity and replication of the virus in different tissues. with the emergence of highly pathogenic prrsv, there is a need for more information on the immunopathogenesis of different strains of prrs, particularly to develop more effective vaccines. porcine reproductive and respiratory syndrome (prrs) is caused by an arterivirus, prrs virus (prrsv) (king et al., ) . two genotypes of prrsv have been identified: ( ) european ortype genotype (prrsv- ), the prototype of which is lelystad virus (lv); and ( ) north american or type genotype (prrsv- ), the prototype of which is the reference strain atc . significant antigenic and pathogenic differences have been reported between and within genotypes (stadejek et al., (stadejek et al., , darwich et al., ) , correlated with a lack of cross-protection by vaccines (geldhof et al., ) . highly pathogenic strains of prrsv (hp-prrsv) have been identified within both genotypes (xiao et al., ; hu et al., ) . the respiratory form of prrs primarily affects growing and finishing pigs, causing interstitial pneumonia, which induces respiratory signs and increases susceptibility to infection with other pathogens (rossow, ) . although infection with prrsv may be subclinical, clinical disease becomes evident when secondary infections are present (drew, ) ; prrsv thus contributes to the porcine respiratory disease complex (prdc) (opriessnig et al., ) . in , the appearance of disease outbreaks caused by atypical or hp-prrsv strains was reported in the usa (halbur and bush, ) . these outbreaks were characterised by increased abortions ( - %), sow mortality ( - %) and preweaning mortality, primarily due to respiratory disease. hp-prrsv strains have been isolated in china and south east asia (tian et al., ; feng et al., ) and eastern europe (karniychuk et al., ) . infection with hp-prrsv strains is associated with severe clinical signs, pulmonary lesions and aberrant host immune responses (xiao et al., ; amarilla et al., ; hu et al., ) . this paper reviews some aspects of the immunopathogenesis of prrs and demonstrates how prrsv replication affects the host pulmonary microenvironment, leading to opportunistic secondary infections. prrsv damages the pseudostratified ciliated epithelium of the respiratory tract, impairing the mucociliary transport system and preventing the removal of microorganisms from the respiratory system (done and paton, ; halbur et al., ) . the primary target cells for replication of prrsv are porcine alveolar macrophages (pams) (duan et al., ) , which are responsible for phagocytosis of microorganisms in the alveoli (cheung et al., ) . replication of prrsv in pams directly impairs their basic functions, including phagocytosis, antigen presentation and production of cytokines (thanawongnuwech et al., ; de baere et al., ) . prrsv induces necrosis or apoptosis in pams (costers et al., ) and also induces apoptosis of lymphocytes and macrophages in the lungs and lymphoid organs, impairing the host immune response (labarque et al., ; gómez-laguna et al., b) . prrsv increases the susceptibility of pigs to secondary bacterial infections and other viral infections (van reeth et al., ; thacker et al., ; brockmeier et al., ; drew, ; halbur et al., ; wills et al., ; renukaradhya et al., ; díaz et al., ) . however, under experimental conditions, secondary infections do not always establish in pigs infected previously with prrsv (drew, ) . concurrent infection with prrsv and porcine circovirus type (pcv ) is associated with more severe disease and higher piglet mortality (allan et al., ; harms et al., ; rovira et al., ; opriessnig et al., ) . mycoplasma hyopneumoniae potentiates prrsv-induced disease (thacker et al., ) . there is a high frequency of concurrent infection with prrsv and streptococcus suis, m. hyopneumoniae or pcv in the field . factors involved in increased susceptibility of pigs to secondary infections include the pneumovirulence of prrsv (martínez-lobo et al., ) and the ability of the virus to persist (> days) in lymphoid organs, mainly the tonsils and lymph nodes (wills et al., ) . these features are influenced by differences in antigenicity and virulence among prrsv genotypes, as well as the genetic background of the host. prrsv- exhibits higher pneumovirulence than prrsv- , without marked differences in systemic clinical signs, viral load or viral distribution (martínez-lobo et al., ) . however, hp-prrsv, whether hp-prrsv- or hp-prrsv- , is associated with more severe clinical signs, as well as increased severity of lung lesions and viral replication (amarilla et al., ; hu et al., ) . the mononuclear phagocyte system of the lung consists of pulmonary alveolar macrophages (pams), pulmonary interstitial macrophages and, in pigs and some other species, pulmonary intravascular macrophages (pims) (longworth, ) . pams are free in the alveolar spaces, where they phagocytise inhaled particles. pims are found within the pulmonary capillaries, adherent to endothelial cells; they remove foreign particles from the blood and are able to migrate to sites of injury. pims release pro-inflammatory mediators, such as metabolites of arachidonic acid and cytokines that modulate pulmonary microvascular physiology (bertram et al., ; chitko-mckown et al., ; carrasco et al., ) . pams represent the main cellular target for prrsv replication, although the virus is also able to replicate in other macrophage subpopulations (duan et al., ) . in addition, prrsv replicates in monocyte and bone marrow derived dendritic cells (dcs) in vitro, although such replication has not been demonstrated clearly in vivo (loving et al., ; wang et al., ; chang et al., ; flores-mendoza et al., ; gimeno et al., ) . the main cell surface receptors for prrsv attachment, internalisation and uncoating are heparan sulphate, sialoadhesin and cd (delputte et al., ; van gorp et al., ) . prrsv replicates in pams and pims in vitro, affecting their bactericidal functions (thanawongnuwech et al., ) . since one of the major functions of pims is the clearance of bacteria from blood (winkler and cheville, ; staub, ) , viral replication in pams and pims may affect colonisation of the lungs by secondary viral or bacterial pathogens, as well as haematogenous bacterial dissemination. prrsv has a higher tropism for pams than for septal macrophages (pims and interstitial macrophages) (gómez-laguna et al., b; amarilla et al., ) . whereas prrsv replicates mainly in pams, expression of pro-inflammatory cytokines has been observed mainly in septal macrophages, pointing to different roles of pams and septal macrophages in prrs. the main function of pams is to provide the first line of phagocytic defence against microbial invasion (cheung et al., ; de baere et al., ) . septal macrophages, which mostly are activated indirectly by the replication of prrsv in bystander cells, may modulate local inflammatory and immune responses through secretion of cytokines (gómez-laguna et al., b) . sialoadhesin, but not cd , downregulates phagocytosis in prrsv-infected pams (de baere et al., ) . prrsv increases the susceptibility of the host to a wide range of viral and bacterial respiratory pathogens, resulting in increased severity of clinical signs and lung lesions. concomitant infections with prrsv and swine influenza virus, porcine respiratory coronavirus (prcv) or pcv are common. more severe clinical signs and growth retardation occur in co-infected pigs than in pigs infected with prrsv alone (van reeth et al., ; harms et al., ; jung et al., ; renukaradhya et al., ; opriessnig et al., ) . in pigs dually infected with prrsv and prcv, exacerbation of clinical signs is associated with an impaired innate immune response in the lungs, specifically a decrease in natural killer (nk) cell-mediated cytotoxicity due to decreased expression of interferon (ifn)a. the adaptive immune response is also impaired, leading to enhanced apoptosis of pams as a consequence of increased concentrations of interleukin (il)- and il- (jung et al., ; renukaradhya et al., ) . modulation of the innate immune response in prrsv-infected pigs under field conditions is associated with a decrease in cytotoxicity, but not the percentage, of nk cells (dwivedi et al., ) . functional modulation of nk cells is associated with increased plasma concentrations of il- , il- and il- , suggesting a role for these cytokines in modulating the host immune response (dwivedi et al., ) . regulatory t cells (tregs) induced by prrsv- , but not prrsv- , impair the host immune response (cecere et al., ; silva-campa et al., . although prrsv- increases the percentage of tregs, its effect on the immune response in vivo is not yet clear. the susceptibility of prrsv-infected pigs to secondary bacterial pathogens has been linked to upregulation of cd and lipopolysaccharide (lps)-binding protein (lbp), which cooperate in the recognition and signalling of lps. enhanced expression of these molecules in the lungs increases susceptibility to secondary bacterial infection. although there is upregulation of cd in prrsv-infected septal macrophages and pams, most cd enhancement in the lungs of prrsv-infected pigs is probably due to infiltration of cd -positive monocytes into the pulmonary interstitium; these may later differentiate into septal macrophages (van gucht et al., ; qiao et al., ) . prrsv and bacterial endotoxin act synergistically to amplify the inflammatory response of infected macrophages (qiao et al., ) . higher expression of pro-inflammatory cytokines is observed in septal macrophages of prrsv-infected pigs (gómez-laguna et al., b) . these findings suggest a role for septal macrophages in the immunopathogenesis of secondary bacterial infections in prrs through upregulation of cd and activation of a cytokine cascade (fig. ) . acute phase proteins (apps) are involved in the acute phase response and are synthesised mainly by hepatocytes in response to pro-inflammatory cytokines (eckersall, ; petersen et al., ) . apps can be classified as 'positive' or 'negative', depending on whether their serum concentrations are increased or decreased, respectively, in the acute phase response. haptoglobin (hp), creactive protein (crp), serum amyloid a (saa) and 'pig-major acute protein' (pig-map) are the main apps in pigs (petersen et al., ) . apps induce pro-inflammatory reactions and fever, but overexpression can also induce an anti-inflammatory state (ceciliani et al., ; petersen et al., ) . a lack of significant changes in serum concentrations of proinflammatory cytokines in prrs may point to a strategy by which prrsv is able to escape from the host immune response, thereby preventing activation of hepatocytes to induce an efficient acute phase response. early expression of hp and pig-map is observed in prrs, whereas serum concentrations of crp and saa exhibit a delayed and variable enhancement (gómez-laguna et al., c) . crp is involved in complement activation and opsonisation, as well as inducing production of cytokines by macrophages (ceciliani et al., ; petersen et al., ) . saa is chemotactic for monocytes, t cells and polymorphonuclear leucocytes (ceciliani et al., ; petersen et al., ) . the late increase in crp and saa may contribute to the inefficient early immune response in prrsv-infected pigs. hp modulates the immune response through complex interactions with a range of effectors. in experimental infections with prrsv, there is an increase in serum concentrations of hp, along with increased expression of il- and tumour necrosis factor (tnf)-a (asai et al., ; gómez-laguna et al., c) . hp interacts with cd , the receptor for prrsv, increasing expression of the anti-inflammatory cytokine il- (díaz et al., ) . cd removes haemoglobin-hp complexes circulating in the blood (kristiansen et al., ) , decreasing the amount of haemoglobin available for bacterial pathogens and reducing oxidative stress. hp may play a role in the pathogenesis of prrs through modulating the immune response and inducing anti-inflammatory cytokines, such as il- . samples of saliva and meat juice are alternatives to serum for measuring and monitoring apps during prrsv infection in pigs (gutiérrez et al., ; gómez-laguna et al., a) . pen-based oral-fluid samples also represent a reliable, cost-effective approach to prrsv surveillance in pigs (prickett et al., ; kittawornrat et al., ) . in contrast to other viral respiratory diseases of pigs (carrasco et al., ; khatri et al., ) , production of pro-inflammatory cytokines is limited in prrs (van reeth et al., ) . low expression of pro-inflammatory cytokines, both at mrna and protein levels, has been reported in pigs infected with prrsv- and prrsv- (thanawongnuwech et al., ; gómez-laguna et al., c; renukaradhya et al., ; garcía-nicolás et al., ) . this may be related to the typically mild respiratory and systemic clinical signs in experimental prrsv infections, in which absence of fever is correlated with development of more severe interstitial pneumonia (van reeth et al., ; van gucht et al., ; gómez-laguna et al., ). there appears to be a correlation between the virulence of the prrsv strain, the severity of clinical signs and the expression of pro-inflammatory cytokines; in pigs infected with hp-prrsv, marked inappetence and severe respiratory signs are related to severe interstitial pneumonia and high levels of expression of il- a in the lungs in comparison with other classical prrsv strains (amarilla et al., ; hu et al., ) . table summarises the main pro-inflammatory cytokines that play a role in prrs. in studies of other respiratory diseases of pigs, efficient activation of the inflammatory response occurs only locally in the lung, whereas there are no substantial alterations in serum concentra- table cytokines examined in porcine reproductive and respiratory syndrome (prrs) and their role in the immunopathogenesis of the disease. role tions of pro-inflammatory cytokines (baarsch et al., ; conn et al., ) . increases in expression of il- a, il- and tnf-a in the lungs of pigs infected with prrsv- are correlated with the development of interstitial pneumonia (gómez-laguna et al., b). as discussed above, prrsv impairs the host immune response (darwich et al., ) . expression of prrsv antigens is correlated with expression of regulatory cytokines, such as il- and transforming growth factor (tgf)-b, in the lungs of pigs (gómez-laguna et al., b , a ; table ). the lack of substantial changes in serum concentrations of pro-inflammatory cytokines may reflect a strategy by which prrsv evades the host immune response. therefore, samples of blood and/or serum do not necessarily reflect the events occurring in lungs of prrsv-infected animals at a given time, although these are very accessible samples. differential expression of pro-inflammatory cytokines has been observed in lymphoid organs of prrsv-infected pigs. whereas a marked increase in expression of tnf-a and il- a was observed in the mediastinal lymph nodes, there was a limited increase in expression of these cytokines in the tonsils and retropharyngeal lymph nodes (barranco et al., ) . prrsv isolates have been classified on the basis of whether they induce tnf-a, il- , both of these cytokines or neither of these cytokines (gimeno et al., ) . tnf-a plays an important role in the inflammatory response; this cytokine may act as an antiviral cytokine, protecting cells from infection against viruses or enhancing selective elimination of virus-infected cells through an ifnindependent mechanism. recombinant porcine tnf-a inhibits prrsv replication in vitro and prrsv-infected pams have reduced expression of tnf-a (lópez-fuertes et al., ) . several prrsv strains are weak inducers of tnf-a and also impair tnf-a production by inhibiting the extracellular signal-regulated kinase (erk) signalling pathway; this mechanism may contribute to the virulence of hp-prrsv (hou et al., ) . promoter and post-transcriptional downregulation of tnf-a has been associated with prrsv non-structural proteins (nsps) a, b and (chen et al., ; subramaniam et al., subramaniam et al., , darwich et al., ) . specific amino acid residues of nsp a and nsp b have been identified that affect tnf-a promoter activity; substitution of specific nsp b amino acids is potential tool for the generation of attenuated prrsv vaccines (subramaniam et al., ) . the limited expression of tnf-a in experimental infections with some prrsv strains may be a mechanism by which these strains impair the host immune response and prevent viral clearance (table ; fig. ). il- is a regulatory cytokine that, among other functions, inhibits the synthesis of pro-inflammatory cytokines (moore et al., ) . a role for il- in inhibiting or counterbalancing the ifn-c response and restricting prrsv replication has been proposed in infections with some prrsv strains (bautista and molitor, ; díaz et al., ) . thus, in the same way that il- inhibits the production of ifn-c, it may also suppress the pro-inflammatory response in prrsv-infected pigs (table ; fig. ) . a different behaviour of regulatory cytokines, such as il- or tgf-b, has been observed in prrsv-infected pigs. there is a significant correlation between prrsv antigen expression and the expression of regulatory cytokines, such il- and tgf-b, in the lungs, but not in the lymphoid organs, of infected pigs (barranco et al., ; gómez-laguna et al., b , a . one hypothesis suggested by these findings is that cells recruited to the pulmonary parenchyma are modulated to secrete il- and/or tgf-b, among other immune mediators. an alternative hypothesis is that the expression of regulatory cytokines plays a key role in the modulation of the inflammatory response within the lung, reducing infiltration and proliferation of inflammatory cells (spight et al., ; charavaryamath et al., ; backus et al., ) . il- is expressed mainly by septal macrophages and, to a lesser extent, pams and other cells, whereas tgf-b is expressed mainly in pams and secondarily in septal macrophages (gómez-laguna et al., b , a . expression of regulatory cytokines by different subsets of lung macrophages may impair bacterial killing by pams and pims, as well reduce the efficiency of the local host immune response by septal macrophages. production of tgf-b occurs in recall responses to some prrsv- strains and is more marked after homologous reinfection, suggesting the presence of tgf-b producing t cell clones dependent on the prrsv strain (díaz et al., ) . the imbalance between pro-and anti-inflammatory cytokines may modulate the expression of cd , which is one component of a complex of receptors required for prrsv entry, including heparan sulphate and sialoadhesin (delputte et al., ; van gorp et al., ) . expression of cd is upregulated by il- and il- , promoting prrsv entry and replication, but downregulated by tnf-a, tgf-b and ifn-c (buechler et al., ; sulahian et al., ; pioli et al., ; weaver et al., ; patton et al., ) . other mediators may regulate expression of cd (table ). there is differential expression of cytokines by different prrsv isolates (gimeno et al., ) and within different lymphoid organs ( barranco et al., ) , indicating that analysis of the expression of pro-and anti-inflammatory cytokines is a useful tool in the study of the immunopathogenesis of different prrsv strains. prrsv impairs local immune responses in the lungs of pigs by a range of mechanisms, including impairment of the mucociliary transport system, decreases in the function and number of pams, induction of apoptosis of immune cells and creating an imbalance between pro-and anti-inflammatory cytokines, which may allow the virus to persist in the host. prrsv decreases the bactericidal activity of macrophages, resulting in increased susceptibility to secondary infections. the variability in cytokine profiles induced by different prrsv strains, as well as the emergence of hp-prrsv in asia and eastern europe, highlight the need to evaluate differences in immunobiology among prrsv strains. prrsv is able to modulate the immune response by increasing the expression of hp in the early stages of infection, which may interact with the viral receptor cd and induce the synthesis of anti-inflammatory mediators, such as il- . there is a need for improved knowledge of the immune response in prrsv infections in order to improve the efficacy of vaccines to control prrs. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. this work was financially supported by the spanish ministry of education and science (project number agl - /gan). mediators involved in regulation of cd receptor. upregulation acute phase response in porcine reproductive and respiratory syndrome virus infection use of saliva for haptoglobin and c-reactive protein quantifications in porcine respiratory and reproductive syndrome affected pigs in field conditions update on abortion storms and sow mortality. swine health and production comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus efficacy of antimicrobial treatments and vaccination regimens for control of porcine reproductive and respiratory syndrome virus and streptococcus suis coinfection of nursery pigs experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus highly pathogenic porcine reproductive and respiratory syndrome virus impairs lps-and poly(i:c)-stimulated tumor necrosis factor-alpha release by inhibiting erk signaling pathway pathogenicity and distribution of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate . th report of the international committee on taxonomy of viruses swine influenza h n virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h n influenza virus porcine reproductive and respiratory syndrome virus (prrsv) in serum and oral fluid samples from individual boars: will oral fluid replace serum for prrsv surveillance? identification of the hemoglobin scavenger receptor apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines the comparative pathology of pulmonary intravascular macrophages porcine reproductive and respiratory syndrome (prrs) virus down-modulates tnf-a production in infected macrophages differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus comparative pathogenicity of type and type isolates of porcine reproductive and respiratory syndrome virus (prrsv) in a young pig infection model gradual development of the interferon-c response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination interleukin- and the interleukin- receptor polymicrobial respiratory disease in pigs modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages application of acute phase protein measurements in veterinary clinical chemistry tgf-b regulation of human macrophage scavenger receptor cd is smad -dependent detection of porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs the scavenger receptor cd : regulation, promoter structure and genomic organization porcine reproductive and respiratory syndrome experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination porcine reproductive and respiratory syndrome virus (prrsv) infection status in pigs naturally affected with post-weaning multisystemic wasting syndrome (pmws) in spain european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells porcine reproductive and respiratory syndrome virus induces cd + cd + cd + foxp + regulatory t cells (tregs) induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus immunoregulatory effects of regulated, lung-targeted expression of il- in vivo porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe pulmonary intravascular macrophages amino acid residues in the non-structural protein of porcine reproductive and respiratory syndrome virus involved in down-regulation of tnf-a expression in vitro and attenuation in vivo porcine reproductive and respiratory syndrome virus non-structural protein suppresses tumor necrosis factor-alpha promoter activation by inhibiting nf-jb and sp human monocytes express cd , which is upregulated by il- and identical to p mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia immunohistochemical staining of ifn-c positive cells in porcine reproductive and respiratory syndrome virus-infected lungs effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr- ) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark regulation of surface cd expression and cellular effects of receptor mediated hemoglobin-haptoglobin uptake on human monocytes and macrophages sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus interaction between porcine reproductive-respiratory syndrome virus and bacterial endotoxin in the lungs of pigs: potentiation of cytokine production and respiratory disease porcine reproductive and respiratory syndrome virus infection increases cd expression and lipopolysaccharide-binding protein in the lungs of pigs differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability upregulation of human monocyte cd upon activation of cell-surface toll-like receptors duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine postnatal colonization of porcine lung capillaries by intravascular macrophages: an ultrastructural, morphometric analysis aberrant host immune response induced by highly virulent prrsv identified by digital gene expression tag profiling key: cord- -vq fh authors: stadejek, tomasz; larsen, lars e.; podgórska, katarzyna; bøtner, anette; botti, sara; dolka, izabella; fabisiak, michał; heegaard, peter m.h.; hjulsager, charlotte k.; huć, tomasz; kvisgaard, lise k.; sapierzyński, rafał; nielsen, jens title: pathogenicity of three genetically diverse strains of prrsv type in specific pathogen free pigs date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: vq fh studies from eastern european countries proved that porcine reproductive and respiratory syndrome virus type (prrsv- ) harbours high genetic diversity and that genetically divergent subtypes – circulate in this area. in the present study, we compared the pathogenicity of two different prrsv- subtype strains and a strain representing prrsv- subtype . four groups of -week-old specific pathogen free pigs were either infected with subtype strain ili , subtype strain or bor , subtype strain , or mock inoculated. the most pronounced clinical signs were observed in pigs infected with bor . pigs from both subtype strain infected groups exhibited significantly elevated mean body temperatures on dpi compared to the other two groups, the difference remaining significant up to dpi for the bor group, only. the pigs in the latter group also displayed significantly highest levels of early viremia together with the most rapid app response. overall, the results indicated that bor strain can be considered a highly pathogenic strain, similarly to subtype strains lena and su -bel, while the virulence of the other subtype strain ili was intermediate between bor and subtype strain. porcine reproductive and respiratory syndrome virus (prrsv) belongs to the arteriviridae family within the order nidovirales (faaberg et al., ) . prrsv is the cause of severe respiratory and reproductive disease in swine worldwide. the virus emerged as a swine pathogen in north america and europe nearly simultaneously in the period - (zimmerman et al., . quickly, nucleotide sequence comparisons of the prototypical isolates lelystad virus and vr- revealed that the european (eu) and north american (na) isolates were only distantly related. later, the eu and na genotypes were officially designated as type (prrsv- ) and type (prrsv- ), respectively (faaberg et al., ) . following the expansion of our understanding of arterivirus evolution and discovery of many new genera in the family of arteriviridae, it was recently proposed to separate prrsv- and prrsv- into two different species (kuhn et al., ) . the results of bioinformatic analysis suggested that prrsv existed at least years back in time (forsberg, ) , and the most recent common ancestor of prrsv- emerged in - , or even earlier (nguyen et al., ) . initially, prrsv- was thought to be genetically homogenous, but studies from italy, lithuania, latvia, belarus and the russian federation established that prrsv- is even more diverse than prrsv- (forsberg et al., ; le gall et al., ; stadejek et al., stadejek et al., , stadejek et al., , stadejek et al., , suarez et al., ) . previously, it has been suggested that the high genetic diversity in prrsv- orf and orf sequences warranted definition of subtypes (stadejek et al., (stadejek et al., , . currently, subtypes (lelystad viruslike), , and are recognized and tentative evidence has been found for potential additional subtypes (stadejek et al., ) . in contrast, multiple genetic clades have been defined in prrsv- but they comprise a degree of sequence diversity found in prrsv- subtype lineages alone (shi et al., ) . the genetic subtyping or clustering the strains of prrsv may be affected by genetic recombination between the strains of the same genotype, and the classification of viruses based on the analysis of small genetic fragments should not be overrated (brar et al., ; franzo et al., ; lu et al., ; martin-valls et al., ; shi et al., ; zhao et al., ) . it has been shown that, in terms of virulence, antigenic characteristics and immunological responses following experimental infection, the belarusian lena subtype strain differed from subtype strains from western europe being generally more pathogenic for pigs (karniychuk et al., ; weesendorp et al., a weesendorp et al., , . later, another subtype belarusian strain su -bel, which was characterized in vitro and in in vivo challenge experiments also proved to be significantly more virulent than western european strains (morgan et al., (morgan et al., , salguero et al., ) . at present, no data are available about the biological characteristics of east european prrsv- strains from other genetic subtypes, e.g. of subtype strains, which compose the second most prevalent cluster of prrsv- , present from lithuania and belarus in the west, to khabarovsk krai in the russian far east in the east (stadejek et al., ) . the aim of the present study was to compare the pathogenicity of different prrsv- strains by inoculation of groups of specific pathogen free pigs. based on orf sequence analysis, two strains from belarus and russian federation and one strain from denmark were classified as subtypes and , respectively. three prrsv- strains isolated in porcine alveolar macrophages (pam) cultures derived from prrs-negative pigs were used in this study. the strain was isolated from a danish pig in (botner et al., ) . the strain ili was isolated in from lung tissue of a russian weaner pig. the strain bor was isolated in from lung tissue of a belarusian pig that died with respiratory disease symptoms. in order to prepare the various inocula, the strains were passaged and titrated in pam cultures. the inoculum of was a th passage (titre of . log tcid /ml). the inoculum of ili was a rd passage (titre of . log tcid /ml). the inoculum of bor was a nd passage (titre of . log tcid /ml). the challenge experiment performed at the bsl animal facilities of dtu national veterinary institute was carried out in accordance with the danish legislation on animal experiments (lbk nr (lbk nr - / / and eu regulations on the use of laboratory animals for research. the pigs used in the experiment were procured from the institute's high health pig production herd, which was tested free from infection with the following pathogens: encephalomyocarditis virus, hepatitis e virus, porcine circovirus type and type viruses, porcine cytomegalovirus, porcine epidemic diarrhoea virus, porcine parvovirus type , porcine respiratory coronavirus, prrsv- and prrsv- , influenza a virus, transmissible gastroenteritis virus, actinobacillus pleuropneumoniae, bordetella bronchiseptica, brachyspira hyodysenteriae, brachyspira pilosicoli and brucella spp., using in-house standard diagnostic methods. twenty-eight -week-old pigs were randomly divided into four groups that were housed separately. after weeks of acclimatization, the pigs (now weeks of age) were inoculated intranasally (i.n.) with ml of virus inoculum in each nostril. group (pigs - ) was inoculated with the strain , group (pigs - ) was inoculated with ili and group (pigs - ) was inoculated with bor . group (pigs - ) was mock-inoculated with ml of eagle's medium in each nostril and served as a prrs negative control group. individual pigs were subjected to daily clinical examination and rectal body temperatures were recorded starting from − day post infection (dpi). in order to obtain a semi-quantitative measure for comparison of clinical disease between the groups, a scoring system developed by mittelholzer et al. ( ) and adapted to prrs experiments was applied. all pigs were evaluated for overall well-being, respiration, eye disorders and appetite. each parameter was scored as (normal condition), (mild disorder), (moderate disorder) or (severe disorder). the scores for individual pigs were added up to a cumulative clinical score (cs) per day. non-stabilized blood samples were collected from the anterior vena cava in ml vacutainers (venoject; terumo europe, leuven, belgium) on − , , , , , , dpi and at euthanasia. serum was isolated (left to coagulate for min and centrifuged at rpm for min at °c) and stored in aliquots at − °c for subsequent real time rt-pcr analysis, or at − °c for antibody and acute phase protein (app) measurements. in parallel to the blood samplings, nasal swab samples were collected on the same days from right nostrils. the swabs were placed in ml pbs (ph . ) and stored at − °c until further analysis with real time rt-pcr. euthanasia of the majority of the pigs was performed on dpi (pigs , , , and ), dpi (pigs , , , , , ) , or dpi (pigs , , , , , ) by intravenous injection of pentobarbiturate ( mg/kg) followed by exsanguination by cutting arteria axillaris. in order to get additional information on the dissemination of virus in individual pigs during the course of infection, pigs representing the various groups (pigs , , , and ) were euthanized on dpi. at necropsy, all pigs were subjected to macroscopic evaluation of respiratory tract lesions, and lung tissue samples were collected for real time rt-pcr and histopathological analysis. lung sections for real time rt-pcr were stored in rnalater according to instruction from the supplier (qiagen) and sections for histopathological evaluation were fixed in % buffered formalin. total rna was extracted from μl serum and nasal swabs in pbs with qiasymphony rna kit automated on qiasymphony sp extraction robot with the protocol ct v , according to instructions provided by the supplier (qiagen, denmark). lung tissue samples stored in rnalater (qiagen) were initially prepared as a % homogenates in rlt buffer (qiagen) containing % β-mercaptoethanol (sigma-aldrich) by homogenization on tissuelyser ii (qiagen, denmark) at hz for min and clarified by centrifugation for min at , rpm. total rna was extracted from μl lung tissue homogenate using rneasy mini kit (qiagen) with the large sample protocol v automated on the qiacube (qiagen) according to instructions from the supplier. the rna was stored at − °c until use. in order to quantify prrsv load in sera, nasal swabs pcr primers and a probe were designed based on orf sequences of , ili and bor . the sequences of the primers and the probe were as follows: fw: ′-ttygggttcachgtcgcag- ′; rev: ′-gaccttcgatarttcgggag- ′; probe: ′-fam-cagagcgcgaacggagaakcgcg-bhq - ′ and were synthesized by eurofins genomics (germany). the pcr reactions contained nm of each primer and nm probe, x qiagen onestep rt-pcr buffer, . mm dntp each nucleotide, μl qiagen onestep rt-pcr enzyme mix and μl rna in a total volume of μl. amplifications were performed on rotor-gene q (qiagen ® ) with the following temperature profile: min at °c for reverse transcription followed by min at °c, and cycles of s at °c, s at °c and s of °c. fluorescent signals were collected during the extension step at each cycle in the green channel and analyzed with rotor-gene q software version . . (qiagen) setting ntc threshold at % and the normalized fluorescence threshold limit at . for cycle threshold (ct) value determination, starting normalization from cycle . samples were tested in duplicates and were considered positive when both replicates had ct below . the efficiency of pcr amplification of all three prrsv strains was between and %. quantification of viral load in experimental samples was performed against a standard curve constructed from a -fold dilution series of . log tcid /ml of the strain. the standard curve had a pcr efficiency of % (r = . ; slope − . ) covering dilution steps corresponding to − . e+ tcid /ml equivalents (equal to . - . log tcid equivalents). antibodies in sera from all pigs were analysed using a prrsv genotype discriminating immunoperoxidase monolayer assay (ipma) (sorensen et al., ) and a genotype discriminating blocking elisa (sorensen et al., ) . for ipma, the serum was initially diluted : and then tested using a fivefold dilution series ( : - : ). the results were expressed as the highest dilution generating a positive signal (titre). in elisa, the serum samples were tested diluted : and the results were expressed as blocking percentage (od%). a sample was considered positive if the od% was below . the concentration of haptoglobin (hp) in serum was determined by a sandwich elisa using an in-house mouse anti-porcine hp monoclonal antibody as catching antibody and a commercial rabbit anti-human hp detection antibody (dako) as previously described (sorensen et al., ) . the serum concentration of c-reactive protein (crp) was analysed by a sandwich elisa using dendrimer-coupled cytidine diphosphocholine (a crp-binding ligand) in the coating layer as described by heegaard et al. ( ) employing polyclonal rabbit antihuman antibodies with cross-reactivity towards porcine crp (dako) followed by peroxidase-conjugated goat anti rabbit antibody for detection (dako). lung sections fixed in % buffered formalin were dehydrated through a graded ethanol and xylene baths and embedded in paraffin wax. sections of - μm were stained with haematoxylin and eosin (he) and microscopic lesions were scored as , no lesion; , mild; , moderate or , severe (table ). the evaluation included interstitial pneumonia infiltration of eosinophils, and hyperplasia of lymphoid follicles. interstitial changes were evaluated at magnification x (objective lens) and x (eyepiece). infiltration of eosinophils was assessed by the average number of cells observed in different highpower fields (hpf), using a magnification of x (objective lens) and x (eyepiece) on the area of about . mm (area of one field of view). hyperplasia of lymphoid follicles in lung parenchyma and balt was observed at magnification of x (objective lens) and x (eyepiece) in one field of view. only visible and activated lymphoid follicles were counted. the details of scoring are presented in table . the microscopic evaluation was performed in a blinded fashion using a standard light microscope olympus bx and cellsens software (olympus). statistical analysis to compare mean viremia, nasal shedding and acute phase proteins concentration between groups was performed at each sampling point using a one-way anova followed by post-hoc tukey's test. if the assumptions of normality or equality of variances were not fulfilled (evaluated by shapiro-wilk and levene's test respectively) non-parametric kruskal-wallis anova test was applied. clinical scores and microscopic lesions scores were compared based on kruskal-wallis anova test. calculations were performed with statistica (statsoft) software. differences were considered statistically significant at p < . . analysis of body temperature was performed using graphpad in stat version . (graphpad software, san diego, ca). student's t-test was used for comparison between means of the infected groups and the control group. no clinical signs were observed in the uninfected control pigs. in groups and , pigs showed mild lethargy on a few days, only (dpi , and , respectively) reflected by cs values of . all pigs in group exhibited varying degrees of clinical signs characterized by mild lethargy, increased respiratory rate, conjunctival hyperaemia and reduced feed-intake. starting from dpi , cumulative cs values of to were observed for an day period with highest scores ( - ) observed in pigs, all infected with bor . between dpi and , cumulative cs in group was significantly higher compared to other groups (p < . ). body temperatures (bt) of all pigs remained within the range of . °c to . °c on dpi - - , and bt did not exceed . °c in any of the controls post inoculation. thus, bt higher than . °c were considered to represent fever. in group , only pig had fever of . °c on dpi. pigs from groups and exhibited significantly elevated mean bt on dpi compared to pigs from group and (p < . ) (fig. ) . hereafter, the mean bt of group was higher than the controls but not statistically significant at any time point. in group , fever with bt ranging between . °c and . °c was recorded in individual pigs for to days in the period from to dpi. two pigs ( and ) in group did not show elevated bt. in group , all pigs had fever of . °c to . °c between dpi and dpi. the duration of fever period was to days for pigs ( and ) and to days for pigs ( , , , ) with the highest bt recorded for the latter pigs. the mean bt for group pigs were significantly higher compared to the controls from dpi to , the highest difference seen on dpi (p < . ) prrsv was detected in serum in all infected pigs at dpi and the viremia persisted until dpi. all pigs from groups and and one pig from group had low level of viremia at dpi (fig. ) . viremia was significantly higher in group infected with bor , at dpi and dpi, when in pigs and it reached about × tcid genomic copy equivalent/ml. on dpi viremia in groups and was significantly higher than in group (p < . ). no viremia was detected in group . prrsv could not be detected in nasal swab samples from any of the control pigs but in most pigs in the infected groups at , and dpi (not shown). at dpi only out of pigs from group had virus positive nasal swab samples compared to and pigs in groups and , respectively. in pig from group virus was detected also at and dpi. there were no significant differences between the groups in the virus detection levels in nasal swabs, the highest observed at and dpi. prrsv was detected in lungs from all inoculated pigs tested with varying ct-values of . ± . (group ), . ± . (group ) and . ± . (group ). however, the differences between the groups were not significant. prrsv could not be detected in lung tissue samples from control animals. all animals seroconverted at - dpi in both the elisa and ipma tests. no differences were seen in the serological profile between the three different strains used for challenge (data not shown). an acute phase protein crp response was observed as early as at dpi, peaking at dpi , in all prrsv infected groups (fig. a) . globally, during the study the mean crp concentration ranged from . μg/ml ( ± . ) to . μg/ml ( ± . ). bor infected pigs showed earliest increase in crp levels. in ili infected pigs, the increase of crp level was slower and peaked at slightly higher level than in bor infected pigs. hp levels ranged from to . mg/ml ( ± . ). an remarkably early response at dpi only in bor infected pigs (p < . ) (fig. b) . no gross lesions of the lungs were observed during necropsy at , , or dpi. microscopically, varying levels of lung lesions were observed in all pigs except one control animal ( table ). the lesions included proliferation of pneumocytes type ii, mononuclear inflammatory cell infiltration (mainly lymphocytes and macrophages) and thickening of alveolar septa. in addition, areas of fibroblast proliferation and fibrosis were observed in lung parenchyma ("honeycomb" pattern). the most affected were lungs of pigs (necropsied at dpi), and (necropsied at dpi), infected with the strain bor , where fibrosis and cellular infiltration were particularly severe, and alveolar lung structure was completely lost. similar severity was observed in pig (necropsied at dpi) infected with the ili strain. fig. . body temperature in pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. statistically significant difference at dpi between groups , and groups , as well as differences between group and remaining groups between dpi - were marked with asterisk (p < . ). fig. . viremia in pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. different superscripts denote significant statistical differences (p < . ). t. stadejek et al. veterinary microbiology ( ) - greater differences existed between the experimental groups in regard to eosinophil infiltration, where again the lungs from the bor infected pigs , and , as well as the ili pig were most affected. eosinophils accumulated around vessels and in the areas with severe fibrosis of the lung parenchyma. occasionally, degranulated eosinophils were also visible. also, a small amount of eosinophils was observed in healthy pigs. however, they were evenly distributed and degranulation was not observed. hyperplasia of lymphoid follicles was higher in bor infected pigs than in and ili infected pigs. the highest score was noted in lungs from bor infected pigs , and , where more than activated lymphoid follicles were observed. in particular, numerous lymphoid follicles were observed in lungs with a high degree of fibrosis and severe cellular infiltration. the most severe interstitial pneumonia, eosinophil infiltration and hyperplasia of lymphoid follicles were observed in the bor infected pigs and that were necropsied at dpi. in the present study, we compared clinical signs, virological parameters, seroconversion, app response and histopathological lesions in pigs infected with either the danish strain from subtype , the russian strain ili from subtype , and the belarusian strain bor from subtype , all belonging to prrsv- . the results indicated that bor strain can be considered a highly virulent strain, similarly to subtype strains lena and su -bel (karniychuk et al., ; morgan et al., ; weesendorp et al., a weesendorp et al., , . the infection with the danish strain did not cause significant clinical signs which is in agreement with the results of earlier studies on prrsv- subtype strains from the netherlands, united kingdom, belgium or korea (frydas et al., ; han et al., han et al., , karniychuk et al., ; morgan et al., ; nielsen and botner, ; weesendorp et al., a) . the mild clinical signs combined with the slightly elevated body temperatures including fever for a few days in pigs infected with the russian ili strain indicate that this is more virulent than , but less virulent than bor , since the pigs infected with the latter strain exhibited more clinical signs, significantly higher body temperatures and more days with fever, together with earlier and higher level of viremia. clinical symptoms of prrsv infection in the field depend on a variety of modifying factors such as differences in genetic susceptibility, environmental factors, immune status, management, virus strain differences or coinfections with other pathogens (zimmerman et al., ) . . concentration of c-reactive protein (a) and haptoglobin (b) in serum from pigs infected with prrsv- strain (group ), strain ili (group ) and strain bor (group ), and uninfected control pigs (group ). data are expressed as mean ± sd. different superscripts denote significant differences (p < . ). t. stadejek et al. veterinary microbiology ( ) - the generally mild clinical disease observed in the present study likely resulted from the use of pigs with a very high sanitary status thus avoiding exaggeration of secondary infections contributing to more overt clinical signs observed in conventional pigs. viremia is one of the measures of prrsv virulence and immediately easier to compare between experiments than clinical signs. in the present study, using quantitative real time rt-pcr, viremia was detected in all challenge groups from dpi to the end of sampling at dpi. at this stage, all pigs infected with and bor were viremic compared to only one pig infected with ili strain. the significantly higher level of viremia detected in pigs infected with bor at and dpi supports the higher virulence of this strain. however, the occurrence of viremia was analysed on predetermined days only, and as such the picture of viremia may not represent its complete dynamics. the analysis of app response performed in the present study provided an important insight into one of the less studied aspects of prrsv infection. apps are part of the systemic acute phase response and important components of the innate immune system. the concentration of app in serum is altered in animals subjected to inflammation, infection or stress. in swine, hp and crp are the main apps. hp is a major antioxidant protective agent mediating removal of free hemoglobin (alayash et al., ) . crp participates in the systemic response to inflammation. its plasma concentration increases during inflammatory states, within hours after tissue injury or infection (black et al., ) . the present work showed that the level of app response can be variable towards different prrsv- strains, likely reflecting the differences in their virulence. similar differences in apps profiles displayed by four prrsv- , subtype strains were recently described by saco et al. ( ) who identified strong correlation between app (in particular hp) concentration in serum and the severity of clinical signs. clinical scores in pigs exposed to eu- prrsv- strain inducing highest hp response, and to ja prrsv- strain inducing highest crp response, where highest (saco et al., ) . in our study, bor infected pigs showed the earliest increase in crp levels but both bor and ili infected pigs had significantly higher levels of crp at dpi compared to pigs infected with the strain. striking differences were observed in hp levels where bor pigs showed an unusual early response at dpi while the response in other groups was absent or minimal (fig. ) . it corresponds well with the previously reported greater and more common responsiveness of crp than hp towards prrsv infection (heegaard et al., ; saco et al., ) . this observation indicates that bor infection is able to stimulate a rapid hp response, possibly by activating neutrophils to release pre-stored hp. such a mechanism has been described for hp (theilgaard-monch et al., ) but not for crp. this finding is another indication that bor is more virulent than ili (and ). it was proposed that the variability in clinical and app response may be related to the different abilities of different prrsv isolates to impact interferon and other cytokines production. in prrsv infections, increased il- levels have been described, while the data on il- β and tnf-α are conflicting (borghetti et al., ; darwich et al., ; weesendorp et al., b) . taking into account that the synthesis of apps is mainly driven by these three pro-inflammatory cytokines, it is expected that more virulent isolates stimulating higher levels of cytokines will also induce stronger acute phase response and more severe clinical symptoms, as in the case of bor , and to a lesser extent ili . results of pathology evaluation were limited in this study as pigs were only necropsied at , , or dpi, rather late in the course of infection at stages when advanced recovery from clinical disease was observed. nevertheless, despite wide range of lesions in each of the experimental groups, bor infected pigs (especially those necropsied at dpi) had generally most evident microscopic lung lesions. as with clinical signs, the lack of gross lesions together with the minor microscopic lesions could result from lack of co-infections in the used pigs of very high sanitary status. the role of eosinophils in lung pathology remains unclear, but the long term consequences of eosinophil activation can result in increased pathological changes in the lungs. these cells produce eosinophilic cationic protein (ecp), which can damage the rna virus, they are also able to present antigens to other cells and capable of phagocytosis and cytotoxicity (giembycz and lindsay, ; gleich et al., ; jacobsen et al., ) . on the other hand, eosinophils may modify inflammation in the lungs through cytokines release and they can also promote fibrosis of the lungs by producing factors participating in transformation of fibroblasts into fibrocytes and myofibrocytes (akuthota et al., ; giembycz and lindsay, ; jacobsen et al., ) . in general, the amount of eosinophils in lung tissues was small but highest in pigs infected with bor strain. in summary, our results suggest that the prrsv- strain bor is more virulent than ili (and ), showing a more rapid and more quickly developing infection, as also supported by the detected early crp and hp responses. the results indicate that the strain bor can be considered a high pathogenic prrsv- virus, similarly to previously evaluated belarusian lena and su -bel (karniychuk et al., ; morgan et al., ) . pathogenicity of bor for conventional pigs, as well as a deep genetic characterization of this strain, remains to be assessed. more efforts should be made in europe and elsewhere to study complete genomes of prrsv- and to study the genetic characteristics of unusually virulent strains, especially from eastern european genetic subtypes. assistance. likewise, bent eriksen and co-workers are acknowledged for taking care of the animals. eosinophils as antigen-presenting cells in allergic upper airway disease haptoglobin: the hemoglobin detoxifier in plasma c-reactive protein cytokine expression, glucocorticoid and growth hormone changes after porcine reproductive and respiratory syndrome virus (prrsv- ) infection in vaccinated and unvaccinated naturally exposed pigs isolation of porcine reproductive and respiratory syndrome (prrs) virus in a danish swine herd and experimentalinfection of pregnant gilts with the virus genomic evolution of porcine reproductive and respiratory syndrome virus (prrsv) isolates revealed by deep sequencing certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology family arteriviridae the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe divergence time of porcine reproductive and respiratory syndrome virus subtypes phylodynamic analysis of porcine reproductive and respiratory syndrome virus (prrsv) in italy: action of selective pressures and interactions between different clades different clinical, virological, serological and tissue tropism outcomes of two new and one old belgian type subtype porcine reproductive and respiratory virus (prrsv) isolates pharmacology of the eosinophil biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease pathogenesis of korean type (european genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs comparative pathogenicity of three korean and one lelystad type porcine reproductive and respiratory syndrome virus (pan-european subtype ) isolates in experimentally infected pigs a robust quantitative solid phase immunoassay for the acute phase protein c-reactive protein (crp) based on cytidine '-diphosphocholine coupled dendrimers optimal combinations of acute phase proteins for detecting infectious disease in pigs eosinophils: singularly destructive effector cells or purveyors of immunoregulation pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate molecular variation in the nucleoprotein gene (orf ) of the porcine reproductive and respiratory syndrome virus (prrsv) re-emerging of porcine respiratory and reproductive syndrome virus (lineage ) and increased pathogenicity after genomic recombination with vaccine variant analysis of orf and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes and retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance pathology and virus distribution in the lung and lymphoid tissues of pigs experimentally inoculated with three distinct type prrs virus isolates of varying pathogenicity a bayesian phylogeographical analysis of type porcine reproductive and respiratory syndrome virus (prrsv) hematological and immunological parameters of / -month old pigs infected with prrs virus c-reactive protein, haptoglobin and pig-major acute phase protein profiles of pigs infected experimentally by different isolates of porcine reproductive and respiratory syndrome virus host-pathogen interactions during porcine reproductive and respiratory syndrome virus infection of piglets molecular epidemiology of prrsv: a phylogenetic perspective evaluation of a blocking elisa for screening of antibodies against porcine reproductive and respiratory syndrome (prrs) virus blocking elisa's for the distinction between antibodies against european and american strains of porcine reproductive and respiratory syndrome virus the porcine acute phase protein response to acute clinical and subclinical experimental infection with streptococcus suis identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe molecular evolution of prrsv in europe: current state of play phylogenetic relationships of european strains of porcine reproductive and respiratory syndrome virus (prrsv) inferred from dna sequences of putative orf- and orf- genes haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation comparative analysis of immune responses following experimental infection of pigs with european porcine reproductive and respiratory syndrome virus strains of differing virulence phenotypic modulation and cytokine profiles of antigen presenting cells by european subtype and porcine reproductive and respiratory syndrome virus strains in vitro and in vivo lung pathogenicity of european genotype strain porcine reproductive and respiratory syndrome virus (prrsv) differs from that of subtype strains importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome virus (porcine arterivirus) the research leading to these results has received funding from the european community's seventh framework programme (fp , (fp , - key: cord- -e rhioty authors: rowland, raymond r.r. title: the interaction between prrsv and the late gestation pig fetus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: e rhioty porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta during late gestation and productively infects the fetus. virus replication and cytokine responses were measured in tissues of fetuses recovered at – days of gestation, just prior to parturition. at the time of recovery, gross anatomical abnormalities were evident in both infected and non-infected fetuses from the infected dams. virus isolation and immunohistochemistry identified the thymus as the primary site of virus replication. steady state rt-pcr amplification of inflammatory, th and th cytokines, showed elevated ifn-γ and tnf-α mrnas in tissues from infected fetuses, which corresponded to elevated cytokine proteins in serum but not amniotic fluid. further evidence for induction of immunity was found in the hyperplastic response of lymph nodes, which included the development of germinal centers occupied cdw + b cells. collectively, these data support the notion that the immunocompetent fetus is capable of initiating an antiviral response, which is compartmentalized within the infected fetus. furthermore, fetal pathology may not be a direct result of virus replication in the fetus. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus, prrsv, belonging to the family arteriviridae cavanagh, ; nelsen et al., ; wensvoort et al., ) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, ) . the arteriviruses, toroviruses, roniviruses and coronaviruses form a single order, nidovirales. arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (reviewed in snijder and mulenberg, ) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic infec-tion, as well as cause severe and fatal disease (plagemann, ) . infection of adult pigs with prrsv usually produces a non-fatal disease, characterized by flu-like symptoms, a transient elevation in temperature and inappetance (reviewed in benfield et al., ; christianson et al., ) . the reproductive form of prrs occurs following the infection of late gestation pregnant gilts or sows. natural infection of the fetus with prrsv is initiated with the infection of gilts and sows at days gestation. after productive replication on the maternal side, the virus crosses the placenta and productively infects the fetus. the mechanism of transplacental infection is unknown, but could be similar to the infected "trojan horse" macrophage, described for ldv (cafruny and bradley, ) . since the pig fetus becomes immunocompetent at about days of gestation, prrsv infection occurs in an immune environment containing functional b and t cells. accordingly, virus-induced reproductive failure can present clinically as delayed returns to estrus, as well as abortions, mummified fetuses, stillborn and weak-born pigs christianson et al., ; collins et al., ; mengeling et al., ; rossow et al., ; rowland et al., ) . surviving neonates can exhibit the severest form of respiratory disease with mortality sometimes reaching % within three weeks after birth (feng et al., ; rossow et al., ; rossow, ) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs (rowland et al., ) . the purpose of this study was to characterize the interaction between prrsv and the pig fetus by ( ) identifying sites of virus replication, ( ) measuring immune and inflammatory cytokines in different compartments, and ( ) evaluating the response of lymph nodes. experiments involving animals were approved by the kansas state university iacu committee. pregnant sows, obtained from a closely monitored prrsv-negative herd, were challenged at days gestation with a sixth passage isolate of sd- , a typical north american field isolate (rowland et al., ) . the methods for the preparation of the prrsv inoculum on marc- cells and infection of pigs are described in rowland et al. ( ) . virus was cultivated on marc- cells in mem supplemented with antibiotics (pen/step) and % fbs. dams, at days gestation were challenged with approximately tcid of virus diluted in ml of culture medium. one half of the inoculum was administered by intramuscular injection in the neck. the remaining dose was administered intranasally. mock-infected sows were challenged with medium recovered from marc- cells. dams were monitored for clinical signs and blood collected weekly. at between and days of an approximate days gestation period, the dams were euthanized. the uterine horns were immediately removed and the individual fetuses with intact placenta were carefully removed and immediately necropsied. a sample of amniotic fluid was collected prior to removal. the brachial artery of each fetus was severed and blood collected using a disposable syringe and serum stored at − • c. maternal, accessory and fetal tissues were collected and stored in formalin for histological staining and immunohistochemistry (ihc), or storage in rnalater (ambion) for rt-pcr of cytokine mrnas. prrsv-specific antibody was measured in sera using the herdcheck ® prrs elisa (idexx) and performed by personnel at kansas state university veterinary diagnostic laboratory. serology results were reported as a sample/positive (s/p) ratio. an s/p ratio greater than . was considered positive for prrsv antibody. virus isolation (vi) in serum and tissues was performed as described in rowland et al. ( ) . briefly, serum was serially diluted in mem supplemented with pen/step antibiotics and % fbs and placed on well plates of confluent marc- cells. after three days, plates were fixed in % acetone and stained with fitc-sdow- anti-nucleocapsid antibody, diluted in pbs with % fbs (nelson et al., ) . the results were reported as the log of the inverse dilution of the last positive well. virus isolation from tissues was the same except that tissues were weighed and homogenized in hanks balanced salt solution and then centrifuged at × g for min to remove debris. the sequencing of the hypervariable region of orf is described in rowland et al. ( ) . total rna was prepared from serum or infected marc- cells using an rneasy kit (qiagen) according manufacturer's instructions. for pcr, cdna was prepared using mlv reverse transcriptase (promega) and msb as the primer. the sense and antisense primers for the outer amplification were msa, -cttcgtcccttcttttcctcgtgg, and msb, -ccgctctagagccaacgatagagtctgc, respectively. the product was re-amplified with a nested set of sense and antisense primers, a, -accgtgtatgttaccatcacagcc and b, acgggaaagatgacaaaactctcc. thirty-two cycles of amplification were performed for each primer pair. the conditions for both amplifications included a • c denaturing step ( s), a • c annealing step ( s), and a • c ( s) polymerization step. the final pcr product, which contained the last nucleotides of orf , the nucleotide untranslated region (utr), and the first nucleotides of orf was sequenced directly by automated dna sequencing. pcr products were cloned into a pcr . ta cloning vector (invitrogen), propagated in escherichia coli and individual plasmids sequenced using m forward and reverse primers. sequences were analyzed using gene jockey ii software. tissue samples for rt-pcr were immediately placed in rna-later (ambion) and stored at − • c. total rna was extracted from approximately g of tissue using rneasy kit (qiagen) according to manufacturer's instructions. the design of cytokinespecific primers and rt-pcr procedures were performed according to reddy and wilkie ( ) . primer sequences are listed in table . rna was diluted to a final volume of l in nuclease free water. cdna was prepared from l of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase (promega) and random hexamers as primers. the amplification of ␤ m mrna was used as an internal control. pcr amplification of cytokine and control cdnas consisted of cycles ( s at • c, s at • c, and s at • c) and dna products electrophoresed on a . % agarose gel and visualized using ethidium bromide. the identity of the dna products was confirmed by dna sequencing. tissue samples were collected and immediately placed in % buffered formalin. paraffin-embedded thin sections were mounted on slides, deparaffinized and stained with hematoxylin for the detection of prrsv antigen, slides were incubated for min with a : dilution of mab sr- anti-nucleocapsid antibody (rural technologies). other antibodies included a polyclonal anti-human cd and b cell antibodies, anti-cdw and anti-cd ␣. bound antibody was detected with biotinylated goat anti-mouse or anti-rabbit ig followed by avidin-hrpo and dab chromagen (ventana medical). slides were counterstained with hematoxylin. the experiment incorporated four prrsv-infected and twomock-infected dams. all maternal serum samples were vi-negative and seronegative for prrsv prior to infection. between one and two weeks after virus challenge, all infected sows were vi-positive in serum, confirming the presence of an active infection. by the time of necropsy, the concentration of circulating virus in the dams had dipped to below detectable levels by vi. a total of viable fetuses were recovered from the four infected dams (see table for summary). two fetuses were dead and partially autolysed and not subjected to further study. ( %) of the viable fetuses were positive for prrsv by vi. since dams were vi-negative in blood at the time of necropsy, it was concluded that the presence of virus serology results, shown in parentheses, are presented as the s/p ratio. s/p ratios greater than . were considered positive for prrsv antibody. dead fetuses were partially autolysed and not tested. gross pathology key: * , partially mummified; * , non-viable fetus or relatively low quantity of amniotic fluid; * , necrotic placenta; * , merconium-and/or blood-stained amniotic fluid; * , small, underdeveloped fetus. in the fetal circulation was the result of virus replication in the fetus and not from contamination with maternal blood. (the virus isolation technique is not subject to false positives from minute amounts of cross-contamination with viral protein or rna.) the number of infected fetuses in each litter varied from no infected fetuses (dam no. ) to five of ( %) infected fetuses for dam no. . fetuses that were vi-negative in serum were confirmed as prrsv-negative by vi-negative results in placenta, lung, lymph nodes and thymus (data not shown). one fetus, - , was seropositive for prrsv (s/p ratio = . ). since there is no maternal transfer of antibody from mother to fetus (tizard, ) , it was concluded that this fetus generated an antibody response to prrsv in utero. of the infected fetuses showed some form of gross pathology, including growth retardation (two fetuses) or reduced amounts and/or merconium-stained amniotic fluid (five fetuses). ongoing virus replication in the fetus as the source of these gross pathological changes is questionable, since non-infected fetuses from infected dams exhibited similar changes. for example, fetuses from dam no. (see fig. ) were either merconium stained (two fetuses), non-viable, possessed reduced amniotic fluid levels (four fetuses) or small (one fetus). except for one autolysed fetus, the fetuses from the two control dams showed no evidence of gross pathology (data not shown). rna viruses frequently exist as a heterogeneous population, frequently referred to as a quasispecies. the appearance or disappearance of individual viral sequences within a quasispecies population is often used as evidence to support the existence of positive or negative selection during infection (elena et al., ; forns et al., ; tsibris et al., ) . mutations that appear in the prrsv genome are useful as markers to identify and follow the appearance and disappearance of viruses within the population (allende et al., ; rowland et al., ) . dna sequencing of orf pcr products, amplified directly from the sera of infected dams, table frequency of t at nucleotide position of orf . consensus nucleotide at position of orf t:c ratio in cloned pcr products (percent) t / ( ) fetus - t / ( ) fetus - t / ( ) fetus - c / ( ) fetus - t / ( ) fetus - t / ( ) identified one dam, no. , which possessed a virus with a mutation within the hypervariable region of orf . the change detected by sequencing the pcr product was a c to t (u for rna) nucleotide transition at position that resulted in a non-conserved amino acid change from threonine to isoleucine in gp . the t- mutation was not detected after sequencing the orf pcr products from the other dams (see table ). pcr products were cloned into a ta plasmid and the individual plasmids sequenced. even though t- was detected in the pcr product from dam no. , the sequence of individual clones showed that two of the five sequences possessed a c at position mutation, which indicated that viruses with the wild-type orf sequence were still present in the population. whole pcr and plasmid-cloned pcr products were sequenced for the five infected fetuses from dam no. . the frequency of the t- mutation ranged from % (fetus - ) to % (fetuses - and - ). these results indicate that the fetus is capable of selecting for a particular virus population, which either arises in the dam or fetus. therefore, fetal infection is a potential source of prrsv diversity. during acute infection of the postnatal pig, the largest quantity of virus and greatest number of cells supporting virus replication are found in the lung, a consequence of targeting alveolar macrophages. during the later stages of prrsv infection, secondary lymphoid organs, including tonsil and lymph nodes, become sources of virus replication (allende et al., ; rossow, ; rowland et al., ; . virus replication in fetal tissues was assessed using a combination of virus isolation and ihc detection of nucleocapsid antigen in formalin-fixed tissues. as summarized in table , virus was isolated from all tissues from infected fetuses, including placenta, umbilical cord, heart, lung, spleen, lymph nodes and thymus. overall, the thymus contained the largest quantity of virus. nine of ten fetuses yielded measurable amounts of virus with five of the ten fetal thymuses producing titers greater than . . for lung, of fetuses were vi-positive and only one fetal lung yielded a virus titer greater than . . the recovery of virus from a tissue can represent virus in circulating blood. therefore, to determine if cells in the thymus and other tissues were a source of prrsv, tissue thin sections were stained with prrsv anti-nucleocapsid antibody. the ihc staining procedure included two sets of negative controls: tissue thin sections from non-infected fetuses and from infected fetuses stained with only secondary antibody. both controls were negative for staining (data not shown). the results in table showed the largest number of positive tissues for the thymus ( of positive), followed by spleen ( of positive) and lymph node ( of positive). within the thymus, antigen-positive cells were located in both medullar and cortical regions (data not shown). prrsv antigen-positive cells were not detected in lung or tonsil. taken together, the virus titration and ihc results identify the thymus as a principal source of virus replication in the prrsvinfected fetus. in the post-natal pig, prrsv infection results in distinct pathology in the lung, including the appearance of interstitial pneumonia. representative lymph node and lung tissues from non-infected and infected fetuses are shown in fig. . there was no discernable difference between lungs from infected and non-infected fetuses (compare fig. panels c and d) . lymph nodes from prrsv-negative fetuses appeared largely undeveloped and devoid of well-defined germinal centers. in contrast, the lymph nodes from prrsv-infected fetuses appeared much more pronounced and enlarged. at the microscopic level, the increased lymph node volume was associated with increased numbers of cells and the formation of distinct germinal centers (see fig. panels a and b) . the overall appearance is consistent with an antigen-activated lymph node. to determine the source of the increased cell volume, formalin-fixed thin sections of lymph nodes were stained with t cell-specific (cd ) and b cell-specific (cdw and cd ␣) antibodies. representative results for mandibular lymph nodes from infected and non-infected fetuses are shown in fig. . the lymph nodes from both infected and non-infected fetuses showed exten- sive areas of staining with anti-cd , indicating the presence of t cells. lymph nodes from non-infected fetuses were negative for cwd staining, but possessed some regions that were positive for cd ␣. the b cell receptor for antigen (bcr) signal transduction complex is composed of a heterodimer of ig␣ and ig␤ chains, which are also known as cd ␣ and cd ␤, respectively. cd ␣ staining in the lymph node from non-infected fetuses is consistent with the presence of pro-and pre-b cells (lee et al., ) . the principle difference between infected and non-infected fetuses was found in an overall increase in cd ␣+ cells as well as the appearance of cdw + cells, which were associated with germinal centers. cdw is beta-galactoside alpha- , -sialyltransferase, which is up-regulated in activated b cells (erikstein et al., ) . the absence of cdw staining in non-infected fetuses is consistent with the overall quiescent nature of the non-stimulated fetal immune system. the up-regulation of cdw after prrsv infection is consistent with b cell activation and the formation of mature germinal centers in response to infection. it should be noted that infected fetuses showed different degrees of staining, a likely consequence of the different stages of fetal infection; i.e. less staining was the result of early infection. together, the overall morphology and lymphocyte marker expression results indicate that the increased volume in the lymph nodes of infected fetuses is largely the result of increased numbers of mature activated b cells, which occupy germinal centers. immune cytokines are important factors in antiviral immunity and can influence the outcome of pregnancy (arck et al., ; basurko et al., ). the detection of cytokine gene expression in tissues was performed using a steady state rt-pcr procedure. rt-pcr was performed on rna isolated from four fetuses randomly chosen from the two mock-infected dams and four randomly selected prrsv-infected fetuses. the tissues selected for rt-pcr amplification were lung, lymph node and placenta. lung and lymph node represent sites cytokine alterations in the post-natal pig. placenta was selected as an accessory tissue located at the fetal maternal interface. amplification of mrnas included cytokines associated with inflammatory (il- , il- ), th (il- , ifn-␥, il- ) and th /regulatory (il- , il- ) responses. the amplification of ␤ m mrna was included as an internal control. the determination of a cytokine response was based on the presence or absence of a pcr product. il- , il- , il- and il- products were detected in tissues from both control and infected fetuses. because of the qualitative nature of the pcr method, it was not possible to accurately determine quantitative differences between control and infected fetuses; and therefore, these cytokines were not subjected to further study (data not shown). il- mrna was not detected in any of the selected tissues for control and infected fetuses. marked differences in expression were observed for tnf-␣ and ifn-␥ mrnas. the results for ifn-␥ and tnf-␣ from lung, mandibular lymph node and placenta are presented in fig. . the results for il- are also shown. the internal control mrna, ␤ m, was amplified from all tissues, indicating that the rna was intact. il- was amplified from lung and mandibular lymph nodes from two of the four control fetuses and from all infected fetuses. the presence of il- was not unexpected, since increased il- production is associated with fetal t cell responses (lin et al., ; rainsford and reen, ) . as shown in fig. a , ifn-␥ and tnf-␣ pcr products were not detected in lung, lymph node or placenta from the non-infected fetuses. however, ifn-␥ pcr products were obtained for lung and lymph nodes from infected fetuses. one infected fetus, - , yielded a faint ifn-␥ product for placenta. for infected fetuses, tnf-␣ mrna was amplified from lung, but not lymph node or placenta (fig. b) . to determine if cytokine gene expression was the direct result of prrsv infection, rt-pcr for ifn-␥ and tnf-␣ was performed on the same tissues from fetuses of infected dam no. , which produced only prrsv vi-negative fetuses (see fig. ). the analysis of mrna expression in lungs and lymph nodes from six fetuses from dam no. fig. . cytokine gene expression in fetal tissues. rt-pcr for cytokine mrnas was performed on lung (l), mandibular lymph node (n) and placenta (p) for control (panel a) and infected (panel b) fetuses as described in the text using the primers in table . amplification of ␤ -microglobulin mrna was used as an internal control. pcr products were electrophoresed on a . % agarose gel and stained with ethidium bromide. showed only the amplification of the ␤ m, but not tnf-␣ or ifn-␥ mrnas (data not shown). similar results were obtained from virusnegative fetuses located immediately adjacent to infected fetuses (data not shown). in order to confirm that tnf-␣ and ifn-␥ were produced during infection, cytokine protein levels were measured in fetal sera and amniotic fluid from infected fetuses and fetuses from mock-infected dams. as shown in fig. , the concentration of tnf-␣ in serum for prrsv-infected fetuses ranged between and pg/ml compared to less than pg/ml for control fetuses. even though the maximum quantities obtained for infected fetuses were near the lower limit of detection for the elisa test, it was clear that tnf-␣ was elevated during infection. detectable concentrations of tnf-␣ (> pg/ml) were found in amniotic fluid from only two control and two infected fetuses. compared to fetuses from mockinfected dams, ifn-␥ concentrations were detected in sera, with values ranging from a low of to more than pg/ml. a maximum level of pg/ml was obtained for a single non-infected fetus. ifn-␥ was not detected in amniotic fluid from either the infected or control fetuses. the presence of ifn-␥ and tnf-␣ in serum, but not amniotic fluid, further supports the notion that the ifn-␥ and tnf-␣ responses are primarily restricted to the prrsv-infected fetus and do not extend to the accessory tissue compartments. this study characterizes the unique biology associated with the interaction between prrsv and the late gestation fetus. consistent with the body of published literature, the fetuses from the infected dams obtained in this study exhibited several anatomic pathological features typical of prrsv infection of the fetus, including lesions associated with the accessory organs, such as umbilical cord and amniotic sac (lager and halbur, ; mengeling et al., ) . one interesting observation from this study was the apparent absence of a correlation between the presence of gross abnormalities and productive fetal infection. for example, several fetuses from dam no. exhibited several types of gross pathology, including death, growth retardation, or merconium/blood stained amniotic fluid. there were also examples of productively infected fetuses that showed no evidence of gross pathology (see fetuses - , - , - , - , - in fig. ). the mechanism for fetal pathology remains unclear, but the results suggest that the source pathology is likely result of the infection of tissues on the maternal side and damage to maternal tissues or production of maternal factors that affect the fetus. in this study we did not observe lesions in the myometrium or placenta; however, stockhofe-zurwieden et al. ( ) reported prrs virions budding from maternal vascular endothelial cells at the maternal-fetal interface. lager and halbur ( ) reported damage to the myometrium during prrsv infection. virus-associated lesions in the myometrium are observed for horses infected with eav (coignoul and cheville, ) . the appar-ent discrepancy between pathology and infection helps to explain the stealthy nature of the prrsv. a significant number of apparently healthy, but infected fetuses, are likely go on to become healthy growing pigs with the capacity to shed virus. rna viruses often exist as a population of closely related sequences, frequently referred to as a quasispecies. the appearance or disappearance of individual gene sequences in the population over the course of infection is used as evidence for changes in fitness that result from selection. prrsv variants with mutations in orf typically appear during the serial passage of virus in culture and during infection of pigs allende et al., ) . the significance of mutations in orf as a source for increased fitness during infection is not completely understood; but is useful for following the fate of individual prrsv subpopulations over the course of a long-term infection . in this study, one dam, no. , showed evidence of a mixed prrsv infection as indicated by the presence of two different orf sequences, which were distinguished from each other by a nucleotide substitution at position in orf . the c to t change was observed in approximately % of the cloned plasmid products obtained from dam no. (see table ). however, the same frequency was not transferred to the individual fetuses, which included two fetuses that possessed only viruses with the t- mutation and a single fetus with a virus population dominated by c at position . these results suggest that fetal infection can alter the selection of prrsv variants and may represent a source of prrsv genetic diversity. to identify the targets of virus replication in the fetus, a variety of tissues were assessed for the presence of virus and virus-infected cells. vi, as opposed to more sensitive approaches, such as pcr, was selected as the means for measuring virus, because the relative insensitivity of vi avoids the possibility of false positive pcr results that might result from small amounts of contaminating maternal material. within the group of selected tissues, virus could be isolated from all tissues. however, the most consistent source and largest quantity of virus were obtained from the thymus. collectively, these data identify the thymus as a primary site of virus replication in the prrsv-infected fetus and confirm an earlier observation for prrsv by benson et al. ( ) . the specific cell population in the thymus targeted prrsv replication remains unknown. the natural predisposition of maternal immunity towards th like responses aids in protecting the fetus by blocking cell-mediated th -related allorejection responses (arck et al., ; entrican, ; raghupathy, ; recently reviewed in challis et al., ) . the negative influence of th cytokines on fetal development can be demonstrated experimentally in mice, which show that a single injection of il- or ifn-␥ into certain strains induces fetal resorption (lala, ; chaouat et al., ) . tnf-␣, when infected into mice is abortifacient (clark et al., ) . th cytokines can also have important long-term impacts. for example, newborn mice that survive maternal infection with influenza virus exhibit behav-ior similar to hyperanxiety and autism. the behavioral changes are a consequence of the altered distribution of dopamine and glutamine receptors during fetal brain development. the effect of influenza virus infection on the fetal brain can be mimicked by the administration of poly i:c, an inducer of ifn (shi et al., ) . furthermore, the addition of ifn-␥ to hippocampal neuron cultures alters the distribution of glutamate receptors, sufficient to affect synaptic activity between neurons (vikman et al., ) . virus infections during pregnancy present an interesting paradox: those cytokines that protect the fetus from viral infection, tend to inhibit fetal development or potentiate rejection of the fetus; while those cytokines that maintain and promote fetal development are associated with the inhibition of antiviral immune responses. because of the potential negative impact of th cytokines on fetal outcome, there is a natural predisposition for the fetus to block the induction of th -associated cytokines. for example, the ifn-␥ response in the developing fetus can be blocked by a combination of factors, including ( ) defects in the capacity of fetal dendritic cells to express mhc class ii and synthesize il- , ( ) hypermethylation of the ifn-␥ gene in fetal t cells, ( ) the absence of target macrophages, and ( ) the presence of an immune environment dominated by th /regulatory cytokines (goriely et al., ; langrish et al., ; melvin et al., ; marodi et al., ; murphy et al., ; prescott et al., ; white et al., ,) . with this in mind, there are examples of viruses, such as epstein-barr virus (ebv), which are capable of stimulating antigen-specific ifn-␥ responses in human umbilical cord blood lymphocytes (ito et al., ; wilson and morgan, ) . these and other data provide a description of the fetus as capable of initiating a robust antiviral th immune response (chaouat et al., ; chipeta et al., ; murphy et al., ) . in this study ifn-␥ and tnf-␣ mrnas were identified in tissues from infected fetuses. rna message was not identified in tissues of fetuses from mock-infected dams or non-infected fetuses from infected dams. this indicates that altered gene expression is the direct result of fetal infection. up-regulated expression was confirmed by the presence of detectable levels of ifn-␥ and tnf-␣ proteins in serum. furthermore, the results indicate that inf-␣ and ifn-␥ cytokines response are compartmentalized within the fetus and do not extend to other compartments, such as amniotic fluid or placenta, thus lessening the probability of allorejection by the dam. from these data, it is apparent that the fetus is capable of initiating a th -like response; however, the capacity of ifn-␥ and tnf-␣ to control prrsv infection in the fetus is not known. additional evidence for induction of virus-specific immunity was found in the development of germinal centers containing activated (cdw +) b cells and seroconversion in at least one fetus. pro-inflammatory cytokines, such as tnf-␣ and ifn-␥ can contribute to pulmonary distress through the activation of alveolar macrophages and other cell populations. the increased quantities of ifn-␥ and tnf-␣ found in the lungs of prrsv-infected fetuses may not be sufficient to cause pulmonary damage to the fetal lung, primarily because fetal macrophages have a reduced capacity to respond to inflammatory stimuli. in addition, il- , up-regulated in response to prrsv infection, is a potent antagonist of ifn-␥ and tnf-␣ activation of macrophages (burchett et al., ; marodi et al., ; johnsen et al., ; thanawongnuwech et al., ) . however, within days after birth, adult macrophages, including mature alveolar and intravascular macrophages emerge into an environment already enriched in ifn-␥ and tnf-␣. the outcome is the rapid recruitment, activation, and cytopathic killing of large numbers of virus-permissive macrophages (choi and chae, ) . this scenario as a cause of severe interstitial pnuemonia in the prrsv-infected newborn requires further investigation, but has obvious implications in the etiology of postnatal pulmonary complications following virus infections of the fetus. porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection murine t cell determination of pregnancy outcome maternal and foetal consequences of dengue fever during pregnancy characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) porcine reproductive and respiratory syndrome virus a comparison of virus isolation, immunohistochemistry, fetal serology, and reverse-transcription polymerase chain reaction assay for identification of porcine reproductive and respiratory syndrome virus transplacental infection in the fetus diminished interferon-gamma and lymphocyte proliferation in neonatal and postpartum primary herpes simplex virus infection trojan horse macrophages: studies with the murine lactate dehydrogenase-elevating virus and implications for sexually transmitted virus infection nidovirales. a new order comprising coronaviridae and arterivirdae inflammation and pregnancy a brief review of recent data on some cytokine expressions at the materno-foetal interface which might challenge the classical th /th dichotomy control of fetal survival in cba × dba/ mice by lymphokine therapy neonatal (cord blood) t cells can competently raise type and immune responses upon polyclonal activation expression of tumour necrosis factor-alpha is associated with apoptosis in lungs of pigs experimentally infected with porcine reproductive and respiratory syndrome virus pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid gestation sows and fetuses experimental reproduction of swine infertility and respiratory syndrome in pregnant sows ecology of danger-dependent cytokine-boosted spontaneous abortion in the cba x dba/ mouse model. i. synergistic effect of lps and (tnf-alpha + ifn-gamma) on pregnancy loss pathology of maternal genital tract, placenta, and fetus in equine viral arteritis isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs the two faces of mutation: extinction and adaptation in rna viruses immune regulation during pregnancy and host-pathogen interactions in infectious abortion cell cycle-dependent regulation of cdw (betagalactoside alpha- , -sialyltransferase) on human b lymphocytes in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii quasispecies in viral persistence and pathogenesis of hepatitis c virus deficient il- (p ) gene expression by dendritic cells derived from neonatal monocytes changes in intracellular cytokine levels in newborn and adult lymphocytes induced by hsv- cytokine mrna profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of ifn-gamma and il- after viral clearance gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus interruption of murine pregnancy by activation of antigen-nonspecific killer cells in the endometrium with endomethacin, high dose il- , or a combination neonatal dendritic cells are intrinsically biased against th- immune responses molecular cloning and expression analysis of pig cd alpha synthesis of t helper -type cytokines at the maternal-fetal interface cytokine receptor signalling in neonatal macrophages: defective stat- phosphorylation in response to stimulation with ifn-gamma hypomethylation of the interferon-gamma gene correlates with its expression by primary t-lineage cells temporal characterization of transplacental infection of porcine fetuses with porcine reproductive and respiratory syndrome virus comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure interferon gamma in successful pregnancies porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents differentiation of united states and european isolates of porcine reproductive and respiratory syndrome (prrs) virus using monoclonal antibodies lactate dehydrogenase-elevating virus and related viruses transplacental priming of the human immune system to environmental allergens: universal skewing of initial t cell responses toward the th cytokine profile pregnancy: success and failure within the th /th /th paradigm interleukin , produced in abundance by human newborn t cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation quantitation of porcine cytokine and beta -microglobulin mrna expression by reverse transcription polymerase chain reaction porcine reproductive and respiratory syndrome lymph node lesions in neonatal pigs congenitally exposed to porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with aminopurine maternal influenza infection causes marked behavioral and pharmacological changes in the offspring the molecular biology of arteriviruses uterine and placental alterations in pregnant sows associated with the porcine epidemic abortion and respiratory syndrome (pears) immunohistochemical staining of ifn-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lung immunity in the fetus and newborn. in: veterinary immunology: an introduction, chapter quantitative deep sequencing reveals dynamic hiv- escape and large population shifts during ccr antagonist therapy in vivo interferongamma-induced changes in synaptic activity and ampa receptor clustering in hippocampal cultures differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd ro-t cells primary immune responses by cord blood cd (+) t cells and nk cells inhibit epstein-barr virus b-cell transformation in vitro this work was partially supported by the usda national research initiative for competitive grants program grants # - - . key: cord- -l si jpn authors: xu, ao-tian; zhou, yan-jun; li, guo-xin; yu, hai; yan, li-ping; tong, guang-zhi title: characterization of the biochemical properties and identification of amino acids forming the catalytic center of c-like proteinase of porcine reproductive and respiratory syndrome virus date: - - journal: biotechnol lett doi: . /s - - - sha: doc_id: cord_uid: l si jpn the non-structural protein (nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) functions as a c-like proteinase ( clpro) and plays a pivotal role in gene expression and replication. we have examined the biochemical properties of prrsv clpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-prrsv strategies. the c-like proteinase ( clpro) of porcine reproductive and respiratory syndrome virus (prrsv) was expressed in escherichia coli and characterized. the optimal temperature and ph for its proteolytic activity were °c and . , respectively. na(+) ( mm) and k(+) ( mm) were not inhibitory to its activity but cu( +), zn( +), pmsf and edta were significantly inhibitory. his( ), asp( ) and ser( ) residues were identified to form the catalytic triad of prrsv clpro by a series of site-directed mutagenesis analysis. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome (prrs) is an important disease throughout the world, leading to significant economic losses in pig production (neumann et al. ; tong et al. ) . prrs virus (prrsv) was identified as the etiological agent of the disease (thiel et al. ) . prrsv genome is a positive-strand rna of approximately , nucleotides, comprising open reading frames (orfs). orf is comprised of two large overlapping orfs, orf a and orf b (meulenberg et al. ; thiel et al. ) . orf a is translated directly whereas orf b is translated by a ribosomal frame shift, yielding a large orf ab polyprotein that is cleaved by virus-encoded proteinases into products related to the virus transcription and replication (allende et al. ) . after the autocatalytic release of nonstructural protein a (nsp a), nsp b and nsp , the remainder of the orf ab polyprotein is cleaved into at least nonstructural proteins (nsp - ) by nsp (ziebuhr et al. ) . prrsv nsp belongs to the c-like serine proteinases ( clsp), or c-like proteinase ( clpro). the predicted catalytic triad of prrsv clpro ao-tian xu and yan-jun zhou contributed equally to this study. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. (snijder et al. ) . the three-dimensional structure of prrsv clpro has recently been determined by x-ray crystallography (tian et al. ) , which makes it possible to identify amino acid residues on its active site by using site-directed mutagenesis assay. ascribing to its important role in the virus life circle, prrsv clpro has been suggested as a promising target for antiviral drug design (tian et al. ). the objectives of the present study were to characterize the biochemical properties of prrsv clpro and identify amino acid residues to be essential for maintaining its proteolytic activity. prrsv strain, cell culture and protein expression system prrsv hun strain (tong et al. ), marc- cells, pet- a (?) vector, e. coli dh a, and bl (de ) were used in the present study. viral rna was isolated from marc- cells infected with prrsv and reverse transcribed using oligo (dt) primer. the cdna encoding the clpro was amplified by pcr from the reversely transcribed mixture with the primer pairs cl-f/ cl-r, in that ndei and xhoi restriction sites were introduced at the -and -ends, respectively. the pcr product was placed downstream of the t promoter, and the resultant plasmid designated pt hispro. similarly, the cdna encoding the nsp that covered the last residues of nsp and the whole sequence of nsp was amplified with the primer pairs nsp -f/nsp -r, in that kpni and hindiii restriction sites were introduced at the -and -ends, respectively. the pcr product was cloned into the pet a vector, and the resultant plasmid was designated as pet a-nsp . the ser to tyr mutation of the clpro was introduced into pet a-nsp with the primer pairs ser ? tyr-f/ser ? tyr-r by a modified pcr-based site-directed mutagenesis method (fisher and pei ) , and the resultant plasmid was designated as pet a-nsp (ser to tyr). the primers used in the study are listed in supplementary table . expression and purification of prrsv clpro and pet a-nsp (ser to tyr) the plasmids pt hispro and pet a-nsp (ser to tyr) were transformed into e. coli bl (de ). the transformed cells were grown at °c until the concentration at a reached to . - . and then induced with iptg for h. the purification of recombinant proteins was performed using ni-nta column. several single amino acid substitutions were introduced into the plasmid pt hispro by a modified pcr-based site-directed mutagenesis method (fisher and pei ) . the primers used for site-directed mutagenesis are given in supplementary table . the pt hispro-derived plasmids with amino acid substitutions were transformed into e. coli bl (de ). these recombinant proteins were also purified using ni-nta column. the proteolytic enzyme ( lm) and substrate ( lm) were reacted in ll of mm tris/hcl buffer (ph . ) containing mm nacl for h at °c. details of the reaction mixtures are described in the respective sections. the reaction was stopped by the addition of a quarter volume of sample buffer. the proteins were analyzed by . % (v/v) sds-page. to estimate the efficiency of proteolytic cleavage, the densities of individual stained bands were scanned and calculated using bandscan version . . the data of cleavage efficiency were expressed as the average values of three replicate experiments. to express prrsv clpro, the plasmid pt hispro encoding the proteinase with his -tag at n-terminus was constructed (fig. ) . the clpro had a calculated molecular mass of . kda. to observe proteolysis at the cleavage site between the nsp and nsp , pet a-nsp (ser to tyr) mutant protein with calculated molecular mass of . kda) was used as the substrate, which included the n-terminal pet a vector leader fragment, and nsp fragment with the tyr substitution of predicted active site ser (fig. ) . although this protein had the clpro sequence, autocatalytic cleavage did not occur because of the ser to tyr mutation. if the substrate had been cleaved at the nsp and nsp junction (e/g) by the active clpro, pet a-nsp moiety of . kda and nsp (ser to tyr) moiety of . kda would be expected. optimal temperature and ph for proteolytic activity the proteolytic activity was optimal at °c ( fig. ) . at , , , , and °c, the proteolytic activity was measured as , , , , and % of that at °c, respectively. the proteolytic activity was maximal at ph . (fig. ) ; the activities were at ph . , . , . , . , . , . , . and . , and were , , , , , , , and % respectively, of that at ph . . these results indicated that the proteolytic enzyme had a relatively low tolerance for ph variation. to provide further evidence that the proteolytic activity that was observed was mediated by the clpro rather than by residual e. coli proteinase, the clpro with the mutation of ser to tyr was incubated with the substrate for h at the optimal temperature and ph. although the substrate contained the sequence recognized by wild-type clpro, it was not cleaved by the mutant proteinase (fig. , lane ), demonstrating that the proteolytic activity was mediated by prrsv clpro. moreover, the ser to tyr mutation caused a complete loss of fig. schematic representation of the prrsv genome and construction of expression plasmids. orf a and b polyproteins are predicted to be cleaved into nsp a, nsp b and nsp through nsp . the plasmid pt hispro was designed to encode prrsv clpro. the plasmid pet a-nsp was designed to cover the last residues of nsp and the whole sequence of nsp fig. effect of temperature on proteinase activity. the enzyme ( lm) and substrate ( lm) were incubated in mm tris/hcl (ph . ) buffer containing mm nacl for h at the indicated temperatures cleavage activity, confirming the importance of ser residue as a functional element. in addition, no cleavage activity was observed in other two control reactions without either the substrate or the enzyme (fig. , lane , ) . the enzyme was relatively insensitive to na ? between and mm. more than % activity was retained even at mm na ? (fig. a) . in contrast with this finding in prrsv, high concentrations of na ? strongly inhibited the proteolytic activity of clpros of the chiba strain of norovirus and equine arteritis virus (someya et al. ; van aken et al. ) . k ? ( mm) did not inhibit proteolytic activity either (data not shown). neither did mn ? , mg ? or ca ? at mm ( , , and %, respectively), suggesting that prrsv clpro might be located or work in a confined environment inside infected cells rather than spreading throughout the whole cells. further study is necessary to elucidate where and how prrsv clpro works in infected cells. cu ? and zn ? at mm decreased the proteolytic activity to or % (fig. b) . cu ? and zn ? inhibit several viral proteinases, such as clpros of severe acute respiratory syndrome-associated coronavirus and norovirus (hsu et al. ; someya et al. ) . these results may facilitate designing anti-prrsv drugs that contain metal ion-conjugated compounds, which have been used in cl protease inhibitors for sars-cov (hsu et al. ). effect of proteinase inhibitors on proteolytic activity pmsf inhibited the proteolytic activity of prrsv clpro up to % (fig. ) . edta showed even higher inhibitory effect ( % inhibition) than pmsf. the proteolytic activity of exfoliative toxin a, which is also a serine proteinase, has been reported to depend on cu ? (sakurai and kondo ) . the proteolytic activity of prrsv clpro may also depend on the presence of some divalent cations. antipain, leupeptin, tpck, e- and benzamidine did not show significant inhibitory effect. fig. effect of ph on proteinase activity. the enzyme ( lm) and substrate ( lm) were incubated for h at °c in the following buffers ( mm) containing mm nacl: citrate (ph . - . ); tris/hcl (ph . - . ); sodium bicarbonate ( . ) fig. proteolytic reaction is mediated by prrsv clpro. the enzyme ( lm) and substrate ( lm) were incubated for h at °c in mm tris/ hcl (ph . ) buffer containing mm nacl. lanes through , clpro with ser to tyr mutation, substrate alone, wild-type clpro alone and wild-type clpro mutational analysis of the catalytic site seven single amino acid substitutions (his to pro, his to asn, asp to ala, asp to glu, ser to phe, ser to cys and ser to pro) were introduced into prrsv clpro by site-directed mutagenesis. the proteolytic activities of these mutant proteins were measured in the trans-cleavage assay described above. as expected, all the substitutions of amino acids his , asp and ser abolished the proteolytic activity (fig. ) . in a previous study, substitution of the predicted nucleophilic ser with ala in prrsv clpro produced an inactive enzyme (tian et al. ). the x-ray crystal structure of prrsv clpro revealed that the protein folded into two b-barrel structures (domains i and ii), typical of the fig. effect of nacl concentrations (a) and divalent cations (b) on proteinase activity. the effect of metal ions was examined by preincubating the enzyme with the compound of interest for h at °c. thereafter, the substrate ( lm final concentration) was added, and the extent of cleavage was calculated after h incubation at °c. the activity was compared with that using mm tris/hcl buffer (ph . ) without cations. (a) the reaction mixtures with the indicated nacl concentration. (b) the reaction mixtures with mm of the indicated inons. lane c, reactions without cations fig. effect of proteinase inhibitors on proteolytic activity. the clpro ( lm) in mm tris/hcl buffer (ph . ) containing mm nacl was preincubated with different proteinase inhibitors ( mm final concentration) for h at °c. thereafter, the substrate ( lm final concentration) was added, and the extent of cleavage was calculated after h incubation at °c. the cleavage activity in the absence of inhibitors was defined as %. lane c, reaction without proteinase inhibitors. lanes through , reactions with antipain, pmsf, leupetin, tpck, benzamidine, e- and edta catalytic domain of chymotrypsin-like proteinases. the canonical catalytic site was located at the opening of the cleft between domains i and ii and consisted of residues his , asp and ser (tian et al. ). taken together, these results supported the prediction that his , asp and ser formed the catalytic triad of prrsv clpro (snijder et al. ) . north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions modification of a pcr-based sitedirected mutagenesis method evaluation of metalconjugated compounds as inhibitors of cl protease of sars-cov lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states characterization of staphylococcal exfoliatin a as a metallotoxin, with special reference to determination of the contained metal by radioactivation analysis the arterivirus nsp protease is the prototype of a novel group of chymotrypsin-like enzymes, the c-like serine proteases characterization of the norovirus c-like protease molecular characterization of positive-strand rna viruses: pestiviruses and the porcine reproductive and respiratory syndrome virus (prrsv) structure and cleavage specificity of the chymotrypsin-like serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) highly pathogenic porcine reproductive and respiratory syndrome expression, purification, and in vitro activity of an arterivirus main proteinase virus-encoded proteinases and proteolytic processing in the nidovirales mutational analysis of the predicted active site residues of prrsv clpro. the substrate was incubated for h at °c with wild-type prrsv clpro or mutants in mm tris/ hcl (ph . ) buffer containing mm. lane c, wild-type prrsv clpro; h p, his to pro mutant; h b, his to asn mutant key: cord- -fe sacjj authors: butler, j. e.; lager, k. m.; golde, william; faaberg, kay s.; sinkora, marek; loving, crystal; zhang, y. i. title: porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic date: - - journal: immunol res doi: . /s - - - sha: doc_id: cord_uid: fe sacjj porcine reproductive and respiratory disease syndrome (prrs) is a viral pandemic that especially affects neonates within the “critical window” of immunological development. prrs was recognized in and within a few years became pandemic causing an estimated yearly $ , economic loss in the usa with comparative losses in most other countries. the causative agent is a single-stranded, positive-sense enveloped arterivirus (prrsv) that infects macrophages and plasmacytoid dendritic cells. despite the discovery of prrsv in and the publication of > , articles, the control of prrs is problematic. despite the large volume of literature on this disease, the cellular and molecular mechanisms describing how prrsv dysregulates the host immune system are poorly understood. we know that prrsv suppresses innate immunity and causes abnormal b cell proliferation and repertoire development, often lymphopenia and thymic atrophy. the prrsv genome is highly diverse, rapidly evolving but amenable to the generation of many mutants and chimeric viruses for experimental studies. prrsv only replicates in swine which adds to the experimental difficulty since no inbred well-defined animal models are available. in this article, we summarize current knowledge and apply it toward developing a series of provocative and testable hypotheses to explain how prrsv immunomodulates the porcine immune system with the goal of adding new perspectives on this disease. north american-like (prototype vr- ). the virus is a member of the family arteriviridae in the order nidovirales, which includes lactate dehydrogenase-elevating virus of mice (ldv), simian hemorrhagic fever virus (shfv), equine arterivirus (eav) and the recently described wobbly possum disease virus (wpdv) [ , ] . as the name implies, except for wpdv that appears to be only neurologic, they are associated with some form of vasculitis. the virus can be transmitted across the placenta to infect the fetus [ , ] despite the fact that the porcine placenta is impermeable to maternal antibodies [ ] . prrs is the number one disease problem in major swine producing areas around the world. it is estimated to cost the industry million dollars a year just in the usa with proportional losses recognized in other countries. this is attributed to the remarkable ability of prrsv to: ( ) infect swine at all stages of production, ( ) be shed in the semen of boars for extended periods of time, ( ) be easily transmitted between farms, ( ) tolerate a high mutation rate, and ( ) negatively modulate the host's immune response. prrs has been a troubling disease because of its persistence and because [ years of research has failed to produce an efficacious vaccine. this has been somewhat surprising since eav infections are resolved in - days and a number of efficacious vaccines are available [ ] . the rapid resolution of eav is reminiscent of the pattern of sterilizing immunity seen with porcine influenza even in germfree (gf) piglets, so it is not simply a case of neonatal incompetence. rather, prrsv is more similar to ldv in which both the virus and the antibody response persist in mice [ ] . as implied by its name, prrs causes two separate pathologies: fetal abortion and respiratory disease in young and older pigs. there is some evidence that prrsv replicates predominately in the thymus, which results in thymic atrophy [ , , ] . this feature separates prrsv from both eav and ldv. while this is especially pronounced with highly pathogenic strains (hp-prrsv) [ , ] , it is not necessarily the case for all isolates. more than , papers have been published on prrs, nearly all of which describe studies using conventional animals [ , , [ ] [ ] [ ] . most initial studies focused on adaptive immunity, although it is well recognized that viral infection also affects the innate immune system [ ] . few studies have focused on immune dysregulation by prrsv, but recent work describes how prrsv can suppress innate immunity (''the innate immune response to prrsv'' section). murtaugh and genzow propose that ''identification of the viral structures that elicit the protective immunity in pigs and factors that modulate the efficacy of protection in vivo is essential to rational development of immunological tools to prevent and control prrs.'' this focus is very important but as general guderian advised hitler in ''if what you are doing is not working, try something different'' [ ] . what is lacking in prrsv research is a greater effort to determine the mechanisms, whereby the virus modulates the porcine immune response. in this review, we describe testable hypotheses to explain how this virus modulates the host immune system. both prrsv and ldv are immune modulatory and although not retroviruses, may have more in common with hiv than eav. ldv elevates igg levels in mice with little production of virus-specific antibodies [ , ] , which is almost identical to what is seen in isolator piglets infected with prrsv [ ] (''the effect of age, rearing, complement and the role of mucosal immunity'' section). polyclonal b cell activation is often associated with autoimmunity and is common to a number of viral infections that are genetically unrelated to the arteriviridae [ ] . many viral infections such as bovine viral diarrhea virus [ ] interfere with ''normal'' immune processes, which prolong the replication window for the viruses and thus increase the opportunity for contagious spread. thus, virus classification may be a poor predictor of the effect of a virus on the immune system. with rare exception, interference with the immune response is not the cause of death; good parasites rarely kill their host. rather, secondary bacterial infections are more likely to cause death in prrsv-infected conventional animals [ , , , ] . renukaradhydad et al. [ ] showed that coinfection with prcv (porcine respiratory coronavirus) reduced nk cell function more than prrsv alone and dual infection caused more pathology [ ] . likewise, prrs decreased the efficacy of siv vaccination and increased clinical disease [ ] , and mycoplasma hyopneumoniae infection significantly prolonged and increased the severity of prrs [ ] . as implied in the name of the disease, the clinical manifestations of prrs involve reproductive failure in sows and respiratory disease in young and growing pigs. historically, field reports described ''uncomplicated'' prrsv infections in young pigs as a mild-to-moderate pneumonia recognized clinically as an increased respiration rate at rest that would become labored with exertion. these observations were readily demonstrated experimentally. reproductive failure, which became the hallmark sign of prrs, included abortion ''storms'' and a sudden increase in dead fetuses and weak-born pigs that would affect most of the sows in the herd. in experimental sow infections during late gestation, fetal death and weak-born pigs are a predictable outcome, but prrsv-induced abortions are uncommon. the course of clinical disease following prrsv infection has been well chronicled. in the hundreds of animal experiments that have been reported since , it has become clear that there is considerable variation in clinical responses. most of this is attributed to the use of different prrsv isolates, and collectively, it appears that the isolates from the early s are less pathogenic than isolates from the late s and certainly much less pathogenic when compared to asian hp-prrsv. although differences in viruses may be a major factor in clinical variability, differences do occur when using the same virus under similar conditions suggesting that the host is also an important variable. fortunately, there is considerable knowledge and expertise in prrsv genetics to allow this to be further tested (''prrs the virus'' section). at this time, variation in clinical response is attributed to genetics, age, and coinfections [ ] . based on early field reports and experimental data, swine become more resistant to clinical disease with age, and boars and sows exhibit fewer clinical signs. this is not completely accurate since there is growing evidence that as prrsv mutates overtime, it may gain in virulence. why adults are more resistant to clinical disease and more likely to resolve the disease with vn antibodies [ ] is unclear, but it may reflect the less well-developed immune system of neonates (fig. ) . likewise, how the virus develops a chronic infection in the boar and is shed in the semen for extended periods of time is not known. current swine husbandry practices are almost completely dependent on the use of artificial insemination resulting in a population of boar studs that may supply semen to tens of thousands of sows. this practice dramatically magnifies the danger of using prrsv-contaminated semen. similarly, the concentration of sows in large buildings certainly contributes to possible horizontal transmission of virus and subsequent clinical and economic affects. at a cellular level, prrsv antigens and nucleic acids have been demonstrated in cells of the monocyte and dendritic cell lineage in a variety of organs. prrsv in the lung is often associated with lesions; however, the presence of virus and lesions is less frequent in other organs. the observations support a tropism of the virus for the lung, which could lead to pneumonia. however, when compared to other swine pathogens, the presence of prrsv in the lung and other organs seems minimal in relationship to clinical disease. one explanation for this may be that the pathogenic mechanism(s) of prrsv is(are) not necessarily a simple cytolytic effect on a tissue with influenza a that infects airway epithelia. instead, prrsv may just affect a smaller group of cells that have important regulatory controls, which could lead to a variety of diseases most likely those of hematopoietic/lymphoid tissues. the behavior of good parasites like viruses is to cause a delay in their eviction to allow for reproduction and transfer of their offspring to another host. others may revert to a low virulence state and continue to survive in the host. viruses such as those in the herpes family that are persistent for life have all evolved mechanisms that dysregulate the immune system. few investigative groups have seriously focused on immune dysregulation during prrsv infections. a great many viruses foil antigen presentation by interfering with mhc expression. rapid reduction of mhc adaptive immunity fig. the critical window of immunological development. neonates are vulnerable during this period since their adaptive immune system is undeveloped, and they depend on innate and passive immunity. within this period, healthy gut colonization takes place which drives the development of adaptive immunity and both oral tolerance and immune homeostasis develop. in some mammals, passive maternal antibodies are provided in utero as well as post-natally through suckling. the colors are a result of blending overlapping events. modified from butler and sinkora [ ] class i surface expression is a common feature of viral infections and is seen with foot-and-mouth disease virus [ ] . in epstein barr virus (ebv) infection, degraded peptides from the ebna- nuclear antigen are not degraded, and so, these peptides are not presented [ ] . something similar happens with presentation of peptides derived from a -kda transcription factor in human cytomegalo virus (hcmv) [ ] . while the complex mechanism in these two examples is incompletely understood, there is better data for several other herpes viruses that inhibit the tap complex. tap is required for the transport of cytosolic peptides (including those derived from a virus) across the er. this step is required in their eventual presentation to cd t cells. tap inhibition is found in herpes infection of swine, dogs, and cattle but not in rodents or lagomorphs [ ] . an adenovirus protein (e ) retains degraded peptides in the er and thus also prevents their presentation to t cells [ ] . in hcmv, several gene products target mhc i for proteasome degradation [ ] . in hiv, the nef and vpu proteins downregulate expression of surface mhc i [ ] . in both human and bovine papilloma viruses, the gene product e is believed to interfere with the processing of cellular proteins and could thus affect presentation of peptides [ ] . viruses may also interfere with mhc ii expression that is induced by ifn [ ] . viral infection also disrupts cell cycling and interferes with cytokine and chemokine production and also cytokine action. the list of examples is long but in general, il- , il- , both type i and ii interferons are affected. as reviewed above, interference with innate cytokine synthesis may be especially important. these effects have been reported for a wide variety of viruses including pox viruses, herpes viruses, adenoviruses, and others. this further indicates that immune dysregulation is widespread among viral infection and that many families are involved indicating that it is a feature of the type of particular pathogens and but not their place in phylogeny. viral gene products also interfere with effector functions of the immune system. for example, they can interfere with apoptosis, and in swine, fmdv has been shown to inhibit the natural killer (nk) cell response to infection [ ] . it is known that adenoviruses can cause lysosomal degradation of fas that is part of the complex used by cytotoxic t cells and nk cells to induce apoptosis of virus-infected cells [ , ] . more than viral genes affect this part of the anti-viral defense [ ] . infecting viruses may also interfere with virus neutralization. the mechanism of viral neutralization has been a matter of conjecture for[ years. do neutralizing antibodies bind those viral epitopes that prevent their recognition by the receptors on potentially permissive cells or do they inhibit the fusion of the viral membrane with the endocytic membrane? if it is simple blocking, multiple antibodies appear to be needed since as many as % of such viral epitopes must be antibody bound to prevent infection [ , ] . is simple blocking by antibodies enough or is help needed from an immune complex? in the case of eav, adding fresh serum as a source of complement, greatly increased the effectiveness of vn. covalent binding of c and c can facilitate clearance by cells that express complement receptors. in addition to merely facilitating clearance, complement-containing immune complexes can augment b cell activation [ ] , whereas igg complexes without complement can downregulate b cell responses through crosslinking to fccriib [ ] . non-neutralizing antibodies may also act as a trojan horse in facilitating virus uptake through fccrs, a process dubbed as antibody-dependent enhancement that can increase infectivity - fold [ ] . recently, attention is being given to another immunosuppressive player in cancer and persistent viral infection. myeloid-derived suppressor cells (mdsc) were first described from a mouse model of lung cancer in which these cells inhibited t cell proliferation [ ] . these cells function through reactive oxygen species (ros), inos and arginase- [ ] . acting through ros, tcr can become nitrated preventing peptide binding [ ] . ros-dependent suppression of cd ? and cd ? t cells by mdsc in hcv infections [ ] . current understanding suggests that mdsc also inhibit nk cell function. mdsc suppression is also known for hiv, vsv, and vaccinia [ ] . since prrsv can be persistent, a role for mdsc should not be ignored. if viral neutralization is complement dependent, viruses that interfere with this mechanism can prolong their replication time in the host. there is evidence that vaccinia, cowpox, and variola secrete proteins that block c convertase action [ , ] . while the mechanism involved is unclear, herpes viruses can also inhibit complement activation [ , ] . it has been known for some time that many viruses that cause persistent infection including ldv and prrsv are strong polyclonal b cell activators and often lead to the appearance of autoantibodies, a symptom that the preimmune repertoire has been expanded [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . tumorigenic viruses like ebv that target b cells give rise to elevated levels of monoclonal antibodies not directed to ebv [ ] . in these cases, immunoglobulin (igg) levels are a poor indicator of the anti-viral response. the innate immune response to prrsv host innate immune responses play a key role against early viral infection. host pattern recognition receptors for rna viruses include rig (retinoic-acid-inducible gene)-i-like receptors (rlrs) and toll-like receptors (tlrs) [ , ] . activation of rlr and tlr signaling pathways leads to activation of interferon regulatory factor (irf- ), irf , and nf-jb, followed by induction of type i ifns (i.e., ifna and b) and expression of inflammatory cytokines. type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [ , ] . prrsv is sensitive to type i ifns, and the sensitivity is confirmed in vivo. pigs that were inoculated with recombinant adenovirus for ifn-a expression and challenged with prrsv day later had reduced lung lesion and delayed viremia and antibody response [ ] . the presence of ifn-a at the time of infection alters innate and adaptive immune responses to prrsv [ ] . prrsv appears to inhibit synthesis of type i ifns in pigs, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifna [ , ] . ifn-a could not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least -fold lower than that of prcv [ ] . prrsv infection of pulmonary alveolar macrophages (pams) does not lead to ifn-a production [ ] . plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifn-a in vivo. prrsv also fails to induce porcine pdcs to produce ifn-a, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn-a [ , ] . however, nf-jb activation occurred in the presence of prrsv. loving et al. [ ] showed that prrsv replicated in monocyte-derived dcs but not lung dcs and that dc response to prrsv was merely limited to ifn-b transcription but no ifn-alpha transcription. prrsv replication in marc- cells significantly inhibits the doublestranded rna-induced type i ifn transcription [ ] . the prrsv proteins that are found to be antagonists of ifn induction include nsp , nsp , nsp , and n (see review [ ] ). nsp has been studied in more detail than the others. nsp is self-cleaved into nsp a and nsp b subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-b expression [ ] . beura et al. [ ] showed that nsp b inhibited double-stranded rna (dsrna)-induced irf phosphorylation and nuclear translocation. however, kim et al. [ ] showed that nsp inhibited irf association with creb-binding protein (cbp) in the nucleus but had no effect on irf phosphorylation and nuclear translocation. the discrepancy is possibly because an nsp b that is -residue longer than its authentic form was used in the beura's study. another possible reason is that different prrsv strains were used. nsp inhibits ifn induction by blocking irf activation, and the ovarian tumor (otu) protease domain interferes with the nf-jb signaling [ ] . nsp also inhibits the antiviral function of isg by the deubiquitinase activity of the out domain [ ] . nsp , an endonuclease, is also an ifn antagonist [ ] . the ifn antagonizing activity is not restricted to nonstructural proteins. nucleocapsid (n) protein inhibits ifn-b induction by interfering with dsrna-induced irf activation [ ] . the multiple components of nsps interfere with ifn induction. the nsps are early proteins, and n is a late one, which may play roles at different stages of viral replication. prrsv interferes not only with ifn induction, but also with ifn-activated signaling. ifns bind to their receptors on cell surface and activate jak/stat signaling, resulting in the expression of ifn-stimulated genes (isgs) [ ] . prrsv inhibits the ifn-activated jak/stat signal transduction and isg expression in both marc- and pam cells [ ] [ ] [ ] . prrsv replication in marc- cells suppresses jak/stat signaling stimulated by addition of ifn-a [ ] . prrsv infection of pam cells also blocks jak/stat signaling, while a vaccine strain ingel-vac prrs mlv has little effect, possibly due to its less efficient replication in the primary cells [ ] . nsp b inhibits the jak/stat signaling via inducing the degradation of karyopherin-alpha (kpna , also called importin-alpha ), which is known to mediate the nuclear import of stat [ ] . prrsv infection of marc- cells also reduces kpna expression. besides nsp b, other prrsv proteins including nsp , nsp , gp , and n were also found to be able to inhibit ifn signaling [ ] . prrsv field isolates have variable suppressive effect on ifn-a induction in pam cultures, and the suppression was found at post-transcriptional stage [ ] . this is not unexpected as prrsv strains are divergent in genomic sequences (''prrs the virus'' section). prrsv infection of monocyte-derived dendritic cells (mo-dc) induces the transcription of ifn-a/b but no detectable ifn-a in culture supernatant, suggesting a blockage at post-transcriptional stage [ ] . prrsv infection of marc- cells inhibits ifn expression by interfering with the rlr signaling pathway [ ] . a variety of type and prrsv were found to stimulate ifn-a secretion by pdc via tlr- pathway, and the effect did not require live virus [ ] . the suppressive effect on pdc was thought to be strain dependent. a novel isolate, a mc , induced ifns in both marc- and pam cells, and virus replication was needed for ifn induction [ ] . type ifns and isgs were detected in a mc -infected cells. a mc infection of pigs resulted in higher level neutralizing antibody than a mlv vaccine strain that is highly homologous in sequence [ ] . variable effect on ifn signaling among prrsv strains was also found [ ] . among six prrsv strains (vr- , ingelvac prrs mlv, vr- , nvsl - , mn , and lelystad) tested, all but mn inhibited ifn signaling in marc- cells, and all but mlv and nvsl blocked the ifn activation in pams. nsp b from the six strains were cloned, and all but mlv nsp b inhibited ifn signaling when overexpressed [ ] . there is good agreement that prrsv infections are not resolved rapidly in piglets, e.g., not in - days, in contrast to infections with swine influenza, fmdv, or eav in horses [ , , ] . further, the carrier state may exist for up to days [ ] , and viral rna can be detected out to dpi [ , ] . antibodies to prrsv can be detected as early as week after infection [ ] (fig. ), yet viral neutralizing (vn) antibodies are not usually detected prior to weeks [ , ] (fig. ) . maximum titers may not be reached until - weeks dpi, and the peak titers are usually modest [ , ] . igg antibody levels appear to peak at - dpi in piglets but persist at lower levels thereafter [ ] . some reports indicate that viremia and viral replication can persist even in the presence of vn antibodies [ , ] , and viremia can be resolved before vn antibodies are detected [ , , ] . in the case of prrsv, ldv, and eav, gp is considered the most important neutralizing epitope in vn [ , [ ] [ ] [ ] . focus has been on the hydrophilic ectodomain of gp [ ] . however, gp has numerous glycosylation sites that might influence the avidity and specificity of antibodies to gp . in general and because of the high frequency of mutation in rna viruses, there is considerable variation in gp among various strains of prrsv (''prrs the virus'' section). thus, the concept of the dependence of antibodies to gp for vn is complicated. using recombinant polypeptides, li and murtaugh [ ] showed that the titer of antibody to the gp ectodomain did not correlate with the vn antibody titer. vane et al. [ ] used peptide-specific antisera to show that the largest number of antigenic sites was associated with gp and no neutralizing targets were associated with either gp or m. using chimeric viruses, lu et al. [ ] showed that gp and m were not responsible for tissue tropism. furthermore, other studies have shown that viremia is resolved before vn antibodies appear [ ] (fig. ) and animals are protected from the european variant without them [ ] . evidence suggests that recognition may depend on strain variants/types. mabs to gp recognize the european variant but not the north american variant [ ] . in spite of these often contradictory reports, the bulk of the evidence supports the view that vn neutralizing antibodies are important for protection [ , , , ] . unfortunately, the mechanism of vn for prrs has not been researched. as regards vn antibodies to prrsv, there are some concerns about work already published. one concern is the amount of data available and from what experimental animal group they was obtained. if vn depends on labor intensive culture studies, it is likely that data currently available are from a few time points and a few animals. whatever viral epitopes or whole virus variants are used, a high throughput microtiter system should be adapted. it would be a shame if the current belief in poor vn activity is a consequence of selected and limited sampling. one can also question the methods used. in most studies, vn is tested using a lab strain virus and marc cells to which the virus has become adapted in vitro. this is a valid assay for the cell line and the prrsv strain used but does it test whether neutralization has occurred in vivo in infected animals in which different target cells and virus variants are interacting? the failure of swine to develop a sterilizing immune response has raised the issue of whether this virus produces fig. in piglets, the appearance of neutralizing antibodies is delayed, but other antibodies appear shortly after infection. from lopez et al. [ ] suppression or tolerance [ ] . some have reported the presence of cd ? cells with a suppressor phenotype (cd ? cd ? foxp ? ) after infections with prrsv [ , ] . silva-campa et al. [ ] showed that porcine cells with the treg phenotype make il and tgfb, confirming their analogous function to those in mice. it is known that pulmonary dendritic cells can induce tolerance through il- [ ] . however, in a three virus study using isolator piglets, an increase in cd cells with a suppressor phenotype was not associated with prrs [ ] . few studies have experimentally tested whether prrsv is functionally immunosuppressive while many show inhibition of type i interferons by prrsv (''the innate immune response to prrsv'' section). if tregs in conventional animals are functional, they appear not to interfere with the antibody response to klh in prrsv-infected pigs [ ] . the thymic atrophy caused by prrsv can result in subnormal levels of double-positive thymocytes drives t cell development and loss of peripheral cd cells [ , ] . some coinfection studies suggest that prrsv can interfere with protective responses to other viruses (''history and discovery of the causative virus'' section), which is supported by extensive field reports of synergy between prrsv infections and endemic infections within herd. infections with asian hp-prrsv elevate a large number of cytokines associated with both innate and adaptive immunity, both pro-inflammatory and otherwise [ ] . this ''cytokine storm'' suggests that prrsv affects many pathways leading to innate and adaptive responses or their suppression. an element in the kinetics of prrsv infection is the age of the host. klinge et al. [ ] showed that prrsv antibodies are detected at the same time in infected piglets and adults, yet viremia is immediate and resolved in sows, but develops late and remains persistent in piglets (fig. ). the delayed increase in viremia in piglets is correlated with a delay in the infection-induced increase in il- ; the increase in this suppressive cytokine seems correlated with viral replication, but not the time of infection. the muchcited viral persistence seems to be a feature of piglets since, except for boars, the virus does not persist in swine infected later in life [ ] (fig. ) . furthermore, the presence of vn antibodies in older pigs is correlated with elimination of the virus [ ] . by contrast isolator piglets appear much more susceptible to b cell immune dysregulation (''response to prrsv infection in germfree piglets'' section) and prrsv is most immune dysregulatory during the critical window of immunological development before immune homeostasis has been established ( fig. ) . figure shows that viremia persists in piglets but not in adults. figure shows that antibodies detected by elisa appear early but the appearance of those with vn activity is delayed. this could reflect a difference in sensitivity between elisa-based assays and vn assays. early protection to all infections depend on innate immunity which then raises the question of whether persistence of viremia in piglets (fig. ) reflects suppression of innate responses in piglets (''the innate immune response to prrsv'' section). while this may initially be critical, there is still too little information to conclude that the adaptive immune response is not impaired. there are reports that the amnestic antibody response to prrsv is poor or absent [ ] , yet little is known about t helper and memory cells in response to prrsv infection. t cell recognition of viral epitopes has been described [ , ] , but a tetramer assay system for these epitopes has not been developed for prrsv. despite the fact that so many viruses interfere with class i presentation, little attention has been given to prrs. overall, there is insufficient information as to whether the b cell or the t cell systems are most affected by prrsv and about the extent to which one or the other is impaired. the genetic variability of prrsv (''prrs the virus'' section) could also be a major player in the puzzle that has confounded investigators for [ years. hard evidence for escape mutants during infection is lacking but heterologous challenge studies indicate immunity to one strain does not confer immunity to all [ ] . in conventional herds, persistence might be due to re-infection with extrinsic variants for which crossprotection is absent. a particularly useful observation comes from so-called herd closure [ , ] . this essentially involves immunizing adult animals in a virus-free herd and then isolating them from exposure to outside animals. that these animals remain prrsv-free suggests that: ( ) vaccinated adult swine can develop sterilizing immunity if isolated from other animals and ( ) escape mutants are unable to establish a re-infection in such herds. however, these experiments have not been performed with asian hp-prrsv or with very young piglets whose immune system is just developing (fig. ). more than vaccines have been developed for prrs, although no single product has been totally successful [ ] . these vaccines and their efficacy are the subject of another review (k.m. lager, submitted). the functional, cellular response in adaptive immunity is characterized by the activation and expansion of antigenspecific, mhc-restricted cytotoxic t lymphocytes (ctl). in general, this is the primary effector function and most efficient immunity against viruses in mammalian species as university of iowa immunology ( ) : - because ctl kill virus-infected cells and arrest the generation of new viral particles. the role of this aspect of the immune response in prrsv infection is poorly understood. costers et al. [ ] published that induction of virusspecific ctl in prrsv-infected swine is very weak and slow to develop. they analyzed this by using prrsvinfected autologous cells as targets of ctl killing. by comparison, these authors show a strong response of similar pigs infected with pseudo rabies virus (prv) in ctl assays using prv-infected target cells. in chronic viral infections, the regulatory element ppp r d plays a significant role in ctl dysfunction [ ] . other in vivo studies have not tested for the predicted prrsv epitopes that would induce ctl responses [ ] and have used nonswine animal models. this complicates interpretation of the small literature available on this subject. furthermore, analysis of ctl induction is complicated by the nature of this effector function. experimentally, ctl killing is measured by analysis of these cells killing virus-infected cells in vitro in an antigen-specific, mhc-restricted manner. in most cases, the virus also kills the virus-infected cells. provided it is allowed by the in vitro system, killing takes days to occur. thus, new approaches are needed. the role of c/d t cells in prrs is unclear. several reports describe that c/d cells are affected by prrsv and other viral infections [ , , ] . the latter shows that c/d t cells behave similarly to cytotoxic and nk cells. in isolator piglets, only the subset of cd ? cd ? c/d t cells was increased, which is the only subset is known to be cytotoxic [ ] . the paucity of information at this point is insufficient to construct a meaningful hypotheses regarding the role of c/d t cells in prrs. however, depleting them in vivo using mabs could determine whether they play a role in either disease resolution or pathology. prrsv affects lymphocyte development in thymus prrsv infection can cause an acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus. thus, development of a protective, adaptive immune response to prrsv may be impaired because prrsv infection negatively impacts circulating and developing lymphocyte populations, and reconstitution of the peripheral lymphocyte pool can be impaired. lymphopenia appears soon after infection [ , , , ] and follows an influx of macrophage-like cells in the thymus and secondary lymphoid organs that contain prrsv [ , ] . there is also a loss of immature t cells in the thymus [ , , ] accompanied by significant lymphadenopathy [ , , - , , ] . it seems important to connect these observations to understand how prrsv affects the development of prrsv-specific immunity. the two mechanisms on which the animal relies to return balance to the circulating t cell pool are thymopoiesis and homeostatic proliferation of peripheral cells [ ] . homeostatic proliferation, or expansion of the existing peripheral t cell pool, is the primary means for reconstitution following peripheral depletion. in mice, both peripheral memory t cells and naïve t cells undergo homeostatic proliferation, though at different rates (fast vs. slow, respectively) and with differing signal requirements (mhc, il- , etc.). naïve t cells undergo slow homeostatic proliferation in secondary lymphoid organs (such as lymph nodes) that is dependent on il- and self-peptide:mhc presentation by an apc [ ] . this type of proliferative recovery has been implicated in autoimmunity because of preferential expansion of t cells with greater specificity and stronger avidity for self, which has been observed following administration of lymphodepleting drugs [ ] . prrsv infection has been shown to result in production of autoantibodies [ , , ] , which may be related to the expansion of autoreactive t cells and/or the failure of the pre-immune repertoire to diversify (''response to prrsv infection in germfree piglets'' section). memory t cells can proliferate outside secondary lymphoid organs, and the signal does not require mhc contact. collectively, the noted lymphadenopathy associated with prrsv infection may be the result of homeostatic proliferation of peripheral t cells, and possibly b cells, to repopulate the peripheral pool. if lymphoid hyperplasia is the result of homeostatic proliferation, it requires determining why the cells do not egress from the lymph node. in addition to proliferation of existing t cells, newly developed thymic emigrants can contribute to restoring the peripheral pool to a normal level following a lymphopenicinducing event. however, reports indicate a loss of t cells in the thymus following prrsv infection [ , ] . development of t cells in thymus is well described in textbooks, and at a certain stage, cd ? cd ? cells (double-positive,dp) interact with cortical thymic epithelial cells (ctec) to scan for positively selecting antigens. positive selection occurs when the t cell receptor has an intermediate affinity/avidity interaction with self-peptide presented by mhc on the ctec. positively selected cells then commit to the cd or cd lineage (single-positive, sp) and rapidly relocate to the medulla where they sample antigen presented by medullary tecs (mtec) and/or dendritic cells. these dp cells should not be confused with those dpc cells in the periphery of normal pigs [ ] . medullary tecs are unique in the expression of autoimmune regulator (aire) gene, which controls the expression of tissue-restricted antigens. tissue-restricted antigens (i.e., self-proteins) are picked up by neighboring thymic medullary dendritic cells for presentation to developing sp t cells, which drives t cell selection. if a high affinity/avidity signal through the t cell receptor at this stage is received, cells die by negative selection to prevent release of autoreactive cells into the periphery, which is referred to as central tolerance [ ] . mature naïve t cells, presumably those that only recognize foreign antigen, are then released into the periphery. various groups have shown a population of macrophage-like cells in the thymus stains for prrsv antigen by immunohistochemistry [ , , ] . in addition, reports have highlighted the negative impact of prrsv infection on thymic cellularity [ , ] , primarily as a loss of cd / cd dp cells in the thymus of prrsv-infected pigs [ ] . the loss of developing t cells in the thymus likely affects the number and nature of newly developed t cells exiting the thymus during prrsv infection. the presentation of prrsv antigens in the thymus may also induce tolerance (loss of naïve cells that would recognize prrsv antigen) and provide a mechanism for the reported increase in regulatory t cells after prrsv infection [ ] . these data together give support to the notion that infection of apcs in the thymus has a detrimental effect on the development of naïve t cells, and this likely has a negative impact on the development of a protective immune response to clear the virus from the pig. some of the lymphopenia that occurs shortly after birth may reflect the rapidly expanding blood volume but whatever the cause, it is not due to a selective depletion of t cells [ ] . in young pigs, prrsv induces a reduction in circulating lymphocytes early after infection, but not in age-matched controls (c. loving, pers com). since the decrease in circulating lymphocytes occurs before obvious phenotypic changes in the thymus, the lymphopenia is: ( ) not due to thymus infection by prrsv, ( ) an effect by prrrv on the peripheral t cell compartment, or ( ) a red herring in the quest to understand how prrsv dysregulates the piglets immune system. it is unclear if the drop in circulating lymphocytes is related to the lymphadenopathy observed later in the infection, but could be a compensatory attempt to repopulate the peripheral lymphocyte pool. response to prrsv infection in germfree piglets ''isolator piglets'' are recovered by caesarian surgery and reared in germfree isolators [ , ] . these animals have not encountered gut flora, which drives development of adaptive immunity through stimulation of toll-like receptors [ , ] (fig. ) . furthermore, they obtain no passive maternal antibody in utero and receive no colostrum that could protect them from pathogens or interfere with immune responsiveness [ ] . finally, isolator piglets have no exposure to other pathogens or to other strains of prrsv. the response of isolator piglets is intrinsic and not modulated by other pathogens, subclinical infections, maternal antibodies, or exposure to other environmental factors. these piglets provide the best in vivo opportunity to identify the direct in vivo effects of prrsv on the neonatal immune system. isolator piglets can also be considered as ex vivo fetal piglets and, therefore, a good model to study prrsv-infected fetuses. since the adaptive immune system is not developed in fetuses, their intrinsic response is either innate or driven by fetal infections that promote development of adaptive immunity (fig. ) . rna viruses are often sensed by intracellular by toll-like receptors which sense either positive or negative single-stranded rna or double-stranded rna (a recognized adjuvant) generated as part of viral replication. these molecules can drive development of adaptive immunity as shown with swine influenza [ ] . fetal piglets are immunocompetent as early as days of gestation (dg) [ ] and have lymph nodes, an active bone marrow, ig gene class-switch recombination has occurred, and the ileal peyer's patches are especially well developed. while some changes are likely to occur between dg and birth (dg ), these have not been identified. when fetuses are confronted with prrsv, they respond in the same manner as isolator piglets [ ] (see below). studies using prrsv-infected isolator piglets [ , , , ] have revealed a number of features about the immune response to prrsv that may provide clues as to how this virus modulates the host immune system. immediately obvious is hypergammaglobulinemia, lymphoid adenopathy, and the appearance of autoantibodies [ ] (fig. ) . polyclonal b cell activation, hypergammaglobulinemia, and the appearance of autoantibodies are also seen in infections by unrelated viruses [ ] . polyclonal b cell activation is also a feature on ldv infection in mice, a related arterivirus that is also persistent [ ] . autoantibodies in prrsv-infected isolator piglets to golgi proteins [ ] are also a feature of ldv infections [ , ] and may be in part due to the site of morphogenesis of arteriviruses [ ] . in addition to hypergammaglobulinemia and autoimmunity, prrsv-infected isolator piglets exhibit abnormal antibody repertoire and b cell development. measured as a repertoire diversification index, the values are in the range of . , not significantly greater than for fetal piglets or sham control isolator piglets but - fold less than sivinfected isolator piglets and conventionally reared piglets (pic; fig. a ). sequence analyses revealed that the cdr binding sites of the ig from prrsv-infected piglets are even more hydrophobic than in newborns and sham controls while those for siv and pic are shifted to the hydrophilic region (fig. b) [ ] . hydrophobic binding sites are incompatible with antibodies that recognize glycoproteins and are a feature of the pre-immune antibody repertoire [ ] . in these animals, b cell differentiation is extremely rapid and cells representing the activated b cell stage are nearly undetectable indicating that b cells rapidly become plasma cells [ ] . comparative cellular studies of isolator piglets infected with prrsv and siv failed to reveal any evidence of immune suppression, i.e., lack of evidence for elevation of fox p cd ? , cd ? t cells. however, cells with a suppressor phenotype were observed in parallel studies using pcv -infected piglets [ ] in which functional immune suppression has been reported [ ] . accepting the fact that the effect of a viral, bacterial, or fungal infection in germfree reflects a direct effect of the pathogen, our data suggest that dysregulation of b cell differentiation is one of the principal feature of neonatal infections with prrsv during the critical window (fig. ) . (figs. , ) . by contrast, adult animals make good vn antibodies and eliminate the infection [ ] . some additional support comes from studies using homologous variants [ ] . osorio et al. [ ] demonstrated that passively administered ig-containing vn antibodies obtained from convalescent sows could provide sterilizing immunity in piglets although a follow-up study showed that while viremia was ablated, viral replication persisted in some tissues [ ] . in the same studies, passive administration of non-neutralizing anti-prrsv serum had little effect although the mechanism of vn was not described. it would be wise to know whether active complement was also transferred. since prrsv is a respiratory infection, it would also seem important to know whether passive antibodies would have reached the respiratory tract. it is known that parenteral and oral vaccination of the sow generates passive antibodies that are protective against tgev [ , ] . these and other studies support the view that effective antibodies were made by adults [ , , , ] . tgev is a gastrointestinal infection, so ingestion of passive maternal antibodies, via milk and colostrum, has access to the site of infection. by analogy to ww ii: ''you need to stop them on the beaches.'' the respiratory tract, especially the upper portion, is the domain of the mucosal immune system. thus, parenterally administered passive antibodies to prrsv are unlikely to reach mucosal sites. this may explain why follow-up studies by lopez et al. [ ] showed that virus still replicated in some tissues. the differences among result obtained using isolator versus conventional piglets might provide clues as to the nature of the apparent neonatal immune dysregulation. while lymph node adenopathy and some thymic atrophy are common to both groups, the extraordinary hypergammaglobulinemia of all isotypes and b cell expansion has only been consistently reported for gf isolator piglets (fig. ) . this may in part be due to the fact that investigators who studied conventional piglets rarely measure ig levels in serum or bal. such measurements in conventional piglets would be difficult to interpret since conventional piglets would have ingested maternal ig through suckling. conventional piglets used in these studies would be from prrsv-free herds, so very little of the ingested and absorbed ig would be prrsv specific and therefore not protective. this may explain why the extent of the disease is similar. both groups of animals make virusspecific antibodies but because of the extraordinary hypergammaglobulinemia seen in isolator piglets, and because absorbed ig are from prrs-free sows, only a tiny proportion would be virus specific [ ] . however, knowing how many cells are virus-specific relative to other viral infections would be a much more useful parameter for comparing both groups. the b cell clonal analysis done with isolator piglets showing selected expansion of the pre-immune repertoire has not been performed in studies of conventional piglets. the opposite is true for cytokine studies. however, cytokine studies in conventional piglets might be misleading because of undetected secondary infection or the effect of regulatory elements in colostrum or the impact of gut colonization [ ] . while the impact of normal gut flora can impact cytokine levels in conventional animals, investigators typically compare their data to control littermates raised in the same environment, so this should play little role. however, the lack of gut colonization of isolator piglets might be in part responsible for the differences in the degree of hypergammaglobulinemia, since elements received via colostrum could establish immune homeostasis which might dampen polyclonal b cell activation and proliferation [ ] . in limited studies, no differences were found between isolator piglets colonized with benign escherichia coli and their colonization-free littermates [ ] . however, studies in mice and rabbits indicate that all colonizers are ''not created equal'' [ ] , so results obtained using only e. coli could be misleading. difference in the innate immune response in isolator versus conventional piglets has not been reported. in summary, prrsv infections that result in fetal abortion, b cell dysregulation in isolator piglets suggests that piglets are more susceptible during the critical window of immunological development (fig. ). since siv infections are rapidly resolved even in gf piglets, it suggests that age-related neonatal immune incompetence cannot alone explain the persistence of prrsv. this would appear to shift blame to active immune dysregulation. while siv is quickly evicted, one must remember it infects primarily epithelial cells, not cells of the hematopoietic/ immune system. thus, siv infections would theoretically provide less opportunity for immune dysregulation of the developing neonatal immune system. in any case, investigators need to be careful about assuming that what happens in piglets, also happens in adults. the prrsv genome as indicated previously, prrsv is a member of the family arteriviridae, in the order nidovirales, which also includes university of iowa immunology ( ) : - the viral families of coronaviridae, inclusive of coronavirinae and torovirinae, and roniviridae [ ] . the nidovirales order (latin: nested set) contains viruses with similar genomic organization and replication strategy. the arterivirion contains a polyadenylated molecule of singlestrand, positive-sense rna (which is itself infectious) that varies in length for prrsv ( , - , bp) and eav ( , - , bp), but not as yet in complete published genomes for shfv ( , bp) and wpdv ( , bp). the particles are roughly spherical with an average virion diameter of nm and consist of a helical nucleocapsid surrounded by a lipid bilayer containing several proteins [ , ] . all arteriviruses replicate in alveolar macrophages of their respective host, apart from wpdv, for which the host cell type is not known. except for wpdv, which was only recently genetically characterized [ ] , each individual arterivirus species consists of many diverse genomes. prrsv has been most studied in terms of host pathogenesis. there are two recognized prrsv genotypes: type or european-like (prototype lelystad) and type or north american-like (prototype vr- ) [ ] . the two main genotypes share approximately % nucleotide identity, but each may vary more than % in nucleotide sequence. the genome length of type ( , - , bp) not only differs from type ( , - , bp), but discrete sections of the genomes are different as well. prrsv rna includes a untranslated region (utr) of - (type ) or - (type ) followed a large replicase gene of variable length processed into at least recognized nonstructural proteins (nsp a, b, ( tf, n), - a, b- ) by self-encoded proteases. the proteases include papain-like protease (plp) a and plp b in nsp , plp in nsp , and a serine protease (sp) in nsp [ , , ] . presently, most of the cleavages have been defined using eav. plp a and b, and plp cleave once cotranslationally, directly downstream of the respective enzyme. sp completes the remaining cleavages. nsp harbors the core rna-dependent rna polymerase (rdrp), nsp is a helicase, and nsp contains a mn ?dependent rnase that cleaves at u stretches (nendou) and is involved in rna replication [ ] . downstream of the replicase gene is overlapping open reading frames (orfs) enumerated as orf encoding for glycoprotein (gp) , orf b encoding non-glycosylated envelope protein e, orf encoding gp , orf encoding gp , orf a encoding non-glycosylated protein a, orf encoding gp , orf encoding the non-glycosylated membrane protein m, and orf encoding the nucleocapsid protein n. since these orfs overlap, mutations to one coding sequence may affect adjacent orfs. they are transcribed as a nested set of at least six subgenomic rnas (sgrnas) in infected cells. all of the downstream orfs encode structural proteins [ , ] . as mentioned above, type prrsv differs in the length of most structural orfs when compared to type viruses. a remarkable feature of the prrsv genome has been the rate of mutational diversification. it has been estimated that prrsv rna may have evolved at a higher rate ( - /site/ year) than other rna viruses ( - - - /site/year) [ ] although another investigator estimates the rate is similar to other rna viruses [ ] . the frequency of mutation includes not only simple mutation, but also is accounted for by a high rate of recombination [ ] [ ] [ ] [ ] . it is estimated that there now exist as many as four major subtypes of type prrsv, based on orf and orf phylogeny [ , ] . even more subtypes, as many as nine, have been identified for type prrsv when based on orf . the husbandry of commercial swine, with large numbers of hogs from different source herds and artificial insemination with boar stud semen, is believed to have accelerated the evolution of prrsv [ ] . there is also ample evidence that two or more prrsv strains may infect an individual pig [ , ] . the combination of husbandry with genetic mutation and recombination between different viral strains has made the study of prrsv evolution challenging. the major envelope proteins of prrsv consist of gp and m [ , ] . gp forms a heterodimeric complex with m linked by a disulfide bond [ ] . both gp and m are thought to traverse the viral envelope three times and have only a small extravirion domain and a longer intravirion domain, much as was shown for ldv and eav [ , ] . gp is the most variable structural protein, and the predicted ectodomain after signal sequence cleavage is approximately residues [ , ] . within these amino acids, two hypervariable regions surround a quite conserved region, which contains the completely conserved cysteine disulfide-linked to m and two potential n-glycosylation sites [ , ] . the conserved domain has been shown to harbor a neutralization domain, and the n-terminal sequence has been termed a decoy epitope that is not neutralizing [ , , , [ ] [ ] [ ] [ ] [ ] . however, since the conserved domain is surrounded by complex oligosaccharides, it is shielded from neutralizing antibodies [ , ] . the m protein, which is believed to act as glue to bring all virion components together, has also been implicated in neutralization [ ] [ ] [ ] . in addition, two of the minor glycoproteins (gp and gp ) have also been shown to harbor neutralizing epitopes [ , , [ ] [ ] [ ] [ ] [ ] . as shown for eav, gp :gp :gp are thought to be disulfidelinked heterotrimers on the extravirion of prrsv and are thought to be in very low amounts compared to gp [ , ] . although the minor glycoproteins may play a role in neutralization of some or all prrsv strains, there is little else known about the viral functions these proteins perform in prrsv [ ] [ ] [ ] . the phosphorylated n protein encapsidates the rna genome, probably in a helical conformation [ , ] , and is most likely involved in capsulation and budding from the endoplasmic reticulum as was shown for eav [ ] . the swine host synthesizes the most antibodies to the abundant n protein, which are non-neutralizing [ ] . replicase proteins that have been shown to induce high levels of antibody are nsp , nsp , and nsp [ ] . nsp has also been shown to harbor many b cell epitopes from different prrsv strains [ , [ ] [ ] [ ] and has recently been shown to be incorporated into the virion [ ] . several infectious clones of prrsv have been produced [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of the clones were developed using type viruses. these infectious clones represent only a fraction of the variability seen in the field, but are extremely useful in probing the genome for dispensable regions [ , , , ] , insertion of foreign genes to develop diva viruses [ , , ] , investigation of structurefunction relationships [ , , , [ ] [ ] [ ] [ ] , examination of host virulence [ , , [ ] [ ] [ ] , and/or the probing of host response [ , , ] . there are also several studies using chimeric viruses, either within or between certain arteriviruses. some chimeric studies have led to the conclusion that the minor glycoproteins, not gp , are important for tropism in cell culture [ , [ ] [ ] [ ] and that the m protein is also not involved [ ] . other investigators have explored combining different regions of type prrsv with type to examine viability [ , ] or to explore the effect of nglycosylation differences between strains [ ] . in an attempt to develop broader crossneutralizing antibody, researchers have mixed regions of the prrsv genome from different strains, creating a panel of chimeric viruses to explore changes in the virus as well as the swine host antibody response [ ] . the same investigators used this technique to attenuate a strain of prrsv [ ] . lastly, researchers have attempted to define regions of the prrsv genome responsible for attenuation/virulence [ , ] or to act as vaccines [ ] . these studies have led to the knowledge that it appears that attenuation, as well as virulence, is multifactorial, involving two or more regions that can differ based upon the lineage of virus used for study. the main lesson learned from these studies is that each strain of prrsv, derived from field isolates or those with defined mutations, harbors individual characteristics that influence the specific pathogenesis seen. these characteristics include viral replication rate, the amount of specific subgenomic messages, the relative ability to process viral replicase proteins, the amount of n-glycans displayed on the virion, the amount of each individual viral protein, the relative interaction rate between viral proteins, and the relative ability of each strain to inhibit type i interferon and to induce humoral and cellular immunity. added to these viral causes of pathogenic differences under defined clinical conditions are the host response to each individual viral strain, host genetics, climate effects, and herd immunity, among other factors. the need for new experimental tools and approaches advances in science have mostly succeeded because the experiments employed were focused on testing a specific hypothesis and because they were designed so that the number of variables was minimized. naturally, this is much more difficult in biology because of the complexity of living systems and because many variables are unknown when the study begins. the image that emerges from the cumulative literature on prrs is that many: (a) represent a category that is often derogatorily referred to as fishing expeditions, i.e., exploratory research, (b) are repetitious of other work already done or represents near re-publication of the same work in another journal, and (c) are noncomparative studies. the work appears to be driven by the pressure to produce a vaccine, not to understand how prrsv modulates the immune system. the combination of swine and prrs offers a particular challenge to immunologists. prrsv does not replicate in mice, there are no practical inbred strains of swine, immunological reagents are limited, and producing stable cells lines has proven to be difficult. most studies have been done using conventionally reared piglets, which represents a complex model as illustrated in the following hypothetical example. consider pigs infected with prrsv and noninfected controls. since pigs are outbred, difference in responses can be genetic. if they are conventional, each animal in each group has not had the same experience since it may have a different mother, and its passive immune experience could differ in terms of colostral regulatory factors obtained and their dosage. suckling patterns differ within a litter giving rise to the often used ''hind teat'' syndrome. if you split the litter, you must then move some piglets to surrogate mothers, which introduces another set of variables. gut colonization plays university of iowa immunology ( ) : - an important role in development of adaptive immunity [ , ] , and colonizers do not have an equal effect [ ] . colonization typically occurs by contamination at the birth canal and thereafter by contact with the mother through suckling or contact with her feces. assuming that each newborn piglet in each experimental group encounters the same environmental experience is extremely difficult to prove. all of these assumes they have the same living conditions and have no contact with other animals that not part of the study. the ''closed herd'' studies cited earlier is an example of how this latter aspect can be properly controlled. conventional animals almost invariably contact other microorganism, some that are pathogens and some that are merely commensals. while experimenters may control for serious pathogens, they typically do not control for subclinical infection or for differences in the make-up and effect of benign colonizers. all of these may affect how a young pig responds to an experimental infection with prrs or a prrs vaccine. the literature shows that animals studied differ in age and there appears to be an age factor in their immune responsiveness and in the persistence of the virus (''the effect of age, rearing, complement and the role of mucosal immunity'' section). if the purpose of a study is to understand how a virus affects the immune system, conventional piglets are probably a poor choice. if on the other hand, the goal is only to test a vaccine under farm conditions, then the approach is fine. after all, the sabin and sauk vaccines and many successful bacterial vaccine before them prevented the spread of many horrible diseases but it would take decades to understand the etiology of the disease and just why these vaccines worked. the story of prrs is more like the story of hiv; the old time vaccine recipes do not work, and so, it is now time to understand the etiology of the viral infection and how it interferes with its immune-based eviction. while there is no mouse model for prrs, there is a mouse model for ldv. the superficial similarities in outcome are such that one wonders why the ldv model has not been used more for prrsv given the vast number of immunological reagents that are available for mouse immunology. assuming that for other reasons, ldv is not a good model, then perhaps the next approach would be to compare how siv, prrsv, and fmdv affect the porcine response in a controlled in vivo setting such as the isolator piglet. one glance at the literature reveals that compared to their counterparts in mainstream immunology/virology, those in the veterinary field are at a disadvantage. one obvious problem is the lack of reagents for work on the swine immune system. however, the literature also suggests an apparent reluctance to employ some of the -year-old technologies already available. notably, simple assays like quantification of igs are rarely used, as are immunohistochemical assays that measure ig-containing cells and elispots that measure isotypic distributions, antigenspecific b cells, and cytokine secretions. while elispot and pcr assays have been used in prrs research, neither of these methods provide data on where the cells responsible are located within the geography of the organs studied. refining these to single cells in situ assays as used in other species would provide more useful information. single cell sorting and recovery of rna by micromanipulation are also available. given the many studies done in conventional piglets that refer to the lack of vn early in development of prrsv infection, why there are no assays to determine the mechanism of vn to test if complement is required or if antibody affinity is important is puzzling. likewise for a disease that affects the respiratory tract, the lack of studies on the mucosal/local immune response to prrsv is conspicuous. while using more controlled in vivo studies can help to understand prrs, they cannot address questions about what prrsv does at the cell and molecular level. without in vitro studies, it will be difficult to understand how prrsv affects the host immune system. as mentioned above, the lack of stable cell lines presents a real problem. this can partially explain why there are no mixed culture studies to determine whether mhc i is downregulated by prrsv and how infected macrophages or the virus itself affects t and b cells and their interactions. even a question still exists as to the exact cell population that can be infected. for example, does prrsv infect lymphocytes or only macrophages/dendritic cells? if this should occur, lymphocytes are present at all different stages of development, and if a particular viral receptor is needed, it may not be present at all times during lymphocyte differentiation. since porcine cell lines immortalized at each stage of lymphocyte development are not available, the question is more difficult to answer. it may also be dangerous to use only laboratory strain for infection studies and only established cell lines to which the strain has been adapted. for example, marc cells used to propagate prrsv do not show downregulation of type ifn, while this is not true for pdcinfected in vivo. to address whether the remarkable polyclonal b cell proliferation seen in gf isolator piglets is the direct effect of the virus, studies involving t-b cell interactions or contact between b cells and infected macrophages are needed. the same applies to cytokines: what cells are making which cytokines and where are these cells histologically located since cytokines typically act at short distances? especially useful for these studies would be engineered prrsv mutants lacking the ability to make certain gene products. the wealth of information on the prrsv genome, the many variants, and engineered mutants, provide a rich resource of research material (''prrs the virus'' section). in the last two decades, which covers the same period in which prrs has been studied, tetramer assays to quantify t cell specificity and involvement have become well established and can now be used with some limitation for cattle and swine. studies that concern innate immunity are already being conducted in vitro (''the innate immune response to prrsv'' section). perhaps the best way to determine how prrsv modulates or dysregulates the immune system is to start with fetal and neonatal animals since the pandemic nature of prrs appears developmentally linked. that the effectiveness of neonatal vaccines is age-dependent is no surprise to any immunologist and forms the basis for the timing of childhood vaccination schemes. while for prrsv and other viruses that cross the placenta, studying the fetal immune response would be wise, but quite impractical. fortunately, in swine and other artiodactyls, newborns are essentially ex vivo fetuses since they can be reared in gf isolators in which maternal regulatory factors and the effects of gut colonization are absent [ , ] . given the experimental ''cleanliness'' of using isolator piglets (''response to prrsv infection in germfree piglets'' section), why they are so seldom used is surprising. first, there is a matter of expense which is not trivial. second is the rather subjective view that isolator piglets are artifacts because they do not reflect the farm experience and environment. so what is the purpose of prrs research: to simulate the farm experience and produce a vaccine ''in the blind'' or to first understand how the virus affects the host? if the former is successful, the latter usually becomes mute. unfortunately, the latter does not seem to be the case for prrs since the virus was identified [ years ago and the disease has not been controlled. one argument favoring isolator piglets is their use as a model for fetal piglets that are aborted after in utero infection. the most compelling argument for the use of isolator piglets to understand how the virus dysregulates the immune system is that it minimizes the number of variables, always a feature of good experimental design. finally, if prrs is primarily a persistence problem in neonates, the use of isolator piglets automatically confines studies to the critical window of immunological development (fig. ) . all studies in biology must grapple with what is ''normal.'' eviction of the virus shortly after infection might be considered ''normal'', while those that are not might be ''abnormal.'' this reasoning is certainly open to discussion. from a practical position, this is a good starting point if the goal is to understand how certain infectious agents affect the immune system. good experiments cannot be done in a vacuum. a glance of the literature shows that many experimental studies compare virus-infected piglets only with noninfected controls. this overlooks the possibility that the changes observed are common to all viral infections including suppression of nk function, interference with class i presentation, and polyclonal b cell activation. rather, experiments need to be designed in a manner to identify ''prrs-specific'' immune dysregulatory factors. a number of those done in studies on innate immunity have been done comparatively (''the innate immune response to prrsv'' section). coinfection studies are really relevant. for example, renukaradhyad et al. [ ] showed that while prcv reduced nk activity by %, dual infection with prrsv reduced this - %. in nearly all coinfection studies, there was an increase in disease [ , , ] as might be expected resulting in increased morbidity and mortality. it would be surprising if coinfection did not result in more pathology and perhaps a delayed/depressed immune response. thus, such studies would seem unreliable in the identification of virulence factors of prrsv. there are also parallel studies using siv, pcv , fmdv, and tgev to distinguish ''normal'' versus ''abnormal.'' however, these viruses have different cell tropism. are there any other porcine virus that infect macrophages and are eliminated in - days? there is also the issue of virulence. in the case of prrsv, one expects the degree of immune dysregulation to parallel the degree of virulence. hp-prrsv is more virulent because it kills the host in a shorter time or produces more severe clinical symptoms. does it also cause more severe immune dysregulation? if not, then assuming all events seen with vaccine strains of prrsv are due to immune dysregulation could lead in the wrong direction. the purpose of this review was to allow individual specialists to review their area of expertise and then to ask each to contribute a subhypothesis. we then assembled these separate views into global hypothesis. our goal was to especially provide new investigators with a number of testable hypotheses that could explain how prrsv dysregulates the neonatal porcine immune system. prrsv suppresses innate immunity, which delays adaptive immune responses prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [ ] as well as weak cell-mediated immune response [ ] . we hypothesize that the suppression of innate immunity can be an important contributing factor to the modulation of host immune responses because type i ifns promote antigen presentation and natural killer cell functions, enhance antibody production of b cells, and play an important role in the differentiation of both cd ? and cd ? t cells. the prrsv interference with the innate immunity is at multiple levels, from ifn induction, ifn-activated signaling to activity of isgs. therefore, viral-mediated suppression of innate immunity not only inhibits early host defense against the infection, but also interrupts the development of adaptive immunity, especially in the young pigs. this may explain why young pigs develop more severe disease and poorer protective immune response during the critical window of development (fig. ) . therefore, we would suggest comparative studies using siv and tgev to determine at the cytokine/cellular level, if prrsv-infected pams or pdcs alter the signal to t and b cells or even developing thymocytes. using the ifn-inducing prrsv strain a mc could add to the value of the model. we further hypothesize that given the divergence of prrsv strains in sequences and clinical features that experiments utilize various strains and engineered mutants. since type i ifns are proinflammatory, the proper amount at the right site and time may be protective, whereas extreme elevation could result in damaging inflammation. a typical example is that hp-prrsv induces high-level ifn-a, but causes high mortality in pigs [ ] . polyclonal b cell activation resulting in hyperplastic lymph nodes packed with ig-containing cells (igcc) is a hallmark of prrsv-infected isolator piglets. this is paralleled by hypergammaglobulinemia in which de novo-synthesized ig levels can increase as much as , -fold in weeks postinfection although \ % of these are virus specific [ , ] (fig. ) . we assume that the same type of immune dysregulation occurs in conventional piglets, although it may be masked by the high concentration of absorbed passive ig that increase serum ig levels to [ mg/ml. the extraordinary hypergammaglobulinemia simultaneously occurs as b cells rapidly differentiate to plasma cells in a manner in which the intermediate stage of activated b cells (cd ? cd -) is virtually absent [ ] . future studies in both conventional and isolator piglets need to confirm or reject the observation that a very small proportion of specific antibodies characterizes the response to prrsv. if confirmed, it would lend support to the view that rapid b cells differentiation allows little time for diversification of the antibody repertoire. this can be tested after pcr recovery and cloning of the rearranged vdj from various tissues. using labeled probes specific for the nonmutated cdr and cdr regions of the seven porcine vh genes, a repertoire diversification index (rdi) can be calculated as described previously and shown in fig. [ , ] . since the rdi is largely a measure of the degree of somatic hypermutation, it indirectly tests whether gc formation and function have been normal. it would be nice to confirm this in conventional piglets and adult swine, but the data would be uninterpretable since conventional piglets and adult swine have been antigenized through contact with other microorganisms, and changes could not be ascribed to prrsv. suspicion about abnormal gc activity might also explain the findings of mulupuri et al. [ ] . they used in vitro restimulation assays to suggest that there is a poor memory b cell response to prrsv. work by raymond and rowland [ ] identified gc in newborn prrsv-infected piglets using a mab to cdw that has not been validated in swine. the gc and memory cell questions need to be pursued using better reagents and better experimental designs. the delay in development of vn antibodies in prrsvinfected piglets while the anti-viral response continue to rise (fig. ) might be because early antibodies are: ( ) complement dependent for vn, ( ) of low affinity, ( ) specific for non-neutralizing epitopes, or ( ) of the wrong antibody isotype. alternatively, the differences between iddex elisa titers and vn merely reflect differences in assay sensitivity. in a single study, the addition of fresh serum did not improve vn to ldv, but it did improve the efficiency of vn to eav in horses suggesting that vn is complement dependent in horses but not in mice [ ] . this is a simple assay and should be done with sera from prrsv-infected swine. a most likely possibility is that antibody affinity is too low in neonates to perform as effective vn antibodies. in the case of denge virus, at least % of the neutralizing epitopes must be bound by antibodies for vn to occur [ ] . immunochemists over the last years have developed a plethora of methods to determine antibody affinity. most of these were developed to study antibody interactions with defined haptens. these studies established a number of very important principles including the observation that avidity, i.e., the staying power of an antibody, was determined by the ratio of the on-rate to the off-rate. thus, some ''quick and dirty'' methods have surfaced based on the principle that antibodies that remain bound in the presence of denaturants like urea or guanidine hcl are used [ ] , which are of high affinity. using this procedure, the relative affinity of a non-vn serum could be compared to that from adult swine that has vn capacity. should the experiments designed to test the role of complement or antibody affinity give negative results, another approach would be to test the specificity of early antibodies for certain viral epitopes. as reviewed in ''humoral responses of conventional animals'' section, vn antibodies to the lelystad virus preferentially recognize gp . assuming gp is the critical epitope, and affinity has been ruled out; it might suggest that antibodies to gp appear late during infection or that gp is poorly expressed on the virions used in the assay. once bound, the fate of the virus-antibody complex can also depend on the isotype of the antibody, which brings us to the fourth possibility. multivalency such as with pentameric igm can compensate for intrinsic binding site affinity and, therefore, perform much better than nonpolymeric igg so that early igm should provide good vn activity. the subclass of the igg antibody can also play a functional role in the effectiveness of complement-mediated vn. in swine, igg is the most totipotent igg based on its motifs for complement and fccr binding [ ] . however, actual functional comparisons have not been carried out. igg is expressed very early in fetal and newborn piglets but after antigen exposure, other igg subclasses, especially igg replace igg [ , ] . during the period in which vn has been typically measured (fig. ) , there is at least tenfold more igg than igm present, and thus, igg is most likely the antibody in serum that is being measured in current vn tests. to determine which subclass of igg is involved would be extremely difficult. first, all commercially available mabs to swine igg are more or less pan specific [ ] . even if such reagents were available, those which bind the virus would almost certainly be a mixture, so most probably antibodies of all subclasses involved, albeit probably dominated by igg . perhaps the only way to truly test the effector function of the different igg subclass antibodies seems at this point unjustifiable. this would require construction of chimeric antibodies for each subclass each with a binding site that recognizes a neutralizing epitope of prrsv akin to the method we have described for expression and recovery of individual porcine igg subclass proteins [ ] . confirmation of this subhypotheses might explain the initial ineffectiveness of the humoral response to prrsv during the critical window, but it does not explain why the extraordinary b cell expansion occurs and what force is driving this event. these require other subhypotheses and experiments to test them. we hypothesize that prrsv infects a population of antigen-presenting cells that migrate to or are constituent in the thymus of fetal or newborn animals, e.g., tecs, macrophages, and pdc that are engaged in thymocytes development and compromises proper t cell development. the interaction of thymocytes with these infected apcs might result in cytokine production/transcription and other protein transcription, which is abnormal compared with agematched controls. furthermore, the emerging t cell populations could be tested for their ability to recognize peptides derived from prrsv or a control antigens like ovalbumin. contrived in vitro systems should be developed to determine whether t cells developed in prrsv-infected thymi can provide t cell help for antibody responses, activation of macrophages, or can behave as ctls. we propose that the role of ctls in prrsv infection is fundamentally different in the infection of neonatal pigs compared to adults. we propose that the ability of pigs infected in utero or shortly after birth to mount any ctl response against prrsv is compromised by the impaired development of ctl precursors due to reduction of thymic selection. further, t cell selection that does occur could suffer from prrsv antigens being seen as self-antigen, as a result of infection of thymic cells involved in t cell selection. contrarily, in animals infected with prrsv as adults, ctl precursors have developed normally, and even though the infection impairs innate immunity, the presence of virus-infected cells eventually could lead to a protracted development of a moderate ctl response. further, we propose that the dysregulation of b cell function favors expansion of cd helper t cells not those required for induction of ctls. this could also contribute to or be the sole cause of the protracted development of antiviral ctl responses in adult animals. we describe below techniques to test theses hypotheses. first, we can use live, virulent virus in the short (hours long) assays to detect ctl killing. alternatively, avirulent strains of the virus can be used as surrogates, allowing the cell death to be solely a result of ctl killing of the target cell. in other circumstances, viral proteins can be delivered to target cells artificially, by vectors for instance [ ] . since the ctl are from an infected animal and the autologous cells (or mhc matched target cell line) are given the vector expressing viral proteins, the measure of killing is now attributable to the ctl, as there is no live virus. a dominating concept of the immunopathogenesis of prrsv infection is the immunosuppression or dysregulation of the adaptive immune response. as with many livestock studies, there is a body of work describing the antibody response but little analysis of ctls. the single report of ctl function describes a basic analysis of a single strain of virus and concludes there is a low-level ctl response that is protracted in the kinetics of development [ ] . a better understanding of ctl biology in prrsv infection will require a more sensitive assay for ctl function. using tools available today, class i mhc tetramers can be designed and tested to track ctl development and function. for instance, cd a (lamp a) is an integral membrane protein that lines the vesicles that contain the granules that mediate killing by nk cells and ctls. these granules are released by the vesicle membrane fusing with the cell membrane and releasing the contents. as a consequence, cd a is now detected on the cell surface. so, a tetramer-positive, cd a expressing cell is a prrsv-specific ctl that has just killed a virus-infected cell. so, not only is the cell phenotype determines, i.e., prrsv-specific cd t cells but also whether these cells function as ctls. another possibility to explain the decrease in ctls might be the action of mdsc [ , ] . these macrophages accumulate at the site of chronic viral infections and tumors and suppress ctls. therefore, highly infected sites such as thymus, lung, and certain lymph nodes [ , , , ] may harbor these cells. since prrsv targets macrophages, could their infection result in differentiation of myeloid cells to mdsc? with these tools, hypothesis testing can determine whether ctls are efficiently induced, induced but not functional, develop early but are rapidly downregulated, develop late, etc. elevation of p expressing, cd ? , cd ? treg populations reported in prrsv-infected isolator pigs is controversial (''humoral responses of conventional animals'' section). however, if class ii sla tetramers could be used to focus on the prrsv reactive cells in that population exclusively, this antigen-specific population may be highly induced, but masked by the present methods of analysis. however, given the evidence available, a more likely hypothesis is that the normal, t cell differentiation is dysregulated as reflected in the apparent dysregulation of helper t cells that promote excessive b cell proliferation while preventing prrsvspecific ctls from expanding that become activated to kill virus-infected cells. the opportunity to manipulate the prrsv genome provides the opportunity to test whether certain viral genes/proteins are responsible for immune dysregulation. nsp is the most variable protein in the virus, subject to insertion/deletion(s) compared to the prototype type strain, vr- . the fact that the nsp protein is an early protein and also a structural component of virions [ ] suggests that it may be in contact with host macrophages and dcs, and stimulators derived from those and other host cells. it also possesses that a key protease, plp , whose ability to downregulate ifn-a and can act to deubiquinate proteins is well established, has a key role in the viral replication cycle by cleaving the nsp / junction. lastly, this protein is the largest protein of the virus. a prior in vivo study has shown that a specific deletion of aa in nsp of strain vr- resulted in virus (vr- d ) with replication kinetics in -week-old swine about log lower than the parent strain, while other deletions elsewhere in nsp had a more dramatic effect on viral replication (''prrs the virus'' section). it was also shown that swine inoculated with vr- d had no delay in onset of antibodies to the nucleocapsid protein. what was intriguing was that these same animals showed a delay in serum ifn-c and a significant decrease in lymph node enlargement over that seen with vr- . unfortunately, no comparison was completed on the thymic tissue or any other immune response measurement. these prior studies must now be examined using more virulent prrsv strains, and we must delineate the amino acids responsible for immune evasion. two strains that we will develop deletion mutants for and test our hypothesis are type strains mn- and asian hp-prrsv. one can begin by deleting the nucleotides of these more virulent viruses that represent the same region as vr d . however, other regions of nsp may serve to evade immune responses. only the hypervariable regions (aa - ; aa - of vr- ) of the respective viruses have been shown to be mutable, so work should concentrate on those areas and make successive deletions based on nsp secondary structural predictions in the infectious clones of the parent viruses. once developed, these mutants will be used in in vivo studies with conventional and isolator piglets and in in vitro studies. since infected mq and pdcs fail to secrete ifna [ , ] , they would also poorly stimulate the antiviral state, so the first event is to compromise the first line of defense (innate immunity), which would allow spread of the virus. second, the ifna-deficient infected mq may then present to peripheral t cells in lymph nodes and without normal levels of il- from dcs and pdcs, would not favor a th profile and differentiation to ctls. thus, a major element in adaptive antiviral immunity is impaired. rather these events favor a th profile that might cause proliferation of cd helper cells at the expenses of tregs and cd ctls. the suggestion that infected mq and pdcs could induce apoptosis of thymocytes might indicate they could have the same effect on the peripheral t cell compartment. this could create a lymphopenic state. the increase in il- suggests suppression that could account for the increase in tregs [ ] and may be derived from mdsc [ ] . the elevation of tregs might be a delayed event, which would have been overlooked by sinkora et al. [ ] who worked only with isolator piglets. it is still difficult to accept that if adaptive immunity is forced to a th profile, it explains the polyclonal b cell activation and runaway b cell proliferation. the third event is that these infected mq, cdcs, and pdcs move to the developing thymus as apcs where they interact with dp thymocytes in the medulla that for reasons unknown, resulting in atrophy of dp thymocytes. together with help from thymic epithelial cells (nurse cells), prrsv may be therefore recognized as a self-antigen so surviving thymocytes could enter the periphery and recognize prrsv as self, as reported by the wieland for anti-golgi antibodies. in fact, the vasculitis that is a feature or arterivirus infections may be due to self-antibodies that coat the vascular as shown by lemke et al. [ ] . while the loss of dp thymocytes might lead to the loss of emerging t cells and in t cell lymphopenia, there is little evidence to support this. however, the quality and quantity of emerging cd , cd , and tregs might be altered as described above for the peripheral t cell compartment. without functional tregs, activated b cells may initially proliferate out of control as suggested from sinkora et al. [ ] . could an abundance of selfreactive th cells, some of which may crossreact with prrsv, be sufficient to drive rapid differentiation to plasma cells or perhaps il- from infected mq? alternatively, gc may not form or are abnormal, so there is little selection and the resultant plasma cells show little repertoire diversification (fig. ) and therefore poor affinity to viral epitopes so that few which are strongly virus specific. while prrsv-specific vn antibodies can control the peripheral spread of the virus, ctls are needed to eliminate virus-infected cells. in most viral infections, pdcs secrete il- that promotes th cells that can also activate mq to kill their intracellular parasites/viruses. if chronically infected tissues are infiltrated by mdsc, such t cells may be inhibited [ ] . in any case, since events in the thymus might reduce the number of peripheral th helpers, the infection would persist. perhaps of greatest effect is that if the number of virus-specific peripheral cd cells is low, there would be fever potential ctls to attack the infected mq. not trivial is that most scenarios described for ctl involve killing of epithelial cells like in siv. in the case of prrsv, it would involve the killing of infected mq. how easy is that? r d i fig. a antibody repertoire diversification measured as a repertoire diversification index (rdi). prrs = isolator piglets infected with prrsv; gf = germfree controls; c/ v = isolator piglets colonized with benign e. coli or infected with siv; pic = young, helminth-infected conventionally reared pigs (pic). b hydropathicity profiles calculated from sequence analysis of the hcdr region of ig from prrsv-infected piglets compared to pic animals (top) and compared to newborns (bottom). the numbers in parentheses indicate the number of sequences examined. hydrophobic hcdr regions i and ii are a feature of an undiversified pre-immune repertoire whereas region iii is characteristic of a diversified repertoire. from butler et al. [ ] adult model while what we have written above might explain the impact of prrsv on neonates, the literature we have reviewed suggests that a separate model is required for the situation in adult swine. while we may be dealing with one disease at the cellular/molecular level, we may be dealing with two disease models at the organismal level as regards the immunological perspective: one for adults and one for neonates. for all sorts of reasons, we believe that immune homeostasis is developing during the critical window of immune development (fig. ) when most piglets are prrsv infected. when an adult pig is considered, they have already properly developed their t cell repertoire and compartment. that means they have normal levels of cd cells that are potential ctls. likewise, they have th cells to form gc and tregs to prevent uncontrolled b cell expansion. as a result, adult animals mount effective immune responses with vn antibodies and ctls that resolve the disease, regardless of whether the innate response continues to be compromised since host protection is now heavily dependent on de novo adaptive immunity (fig. ) . in fetal and newborn piglets, innate immunity probably plays the major role in immune defense but after development of adaptive immunity, it become compensatory, not primary. this most likely explains why studies like those of robinson et al. [ ] show that prrs is resolved in adults, presumably by both vn antibodies and ctls. thus, the host adaptive response override the negative effect of prrsv on innate immunity in adult animals. this suggests that the principal impact of prrsv is on the fetus and the neonate during the critical window and is thereafter not a serious threat to adults. from the position of vaccinologists, it would seem wise to supply neonatal vaccinates with the ingredients that would promote immunocompetence as summarized in fig. . all of the events described for fetal/neonatal and adult animals are relevant to the common vaccine version of prrsv. however, is the effect of hp-prrsv merely a quantitative difference or does it have a qualitative effect? namely, does hp-prrsv primarily target the thymus so its greatest impact is on t cell cells development? since hp-prrsv has a greater effect than vaccine strain, prrsv on post-natal lymphopenia suggests that hp-prrsv also acts in the periphery. as previously described, failure to produce vn antibodies could be epitope dependent, so that differences between animals with and without vn antibodies could be epitope specificity, not a difference in affinity regardless of the mechanism of vn. the beauty of prrrv genetics is that a large number of variant are available and others can be engineered (''prrs the virus'' section). the availability and expertise of the investigators in this area provide an unusual opportunity for the experimental design of studies to determine how certain viral genes affect immune dysregulation and how epitopes differs in their ability to stimulate protective immune responses. testing the global hypothesis the working hypothesis offers numerous opportunities for testing. exactly, how each step in the scheme is tested is left to the ingenuity of the investigators. suffice to say there is a great need to know the cytokine, co-stimulatory molecule expression and signaling features of prrsvinfected macrophages when acting as apc versus noninfected macrophages both in thymus and in the periphery. do these ifna-impaired macrophages preferentially or inappropriately stimulate certain t cell subsets or do they promote differentiation of mdsc? using engineered mutants, one might determine what genetic features of the virus are responsible for any aberrant signaling. the core protein of hcv promotes mdsc differentiation [ ] . such ''defective mutants'' might also be the basis for future vaccines. likewise, it would be wise to know what signaling events are aberrant in thymocytes from prrsvinfected animals. as the runaway b cell proliferation still lacks an explanation, it would seem important to know whether infected macrophages can explain that part of the puzzle. testing for germinal center formation, antibody affinity and the complement dependence of vn are relatively straightforward. the issue of tregs should be resolved. are those with a suppressor phenotype functionally suppressive? could it be the lack of functional tregs that permits runaway b cell proliferation and differentiation? while tetramer assays could be valuable, they will remain in the distant future given the state of swine genetics. in the meantime, it is reasonable to assume that the same co-stimulatory molecules and signaling pathways that operate in mice also operate in swine so the information gained from studies in pigs can take advantage of the vast resource of information assembled from mouse research. using simplified systems such as in vitro assays and isolator piglets has the best chance of determining how prrsv dysregulates the neonatal porcine immune system. if pandemic prrs is a neonatal phenomenon, developing vaccines that stimulate development and maturation of the adaptive immune system, e.g., probiotic cultures, should also be considered. porcine reproductive and respiratory syndrome epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus identification of a novel nidovirus associated with a neurological disease of the australian brushtail possum (trichosurus vulpecula) virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid-gestation sows and fetuses in vitro infection by procine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii immunoglobulins of the mammary secretions the immune responses to equine arteritis virus: potential lessons for other arteriviruses polyclonal activation of b cells by lactate dehydrogenase-elevating virus is mediated by n-glycans on short ectodomains of the primary envelope glycoprotein the interaction between prrsv and the late gestation pig fetus pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs characterization of thymus atrophy in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus immunological responses of swine to porcine reproductive and respiratory syndrome virus infections immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states natural killer cell frequency and function in pigs selectively bred for high and low antibody and cell-mediated immune response: response to vaccination with modified live transmissible gastroenteritis virus in vivo polyclonal b-lymphocyte activation elicited by murine viruses lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs hypergammaglobulinemia and autoantibody induction mechanisms in viral infection the evolution of bovine viral diarrhea: a review effect of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasturella multocida in pigs secondary infection with streptococcus suis serotype increase the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs porcine reproductive and respiratory syndrome virus-induced suppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs proinflammatory cytokines and viral respiratory disease in pigs vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of siv vaccination effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (prrsv)-induced pneumonia by mycoplasma hyopneumoniae genome-wide associated and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection humoral response to porcine reproductive and respiratory syndrome virus infection with foot-and-mouth disease virus results in a rapid reduction of mhc class i surface expression isolation of cytotoxic lymphocytes from healthy seropositive individuals specific for peptide epitopes from epstein-barr virus nuclear antigen : implications for viral persistence and tumor surveillance selective interference with class i major histocompatibility complex presentation of the major immediate-early proteins following infection with human cytomegalovirus molecular mechanism and species specificity of tap inhibition by herpes simplex virus icp herpes simplex virus inhibitor icp destabilizes the transporter associated with antigen processing (ytap) heterodimer retention of adenovirus e glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation dissociation of type i membrane proteins from the er to the cytosol is sensitive to changes in redox potential the downregulation of cd and mhc-i by primate lentiviruses: a paradigm for the modulation of cell surface receptors interaction of the bovine papillomavirus e qirh rhw clathrin adapter complex. ap- modulation of the major histocompatibility complex antigen expression by viral infection apoptosis regulators from dna viruses rapid and transient activation of cdt cells to interferon gamma production, nk cell-like killing and antigen processing during acute virus infection the adenovirus e / . k- . proteins down modulate the apoptosis receptor fas/apo- by inducing its internalization subverison of the immune system structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development c d of complement as a molecular adjuvant: bridging innate and acquired immunity fc-dependent inhibition of mouse b cell activation by whole anti-mu antibodies measles virus nucleocapsid protein binds to fcgammarii and inhibits human b cell antibody production monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention dengue viremia titer, antibody response pattern and virus serotype correlate with disease severity inhibition of the complement cascade by the major secretory protein of vaccine virus regulation of complement activity by vaccinia virus complement-control protein herpes simplex virus glycoproteins gc- and gc- bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity immune evasion properties of herpes simplex virus type glycoprotein gc lactate dehydrogenaseelevating virus induces anti-golgi apparatus antibodies infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies autoantibodies against golgi apparatus induced by arteriviruses autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against golgi antigens and other subcellular elements polyclonal activation of b cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (prrsv)-induced pigs neonatal infection of mice with lactate dehydrogenaseelevating virus results in suppression of humoral anti-viral immune response but does not alter the course of viremia or the polyclonal activation of b cells and immune complex formation hodgkins disease and the epstein-barr virus the toll-like receptor (tlr )-specific stimulus loxoribine uncovers a strong relationship within the tlr , and subfamily innate immune recognition of viral infection immunomodulatory functions of type i interferons interferon signalling network in innate defence adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus the presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc- cells interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus identification of two autocleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells the cysteine domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf activation in immortalized porcine alveolar macrophages jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation variable interference with interferon signal transduction by different strains of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nsp beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha degradation mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication porcine reproductive and respiratory syndrome virus activates the transcription of interferon alpha/beta (ifn-alpha/beta) in monocyte-derived dendritic cells (mo-dc) porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway impact of genotype and of porcine reproductive and respiratory syndrome viruses on interferon-alpha responses by plasmacytoid dendritic cells induction of type i interferons by a novel porcine reproductive and respiratory syndrome virus isolate enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain antibody response to porcine reproductive and respiratory syndrome virus (prrsv) nonstructural proteins and implications for diagnostic detection and differentiation of prrsv types i and ii studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with foot-and-mouth disease virus porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus antigen-specific b cell responses to porcine reproductive and respiratory syndrome virus infection modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) serum immune response to the proteins of porcine reproductive and respiratory syndrome (prrs) virus protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent antibody production and blastogenesis response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus the challenge of prrs immunology the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain the major envelope proteins gp of a european reproductive and respiratory syndrome virus contains a neutralizing epitope in it n-terminal ectodomain monoclonal antibodies to the gp of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the gp dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies chimeric viruses containing the nterminal ectodomains of gp and m proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus different european-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs posttranslational processing and identification of a neutralization domain of the gp protein encoded in orf of the lelystadt virus passive transfer of virusspecific antibodies confer protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin- and regulatory t-lymphocytes (treg) induction of inducible cd ? cd ? porcine reproductive and respiratory syndrome virus induces cd ? pulmonary dendritic cells producing il- mediate tolerance induced by respiratory exposure to antigen comparative lymphocyte profile and the t and b cell spectratype of germfree piglets infected with three important viruses thymocyte and peripheral blood t lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine identification of immunodominant t cell epitopes present in glycoprotein of the north american genotype of porcine reproductive and respiratory syndrome virus cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenged-exposed with an antigenically distinct isolate control and elimination of porcine reproductive and respiratory syndrome virus effect on total pigs weaned of herd closure for elimination of porcine reproductive and respiratory syndrome virus functional impairment of prrsv-specific peripheral cd ? cd high cell in vivo discovery of immunotherapy targets in the tumour microenvironment identification of cytotoxic t lymphocyte epitopes on swine viruses: multi-epitope design for universal t cell vaccine gamma delta lymphocyte response to porcine reproductive and respiratory syndrome virus efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity hematological and immunological parameters of / -month old pigs infected with prrs virus effects of different us isolates of porcine reproductive and respiratory syndrome virus (prrsv) on blood and bone marrow parameters of experimentally infected pigs experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of the virus envelope genes from genetically divergent strains comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (prrsv) by in situ hybridization in pigs infected with isolates of prrsv that differ in virulence lymphodepletion and homeostatic proliferation: implications for transplantation autologous regulation of naive t cell homeostasis within the t cell compartment a causal link between lymphopenia and autoimmunity characterization of a porcine cd -specific mab that distinguishes cd /cd double-positive thymic from peripheral t lymphocytes antigen presentation in the thymus for positive selection and central tolerance induction the piglet as a model for b cell and immune system gnotobiotic pigs derivation and rearing antibody repertoire development in fetal and neonatal piglets. viii. colonization is required for newborn piglets to make serum antibodies to t-dependent and type tindependent antigens antibody repertoire development in fetal and neonatal piglets. ix. three pamps act synergistically to allow germfree piglets to respond to ti- and td antigens antibody repertoire development in fetal and neonatal piglets. xvi. influenza stimulates adaptive immunity, class switch and diversification of the igg repertoire encoded by downstream cc genes development of the humoral immune response of the pig antibody repertoire development in fetal and neonatal piglets. xxiii. fetal piglets infected with a vaccine strain of prrs virus display the same immune dysregulation seen in isolator piglets antibody repertoire development in fetal and neonatal piglets. xix. undiversified b cells with hydrophobic hcdr s preferentially proliferate in prrs porcine reproductive and respiratory syndrome virus (prrsv) subverts development of adaptive immunity by proliferation of germline-encoded b cells with hydrophobic hcdr s lactate dehydrogenase-elevating virus and related viruses forced enrichment of hydrophobic amino acids in immunoglobulin cdr -h impairs splenic b cell development but not antibody production porcine circoviruses: a minuscule yet mammoth paradox contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs immunization of pregnant gilts with prcv induces lactogenic immunity for protection of nursing piglets from challenge with tgev the mammary gland in mucosal and regional immunity role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire order nidovirales novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses efficient - frameshifting by mammalian ribosomes to synthesize an additional . arterivirus protein the origin and evolution of porcine reproductive and respiratory syndrome viruses arterivirus molecular biology and pathogenesis divergence time of porcine reproductive and respiratory syndrome virus subtypes genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china recombination between north american strains of porcine reproductive and respiratory syndrome virus high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity a bayesian phylogeographical analysis of type porcine reproductive and respiratory syndrome virus (prrsv) molecular evolution of prrsv in europe: current state of play recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo genetic and phenotypic characterization of a united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus characterization of proteins encoded by orfs to of lelystad virus intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus structural proteins of equine arteritis virus the envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography arterivirus structural proteins and assembly signal peptide cleavage from gp of prrsv: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence neutralizing antibody responses of pigs infected with natural gp n-glycan mutants of porcine reproductive and respiratory syndrome virus role of neutralizing antibodies in prrsv protective immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus gp ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain lelystad virus monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection effect of virusspecific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages gp of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro molecular assessment of the role of envelope-associated structural proteins in cross neutralization among different prrs viruses immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the gp or m genes dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion glycosylation of minor envelope glycoproteins of porcine reproductive and respiratory syndrome virus in infectious virus recovery, receptor interaction, and immune response formation of disulfidelinked complexes between the three minor envelope glycoproteins (gp b, gp , and gp ) of equine arteritis virus glycosyl-phosphatidylinositol (gpi)-anchored membrane association of the porcine reproductive and respiratory syndrome virus gp glycoprotein and its co-localization with cd in lipid rafts potential role of porcine reproductive and respiratory syndrome virus structural protein gp in apoptosis inhibition the structural biology of prrsv cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid nuclear localization of non-structural protein and nucleocapsid protein of equine arteritis virus competitive elisa for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant e. coli-expressed nucleocapsid protein as antigen pathogenicity of swine influenza h n virus antigenically distinguishable from classical and european strains serologic marker candidates identified among b-cell linear epitopes of nsp and structural proteins of a north american strain of porcine reproductive and respiratory syndrome virus epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp fragment of the replicase polyprotein contains a cluster of b-cell epitopes monoclonal antibody and porcine antisera recognized b-cell epitopes of nsp protein of a chinese strain of porcine reproductive and respiratory syndrome virus highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein into virions identification of and cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel au-rich sequences restored replication of a -proximal -nucleotide deletion mutant a full-length cdna infectious clone of north american type porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp region identification of nonessential regions of the nsp replicase protein of porcine reproductive and respiratory syndrome virus strain vr- for replication in cell culture infectious clonederived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome infectious transcripts from cloned genomelength cdna of porcine reproductive and respiratory syndrome virus generation of an infectious clone of vr- , a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the nsp -encoding region a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus attenuation of porcine reproductive and respiratory syndrome virus strain mn using chimeric construction with vaccine sequence the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence expression of a foreign epitope by porcine reproductive and respiratory syndrome virus influence of nlinked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies the nsp alpha and nsp papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are nonessential for virus infectivity functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association chinese and vietnamese strains of hp-prrsv cause different pathogenic outcomes in united states high health swine identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones viable porcine arteriviruses with deletions proximal to the end of the genome identification of amino acid residues important for anti-ifn activity of porcine reproductive and respiratory syndrome virus non-structural protein a virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleukin- in vitro and in vivo construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture replacement of the heterologous ( ) untranslated region allows preservation of the fully functional activities of type porcine reproductive and respiratory syndrome virus arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras the isolator piglet: a model for studying the development of adaptive immunity dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load antibody repertoire development in fetal and neonatal pigs. xiii. ''hybrid vh genes'' and the preimmune repertoire re-visited antibody repertoire development in fetal and neonatal piglets. xxi. vh usage remains constant during development in fetal piglets and postnatally in pigs exposed to environmental antigen adaptation o a commercial elisa to determine the igg avidity in sweep experimentally and naturally infected with neospora caninum porcine igg: structure, genetics and evolution linkage haplotype for igg and iga subclass genes antibody repertoire development in fetal and neonatal piglets. xvii. igg subclass transcription revisited with emphasis on new igg resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmacytomas are unknown induction of foot and mouth disease virus (fmdv) specific cytotoxic t cells killing by vaccination acknowledgments the authors acknowledge and thank nancy wertz for her help in assembly of the manuscript and to dr. eric nelson for scientific review of the manuscript. key: cord- -ndrja os authors: arjin, chaiwat; pringproa, kidsadagon; hongsibsong, surat; ruksiriwanich, warintorn; seel-audom, mintra; mekchay, supamit; sringarm, korawan title: in vitro screening antiviral activity of thai medicinal plants against porcine reproductive and respiratory syndrome virus date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: ndrja os background: porcine reproductive and respiratory syndrome (prrs) caused by prrs virus (prrsv) results in economic losses in the swine industry globally. several studies have investigated the use of plant extracts in the prevention and control of prrs outbreaks. thai medicinal plants may be useful for treating prrsv infection in pigs. therefore, we investigated the in vitro anti-prrsv and antioxidant properties of seven thai medicinal plants: caesalpinia sappan linn., garcinia mangostana linn., houttuynia cordata, perilla frutescens, clinacanthus nutans, phyllanthus emblica, and tiliacora triandra. results: using antiviral screening, we observed that t. triandra extract strongly inhibited prrsv infectivity in marc- cells [virus titer . median tissue culture infective dose (tcid( ))/ml (log )] at h post-infection, whereas c. sappan extract strongly inhibited prrsv replication [virus titer . tcid( )/ml (log )] at h post-infection. c. sappan extract had the highest total phenolic content [ . mm gallic acid equivalent/g] and lowest half-maximal inhibitory concentration [ . mg/ml in , -diphenyl- -picrylhydrazyl and . mg/ml in , -azino-bis ( -ethylbenzothiazo-line- -sulfonic acid) diammonium salt]. conclusion: t. triandra extract could inhibit prrsv infectivity, whereas c. sappan extract was the most effective in inhibiting prrsv replication in marc- cells. this study elucidates the antiviral activities of thai medicinal plant extracts in vivo. the results promise that thai medicinal plant extracts, particularly t. triandra and c. sappan extracts, can be developed into pharmaceutical drugs for the prevention of prrs in pigs. porcine reproductive and respiratory syndrome virus (prrsv) is endemic in most pig-producing countries, and it results in enormous economic losses to the swine industry globally [ ] . this enveloped, positive-sense, singlestranded rna virus belongs to the arteriviridae family (order nidovirales), which also includes the equine arteritis virus, mouse lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus [ ] . in general, prrsv infection causes a disease that is characterized by reproductive failure in sows and respiratory infections in growing pigs [ ] , and this disease predisposes pigs to infection by bacteria and other viral pathogens [ , ] . this disease is known as porcine reproductive and respiratory syndrome (prrs) and has become endemic in many countries throughout the world following an epidemic phase [ , ] . its incidence was first reported in thailand in , and since then, several outbreaks have been reported [ ] . it has become a major infectious disease that causes high mortality in swine and production losses in the swine industry in this country. preventative measures such as gilt acclimatization, vigilant biosecurity, and vaccination have been shown to be useful in controlling prrs outbreaks, and supportive treatments are available for alleviating its severity; however, no specific treatment for prrs is available [ , ] . antiviral therapeutics are a critical tool for combating viral infections, particularly in cases wherein no vaccines are available against the circulating virus. thus, pharmacological intervention may represent an alternative approach in controlling prrsv. a number of natural compounds and compositions have been shown to possess antiviral activities against prrsv. gao et al. [ ] showed that cryptoporus volvatus extract exhibited antiviral activity against prrsv infection and replication. pringproa et al. [ ] reported that crude cynodon dactylon extract significantly inhibited prrsv replication as early as h post-infection (hpi). therefore, the antiviral activities of other thai medicinal plants against prrsv should also be investigated. thai medicinal plants such as caesalpinia sappan linn., garcinia mangostana linn., houttuynia cordata, perilla frutescens, clinacanthus nutans, phyllanthus emblica, and tiliacora triandra are known to have antioxidant and antiviral activities. these plants have already been promoted for use in primary health care and have been classified according to their pharmacological actions [ ] [ ] [ ] [ ] [ ] [ ] . therefore, the aim of this study was to determine the antiviral activities of thai medicinal plant extracts against prrsv infection in vitro and to measure their phytochemical contents to develop an alternative anti-prrsv therapy for use in veterinary medicine. prior to determining antiviral activity, we evaluated the cytotoxicity of the seven thai medicinal plant extracts on the viability of marc- cells, and viability is expressed as % cytotoxic concentration (cc ). the results showed that the cc of the seven plant extracts ranged from to μg/ml, and the effect of thai medicinal plant extract concentration on the tested cells increased in a dose-dependent manner (fig. ) . p. emblica extract had the lowest cc of μg/ml. the cc of g. mangostana extract was the second lowest ( . μg/ml) and that of c. sappan extract was μg/ ml. further, t. triandra and h. cordata extracts had cc of μg/ml, whereas c. nutans and p. frutescens extracts had the highest cc ( μg/ml). we treated prrsv with different concentrations of thai medicinal plant extracts that were determined based on their cc values so that these plant extracts did not affect the proliferative activity of marc- cells. the screening results of the inhibition of prrsv infectivity showed the potential of thai medicinal plant extracts to inhibit prrsv infectivity (fig. ) . t. triandra extract significantly inhibited prrsv infectivity in marc- cells at hpi when supplied at a concentration of μg/ ml (p < . ), and the observed virus titer at this concentration was . tcid /ml (log ). interestingly, p. emblica extract at a low concentration of μg/ml could inhibit prrsv infectivity [virus titer = . tcid /ml (log )]. as shown in fig. , immunoperoxidase monolayer assay (ipma) indicated that t. triandra and p. emblica extracts blocked prrsv infectivity in marc- cells, as shown by slight brown staining of cells. different thai medicinal plant extracts were tested in an in vitro inhibitor screening assay to determine inhibition of prrsv replication at three time intervals ( , , and hpi). at various time points after the infection, prrsv in supernatants was quantified for determining virus titer by ipma. results of screening were the same as those of the inhibition test of prrsv infectivity, i.e., prrsv replication was inhibited in a dose-dependent manner (fig. ) . interestingly, as shown in fig. , we found that c. sappan extract had significant potential to inhibit prrsv replication in vitro. as shown in fig. l , few cells that were stained brown showed the efficiency of c. sappan extract at a concentration of μg/ml, and the inhibition of prrsv replication by c. sappan extract was significantly stronger than that by other plant extracts at hpi [ . tcid /ml (log )]. the total phenolic contents of the seven thai medicinal plant extracts were determined using the folin-ciocalteu assay by constructing a standard curve of gallic acid. total phenolic content was the highest in c. sappan extract [mean ± standard error: . ± . mm gallic acid equivalent (gae)/g sample], followed by g. mangostana extract ( . ± . mm gae/g sample), with the lowest total phenolic content was observed in h. cordata extract ( . ± . mm gae/g sample) ( table ) . c. sappan extract had the highest antioxidant activity, with ic values of . ± . mg/ml in , -diphenyl- picrylhydrazyl (dpph) and . ± . mg/ml n , azino-bis( -ethylbenzothiazo-line- -sulfonic acid) diammonium salt (abts) and a reducing power of . ± . mm fe + /g in the ferric-reducing antioxidant power (frap) assay (table ) . p. emblica extract had the second strongest antioxidant activity against free radicals, with ic values of . ± . mg/ml in dpph and . ± . mg/ml in abts and a reducing power of . ± . mm fe + /g sample in the frap assay. prrsv outbreak causes significant economic loss in the swine industry worldwide. the current commercial prrsv vaccines are inadequate to protect pigs from prrsv infections [ ] . medicinal plants have progressively been explored as suitable alternative sources of antiviral agents [ ] . thai medicinal plants have widely been used as a source of herbal medicines because of their high bioactive compound contents that are effective against various diseases. in this study, seven thai medicinal plant extracts were screened for their antiviral activity against prrsv. before determining the antiviral properties of a compound, it is essential that a cytotoxicity assay is performed to determine the concentrations that can be used to avoid cell damage and ensure prrsv selectivity in vitro. in this study, we reported cytotoxicity as cc , which indicates the concentration of a substance that can inhibit virus activity by %. we found that p. emblica extract showed the highest cell toxicity ( . μg/ml). in this study, high-potential plant extracts were found to be c. sappan and t. triandra extracts, with cc of and μg/ml, respectively. antiviral compounds should be highly effective while showing minimal toxicity to normal cells and tissues [ ] . in this study, we investigated the antiviral activity of seven thai medicinal plant extracts against prrsv by assessing the inhibition of prrsv infection and replication in marc- cells. the range of plant extract concentrations was determined based on their cc values. p. emblica extract inhibited prrsv infection in marc- cells and in vitro. p. emblica extract at a concentration of μg/ml inhibited prrsv infectivity at a virus titer of . tcid /ml (log ). in this study, p. emblica extract showed the highest cytotoxicity to marc- cells with cc of < μg/ml. therefore, the antiviral activity of other plant extracts were investigated in this study. we found that t. triandra extract at a concentration of μg/ml significantly inhibited prrsv infectivity at a virus titer of . tcid (log ). while t. triandra extract has been used as anti-inflammatory [ ] , anticancer [ ] , and antimicrobial agents against mycobacterium tuberculosis [ ] , its antiviral activity, particularly against prrsv, has not been investigated previously. therefore, this is the first report to indicate that t. triandra extract could significantly prevent the entry of prrsv into marc- cells. however, t. triandra extract was not found to be effective in inhibiting prrsv replication. all studied plant extracts could inhibit prrsv replication when applied at high [ ] and the antimicrobial properties of c. sappan [ ] have previously been investigated, this is the first study to reveal the inhibitory activity of c. sappan extract on prrsv replication in marc- cells. regarding phytochemical content, c. sappan extract had the highest total phenolic content ( . ± . mm gae/g sample). the total phenolic content of a plant is considered an indicator of its antioxidant capacity because the redox properties of phenolic compounds allow them to act as reducing agents, hydrogen donors, and radical scavengers [ ] . previously, lee et al. [ ] reported that ethanolic c. sappan extract had a total phenolic content of . μg gae/mg. the values of total phenolic content in this study were slightly abu-jafar and huleihel [ ] reported that ethanolic eucalyptus camaldulensis leave extracts had strong antiviral activity against different members of the herpes virus family (hsv- , hsv- , and vzv). ramalingam et al. [ ] reported that the ethanolic extracts of andrographis paniculata have the highest antiviral inhibitory effects against dengue virus in vero cells. the screening of plants as possible sources of antiviral agents has led to the discovery of potent inhibitors of in vitro viral replication, thereby increasing the probability of identifying new bioactive plant compounds [ ] . these findings suggest the appropriate species and concentration of plant extract that could effectively inhibit prrsv replication, with both t. triandra and c. sappan extracts being highly effective in inhibiting prrsv infection in vitro by interfering with viral attachment and inhibiting viral replication and/or virus release, respectively. the modes of action of t. triandra and c. sappan extracts against pprsv require further investigation but are likely to be related to the natural compounds they contain. therefore, it was speculated that both t. triandra and c. sappan extracts are potential candidates for preventing prrsv infection in pigs. however, the plant extracts used for testing antiviral activity was crude extracts. in future, we plan to purify the most effective thai medicinal plant extracts (t. triandra and c. sappan extracts) for screening the active compound that is highly effective against prrsv. thai medicinal plant extracts exhibit antiviral activity against prrsv. t. triandra extract effectively inhibited prrsv infection. and c. sappan extract had the strongest antiviral activity against prrsv replication. these activities can be presumably attributed to the total phenolic contents and antioxidant activities of these plant extracts. although several previous studies have shown the antiviral activity of plant extracts against prrsv, there are no reports on the antiviral activities of t. triandra and c. sappan extracts against prrsv. to the best of our knowledge, this study is the first to report the inhibitory activity of t. triandra and c. sappan extracts against prrsv activity in vitro. further studies are required to elucidate the mechanisms of action of these plant extracts on prrsv. all chemicals used in this study were of analytical grade or higher. ethanol and methanol were obtained from merck (darmstadt, germany). abts, -hydroxy- , , , tetramethylchroman- -carboxylic acid (trolox), dpph, folin-ciocalteu phenol reagent, -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide (mtt), sodium carbonate, and , , -tri-pyridyl-s-triazine were purchased from sigma chemical co. (st. louis, mo, usa). ferric chloride hexahydrate and potassium persulfate were procured from loba chemie pvt (mumbai, india). gallic acid was procured from fluka chemical co. (buchs, switzerland). dulbecco's modified eagle's medium (dmem) was procured from gibco (massachusetts, usa). ethanolic c. sappan, g. mangostana, h. cordata, p. frutescens, c. nutans, p. emblica, and t. triandra extracts were purchased from specialty natural product co. ltd. (thailand). marc- tissue culture cells were grown in dmem containing % fetal bovine serum (gibco) and % penicillin/streptomycin and incubated at °c in a % co atmosphere. to produce inoculated cells, prrsv (vr north american genotype) was propagated in marc- cells, and virus titer was quantified using ipma. the cytotoxicity of the seven thai medicinal plant extracts was determined using the mtt assay. briefly, marc- cells were plated at a density of cells/ well in -well plates and incubated in a % co atmosphere at °c for h. when cells had at least % confluence, the medium was removed and replaced with medium containing two-fold serial dilutions of the plant extracts. in addition, medium without plant extract was used as a positive control. incubation was then continued in a % co atmosphere at °c for h. after this, the medium was removed, μl of freshly prepared mtt solution ( mg/ml) was added to each well, and the plates were incubated at °c for h. then, the medium was replaced with μl dmso to dissolve the crystals, and the plates were incubated at °c for min to dissolve any air bubbles before measuring the mtt signal at an absorbance of nm. results are reported as cc . the inhibition of virus infection assay was performed as previously described [ ] . briefly, the plant extracts at the concentration that was determined in the cytotoxicity test outlined above and at two lower concentrations in two-fold dilution were mixed with prrsv at tcid /ml at a ratio of : and incubated at °c for h. dmso ( %) containing medium mixed with prrsv served as the control. thereafter, the mixture of prrsv and plant extracts as well as controls were inoculated in marc- cells at a density of cells/well in a well plate and incubated at °c for h. subsequently, the medium was removed and replaced with a fresh medium containing % fbs. the plates with marc- cells were cultured under standard conditions for h hpi, and supernatants were collected to quantify virus titer. the inhibition of viral replication assay was performed as previously described [ ] . briefly, marc- cells were plated at a density of cells/well in -well plates and infected with prrsv at a multiplicity of infection of at °c for h. then, prrsv was removed from each well and replaced with the diluted plant extracts at the concentration that was determined in the cytotoxicity test and at two lower concentrations ins two-fold dilution. further, % dmso was mixed to medium as the control. the plates were cultured under standard conditions; supernatants were collected at , , and hpi; and virus titer was quantified. virus titer was further assessed by ipma as previously described [ ] . briefly, cells were fixed with μl of % cold formalin for min at room temperature (rt), washed once with μl of phosphate-buffered saline (pbs) and twice with μl of . % pbs tween- (pbst), and blocked with μl of % bsa in . % pbst for min at rt. after blocking, the cells were stained with μl of anti-prrsv nc protein monoclonal antibody (median diagnostics, gangwon-do, korea) diluted at a ratio of : at rt for min, washed, and incubated with peroxidase-conjugated affinipure goat anti-mouse igg (h + l) (jackson immunoresearch, pennsylvania, usa) diluted at a ratio of : for min at rt. after washing thrice with pbs, the cells were counter stained with , -diaminopentane substrate and examined under a microscope. virus titer is expressed as tcid and was determined using the reed-muench method. the total phenolic contents of the plant extracts were determined using the folin-ciocalteu method [ ] , and their free radical-scavenging activities were determined using the dpph-scavenging and abts-scavenging assays, as previously reported [ , ] . antioxidant activities were determined using the frap assay, according to the benzie and strain method [ ] . differences in antiviral activities among the different concentrations of each plant extract were tested using one-way analysis of variance with tukey's post hoc test for a comparison of means. cc was calculated using regression analysis of dose-response curves for the mtt assay. all statistical analyses were performed using the spss . software (spss inc., chicago, il, usa) with a significance level of p-value of ≤ . . prrs virus receptors and their role for pathogenesis porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) influence of hypericum perforatum extract on piglet infected with porcine respiratory and reproductive syndrome virus epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview spatial epidemiology of porcine reproductive and respiratory syndrome in thailand serological studies and isolation of porcine reproductive and respiratory syndrome (prrs) virus in thailand respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines cryptoporus volvatus extract inhibits porcine reproductive and respiratory syndrome virus (prrsv) in vitro and in vivo in vitro virucidal and virustatic properties of the crude extract of cynodon dactylon against porcine reproductive and respiratory syndrome virus in vitro virucidal and virustatic properties of the crude extract of cynodon dactylon against porcine reprodu in vitro anti-influenza viral activities of constituents from caesalpinia sappan active constituents against hiv- protease from garcinia mangostana evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection a novel substance purified from perilla frutescens britton inhibits an early stage of hiv- replication without blocking viral adsorption antiviral activities of clinacanthus nutans (burm.f.) lindau extract against cyprinid herpesvirus in koi (cyprinus carpio koi) in vitro anti-herpes simplex virus activity of , , , -tetra-o-galloyl-β-d-glucose from phyllanthus emblica l. (euphorbiaceae) in vitro antiviral activity of germacrone against porcine reproductive and respiratory syndrome virus south african medicinal plant extracts active against influenza a virus virucidal activity of garcinia parvifolia leaf extracts in animal cell culture some phytochemicals and anti-inflammation effect of juice from tiliacora triandra leaves chemical constituents and in vitro anticancer activity of tiliacora triandra leaves antimycobacterial activity of bisbenzylisoquinoline alkaloids from tiliacora triandra against multidrug-resistant isolates of mycobacterium tuberculosis in vitro antimicrobial activity of caesalpinia sappan l retracted: antioxidant properties of benzylchroman derivatives from caesalpinia sappan l. against oxidative stress evaluated in vitro antiviral activity of eucalyptus camaldulensis leaves ethanolic extract on herpes viruses infection anti-dengue activity of andrographis paniculata extracts and quantification of dengue viral inhibition by sybr green reverse transcription polymerase chain reaction in-vitro antiviral activities of extracts of plants of the brazilian cerrado against the avian metapneumovirus (ampv) development of an immunoperoxidase monolayer assay for the detection of antibodies against peste des petits ruminants virus based on bhk- cell line stably expressing the goat signaling lymphocyte activation molecule total phenol analysis: automation ans comparison with manual methods use of a free radical method to evaluate antioxidant activity antioxidative activity of mungoong, an extract paste, from the cephalothorax of white shrimp (litopenaeus vannamei) ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. in: oxidants and antioxidants part a: academic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank dr. wolfram spreer of the university of hohenheim for his critical comments on this article and thank enago (https://www.enago.com) for the english language review. authors' contributions kp, sh, and ks contributed to the study design. ca performed the experiments, carried out the statistical analysis, and drafted the manuscript. kp, sh and ks contributed to the statistical analysis and critically reviewed the manuscript. kp, ms, sm, wr, and ks conceived the study, coordinated the work described, and contributed to the manuscript preparation. all authors read and approved the final manuscript.funding ca was supported financially by a ph.d. scholarship of research and researcher for industries projects (rri), thailand science research and innovation, under contract no. phd i . the funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. also, this project was partially supported by chiang mai university. the datasets supporting the results of this article are available in the figshere (https://figshare.com/s/ bfdb d a c ffaf).ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -txfuuu d authors: lim, byeonghwi; kim, sangwook; lim, kyu-sang; jeong, chang-gi; kim, seung-chai; lee, sang-myeong; park, choi-kyu; te pas, marinus f. w.; gho, haesu; kim, tae-hun; lee, kyung-tai; kim, won-il; kim, jun-mo title: integrated time-serial transcriptome networks reveal common innate and tissue-specific adaptive immune responses to prrsv infection date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: txfuuu d porcine reproductive and respiratory syndrome virus (prrsv) infection is the most important viral disease causing severe economic losses in the swine industry. however, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during prrsv infection are poorly understood. we constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of prrsv infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [blns], and tonsils) via rna-seq. three groups with specific expression patterns (i.e., the -dpi, lung, and bln groups) were discovered. the dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza a infection. moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the bln-specific group showed down-regulated ampk signalling related to viral replication. our study may provide comprehensive insights into prrsv infection, as well as useful information for vaccine development. porcine reproductive and respiratory syndrome (prrs) is one of the most important diseases affecting commercial pig productivity in the swine industry worldwide [ ] . prrs is caused by the prrs virus (prrsv), a single-stranded rna virus [ , ] , resulting in severe reproductive losses for breeding pigs and respiratory problems for growing pigs [ ] . vaccination-a solution for this problem-is still limited because of the high mutation rate in the viral proteins and the intrinsic characteristics of prrsv that impede innate immune responses [ ] [ ] [ ] . therefore, to date, several studies have been performed to identify host factors that confer resistance to prrsv infection. some mutations in the guanylate-binding protein and cluster of differentiation (cd ) genes were reported to be associated with prrsv susceptibility [ , ] , and also cd knockout was proved to show full resistance for prrsv infection [ ] . porcine alveolar macrophages (pams) represent the main cellular target for prrsv infection [ ] . prrsv replication is initiated in the cytosol in infected cells following receptor-mediated endocytosis and disassembly, and replication primarily occurs in the lung and lymphoid organs, but not the spleen [ ] . prrsv can modulate host immune responses by down-regulating interferon-β production and suppressing the activity of antigen-presenting cells [ , ] . in addition, serum viral loads have been found to increase for approximately week after infection and then gradually decrease over the course of a month [ ] . in contrast, viral loads in lymphoid organs are reported to reflect high viral replication for a few months [ ] . several recent studies have involved the use of rna sequencing (rna-seq) to identify the functional basis of host responses to prrsv infection. serial blood transcriptomes observed following prrsv infection in commercial pigs showed three main clusters related to immune signalling, dna repair, and cell signalling [ ] . additionally, the blood transcriptomes in prrsvinfected gilts indicated the development of innate immunity at days post infection (dpi) and t cell signalling at dpi [ ] . relatively few prrsv studies in tissues based on rna-seq have been conducted thus far. the lung transcriptomes of prrsv-infected pigs revealed genes that were potentially related to early innate immune responses [ ] . analysis of tracheobronchial-lymph node transcriptomic responses to highly pathogenic prrsv infection revealed that they were mainly associated with cell death [ ] , and the tonsil transcriptomes of pigs revealed that high viral levels activated polarisation of blood cell functions [ ] . however, in most studies, analysis was performed at one or two time points in a single tissue after prrsv infection. therefore, an examination of the dynamic regulatory changes in gene expression levels at serial time points in multiple tissues is needed to gain comprehensive insights into prrsv infection. in this study, we investigated the molecular mechanisms of prrsv infection by integrating the differences in the expression of various genes in tissues responsible for respiration (lungs) and immunity (bronchial lymph nodes [blns] , and tonsils) using rna-seq data at serial time points. differentially expressed genes (degs) were identified at each time point and in each tissue after prrsv infection. then, dynamic molecular networks were constructed to identify tissue-and time-dependent gene expression levels and patterns. marc- cells, which are highly permissive to prrsv infection, were used for virus propagation and functional assays. marc- cells were maintained in rpmi- medium (gibco ® rpmi- , life technologies, carlsbad, ca, usa) supplemented with heat-inactivated % foetal bovine serum (life technologies), mm l-glutamine, and antibiotic-antimycotic (anti-anti, life technologies) containing iu/ml penicillin, µg/ ml streptomycin, and . µg/ml amphotericin b in a humidified chamber at °c under % co conditions. the prrsv- strain ja (genbank: ay . ) was used in this study. weeks-old piglets (n = ) were obtained from a prrsv-negative farm and housed in animal rooms at our facility. after days of acclimation, pigs were intramuscularly challenged with ml of prrsv (ja strain; × tissue culture infectious dose (tcid) /ml), diluted in sterile pbs. all infected pigs were humanely euthanised at , , , , and dpi, respectively. the remaining pigs were humanely euthanised without virus infection as an uninfected control ( dpi) group. a schematic overview of the animal study is shown in figure a . blood was collected at , , , , , and dpi from the euthanised pigs, and serum was separated for viral load detection and serological assays. the lungs, blns, and tonsils were aseptically extracted after euthanasia. these tissues were collected in tubes, snap-frozen using liquid nitrogen, and stored immediately at − °c for rna extraction. all animal experiments were approved by the jeonbuk national university institutional animal care and use committee, republic of korea (approval number - ). viral rna was extracted from µl of each serum sample and g of each tissue sample, using a mag-max ™ viral rna isolation kit (ambion, applied biosystems, life technologies) and a total rna extraction kit (hybrid-rtm, geneall, seoul, republic of korea), respectively, per the manufacturers' instructions. serum and lung viral loads were measured using a prime-q pcv , prrsv detection kit (genet bio, inc., daejeon, republic of korea) with a fast real-time pcr system (applied biosystems, foster city, ca, usa). a standard curve was generated from known titres of prrsv and used to calculate the quantity of prrsv in each sample by converting each cycle threshold value to the tcid /ml-equivalent values. the prrsv titres in lung tissues were measured with marc- cells, using a microtitration-infectivity assay. briefly, tissue homogenates [ % (weight/ volume)] from the extracted lungs were prepared in dulbecco's modified eagle's medium with antibiotics, vortexed for - min, and centrifuged at ~ × g for h at ℃. after centrifugation, each collected supernatant was filtered through a sterile . μm syringe filter and incubated with marc- cells to measure the viral titre. prrsv titres were calculated at to dpi, based on the observed cytopathic effects, and were expressed as tcid /ml. prrsv-specific immunoglobulin g (igg)-type antibodies were detected in the serum using a commercially available elisa kit (bionote prrs ab . , hwaseong, republic of korea) based on the detection of the nucleocapsid protein, according to the manufacturer's instructions. the sample to positive (s/p) ratio of each serum sample was ≥ . , which was considered to be indicative of the presence of prrsv antibodies. total rna was extracted from the lung, bln, and tonsil tissues using the trizol reagent (invitrogen, life technologies) according to the manufacturer's recommendations. total rna concentrations were calculated using quant-it ribogreen (invitrogen, life technologies, carlsbad, ca, usa). to assess the rna-integrity number, samples were run on the tapestation rna screentape system (agilent technologies, santa clara, ca, usa) (additional file ). a cdna library was independently prepared with µg of total rna for each sample using the illumina truseq stranded mrna sample prep kit (illumina, inc., san diego, ca, usa). the first step in the workflow involved removing the rrna from the total rna, using the ribo-zero rrna removal kit (human/mouse/rat; illumina, inc.). subsequently, the figure overview of the study design and the measured phenotypes. a schematic representation of the experimental design in terms of the sample types, target tissues (lungs, blns, and tonsils), and time points ( dpi, dpi, dpi, dpi, dpi, and dpi) after prrsv infection. b serum and lung viral loads and serum antibody levels in prrsv-infected pigs. remaining mrna was fragmented into small pieces using divalent cations under elevated temperature conditions. the cleaved rna fragments were copied into first-strand cdna using superscript ii reverse transcriptase (invitrogen, life technologies) and random primers. this step was followed by second-strand cdna synthesis using dna polymerase i, rnase h, and dutps. the cdna fragments were subjected to an end-repair process, involving the addition of a single ' a' base, after which adapters were ligated. the products were then purified and enriched by pcr to create the final cdna library. the libraries were quantified using kapa library quantification kits for illumina sequencing platforms, according to the qpcr quantification protocol guide (roche, basel, switzerland), and the libraries were validated using the tapestation d screentape system (agilent technologies, santa clara, ca, usa). the indexed libraries were then analysed on an illumina hiseq instrument (illumina, inc.), and paired-end ( × base pair) sequencing was performed. all raw rna-seq data generated in this study were deposited in the ncbi sequence read archive database under the accession number prjna . to select the quality-filtering strategy, a quality check of raw read data was performed for each sample using the fastqc software v . . , and the reads were trimmed with adaptors using the trimmomatic software v . based on the quality results. then, the trimmed reads were re-checked with fastqc and mapped to the reference genome (sus scrofa . , gca_ . ) of the ensembl genome browser (https ://www.ensem bl.org/ sus_scrof a/) as the default option of the hisat v . . programme. raw counts corresponding to the genes in each library were calculated based on the exons in sus scrofa gtf v (ensembl) as the genomic-annotation reference file, using the featurecounts of subread package, v . . . all deg analyses for the obtained raw counts were performed using the edger software package v . . of bioconductor. to reduce statistical bias in the deg analyses, genes were excluded when all samples had raw counts of ≤ . normalisation of the raw counts was performed using the trimmed mean of m-value (tmm) method, and dispersion parameters were estimated and applied using the cox-reid profile-adjusted likelihood method in edger. degs were identified for each time point ( , , , , and dpi; versus gene expression at dpi) for each tissue (lungs, blns, and tonsils) using a negative binomial-generalised linear model, and p-values were corrected for multiple comparisons based on the false discovery rate (fdr). degs were determined based on an fdr of < . and an absolute log fold-change (fc) of ≥ . multidimensional scaling (mds) was performed using the limma function of the r package to identify the similarities among samples. gcn analysis was conducted by filtering out: (i) degs with no significant fdr (fdr < . ) observed at any of the time points in tissues and (ii) degs with a not stringent significant value (absolute log fc ≥ . ), in order to increase the efficiency of network construction. before gcn analysis, significant associations between the filtered genes were calculated using the partial correlation coefficient with information theory (pcit) algorithm [ ] . correlations were estimated to assess coexpression, and the network was constructed using genes with absolute co-expression correlations of ≥ . . gcn visualisation was performed using the cytoscape v . . software, and the resulting network consisted of genes (nodes) and connections (edges). clustering analysis was performed using the log fc values of genes in the constructed network. after determining the optimal number of clusters, the genes were analysed using the k-means clustering algorithm with iterations, using the multi experiment viewer (mev) software. the genes in the constructed gcn were classified as up-and down-regulated genes based on the time point for tissue that showed the maximum fc, and were annotated to the kyoto encyclopaedia of genes and genomes (kegg) using database for annotation, visualization and integrated discovery (david) v . . in addition, enrichment analyses were performed with bps, using gene ontology (go) terms and kegg pathways for the genes in each gcn. go annotations were filtered with the direct option and applied to enrichment analyses with the following cut-offs: p value < . and counts ≥ . next, treemaps for the enriched go terms were visualised using the revigo tool. kegg annotations were also enriched using the same cut-off criteria and are represented by the -log p value and fold enrichment. all data used in the enrichment analyses were annotated in sus scrofa. gsea for gcn group-specific genes were conducted using the gene-ranking method based on gene sets in the kegg database to determine the enrichment scores and statistically significant differences, using the gsea v . . software. all analyses were performed using the log -normalised tmm counts of the selected tissues and time points that showed the largest expression changes in each group. counts corresponding to: (i) dpi in all tissues were used for the dpi-specific group, (ii) dpi in lung tissues were used for the lung-specific group, and (iii) and dpi in bln tissues were used for the bln-specific group. gsea results were visualised as enrichment maps with significant pathways (fdr < . ) following the benjamini-hochberg correction using cytoscape, and their connections indicated the similarities between gene sets. additionally, the core enriched genes of pathways showing the highest normalised enrichment score (nes) were expressed as heatmaps. then, the modulations of responsible gene products (proteins) in the selected representative kegg pathways (determined through gcn and gsea) were confirmed using the clusterprofiler package in the r software. among the genes corresponding to each protein, genes showing the maximum changes were used as the representative values. the ppi network was investigated for the top interactions (identified in this study) with extremely high gene expression levels using the homo sapiens database of string v . . . the viral loads decreased markedly beyond dpi, and lung samples without detectable viral loads were observed during these time points. serum antibodies (igg) were first detected at dpi and their levels increased up to dpi, but slightly decreased at dpi. a total of . billion paired-end sequence reads were produced from tissue samples (from tissues of individuals), and the average of the number of reads produced per sample was . million (additional file ). the reads that passed the trimming process were mapped to pig reference genome . (~ . % identity), and the average unique mapping rate was . %, ranging from . to . %. transcriptome read data were produced using tissues (lungs, blns, and tonsils) at time points ( , , , , , and dpi), as shown in figure a . the transcriptomes produced under prrsv infection showed clear clustering for each tissue type, as determined by mds analysis (figure a ). degs were confirmed by comparing the gene expression levels at each time point ( , , , , and dpi), relative to those at dpi, and overlapping degs among tissues and different time points are shown in a venn diagram ( figure b ). we observed dynamic changes in the gene expression levels in lung and bln tissues at each time point. lung tissues demonstrated a large proportion of up-regulated genes at all time points ( dpi: %, dpi: %, dpi: %, dpi: %, and dpi: %) against dpi, whereas bln tissues mostly displayed a large proportion of down-regulated genes ( dpi: %, dpi: %, dpi: %, dpi: %, and dpi: %). interestingly, we observed a tendency towards gene up-regulation at dpi in all tissues. moreover, the numbers of degs in lung and bln tissues increased markedly at dpi, then sharply decreased, and slightly increased at dpi. tonsil tissues showed relatively subtle changes; therefore, we had difficulties in analysing them at all time points, except at dpi ( genes). gcn construction using genes and significant connections (selected using the pcit algorithm) was performed to integrate the transcriptomes for one respiratory and two immunity-related tissues at all time points (figure ). the locations of genes were marked close to one another when many common neighbours were found. the resulting network was precisely concentrated for three groups ( dpi, lung tissue, and bln tissue), and the groups were linked by some genes. five clusters were identified via clustering analysis using the log fc values of genes in the gcn and were specifically matched to each group. cluster , representing the dpi-specific group, was composed of genes that were up-regulated in all tissues with prrsv at dpi. clusters and , which represented the lung-specific group, contained down-regulated genes at dpi and up-regulated genes at - dpi in the lungs, respectively. the blnspecific group comprised two clusters of strongly downregulated genes ( genes in cluster and genes in cluster ) at and dpi in bln tissues. analysis of cluster showed that the genes were slightly up-regulated at and dpi in blns. additionally, the bln-specific group contained some genes that showed maximum changes in the tonsils. we selected representative up-and down-regulated genes at different time points in different tissues with relatively high absolute fc values in the constructed gcn and categorised them as follows: up-regulated genes and down-regulated genes; significant pathways were identified by performing kegg enrichment analyses using david ( figure a ). the bubble plot of the enriched biological pathways revealed up-regulated genes in kegg pathways that are mainly related to immune responses (i.e., cytokine-cytokine receptor interaction, influenza a, cytosolic dna-sensing pathway, and rig-i like receptor signalling pathways) and down-regulated genes in pathways mainly related to energy metabolism (i.e., nitrogen metabolism, ppar signalling pathway, and ampk signalling pathway). these pathways provided important insights into the specific immune responses of host cells to prrsv infection. functional-enrichment analyses based on the kegg ( figure b -d) and go ( figure e -g) databases were performed to investigate the biological processes (bps) associated with each group following prrsv infection. the kegg pathways of the dpi-specific group were enriched for terms related to viral infection, including influenza a, the rig-i-like receptor signalling pathway, herpes simplex infection, and the cytosolic dna-sensing pathway ( figure b ). in the lung-specific group, kegg terms associated with immune responses, such as cytokine-cytokine receptor interaction, rheumatoid arthritis, and the chemokine signalling pathway, were identified ( figure c) . interestingly, the bln-specific group showed kegg terms related to lipid metabolism such as the ampk signalling pathway, the ppar signalling pathway, glycerolipid metabolism, and the adipocytokine signalling pathway ( figure d ). the illustrated treemaps revealed bps for significant go terms such as defence response to virus and negative regulation of viral genome replication in the dpi-specific group (figure e ); immune response in the lung-specific group ( figure f ); and regulation of epithelial cell proliferation in the bln-specific group ( figure g ). the enrichment results were consistent between the kegg and go terms. to validate the results of specific groups, gsea based on the kegg database were performed using log -normalised tmm counts at each time point for each tissue to identify the maximum gene expression changes for each group, and the gsea results were consistent with the kegg enrichment analyses of degs. gsea using the -dpi data for all tissues revealed many significant pathways related to viral infection, among which influenza a showed the highest nes ( figure a ). the expression levels of core enriched genes in influenza a were visualised by generating a heatmap, and (rsad , ddx , cxcl , mx , rnasel, ifnb , ifih , oas , ifn-alphaomega, il figure b ). gsea using the -dpi data for lung tissues revealed many significant pathways related to immune responses, among which the cytokine-cytokine receptor interaction pathway showed the highest ness (figure a) . the expression levels of core enriched genes involved in cytokine-cytokine receptor interactions were visualised by generating a heatmap, and (tnfrsf , tnfrsf , tnfrsf b, ccl , cd , cxcr , il ra figure b ). the gsea results obtained with the -and -dpi data for the bln tissues could not be used to construct an enrichment map because only a few statistically significant pathways and low similarity were observed among the gene sets. in the ppi network, the top proteins (i.e., those with the strongest interactions with ifnb (ifnb ), and the largest differences in expression levels) were all immunerelated proteins (ifnar , irf , irf , ifnar , stat , irf , socs , irf , stat , irf , ifih , tyk , irf prrsv infection in pigs can cause a complicated disease when functioning as a primary respiratory infectious agent or as a cofactor in porcine respiratory disease complex (prdc), and prrsv was reported to be the most common virus associated with prdc [ ] [ ] [ ] [ ] . in addition, prrsv was reported to inhibit the host immune defence system, which can lead to further infections (secondary/opportunistic pathogens), resulting in more serious and chronic diseases [ , ] . therefore, understanding the functional and regulatory mechanisms of respiratory and immunity-responsible tissues in the host during prrsv infection is essential for preventing and controlling diseases directly linked to animal productivity. in this study, we compared and integrated serial whole transcriptomes for three tissues (lungs, blns, and tonsils) during prrsv infection. interestingly, the lungs had lower viral loads than the serum ( figure b) , even though the lungs showed major symptoms during prrsv infection [ ] . based on mds analysis, a well-defined (see figure on next page.) ; b and e) , lung-specific network (clusters and ; c and f), and bln-specific network (clusters and ; d and g) were used for the analyses. kegg-enriched pathways for the whole gcn network (a) and each specific network (b-d) were visualised by generating bubble and bar plots. a bubble plot corresponding to the whole network was generated for each analysis for the up-regulated (red) and down-regulated genes (blue) (a). data information: significantly enriched pathways represented in the plots met the following cut-off criterion: − log p value > . . go treemaps were created based on the p values associated with the bp terms for each specific network (e-g). lim et al. vet res ( ) : trajectory of transcriptomes related to prrsv infection was identified in each tissue (figure a ). the number of degs increased until dpi, then decreased until dpi, and finally increased slightly at dpi ( figure b ). the drift curves for the degs were very similar to serum and lung viral load curves, confirming that the overall host responses according to prrsv infection may be most active at approximately dpi, although they were not comparable to the serum antibody levels ( figure b) . previous reports showed that viral loads were detectable in lymphoid organs (excluding the spleen) until immediately before viral extinction [ , ] , although no similarity prrsv can infect cells of the macrophage and monocyte lineages in pigs, and thus, it may affect many aspects of tissue remodelling, development, immunity, and pathology [ ] . prrsv can delay innate and adaptive immune responses by inhibiting the production of type-i interferons (ifns), especially ifn-α, which is important for intracellular signal transduction [ , ] . because the lungs are known as the main target of prrsv infection and lymphoid organs can serve as viral reservoirs [ ] , the observation of common degs probably indicates identical host responses among these tissues, whereas unique degs for each tissue may serve specific functions in the respective tissues during prrsv infection. weighted gcns, which indicate specific gene expression changes, have been used as a powerful approach for identifying specific molecular mechanisms at the system level [ ] . we integrated the transcriptomes of multiple tissues and time points by constructing a gcn with stringent degs and a pcit algorithm (figure ). the constructed network revealed a clear separation of one time point group ( dpi), one respiratory responsible tissue group (lungs), and one immunity-responsible tissue group (blns), and each group showed dynamic changes in the host-response system after prrsv infection. in addition, the genes included in the lung and bln groups showed specific differential expression in each tissue, whereas the genes included in the -dpi group were commonly expressed in all tissues. these features suggest that host responses to prrsv infection are mainly regulated through the expression of different genes in the lungs and lymphoid organs (blns and tonsils). furthermore, gene expression levels may be similarly regulated at dpi in three tissues. dynamic changes in these significant gene subsets at dpi may represent biological signals associated with general and early immunological mechanisms in response to virus infection. significant kegg terms related to viral infections and immune signalling were identified through kegg enrichment analyses performed for each up-and down-regulated genes ( and genes, respectively), based on the time point for each tissue with a maximum fc value in the gcn, which was constructed using serial degs ( figure a ). in particular, the enriched terms related to rna viral infection (influenza a, the rig-i-like receptor signalling pathway, measles, hepatitis c virus (hcv), and the toll-like receptor signalling pathway) and innate immune signalling (cytokine-cytokine receptor interaction, the jak-stat signalling pathway, haematopoietic cell lineage, and the chemokine signalling pathway) were found among the highly significant pathways in the dpi-specific group ( figure b ). in addition, gsea results based on the expression levels at dpi in all tissues revealed clustering with kegg terms related to viral infection, and influenza a showed the highest nes (figure a) . previous reports showed that rig-i-like receptors (rlrs), consisting of three proteins (rig-i, mda , and lgp ), are one type of pattern-recognition receptors (prrs) that activate innate immune-signalling pathways by detecting viral rna in the cytosol [ , ] . influenza a viruses are rna viruses that express non-structural protein (ns ), which is important for evading toxic innate immune responses. ns can suppress mrna processing and transport, which inhibits the binding of the viruses to double-stranded rna molecules and restrains rlr activation [ ] [ ] [ ] . these functions are known to interfere with, and delay, both the expression of the cytokine ifnα/β and the initiation of an ifn-induced antiviral state [ ] . it has been reported that both the structural and non-structural proteins of prrsv have polygenic toxicity in hosts [ ] , particularly, nsp tf and nsp n generated by a ribosomal frameshift mechanism affect the suppression of cellular innate immune responses [ ] . these features of prrsv can be very susceptible to ifn-α/β, although they do not exhibit typical innate immune-signalling activation, including type-i ifn responses [ ] . in addition, signalling associated with the rig-i-like receptor and jak-stat pathways, which play important roles in ifn production, can become disrupted by prrsv during acute infection [ ] . additionally, analysing the lung transcriptomes of prrsv (north american strain ch a)-infected pigs (landrace × yorkshire) showed degs associated with inflammatory signalling at dpi [ ] , which is similar to our results. thus, the enriched biological terms for the dpi-specific group found in this study suggest that both prrsv and influenza a virus, both of which cause respiratory illnesses, exhibit similar infection mechanisms, immune evasion, and innate immune signalling. additionally, the rig-i-like receptor is thought to function as the main prr for prrsv during early immune responses. kegg enrichment analysis of the lung-specific group (containing clusters and of the gcn) implicated cytokine-cytokine receptor interactions, cell-adhesion molecules, rheumatoid arthritis, and chemokine signalling pathways in prrsv infection, all of which are related to immune signalling ( figure c ). cluster included several genes related to immune responses, which were especially up-regulated at - dpi in the lungs (figure ). gsea at dpi revealed clustering of kegg terms related to immune signalling (which showed the largest changes in expression levels), among which cytokine-cytokine receptor interactions showed the highest nes ( figure a ). in contrast to the common early immune responses, terms in the lung-specific group were not enriched for innate immune signalling. moreover, lung viral loads were not detected after dpi, and the levels of anti-prrsv antibodies (igg) started to increase after approximately dpi ( figure b ). therefore, based on the viral loads and antibody levels found in this study, we proposed that the immune signalling pathway terms identified in the lung-specific group represent the adaptive immune-response mechanism occurring in the respiratory system. many studies regarding the adaptive immune responses that occur during prrsv were mainly focused on humoral responses associated with various cytokines and the development of cell-mediated immunity (cmi). with regard to humoral responses, it has been reported that non-neutralising antibodies against prrsv proteins (i.e., the n protein and non-structural proteins) are produced beginning at approximately dpi, whereas neutralising antibodies (nas) were detected later after approximately dpi [ , ] . although prrsv-specific t cells were previously observed early in lymphoid tissues (beginning at approximately dpi), the t cell responses did not last long and did not correlate with viral loads [ ] . in addition, another study showed that the activity of ifn-γ-secreting cd + t cells against prrsv was weak and delayed [ , ] . as previous studies have shown, nas (humoral responses) and cellular responses (cmi) involved in adaptive immune signalling against prrsv were detected at abnormally low levels and were delayed, which may reflect the sequential triggering of interference and delayed innate-immune signalling. lymphoid organs (blns and tonsils) were not highlighted in the lung-specific group due to weaker immune responses in these tissues compared to those in the lungs. significant go terms related to immune responses and apoptosis were identified in each tissue at - dpi in the bln in this study (additional file ), but the tonsils tissues were difficult to study in terms of associated mechanisms because of the small number of degs. in agreement with the results of this study, a previous study demonstrated that changes in the expression of proinflammatory cytokines (il- α, tnf-α, and il- ) at to dpi in prrsv (european strain )-infected lymphoid organs (retropharyngeal lymph nodes, mediastinal lymph nodes, and tonsils) were found only in the lymph nodes [ ] . consequently, we discovered that adaptive host immune signalling in response to prrsv infection was relatively active in the lungs, although such signalling was significantly lower in lymphoid organs (blns and tonsils). the bln-specific group contained clusters and (showing down-regulated genes) and was highly enriched for the ampk signalling pathway, the ppar signalling pathway, glycerolipid metabolism, and the adipocytokine signalling pathway, which are related to lipid metabolism ( figure d) . during viral infection, lipid metabolism can be regulated by viruses; it promotes membrane fusions during viral entry and efficient replication [ , ] . among these pathways, several viruses have been reported to inhibit ampk signalling, which plays a major role in cellular-energy homeostasis including lipid production. human immunodeficiency virus (hiv) is known to encode a trans-activating regulatory protein (tat), which has been reported to inhibit phosphorylation of the ampk α-subunit at thr and to concomitantly reduce the phosphorylation of the ampk substrate, acetyl-coa carboxylase [ ] . it was also reported that the hcv proteins ns b and ns a can activate protein kinase b (pkb/akt), which inhibits ampk by phosphorylating ser and thr [ ] [ ] [ ] [ ] . moreover, previous reports showed that hiv, hcv, and influenza a virus replication could be inhibited by -aminoimidazole- -carboxamide ribonucleotide (aicar), which is an ampk activator [ , , ] . marc- and porcine monocyte-derived dendritic cells (mdcs) infected with prrsv (north american strain p -gfp) showed significant suppression by ampk activators (sodium salicylate and u a) [ ] . recently, it was confirmed that ampk activity increased at up to . dpi in pk- cd cells, that pams were infected with prrsv (north american strain wuh ), that replication was inhibited through acetyl-coa carboxylase (acc ), and that fatty acid biosynthesis was reduced by the ampk activator a [ ] . in addition, proteins nsp , nsp , and nsp of prrsv are also well known to induce cell membrane rearrangement for efficient replication [ ] , and it may interact with lipid metabolism such as ampk signalling. in this study, we performed enrichment analyses for the bln-specific group in the gcn, which confirmed that genes related to ampk signalling were only downregulated in bln tissues (figure ) . these results suggest that prrsv regulates ampk signalling to create a suitable environment for viral replication in lymphoid tissues including blns, which function as a reservoir, thereby establishing persistent infection. we examined highly significant pathways including influenza a and cytokine-cytokine receptor interactions through enrichment analyses for each group in the gcn construction and gsea, based on all genes. we also intensively investigated the expression levels of significant genes identified through both analyses. firstly figure b ). all selected genes were associated with antiviral and immune signalling. in particular, rsad (viperin), ddx (rig-i), mx (mxa), and oas ( ′- ′oas and oas) were highly expressed in all tissues at dpi (log fc ≥ . ), and ifnb (ifnβ) and ifn-alphaomega (ifnα) were extremely highly expressed at dpi in the lymphoid organs (blns and tonsils) (log fc ≥ . ). rsad (viperin) encodes a cellular protein that was found to inhibit the replication of various dna and rna viruses, including influenza a [ ] . during influenza a infection, viperin decreased lipid raft formation by reducing the activity of farnesyl diphosphate synthase, which inhibited viral budding and release [ ] . in addition, viperin up-regulation in prrsv (north american strain bb )-infected marc- cells was reported to inhibit viral replication [ ] . rig-i, a prr responsible for type-i interferon responses, is an essential molecule for innate immune signalling that recognises virus-infected cells in mammals, including pigs [ ] . the human mxa protein has been shown to exert antiviral activity against a wide range of rna viruses (including influenza a) and some dna viruses [ ] . moreover, expression of the porcine mx protein was reported to increase at up to dpi in prrsv (american strain snuvr )-infected marc- cells [ ] . oas is known to encode a member of the - a synthetase family, which comprises essential proteins involved in the innate immune responses to viral infection; it promotes viral rna degradation and inhibition of viral replication [ ] . furthermore, it was reported that oas overexpression in pams inhibited prrsv (north american strain bj- ) replication [ ] . ifn-alphaomega encodes a unique isoform found only in a few species such as pigs and cows [ ] , and its antiviral activity against prrsv (north american strain sdsu- -p ) infection was found to differ depending on the target cell [ ] . in addition, ifnb , one of the representative signalling genes involved in antiviral innate immunity in mammals including pigs, showed high antiviral activity in pams, but not in marc- cells, indicating an opposite effect compared to that of ifn-alphaomega [ ] . in this study, we also focused on the top proteins showing high interactions with ifnb through the ppi network (figure ) , some of which (ifnar , ifnar , irf , irf , irf , stat , and stat ) were associated with ifn-mediated immune responses (autocrine and paracrine signalling) and promoted ifn secretion through intracellular pathogen recognition [ ] . generally, pathogens are recognised by prrs, and ifn expression can be induced in the nucleus through the activation and phosphorylation of irf and irf . ifns secreted from cells can bind to the receptors ifnar and ifnar , which initiates signal transduction pathways; subsequently, ifn-stimulated genes are expressed after the formation of the isgf complex (irf , p-stat , and p-stat ). collectively, these findings suggest that the host responses at dpi in all tissues were associated with up-regulated genes related to antiviral signalling that were common to the case of influenza a infection, and immune-related genes specifically expressed in lymphoid organs (blns and tonsils) were identified. in particular, the ifn-alphaomega and ifnb genes, which encode type-i interferons (which were expressed at extremely high levels in lymphoid organs), need to be further studied, so as to clarify their molecular functions in prrsv infection. secondly, cytokine-cytokine receptor interactions (confirmed by gcn-enrichment analysis and gsea) included up-regulated expression of tnfrsf (ox ), tnfrsf ( - bb), tnfrsf b (dcr ), ccl (ccl ), cd (cd ), cxcr (cxcr ), il ra (il ra), tnfsf (rankl), ccr (ccr ), ccl (ccl ), il (il ), and il rb (il rb), which include adaptive immune-related genes ( figure a ). tnfsf and tnfrsf are known to control innate and adaptive immune cells by regulating various mechanisms, inducing the costimulation or co-inhibition of immune responses [ ] . ox is a co-stimulatory receptor expressed in activated t cells after antigen recognition, and interaction with its ligand can promote t cell proliferation and survival, as well as cytokine production. - bb is an inducible co-stimulatory receptor that is mainly expressed in t cells, and interaction with its ligand is essential for t cell development, survival, proliferation, effector function, and memory t cell formation [ ] . rankl is a strongly up-regulated ligand in t cells after antigen receptor stimulation, and it affects bone homeostasis, lymphoid organ development, and t cell-dendritic cell interactions [ ] . in summary, we identified genes affecting t cell maturation, proliferation, and survival with respect to adaptive immune signalling at dpi; however, further studies should be performed to clarify the roles of these genes during prrsv infection. a possible concern regarding the generation of serial data for these respiratory and immune tissues is the use of tissue samples obtained from different animals at each time point. however, the trends observed for serum viral loads and antibody titres ( figure b ) corresponded with those of previous studies [ , , ] that used blood collected from genetically identical pigs from multiple time points, indicating that the pigs used in this study could have had similar responses to prrsv at any given time point. we performed deg profiling at six serial time points with one respiratory and two immunity-responsible tissues during prrsv infection, based on rna-seq data. additionally, three groups with specific expression patterns (i.e., the -dpi, lung, and bln groups) were discovered by integrating the data via gcn construction. our findings suggested the involvement of key signalling pathways through functional-enrichment analyses. at dpi, all three tissues showed antiviral and innateimmune signalling similar to the case for influenza a infection, with the lymphoid organs (blns and tonsils) showing relatively stronger expression levels in response to infection than the lungs. moreover, we observed the adaptive immune responses that were most active in the lung tissues, based on high expression levels of various cytokines, whereas the responses were relatively weak in the lymphoid organs. independently, ampk signalling appeared to be down-regulated specifically in bln tissues, resulting in chronic infection through a direct relationship with viral replication. these results provide an understanding of the host's regulatory mechanisms and should be useful for vaccine development and studying prrsv resistance. furthermore, gene expression can be regulated by epigenetic mechanisms including dna methylations, histone modifications, and non-coding rnas. therefore, we suggest the need for future studies that identify an association between gene expression and epigenetic changes through the integration of multi-omics layers. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . additional file . table s . rna quality scores. table s . overview of data processing. table s control of porcine reproductive and respiratory syndrome (prrs) through genetic improvements in disease resistance and tolerance characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) den besten a, wagenaar f ( ) mystery swine disease in the netherlands: the isolation of lelystad virus porcine reproductive and respiratory syndrome challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology the challenge of prrs immunology immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection polymorphisms in the porcine cd associated with response to prrsv infection gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virus-infected pigs porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf activation in immortalized porcine alveolar macrophages evidence for a major qtl associated with host response to porcine reproductive and respiratory syndrome virus challenge lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero bioinformatic analyses in early host response to porcine reproductive and respiratory syndrome virus (prrsv) reveals pathway differences between pigs with alternate genotypes for a major host response qtl differences in whole blood gene expression associated with infection time-course and extent of fetal mortality in a reproductive model of type porcine reproductive and respiratory syndrome virus (prrsv) infection understanding prrsv infection in porcine lung based on genomewide transcriptome response identified by deep sequencing analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus the effect of prrs viral level and isolate on tonsil gene expression combining partial correlation and an information theory approach to the reversed engineering of gene co-expression networks effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus bordetella bronchiseptica, or a combination of both organisms in pigs immunostimulation, pcv- [porcine circovirus] and pmws mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia porcine respiratory disease complex innate and adaptive immunity against porcine reproductive and respiratory syndrome virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model macrophages as apc and the dendritic cell myth interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus polyclonal activation of b cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (prrsv)-infected pigs integrated transcriptomes throughout swine oestrous cycle reveal dynamic changes in reproductive tissues interacting networks molecular mechanisms of viral inhibitors of rig-i-like receptors rig-i-like receptor regulation in virus infection and immunity influenza virus evades innate and adaptive immunity via the ns protein the influenza virus ns protein: inhibitor of innate and adaptive immunity interplay between influenza a virus and the innate immune signaling influenza a virus lacking the ns gene replicates in interferon-deficient systems the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis nonstructural proteins nsp tf and nsp n of porcine reproductive and respiratory syndrome virus (prrsv) play important roles in suppressing host innate immune responses the presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera the level of virusspecific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load role of neutralizing antibodies in prrsv protective immunity lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (prrsv) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (pbmc) from pigs vaccinated and exposed to natural infection differential expression of proinflammatory cytokines in the lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs lipids at the interface of virushost interactions multifaceted roles for lipids in viral infection sirt regulates tat-induced hiv- transactivation through activating amp-activated protein kinase the hepatitis c virus ns a protein activates a phosphoinositide -kinase-dependent survival signaling cascade hepatitis c virus nonstructural b protein modulates sterol regulatory element-binding protein signaling via the akt pathway insulin antagonizes ischemia-induced thr phosphorylation of amp-activated protein kinase α-subunits in heart via hierarchical phosphorylation of ser / enhanced hepatitis c virus genome replication and lipid accumulation mediated by inhibition of amp-activated protein kinase peroxisome proliferatoractivated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice the ancient drug salicylate directly activates amp-activated protein kinase fatty acids regulate porcine reproductive and respiratory syndrome virus infection via the ampk-acc signaling pathway viperin, a key player in the antiviral response the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from monkey viperin restricts porcine reproductive and respiratory syndrome virus replication rig-i in rna virus recognition human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity expression of interferon-α and mx protein in pigs acutely infected with porcine reproductive and respiratory syndrome virus (prrsv) the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities porcine ′, ′-oligoadenylate synthetase inhibits porcine reproductive and respiratory syndrome virus replication in vitro evolution of the class cytokines and receptors, and discovery of new friends and relatives differential expression and activity of the porcine type i interferon family differential regulation of type i and type iii interferon signaling the tnf family of ligands and receptors: communication modules in the immune system and beyond agedependent resistance to porcine reproductive and respiratory syndrome virus replication in swine emergence of two different recombinant prrsv strains with low neutralizing antibody susceptibility in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all data generated or analysed during this study are included in this published article. all animal experiments were approved by the jeonbuk national university institutional animal care and use committee, republic of korea (approval number - ). not applicable. the authors declare that they have no conflict of interest. key: cord- -cmjfqkjz authors: cruz, jazmina l.g.; zúñiga, sonia; bécares, martina; sola, isabel; ceriani, juan e.; juanola, sandra; plana, juan; enjuanes, luis title: vectored vaccines to protect against prrsv date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: cmjfqkjz prrsv is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. commercially available vaccines are only partially effective in protection against prrsv. moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. therefore, new efficient vaccines are required. vaccines based on recombinant virus genomes (virus vectored vaccines) against prrsv could represent a safe alternative for the generation of modified live vaccines. in this paper, current vectored vaccines to protect against prrsv are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. special attention has been provided to the use of transmissible gastroenteritis virus (tgev) as vector for the expression of prrsv antigens. this vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. a tgev derived vector (rtgev) was generated, expressing prrsv wild type or modified gp and m proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. several strategies are proposed to improve current rtgev vectors expressing prrsv antigens. prrsv is the causative agent of the most important infectious disease affecting the porcine herds worldwide. the immune response to prrsv is poorly understood but, in spite of this, some vaccines are being commercialized. commercial vaccines are mostly modified live vaccines based on attenuated european or north american prrsv strains (i.e., ingelvac ® -prrs from boehringer ingelheim, amervac ® -prrs from hipra, or pyrsvac- from syva labs). nevertheless, some inactivated vaccines are also available (i.e., progressis ® from merial, ingelvac ® -prrs kv from boehringer ingelheim, or suipravac ® -prrs from hipra). modified live vaccines have been preferentially used, as they can establish protective immunity, measured by viral load in blood and tissues. nevertheless, current vaccines against prrsv have several limitations. in general, modified live vaccines protect against challenge with homologous isolates. they could also protect against heterologous viruses (diaz et al., ; zuckermann et al., ) . furthermore, live vaccines provide partial protection against clinical disease but did not prevent infection (osorio et al., ) and, more importantly, they can revert to virulence (botner et al., ; nielsen et al., ) . as the attenuated vaccines induce an immune response resembling that induced by prrsv natural infection, they do not induce high levels of neutralizing antibodies. killed prrsv vaccines, on the other hand, in general, have been less effective in prevention of both infection and disease (ostrowski et al., ) . the innate immune response against prrsv is very weak, probably contributing to the delay in subsequent humoral and cellular immune responses, and also to virus persistence (kimman et al., ). prrsv does not induce interferon (ifn)-␣ production (albina et al., ; calzada-nova et al., ) , a key element in host antiviral response, leading to a minimal production of inflammatory cytokines and activation and recruitment of natural killer (nk) cells (murtaugh et al., ) . prrsv-induced suppression of type i ifn production is due to the interference in the activation of ifn-␤ promoter stimulator (ips- ), located downstream of sensor molecule rna helicase rig-i. the inactivation of ips- avoids ifn regulatory factor (irf) activation and, consequently, type i ifn production (luo et al., ) . therefore, to design an effective vaccine against prrsv, it would be advisable to increase the production of type i ifn. to date, different adjuvants promoting the production of ifn have been tested, in addition to the current vaccines formulations, with limited success (charerntantanakul, ) . a hallmark of the swine humoral response against prrsv is the production of non-neutralizing antibodies detected early in the infection, followed by a low neutralizing antibody (nab) titer that is detected more than weeks after infection (kimman et al., ; murtaugh et al., ) . one possible explanation for the late detection of nabs is the difference on technique sensitivity, as elisa has higher sensitivity than neutralization assays. therefore, the presence of very low titers of nabs early in the infection cannot be completely discarded. early non-neutralizing antibodies are mainly induced by nucleocapsid (n), m and gp proteins, and have been involved in antibody-dependent enhancement of prrsv infection (mateu and diaz, ; murtaugh et al., ) . nabs are induced by gp , gp , gp and m proteins, although the ones recognizing gp are the most relevant for protection (kim and yoon, ; ostrowski et al., ) . two b cell epitopes were identified in gp protein ectodomain: an immunodominant epitope (ide), that has been proposed to act as a decoy epitope, and an epitope critical for neutralization (ecn), that is recognized by nabs (ostrowski et al., ) . several hypothesis have been proposed to explain the delay in nabs induction by gp protein, such as the presence of the ide, and glican-shielding of the ecn (lopez and osorio, ) . the role of nabs in protection was demonstrated by passive transfer of these antibodies . protection of swine against prrsv infection correlated with the level of nabs and it was proposed that an efficient vaccine must induce nab titers of : to prevent prrsv infection (lopez et al., ) . prrsv infection results in a weak and delayed t cell mediated immune response that should be necessary for the elimination of the virus (mateu and diaz, ; murtaugh et al., ) . it has been shown that the induction of ifn-␥ secreting cells, complementing neutralizing antibodies, provides partial protection against prrsv (zuckermann et al., ) . as interleukin (il)- levels inversely correlate with ifn-␥ response, it has been proposed that the expression of il- may be responsible for the suppression of t cell responses (charerntantanakul et al., ; kimman et al., ). m protein is the most potent inducer of t cell proliferation, followed by gp , gp and gp (bautista et al., ) , and may play a role in protection. different vaccine adjuvants have been tested to improve t cell responses to prrsv. nevertheless, in addition to the adjuvants included in vaccine formulation, only il- and cpg oligodeoxynucleotides enhanced protection conferred by current vaccines (charerntantanakul, ). there are three main problems for the development of more efficient vaccines against prrsv: the correlates of protection are not well known, prrsv may induce negative regulatory signals for the immune system, and there is a extremely large antigenic variability in prrsv structural proteins. as indicated above, the prrsv heterodimer gp -m must be the main inducer of protective humoral and cellular responses. nevertheless, minor structural proteins are also required for prrsv virion infectivity (wissink et al., ) and may play a role in protection. also, there is limited information about the t cell epitopes implicated in the induction of a protective t cell response (mateu and diaz, ) . one of the mechanisms used by viruses to suppress or evade the host immune response is the induction of regulatory t cells (treg). porcine treg phenotype is cd + cd + foxp + (kaser et al., ) , as that described for human and mice (belkaid, ) . tregs have been classified in natural and induced. the latter ones can be subdivided in three subtypes: treg (tr ) secreting il- , t helper (th ) secreting transforming growth factor (tgf)-␤, and converted tregs (belkaid, ) . it has been recently described that american type prrsv-infected dendritic cells induced tregs, an effect that was reverted by the addition of ifn-␣. the induced treg population is a th type, as it promotes tgf-␤ but not il- expression (silva-campa et al., ) . in contrast, dendritic cells infected with eu type prrsv viruses did not induce treg cells, although they exhibited an unbalanced ability to stimulate t cell immune responses (silva-campa et al., ) . the impact of treg induction on delayed immune responses after prrsv infection remains to be established, as well as the viral proteins involved in this process. prrsv strains are extremely diverse, even when they belong to the same genotype. among the structural proteins, m protein is the most conserved one, while gp is the most variable one (dea et al., ) . this high antigenic variability represents a problem for the development of universal vaccines against prrsv, as shown by the low efficacy of current vaccines against heterologous challenge. to solve this problem, common critical b and t cell epitopes must be identified (mateu and diaz, ) . nevertheless, it has been reported that the ability of a vaccine to induce a strong cellular immune response may be more important for protection than the genetic similarity with the challenge strain (diaz et al., ) . an additional problem is the difference in the immune responses elicited by prrsv in animals with different host genetic background (lewis et al., ) . therefore, the knowledge of host responses to prrsv infection is required for the development of an efficient vaccine. as mentioned above, both modified live and inactivated vaccines have been developed for prrsv. live vaccines led to better results than killed-virus based vaccines. nevertheless, the live attenuated vaccines have several problems such as incomplete protection, virus shedding and possible reversion to virulence (kimman et al., ). this problem was increased by the use of potentially hazardous methods to control the disease, such as the use of live field virus to vaccinate pigs. vector-based vaccines could represent an advantage to stimulate both humoral and cell immune responses against prrsv, and for the design of a marker vaccine. nevertheless, the results reported to date using viral vectors are not fully satisfactory and new vectors, or antigenic combinations, must be explored. prv, also known as aujeszky's disease virus (adv) is an alphaherpesvirus, classified within the family herpesviridae. prv is the causing agent of pseudorabies that was a worldwide-spread economically important disease. swine is the natural host of prv, but the virus also infects a broad range of vertebrates, including farm animals (pomeranz et al., ) . in order to eradicate the virus, modified live vaccines have been successfully used. all the vaccine strains were ge − phenotype, i.e., have a ge gene deletion. the elimination of ge causes virus attenuation by reducing the virus transmission, but does not reduce virus production in cell culture nor the induction of protective immunity (nauwynck et al., ) . these live attenuated prv have been used as vectors to protect against swine infectious diseases, such as classical swine fever (hooft van iddekinge et al., ) , or porcine circovirus (ju et al., ) . a recombinant prv, based on the attenuated bartha strain, was constructed expressing prrsv gp protein (qiu et al., ) . protection was evaluated by inoculation of -week-old piglets and homologous challenge with prrsv ch- strain. none of the animals, even those inoculated with a commercially available inactivated vaccine, developed anti-gp antibodies before challenge. after challenge, the production of anti-gp antibodies was detected in all animals. nevertheless, none of them produced neutralizing antibodies against prrsv (qiu et al., ) . reduced lung lesions and viremia, and faster virus elimination from tissues was observed in animals inoculated with prv vaccine vector, similar to that found in animals inoculated with the commercial vaccine (qiu et al., ) . alternative recombinant attenuated prv vaccine vectors expressing different combinations of prrsv antigens have also been generated. these vectors expressed gp alone or together with m protein, or modified gp (gp m), containing a pan dr thelper cell epitope (padre) between the decoy epitope and the ecn, recognized by nabs (fang et al., ) , alone or co-expressed with m protein. the gp -m heterodimer was detected in the recombinant prvs co-expressing both proteins, suggesting that prrsv antigenic structures were not changed (jiang et al., c) . the prv co-expressing gp m and m proteins was the most promising candidate in the induction of neutralizing antibodies and lymphocyte proliferation, as tested in the mouse model. as a consequence, the protection conferred by this vector was evaluated in the porcine respiratory model, in relation to the protection provided by a commercially available prrsv killed vaccine. animals inoculated with the recombinant prv expressing prrsv proteins developed nabs before the challenge. furthermore, after challenge, the nab titer was up to -fold higher in animals inoculated with recombinant prrsv compared with those inoculated with the killed vaccine. none of the animals inoculated with the empty prv developed neutralizing antibodies at any time during the experiment (jiang et al., c) . lymphocyte proliferative responses were also higher in animals inoculated with the recombinant prv expressing gp m and m proteins. accordingly, lung lesions and viremia were lower in these animals, indicating a certain protection against the homologous challenge (jiang et al., c) . adenoviruses are currently one of the most extended systems for gene delivery. as vectors, they have high capacity for the insertion of foreign genes (from kb up to kb, depending on the system), and are able to transduce a broad range of cell types (bantounas and uney, ) . different replication-defective recombinant adenoviruses (rad) have been used as vectors for prrsv, both for vaccine development and for analysis of immunogenic properties of prrsv wt or modified structural proteins. a set of rads expressing prrsv gp , m and a m-gly-thr-thr-gp fusion protein were generated. these rads were tested in the mouse model. the rad expressing m-gp fusion protein induced and increased neutralizing antibodies humoral immune response, compared with mice inoculated with rad expressing gp and m proteins independently or empty adenovirus vector . the rad expressing m-gp fusion protein also induced enhanced lymphocyte proliferation and cytotoxic t-lymphocyte (ctl) responses . unfortunately, protection conferred by these vectors was not evaluated in the porcine system. the same authors also generated a set of rads expressing other prrsv structural protein combinations, such as gp , gp or gp alone, and gp -gp , gp -gp or gp -gp -gp fusion proteins . mice inoculated with rads expressing fusion proteins developed higher nab titers and lymphocyte proliferation responses than those inoculated with rads expressing independent prrsv proteins. interestingly, specific ctl responses were higher in mice inoculated with rads expressing gp -gp or gp -gp -gp fusion proteins . in fact, authors selected the recombinant rad gp -gp as the best vaccine candidate for testing protection in pigs. this recombinant induces nabs in vaccinated piglets before challenge, and higher lymphocyte proliferation responses, il- and ifn-␥ production. nevertheless, the rad expressing gp -gp fusion protein did not fully protect against homologous challenge, as only a moderate decrease in lung lesions and viremia was observed wang et al., ) . to improve the efficacy of the rad-based vaccine, heat shock protein (hsp) and granulocyte-macrophage colony stimulating factor (gm-csf) were co-expressed as genetic adjuvants wang et al., ) . a set of rads was obtained, expressing hsp - xgly-gp -gp and hsp - a-gp -gp fusion proteins, with a five glycine or a a protease linker, respectively. piglets inoculated with rad expressing fusion proteins induced higher nab titers, produced higher ifn-␥ levels, and presented reduced lung lesions, than those inoculated with rad expressing gp -gp protein . introduction of a protease between the hsp and prrsv fusion protein resulted in a better production of il- by inoculated animals, and also lower viremia. this could be due to the release of native hsp with higher adjuvant activity . gm-csf has been widely used as an effective mucosal adjuvant (toka et al., ) . intranasal inoculation of vectors expressing gm-csf stimulates ifn-␥ and il- production in lung tissues (bukreyev et al., ) . a rad expressing gm-csf-leu-glu-gp -lys-leu-gp fusion protein was generated. a moderate increase in nab levels was observed before challenge in piglets inoculated with this rad vector, compared with animals inoculated with empty rad or rad expressing gp -gp fusion protein alone. after challenge, animals inoculated with rad expressing the fusion protein containing the adjuvant developed significantly higher nabs than the control animals . lymphocyte proliferation responses and ifn-␥ and il- production were also enhanced in those animals. these enhanced immune responses correlated with a significant decrease in the viremia and lung lesions, indicating that gm-csf enhanced the immunogenicity of rad-based gp -gp vaccine . adenovirus vectors have also been used to evaluate the antigenicity of prrsv structural proteins, such as gp and gp , and the role of gp glycosylation on immune responses (jiang et al., a,b) . unfortunately, these studies have been performed using the mouse model. the effect of ifn-␣ for protection against prrsv has recently been analyzed using rad. piglets were inoculated with a rad expressing porcine ifn-␣ and challenged with prrsv (brockmeier et al., ) . results obtained indicate that the presence of ifn-␣ has a moderate protective effect against prrsv infection. poxviruses are the largest known animal dna viruses. they have been extensively used as expression vectors for vaccination, allow expression of large foreign genes, induce strong cell mediated and humoral immune responses, and safe poxvirus vectors are available (paoletti, ; wang et al., ) . fowlpox was the first poxvirus used as vaccine vector for prrsv (guoshun et al., ) . fowlpox virus (fpv) belongs to the avipoxvirus genus, and its replication is restricted to avian species. nevertheless, attenuated strains of fpv have been used as vectors for poultry and mammals, resulting in strong and protective immune responses (paoletti, ; wang et al., ) . a gp -pro-pro-ser-gp fusion protein, alone or combined with porcine il- , was expressed using recombinant fpv. piglets inoculated with recombinant fpv expressing prrsv antigens induced neutralizing antibodies at dpi, and higher lymphocyte proliferation response, than those inoculated with the empty vector (guoshun et al., ) . vaccinated animals also showed increased ifn-␥ production, compared with non-vaccinated ones. piglets vaccinated with fpv co-expressing prrsv antigens and il- produced higher ifn-␥ amount than those inoculated with fpv expressing gp -gp fusion protein alone (guoshun et al., ) . partial protection was also observed after challenge with an homologous strain, as viremia was decreased in vaccinated animals (guoshun et al., ) . modified vaccinia virus ankara (mva), a member of the orthopoxvirus genus, has also been used as a vector for prrsv (zheng et al., ) . mva was used as the vaccine agent for the prevention of smallpox, and has been extensively used as viral vector for infectious diseases and cancer. mva is highly attenuated, even in immunosuppressed animals, but induces strong humoral and cellular immune responses (wang et al., ) . four recombinant mva viruses expressing prrsv antigens were constructed, expressing gp or m proteins alone, gp -m fusion protein, or coexpressing gp and m proteins (zheng et al., ) . these vectors were tested in the mouse model. mice inoculated with recombinant mva expressing heterologous antigens developed prrsv neutralizing antibodies, with the highest antibody titers found in mice inoculated with the recombinant mva co-expressing gp and m proteins. similar results were obtained when ifn-␥ and il- production was analyzed, indicating a th type cellular immune response (zheng et al., ) . unfortunately, authors did not perform protection experiments in piglets. therefore, the usefulness of mva as vector for prrsv vaccination remains to be determined. expression vectors have been engineered using different viral replicons, by replacing the virus structural genes by heterologous ones. these rna vectors, or replicons, express high levels of the foreign proteins, and replicate but are not packaged into virus-like particles unless structural proteins are provided in trans. therefore, replicons do not spread into neighbor cells and are safe for their use as vaccines (nagai et al., ; rayner et al., ) . an alphavirus replicon derived from venezuelan equine encephalitis virus (veev) has been successfully used as a vaccine against different pathogens, including swine influenza . veev-derived vaccines induce robust humoral, mucosal and cellular immunity (rayner et al., ) . it has been recently described that a veev replicon expressing prrsv gp and m proteins reduced viremia after prrsv challenge and provides partial protection (mogler et al., . replicons from classical swine fever virus (csfv) and vesicular stomatitis virus (vsv) expressing gp and m proteins have also been generated, expressing high levels of prrsv antigens (n. ruggli, personal communication). coronaviruses have several advantages as vectors over other viral expression systems: (i) they are single-stranded rna viruses that replicate in the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely (lai and cavanagh, ) ; (ii) these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes (enjuanes et al., (enjuanes et al., , ; (iii) a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues. since coronaviruses in general infect both respiratory and enteric mucosal surfaces, these viruses may be used to target the antigen to the enteric and respiratory areas to induce a strong secretory immune response; (iv) the tropism of coronaviruses may be engineered by modifying the s gene (ballesteros et al., ; kuo et al., ; sanchez et al., ) ; (v) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available and therefore are suitable to develop safe virus vectors; and (vi) infectious coronavirus cdna clones are available to design expression systems. our group obtained the first infectious coronavirus cdna clone, for tgev. this cdna was propagated as a bacterial artificial chromosome (bac) (almazán et al., ; gonzalez et al., ) . vectors based on this infectious cdna were engineered by cloning foreign genes in the place previously occupied by non-essential genes a and b, leading to high (> g/ cells) and stable (> passages) expression levels of specific heterologous genes (enjuanes et al., ; ortego et al., ; sola et al., ) . foreign gene expression levels were optimized by the study of the transcription-regulating sequences (trss), involved in coronavirus gene expression. our group has generated a set of trss ranging from intermediate to high foreign gene expression levels (alonso et al., ) , a combination of these trss could be used to drive the expression of two or three heterologous genes from just one infectious cdna (i.e., dicistronic or tricistronic vectors). tgev derived vector biosafety was improved by the generation of replicationcompetent, propagation-deficient viruses (ortego et al., ) . porcine respiratory coronavirus (prcv) is a mutant of tgev that replicates in the respiratory tract and causes no or mild clinical signs. prcv is spread worldwide and induces antibodies that can also neutralize tgev (saif et al., ) . therefore, preexisting immunity against the tgev vector could have been a problem. nevertheless, in vivo experiments showed that antibody titers against tgev increased even after two re-infections of pigs with rtgev vector (alonso s., sola i. and enjuanes l., unpublished results). one of the main advantages of recombinant tgev (rtgev) as a vector for prrsv is that tgev is a potent inducer of ifn-␣ in a process that is mediated by the virus transmembrane (m) protein (calzada-nova et al., ; charley and laude, ). in addition, as mentioned above, tgev vectors may present antigens at mucosal sites, eliciting mucosal and systemic immune responses. therefore, rtgev vectors will represent a novel strategy to study the induction of protection against prrsv. prrsv structural proteins gp and m accumulate in the endoplasmic reticulum of infected cells, where they form disulfidelinked heterodimers that are incorporated into the virion. m protein fig. . predicted gp -m heterodimer topology. the prrsv gp -m heterodimer may be anchored in membranes, with both proteins exposing to the surface a short nterminal ectodomain. the gp protein ectodomain contains the protein motives relevant in antigenicity, such as the epitope critical in neutralization (ecn, purple) and the decoy immunodominant epitope (ide, green). signal peptide (red) cleavage is represented by a black arrowhead. both gp and m proteins contain predicted glycosylation sites (yellow), although only gp protein is glycosylated (represented by orange circles). m protein contains in its c-terminal an endoplasmic reticulum retention signal (dark green). homodimers are also detected in infected cells, but are not incorporated into the virus particle (dea et al., ; meulenberg, ) . gp and m proteins are essential for the production of viral particles, although additional minor envelope proteins are required for virion infectivity (wissink et al., ) . according to the accepted topology of the gp -m heterodimer (fig. ) , both gp and m proteins expose a short ectodomain on the virion surface, being involved in receptor recognition. gp ectodomain contains several glycosylation sites, depending on the viral strain. it has been described that gp -m protein heterodimer formation is previous to gp glycosylation (mardassi et al., ) . gp glycosylation sites are close to the ecn epitope, and it has been proposed that the steric hindrance caused by the glycosylation is one of the causes for the potential delay in the production of nabs after prrsv infection (see below). it has been recently described that gp -m heterodimer interacts with the prrsv receptor, porcine fig. . design of rtgev expressing prrsv antigens. scheme of the tgev infectious cdna clone, cloned in a bac (pbac-tgev fl ). after transfection of cells, a full-length virus genome is generated (rtgev). cmv, cytomegalovirus immediate-early promoter; polya, tail of a residues; hdv, hepatitis delta virus ribozyme; bgh, bovine growth hormone termination and polyadenylation sequences. the tgev derived vectors are based on a tgev genome in which non-essential ab genes were deleted (rtgev- ab). genes encoding prrsv heterologous proteins were cloned in this position. expression of the foreign genes was driven by transcription regulatory sequences (trss) from genes a and n. ). an additional mutant, lacking n glycosylation site and decoy epitope, was obtained (n s-ide). in all cases, rtgev viruses were recovered with high titers. (b) st cells were infected with the rtgevs and double immunofluorescence staining was performed. tgev n protein specific monoclonal antibodies and a secondary antibody staining red were used to identify virusinfected cells. expression of gp was detected with rabbit antiserum specific for a gp peptide coupled to a secondary antibody staining green (upper panels). expression of m protein was detected with a rabbit antiserum specific for an m protein peptide, coupled to a secondary antibody staining green (lower panels). the percentage of infected cells expressing prrsv antigens was estimated by the analysis different microscopic fields. sialoadhesin, and that this interaction is dependent on gp glycosylation, most likely at the glycosylation site overlapping with the epitope recognized by neutralizing antibodies (van breedam et al., ) . as described above, gp and m proteins have been involved in the induction of prrsv neutralizing antibodies and a strong cellular immune response, respectively (bautista et al., ; ostrowski et al., ) . these data indicate that the gp -m heterodimer is the most promising antigenic structure that could be used in the construction of an efficacious vaccine against prrsv. a dicistronic tgev cdna encoding prrsv gp and m proteins was engineered (fig. ) . prrsv genes were cloned in the place of non-essential genes a and b. gp expression was driven by the transcription-regulating sequence of gene a (trs a), while m protein was expressed from an optimized trs partially derived from gene n (trs n) (alonso et al., ) . therefore, prrsv genes were expressed from independent subgenomic mrnas. the recovered virus expressed gp and m proteins in % and % of the to study if gp and m proteins expressed by rtgevs also colocalize, confocal microscopy analysis was performed. ma- or st cells were infected with prrsv and the rtgevs, respectively, and double immunofluorescence staining was performed. expression of gp was detected with a monoclonal antibody specific for gp , coupled to a secondary antibody staining red (upper panels). expression of m protein was detected with a rabbit antiserum specific for an m protein peptide, coupled to a secondary antibody staining green (medium panels). as shown in the merge, colocalization of gp and m proteins was observed both in the prrsv and rtgev infected cells (lower panels). mutant gp proteins (gp -n s and gp -ide-n s) expressed by rtgevs also colocalized with m protein. rtgev infected cells, respectively (fig. (b) ). expression levels were maintained even in virus recovered from tissues after infection of piglets with the rtgev. this result substantially advanced the efficacy of previous rtgevs expressing individually prrsv antigens, showing high expression levels of gp , but with limited stability. co-expression of m protein with gp reduced gp toxicity and probably will elicit a better t cell immune response. the protection conferred by this vector was tested in vivo. one-week-old piglets were inoculated with × pfu of the rtgev by three routes: oral, nasal and intragastric. nine weeks later, a challenge was perfomed with × tcid of a virulent european prrsv strain. blood samples were collected at different times post-inoculation, and humoral immune responses were evaluated by elisa. all animals presented a high antibody response against tgev, therefore, the vector infected target tissues as expected. vaccinated animals also showed a clear humoral response against prrsv gp and m proteins. a fast recall of the immune response was observed after the challenge, as vaccinated animals induced higher antibody titers against prrsv antigens and earlier than control ones. nevertheless, the immune response elicited by this rtgev provided very limited protection, and antibody titers decreased before challenge. the lack of protection against challenge was likely due to the relatively low levels of neutralizing antibodies produced before challenge. nevertheless, results using rtgev as a platform were promising, as a humoral immune response against prrsv antigens was elicited. gp antigenicity may be a problem for the obtention of efficient vaccines. therefore, several strategies to change gp antigenic structure were performed (fig. (a) ). in all cases, the gp mutants were co-expressed with m protein using a dicistronic vector, to minimize toxicity problems due to gp production. the ectodomain of gp protein is n-glycosylated. there are three or four predicted glycosylation sites in the gp from the north american strains of prrsv, whereas there are only two sites in the gp protein from european strains (wt, fig. (a) ). the relevance of the n-glycans in gp antigenicity is not clear. it has been proposed that the removal of the glycosylation sites could lead to the improvement of the immune response against prrsv, due to the elimination of the steric hindrance raised by the carbohydrate on the epitope inducing nabs (ansari et al., ) . elimination of the glycosylation sites present only in the north american strains, both in engineered and natural prrsv mutants, led to an increase in the levels of nabs induced by the mutant viruses (ansari et al., ; faaberg et al., ) . nevertheless, it is worth noting that these sites are not present in european prrsv strains. although the elimination of the glycosylation site overlapping the epitope critical for neutralization (g ) (fig. (a) ) often leads to non-infectious viruses (ansari et al., ; wissink et al., ) , natural north american strain mutants lacking this glycosylation site were found ( . % of the sequenced gp proteins). surprisingly, one of this natural mutants elicited lower neutralizing antibody response than the wild-type prrsv strain (faaberg et al., ) . this is in contrast with the data obtained with lactate dehydrogenase-elevating virus (ldv), where deletion of the n-glycan enhanced the nabs response (plagemann and moenngin, ) . elimination of the most conserved n-glycosylation site (g , fig. (a) ) (only . % of the sequenced north american gp proteins lack this motif) led to higher levels of neutralizing antibodies compared with the response elicited by the wild-type virus (ansari , ) . as glycosylation of gp is probably also involved in virus infectivity, it is difficult to analyze the influence of n-glycans on the immunogenicity of the protein. in the rtgev system, prrsv gp and m are not involved in infectivity and, therefore, the relevance of these proteins antigenicity in protection could be analyzed in our laboratory using the rtgev vector. gp mutants lacking glycosylation site g (n s), g (n s) or both (n , s) were generated (fig. (a) ). the mutation asn by ser was selected in all cases, as this substitution most likely introduced little secondary structure modifications. also, some prrsv field strains bear similar asn by ser aminoacid mutations in putative glycosylation sites. all rtgev viruses were recovered with high titers (fig. (a) ). nevertheless, only the n s mutant, lacking the glycosylation site partially overlapping the ecn, was stable ( fig. (b) ). this rtgev vector expressed high levels of gp -n s and m prrsv proteins in % and % of the infected cells, respectively (fig. (b) ). several b cell epitopes have been found in gp protein. an immunodominant epitope is located in the endodomain and, therefore, has probably limited effect on the antigenicity of the ecn epitope, as it is not exposed in the viral surface (dea et al., ; oleksiewicz et al., ; rodriguez et al., ) . a second immunodominant epitope (ide) was described in the ectodomain of gp , close to the ecn (fig. (a) ) (ostrowski et al., ) . it has been suggested that this immunodominant site could be responsible for the delay in the production of nabs against prrsv acting as a decoy epitope. antibodies against ide and ecn epitopes were found in the sera of prrsv infected pigs, appearing at different times post-infection. furthermore, an increase in the titers against ecn correlates with a decrease in the level of antibodies specific for ide (lopez and osorio, ; ostrowski et al., ) . an enhanced immunogenicity of a recombinant gp protein in which a synthetic sequence spacer has been introduced between ide and ecn epitopes, to better display the neutralizing epitope has been reported. the data suggests that ide is in fact acting as a decoy epitope (fang et al., ) . rtgev vectors were engineered expressing gp mutants lacking ide, in order to clarify whether this epitope is acting as a decoy epitope, enhancing the production of prrsv specific nabs. this approach represents an advance over similar constructions made in a prrsv infectious cdna clone, as in this case the deletion of the decoy epitope prevents the recovery of the recombinant virus (ansari et al., ) . two gp modifications were combined within the same construct, expressing gp protein lacking the decoy epitope and the glycosylation site overlapping the epitope recognized by neutralizing antibodies (n s-ide, fig. (a) ). the rtgev virus was recovered with high titer (fig. (a) ), and expressed modified gp and m proteins in % and % of the infected cells, respectively ( fig. (b) ). the data obtained in cultured cells suggest that rtgev vectors expressing prrsv antigens were not fully stable, mainly due to gp protein toxicity resulting in a significant lost of gp expression after - virus vector passages in cell culture. in contrast, m protein expression was fully stable, with at least % of infected cells expressing m protein for more than passages in tissue culture. a decrease in gp expression was also observed after the introduction of modifications in this protein (upper panels, fig. (b) ). again, m protein expression remained constant, independently of gp mutant co-expressed (lower panels, fig. (b) ). the reduction in gp expression could be responsible of the modest results in protection observed with the live rtgev vectors, in comparison to the protection elicited with non-infectious antigens expressed using rtgev vectors. as described above, the rtgev vector expressing prrsv gp and m proteins represents a substantial advance on the efficacy of previous rtgevs expressing prrsv antigens (i.e., gp alone). we postulated that co-expression of m protein with gp reduces gp toxicity by the formation of gp -m heterodimer. to clarify this issue, confocal microscopy analysis was performed (fig. ) . ma- or st cells were infected with prrsv and the rtgevs, respectively, and double immunofluorescence staining was performed. as shown in the merge (fig. , lower panels) , colocalization of gp and m proteins was observed both in the prrsv and rtgev infected cells. this result suggests that the gp -m heterodimer is formed in both cases. the decrease in gp expression levels by the introduction of gp mutations suggested that the modifications could affect heterodimer formation. colocalization of gp and m proteins was also observed when a mutant gp protein (i.e., gp -n s, or gp -ide-n s) was expressed by the rtgev vector (fig. ) , suggesting that a heterodimer was also formed by mutant gp proteins. coimmunoprecipitation of gp and m proteins to fully demonstrated gp -m heterodimer formation is in progress. . . protection conferred by rtgev derived vaccines . . . formulation of a killed vaccine expressing gp mutants with alterations in the glycosylation pattern as a complementary approach, a killed vaccine was developed based on the rtgev-gp -n s-m virus, co-expressing gp lacking the first glycosylation site and m proteins. st cells were infected with this rtgev, and the culture medium was harvested at hpi. soluble antigens were inactivated by incubation with binary ethylenimine (bei), and a vaccine was formulated. groups of six -week-old piglets were intramuscularly inoculated with the formulation to evaluate the protection conferred by this vaccine. a boost was performed weeks after inoculation. six weeks after the first inoculation, animals were challenged by intranasal inoculation with tcid of prrsv/olot strain. blood samples were collected at different times post-inoculation to determine the levels of specific antibodies by elisa. vaccinated animals induced higher and faster antibody titers against prrsv antigens than control animals (fig. (a) , left panel). neutralizing antibody titers were also higher in the vaccinated animals when compared with non-vaccinated animals (fig. (a) , right panel). viremia, gross lesions, and histopathology in the lungs of vaccinated and nonvaccinated animals were analyzed. a clear degree of protection samples were analyzed by enzyme-linked immunosorbent assays (elisas) specific to detect antibodies against tgev, gp and m. to evaluate response against gp , gp protein from prrsv olot strain was expressed and purified from insect cells and used as antigen for the elisa. (b) lung damage caused by prrsv infection. the lungs from animals inoculated with empty rtgev vector, or rtgev expressing gp -n s and m proteins, were analyzed. lung lesions observed in all the pigs, with different degree of severity, included a craneo-ventral consolidation of apical and medial lung lobes. was observed, as the lungs from vaccinated animals showed a significantly lower degree of lung damage than those from nonvaccinated ones (fig. (b) , left panel). furthermore, a reduction in viremia was also observed in vaccinated animals (fig. (b) , right panel). altogether, these data suggested that the elimination of the glycosylation site close to the neutralizing epitope improves protective immune response against prrsv. the protection conferred by rtgev-gp -n s-m was tested in vivo. one-week-old piglets were inoculated with × pfu of the rtgev by three routes: oral, nasal and intragastric. a boost was performed weeks after inoculation. six weeks later, a challenge was performed with × tcid of prrsv/olot strain. blood samples were collected at different times post-inoculation, and humoral immune responses were evaluated by elisa. all the animals produced a high antibody response against tgev (data not shown), therefore, the vector infected target tissues as expected. after challenge, vaccinated animals showed a clear humoral response against prrsv antigens (fig. (a) ). a moderately faster recall response was observed, as vaccinated animals induced higher antibody titers against prrsv antigens and earlier than control animals (fig. (a) ). the protection conferred by this tgev based vaccine was also evaluated. a certain degree of protection was observed, as the lungs from vaccinated animals showed a lower degree of lung damage than those from non-vaccinated ones (fig. (b) ). nevertheless, the immune response was not strong enough to provide full protection, probably because the levels of neutralizing antibodies were similar in vaccinated and non-vaccinated animals (data not shown). to date, rtgev expressing prrsv antigens only provided partial protection. this could be due to the fact that the expression of prrsv antigens by rtgev vectors was not fully stable, mainly due to gp protein toxicity resulting in a significant lost of gp expression in - passages. in contrast, m protein expression was fully stable, with at least % of infected cells expressing m protein for more than passages in tissue culture. the lack of full protection using rtgev expressing prrsv antigens could also be due to the presence of domains in the expressed proteins inducing negative regulatory t cells (treg). as the vector used in the immunization (rtgev) efficiently induced the production of ifn, it is likely that either prrsv gp or m proteins could contain negative signals inducing treg. this negative regulation of the immune response elicited could also be a major cause for the delay in the development of a protective immune response against prrsv. to improve rtgev vector stability different strategies can be developed, such as the expression of small domains of gp containing the epitopes relevant for protection but lacking domains responsible for instability in their expression. alternatively, the generation of a library of point mutants in gp fragments in which the epitopes eliciting negative treg have been eliminated may overcome what we consider the second most relevant limitation in the protection against prrsv. these approaches are currently in progress in our laboratory. an improvement of vaccination strategies against prrsv is required, as current vaccines have limited efficacy. best results have been obtained using modified live vaccines and virus vectored vaccines could represent an advantage to stimulate immune responses against prrsv. the results reported to date using viral vectors are not fully satisfactory and new vectors must be explored. tgev based vector vaccines expressing different prrsv antigenic combinations represent a promising candidate to provide protection against two porcine viruses: prrsv and tgev. the use of rtgev vectors led to promising results, similar to those obtained with other vectored vaccines. nevertheless, as reported for other rna viruses, data obtained indicate that heterologous protein expression stability was limited. therefore, increase of prrsv antigens expression stability, and removal of domains eliciting treg, represent new avenues to improve the development of an efficient prrsv vaccine. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome transcription regulatory sequences and mrna expression levels in the coronavirus transmissible gastroenteritis virus influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism the evolution of adenoviral vectors and their application to gene delivery and rnai in the cns t cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus regulatory t cells and infection: a dangerous necessity appearance of acute prrs-like symptoms in sow herds after vaccination with a modified live prrs vaccine adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuated viral replication and increases the level of pulmonary antigen-presenting cells characterization of the cytokine and maturation responses of pure populations of procine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists adjuvants for porcine reproductive and respiratory syndrome virus vaccines effects of porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on t cell activation and antiviral cytokine production induction of alpha-interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates different europeantype vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs coronavirus derived expression systems coronavirus reverse genetics and development of vectors for gene expression neutralizing antibody responses of pigs infected with natural gp n-glycan mutants of porcine reproductive and respiratory syndrome virus enhanced immunogenicity of the modified gp of porcine reproductive and respiratory syndrome virus stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by the insertion of an intron immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp /gp of porcine reproductive and respiratory syndrome virus and swine il- comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection recombinant adenovirus expressing gp and m fusion proteins of porcine reproductive and respiratory syndrome virus induce both humoral and cell-mediated immune responses in mice analysis of immunogenicity of minor envelope protein gp of porcine reproductive and respiratory syndrome virus in mice influence of porcine reproductive and respiratory syndrome virus gp glycoprotein n-linked glycans on immune responses in mice immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus enhanced immune responses of mice inoculated recombinant adenoviruses expressing gp by fussion with gp and/or gp of prrs virus immunogenicity of a recombinant pseudorabies virus expressing orf -orf fusion protein of porcine circovirus type detection of foxp protein expression in porcine t lymphocytes molecular assessment of the role of envelope-associated structural proteins in cross neutralization among different prrs viruses challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses genetic perspectives on host responses to porcine reproductive and respiratory syndrome (prrs) hsp fused with gp and gp of porcine reproductive and respiratory syndrome virus enhanced the immune responses and protective efficacy against virulent prrsv challenge in pigs role of neutralizing antibodies in prrsv protective immunity protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus the challenge of prrs immunology prrsv, the virus replicon particle prrsv vaccine provides partial protection from challenge replicon particles expressing prrsv gp and matrix reduce viremia following homologous and heterologous challenge immunological responses of swine to porcine reproductive and respiratory syndrome virus infection sendai virus engineering: from reverse genetics to vector development cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations pocine b-cells recognize epitopes that are conserved between the structural proteins of american-and europeantype porcine reproductive and respiratory syndrome virus generation of a replicationcompetent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome transmissible gastroenteritis coronavirus gene is not essential but influences in vivo virus replication and virulence prrsv: comparison of commercial vaccines in their ability to induce protection against current prrsv strains of high virulence passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain applications of pox virus vectors to vaccination: an update lactate dehydrogenase-elevating virus, equine arteritis virus and simian haemorrhagic fever virus, a new group of positive strand rna viruses molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. microbiol protective immunity induced by a recombinant pseudorabies virus expressing the gp of procine reproductive and respiratory syndrome virus in piglets alphavirus vectors and vaccination cd (+) t cells induced by a dna vaccine: immunological consequences of epitopespecific lysosomal targeting immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells engineering transmissible gastroenteritis virus genome as an expression vector inducing latogenic immunity molecular adjuvants for mucosal immunity the m/gp glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner rapid development of an efficacious swine vaccine for novel h n poxvirus expression vectors gm-csf fused with gp and gp of porcine reproductive and respiratory syndrome virus increased the immune responses and protective efficacy against virulent prrsv challenge envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune response of porcine reproductive and respiratory syndrome virus (prrsv) assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge we thank c.m. sánchez, s. ros, and m. gonzález for technical assistance. this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt, bio - ), the national pork board , and the european union (fp , plaprova- , and porrscon- ). s.z., i.s. and m.b. received contracts from the eu. j.l.g.c. received contract from community of madrid. key: cord- - u bm fz authors: sánchez-carvajal, j.m.; rodríguez-gómez, i.m.; ruedas-torres, i.; larenas-muñoz, f.; díaz, i.; revilla, c.; mateu, e.; domínguez, j.; martín-valls, g.; barranco, i.; pallarés, f.j.; carrasco, l.; gómez-laguna, j. title: activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of prrsv- infection with the virulent strain lena date: - - journal: vet microbiol doi: . /j.vetmic. . sha: doc_id: cord_uid: u bm fz porcine reproductive and respiratory syndrome virus (prrsv) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. pulmonary alveolar macrophages (pams) are the main cells supporting prrsv replication, with cd as the essential receptor for viral infection. although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for prrsv virulent strains. this research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by prrsv- strains of different virulence. conventional pigs were intranasally inoculated with the virulent subtype lena strain or the low virulent subtype strain and euthanised at , , and dpi. lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of il- and ifn-γ in sera compared to -infected pigs. extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in lena-infected pigs. lung viral load and prrsv-n-protein(+) cells were always higher in lena-infected animals. prrsv-n-protein(+) cells were linked to a marked drop of cd (+) macrophages. the number of cd (+) and inos(+) cells gradually increased along prrsv- infection, being more evident in lena-infected pigs. the frequency of cd r (+) and foxp (+) cells peaked late in both prrsv- strains, with a strong correlation between cd r (+) cells and lung injury in lena-infected pigs. these results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response. porcine reproductive and respiratory syndrome virus (prrsv) encompasses two species, betaarterivirus suid and betaarterivirus suid (formerly, prrsv- and prrsv- , respectively) (gorbalenya et al., ) , which present a wide inter-and intraspecies viral diversity (balka et al., ; shi et al., ; stadejek et al., ) . since , different outbreaks characterised by high morbidity and mortality rates, fever, haemorrhages, severe lesions in lung and, eventually, in other organs such as thymus or lymph nodes, have been reported associated with virulent prrsv- strains (canelli et al., ; karniychuk et al., ; morgan et al., morgan et al., , ogno et al., ; sinn et al., ; weesendorp et al., ) . several contradictory results about viraemia, tissue viral load, early virus clearance, low frequencies of prrsv-specific ifn-γ secreting cells or prrsv neutralizing antibodies have been reported after infection with prrsv- virulent strains. however, there is consensus on the fact that some strains are more virulent than others (canelli et al., ; ferrari et al., ; frydas et al., ; geldhof et al., ; morgan et al., ; renson et al., ; stadejek et al., ; weesendorp et al., weesendorp et al., , . prrsv replicates predominantly in the lung, causing a mild to severe interstitial pneumonia which may be complicated to suppurative bronchopneumonia due to the increased lung sensitisation to bacterial infections associated with the damage and impairment of the different pulmonary macrophage subpopulations (pulmonary alveolar macrophages, pams; pulmonary intravascular macrophage, pims; and interstitial macrophages) (brockmeier et al., ; thanawongnuwech et al., ) . pams are the main cellular target of prrsv, although interstitial and intravascular macrophages can be infected too (bordet et al., ; duan et al., ; gómez-laguna et al., ) , with j o u r n a l p r e -p r o o f the nucleocapsid protein n (prrsv-n-protein) as the most abundant viral protein during prrsv infection (rowland et al., ) . pams express high levels of cd scavenger receptor (sánchez et al., ; van gorp et al., ) which is essential to support prrsv internalisation and disassembly interacting with gp and gp viral proteins (burkard et al., ; das et al., ; whitworth et al., ) . a soluble form of cd (scd ), that may be released from tissue macrophages and monocytes, has been identified in plasma as potential biomarker for macrophage activity and inflammation (costa-hurtado et al., ; møller, ; pasternak et al., ) . prrsv is known to modulate the host immune response by inducing changes in the frequencies of immune cell subsets in blood (dwivedi et al., ; ferrari et al., ; morgan et al., ; weesendorp et al., ) and in tissues (gómez-laguna et al., ; rodríguez-gómez et al., ) , leading to an enhanced susceptibility to secondary bacterial infections (karniychuk et al., ; renson et al., ; sinn et al., ). an early decrease in the frequency of monocytes, nk cells or cytotoxic t cells linked to a strong inflammatory response in target organs has been described upon experimental infection with prrsv- virulent strains (ferrari et al., ; morgan et al., ; weesendorp et al., ; . in addition, some studies indicate an early overproduction of pro-inflammatory cytokines, such as ifn-γ, il- β or il- , as the main source of pulmonary injury after infection with virulent prrsv- strains (amarilla et al., ; morgan et al., ; renson et al., ; weesendorp et al., ) . nevertheless, other potential mechanisms, such as an imbalance among pro-and anti-inflammatory responses, might predispose to secondary infections contributing to the onset of the porcine respiratory disease complex (prdc) van gucht et al., ) . in this context, overproduction of nitric oxide (no), mainly triggered by inducible no synthase (inos) (akaike and maeda, ) , or upregulation of cd , as the primary lipopolysaccharide (lps) receptor (zanoni and granucci, ) , could contribute to lung inflammation upon infection with prrsv (chen et al., ; lee and kleiboeker, ; van gucht et al., yan et al., ) . by contrast, the transcription factor forkhead box protein (foxp ), is an essential transcription factor for the development of regulatory t cells (tregs) and hence, a useful marker to detect them. this subset could be involved in supressing the activation of other t-cell populations (käser et al., ) . cd receptor (cd r ), expressed on myeloid cells and b cell subsets (poderoso et al., ) , is an inhibitory surface receptor that might deliver inhibitory signals dampening the activation of cells which express it (vaine and soberman, ) . thus, both immune markers might play an important role inhibiting the production of proinflammatory cytokines (elmore et al., ; nedumpun et al., ; singh et al., ; vaine and soberman, ; wang et al., ) , lessening the exuberant lung injury observed with virulent prrsv- strains. whereas all these markers may play a key role in prrsv virulence, there are scarce studies analysing their role in the context of the lung lesion. therefore, the systemic immune response and immunopathology of lung are evaluated in this study with the goal of exploring the role of selected immune markers in the pro-and anti-inflammatory priming of the lung to secondary bacteria after infection with a virulent prrsv- strain (subtype , lena strain) in comparison with a low virulent prrsv- strain (subtype , strain). j o u r n a l p r e -p r o o f the low virulent strain (subtype prrsv- ) was isolated from the serum of a piglet with pneumonia from a prrsv-positive herd located in spain in . the virulent lena strain (subtype prrsv- ) is considered as the prototype of prrsv- virulent strains. lena strain isolation was performed from lung homogenates obtained from weak born piglets from a prrsv-positive herd from belarus in with a high mortality rate, reproductive failure and respiratory disorders (karniychuk et al., ) . viral stocks were produced from the th passage of each strain on pams, titrated by means of immunoperoxidase monolayer assay and expressed as tissue culture infectious doses (tcid )/ml ( strain: . tcid /ml; lena strain: . tcid /ml). the animals and samples used in this study were part of a project to investigate the pathogenesis of the infection with prrsv- strains of different virulence . briefly, fifty-two -week-old male and female piglets (landrace x large white crossbred) were obtained from a historically prrsv-negative farm. all pigs were negative for porcine circovirus type (pcv ), prrsv and mycoplasma hyopneumoniae by elisa and pcr assays (mattsson et al., ; sibila et al., ) . piglets were blocked by weight and sex and randomly assigned to three different groups and housed in separate pens: lena group (n= ), group (n= ) and control group (n= ). after an acclimation period of seven days, piglets were intranasally inoculated with either the low virulent strain or the virulent lena strain (both used at x tcid /ml, ml/nostril, using the mad nasal™ intranasal mucosal atomization device, teleflex, alcalá de henares, madrid, spain). the control group was mock- commencing day prior to inoculation, piglets were daily monitored to evaluate clinical signs (liveliness, respiratory symptoms and anorexia) and rectal temperature. quantification of clinical signs was performed by applying the following clinical score: liveliness (score , no abnormalities; score , reduced liveliness but with response to external stimuli; score , pig prostration; score , agonic pig); respiratory symptoms (score , no abnormalities; score , mild dyspnoea; score , evident dyspnoea; score , evident dyspnoea with tachypnoea; score , evident dyspnoea, tachypnoea and cyanosis); and anorexia (score , eating without abnormality; score , sporadic frequency of eating; score , no eating). the sum of these scores represented the total clinical score per animal and per day. rectal temperatures > . °c were considered hyperthermia. at necropsy, gross lung lesions were recorded and scored by the same pathologist (halbur et al., ) . afterwards, samples from apical, medial and caudal lobes from the right lung were collected and fixed in % neutral-buffered formalin (fisher scientific ltd., loughborough, uk) for histopathological and immunohistochemical studies. four-micron tissue sections were stained with haematoxylin and eosin and blindly graded by two pathologists for the histopathological evaluation. the severity of histopathological j o u r n a l p r e -p r o o f lesions in the lung was scored as previously described by halbur et al. ( ) : , no microscopic lesions; , mild interstitial pneumonia; , moderate multifocal interstitial pneumonia; , moderate diffuse interstitial pneumonia; and , severe interstitial pneumonia. in addition, a similar score was developed considering the diagnosis of suppurative bronchopneumonia : , no microscopic lesions; , mild bronchopneumonia; , moderate multifocal bronchopneumonia; , moderate diffuse bronchopneumonia; and , severe bronchopneumonia. altogether, the final score included the total of both, the interstitial pneumonia score and the bronchopneumonia score, being points the maximum possible score. rna was isolated from sera using nucleospin ® rna virus (macherey-nagel, düren, germany) according to manufacturer's instructions. for lung, rna was purified from tissue homogenate using a combined procedure with trizol™ (thermo fisher scientific, serial -fold dilutions of or lena orf rt-pcr products with known quantities, ranging from to genomic copies/ml were used as standards to generate a standard curve and, therefore, to determine the prrsv genomic copies in sera and lung. the rt-qpcr efficiency (e) was estimated for each strain by a linear regression model. the e value was calculated from the slope of the standard curve according to equation: also, a set of eight serial -fold dilutions of know tcid /ml (starting from tcid /ml) was included in order to determine a relation between ct-values, genomic copies/ml and tcid /ml. an inter-run calibrator sample with a known number of prrsv copies was introduced in each experiment to self-control inter-run variation. the area under the curve (auc) for viremia and lung viral load was calculated using the trapezoidal approach (greenbaum et al., ) . results of viral load in sera and lung are showed in equivalent tcid (eq tcid ) per ml. results were expressed in pg/ml for ifn-γ, il- and il- , and ng/ml for lbp and j o u r n a l p r e -p r o o f scd . the minimum detectable concentrations were pg/ml for ifn-γ, pg/ml for il- , pg/ml for il- , . ng/ml for lbp and . ng/ml for scd . four-micron sections from lung were dewaxed in xylene and rehydrated in descending grades of alcohol until distilled water. then, endogenous peroxidase inhibition was performed in a % h o solution in methanol for min. epitope demasking, primary antibodies dilutions and blocking of non-specific binding are detailed in table . monoclonal primary antibodies were incubated overnight at ºc in a humid chamber. tris buffered saline (ph . ) were used as wash and diluent buffers, respectively. antibody specificity was verified by exchanging the primary antibody by isotype matched reagents of irrelevant specificity. one negative control which consisted of replacement of the primary antibody by bsa blocking solution was included in each immunohistochemical assay to rule out non-specific bindings. the number of immunolabelled cells was quantified in non-overlapping selected high magnification fields of . mm (olympus bx , olympus iberia sau, l'hospitalet de j o u r n a l p r e -p r o o f llobregat, barcelona, spain) and expressed as the mean of the score for each animal per mm . labelled cells were morphologically identified by differentiating among pams, pims and interstitial macrophages. differences between groups were evaluated for approximate normality of distribution by the d'agostino and pearson omnibus normality test followed by the mann whitney's u non-parametric mean comparisons test. correlation coefficients were assessed by the spearman and pearson tests and were considered relevant with r > . and p < . . data analyses and figures were performed by using graphpad prism . software (graphpad prism software . , inc., san diego, ca, usa) and inkscape . software. a p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . *** p ≤ . and **** p ≤ . . (table ) . viraemia and lung viral load were determined by rt-qpcr (efficiency of %; slope = . ; detection limit: copy/µl; slope-intercept = . ; and high linearity, r = . ). all animals were negative by rt-qpcr at day and control pigs remained so all throughout the experiment. in sera, four out of five -infected pigs and all lena-infected pigs were prrsv positive as early as dpi. viraemia was always higher in lena-than in -infected pigs from to dpi (p < . at , , dpi; p< . at dpi), reaching the highest viral load at dpi ( . x eq tcid /ml). the auc for viremia (mean) in lena and group were . and . respectively ( fig. a) . the viral load in the lung displayed a similar kinetics to that of serum for both infected groups, reaching the j o u r n a l p r e -p r o o f maximum lung viral loads at dpi in lena group ( . x eq tcid /ml), whereas group peaked at dpi ( . x eq tcid /ml) (fig. b) . by contrast to sera, prrsv- was just detected in lung in two out of five animals in both infected groups at dpi, being positive all infected piglets from dpi onwards. the auc for lung viral load (mean) in lena group was and . for group (fig. b) . in lena infected-group the statistical analysis revealed a positive correlation among viraemia, lung viral load, temperature, clinical signs score and the number of prrsv-n-protein + cells in the lungs (table ) . a correlation among lung viral load and viraemia and prrsv-n-protein + cells was also observed in -infected pigs (r = . , p < . ; and, r = . , p < . , respectively). prrsv-specific antibodies were first detected at dpi in sera from both prrsv- infected groups (non-significant differences in s/p ratios) (data not shown). a significant increase in ifn-γ serum levels was detected after lena infection at and dpi (maximum mean level of ± pg/ml at dpi) compared to control (p< . ) and (p< . ) groups (fig. c ). maximum il- levels in serum of group were observed at dpi (mean of ± pg/ml), whereas pigs belonging to lena group reached the highest il- levels at dpi (mean of ± pg/ml) (fig. d) . il- , lbp or scd were not detected in serum samples from both control and infected groups throughout the study. both viraemia and lung viral load displayed a positive statistical correlation with ifn-γ levels, which in turn were also correlated with temperature and the clinical signs score in lena infected-pigs (table ) . j o u r n a l p r e -p r o o f the labelling of prrsv-n-protein was mainly observed in pams and in a lesser extent in interstitial and intravascular macrophages (figs. a- b) . in lena-infected pigs, clusters of prrsv-n-protein + macrophages were observed within foci of bronchopneumonia at and dpi (fig. b inset) . a progressive increase in the number of prrsv-n-protein + cells was detected throughout the study in both prrsv- -infected groups, reaching a peak at and dpi in lena and -infected piglets, respectively. this increase was significantly higher in lena than in group (p< . at , and dpi) (fig. e , primary axis). no positive cells were detected in control pigs. (table ) . the labelling against cd was mainly observed in the cell membrane and cytoplasm of monocytes, interstitial and intravascular macrophages and, occasionally, in pams (figs. a- b, insets). whereas no changes were observed in the number of cd + cells in the control group along the study, a gradual increase with maximum expression at dpi was detected in both infected groups (fig. e) . lena-infected pigs showed the highest frequency of cd + cells when compared to control animals (p< . ) in association with the presence of suppurative bronchopneumonia (fig. b ). cd + interstitial and intravascular macrophages were observed infiltrating extensive areas of the interstitium, whereas almost no cd + cells were present in the bronchial wall and alveolar lumen. interestingly, the number of cd + cells in lena-infected piglets displayed a strong positive correlation with the concentration of il- in sera (table ) . the granular intracytoplasmic immunostaining of inos was primarily observed in pams and interstitial macrophages in foci of interstitial pneumonia and bronchopneumonia (figs. c- d). the number of inos + cells followed a similar kinetics in both prrsv- infected groups, with a progressive increase from dpi onwards, reaching a significant increase by the end of the study ( dpi) in lena-infected pigs compared to (p< . ) and control groups (p< . ) (fig. f) . cd r labelling was detected in the cytoplasm of intravascular and interstitial macrophages located inside or surrounding bronchopneumonia foci, with occasional j o u r n a l p r e -p r o o f expression in pams and monocytes (figs. a- b, insets). whereas the number of cd r + cells significantly increased in lena-infected pigs at - dpi (p< . at dpi with respect to group; and p< . at dpi and p< . at dpi with respect to control group), this increase was just detected at dpi in -infected pigs (p< . with respect to control group) (fig. e, primary axis) . control animals presented a scarce number of cd r + cells along the study. for lena infected-pigs a strong positive correlation was observed among the frequency of cd r + cells and the microscopic lung lesions (fig. e ) ( table ) . foxp yielded a nuclear immunolabelling in lymphocytes mainly located in areas of atelectasis and interstitial pneumonia (figs. c- d, insets). although two lena-infected pigs exhibited a higher number of foxp + cells at dpi, the kinetics of positive cells for this immune markers showed a gradual increase along the study in both lena-and infected animals, reaching the maximum at dpi (fig. f ). there were no significant differences in the number of foxpp + cells among infected groups. however, a significant increase of foxp + cells was detected at and dpi in lena-infected pigs compared to control animals (p< . ). prrsv plays a pivotal role in prdc, modulating the host immune response and favouring secondary bacterial infections van gucht et al., ) . virulent prrsv- strains cause more severe clinical signs, higher mortality rates as well as marked lung injury with a higher incidence of bronchopneumonia as opposed to low virulent strains (amarilla et al., ; canelli et al., ; frydas et al., ; j o u r n a l p r e -p r o o f gómez-laguna et al., ; morgan et al., ; renson et al., ; rodríguez-gómez et al., ; stadejek et al., ; weesendorp et al., ) . accordingly, we hypothesise that severe pulmonary lesions observed along infection with virulent prrsv- strains might be associated with a higher decrease in the amount of pams as well as an imbalance between anti-and pro-inflammatory responses with different molecules potentially involved in this process. as previously described (renson et al., ; weesendorp et al., ) , severe systemic and respiratory symptoms as well as hyperthermia were observed in animals infected with virulent lena strain, whereas low virulent strain only caused mild clinical signs and a slightly increase of rectal temperature. furthermore, virulent lena strain caused an earlier and stronger onset of lung lesions due to extensive consolidated areas in the apical and medial lobes which were microscopically linked to suppurative bronchopneumonia as well as severe characteristic interstitial pneumonia. on the other hand, prrsv virulence has been associated with higher virus titre and antibody response in vivo (brockmeier et al., ; lu et al., ) although lena virulent strain elicited a quite higher viraemia than the low virulent strain, no differences were observed in the antibody response in the early phase of infection. similar results have been previously reported by others when comparing lena with low virulent strains (renson et al., ; weesendorp et al., ) , and confirm that prrsv- virulence and specific non-neutralizing antibodies are not associated in the acute phase of infection. response when compared with low virulent strains (amarilla et al., ; liu et al., ; levels of il- at dpi and ifn-γ at - dpi were detected in the sera of lena-infected pigs. increased concentration of il- in plasma is associated with both systemic and respiratory symptoms (van reeth and nauwynck, ) and could play a dual role during virus infection: (i) protecting the host from infection and (ii) inducing inflammation and tissue damage when it is overexpressed (liu et al., ) . it is known that ifn-γ is, mostly produced by activated nk cells, nkt cells, γ/δ t cells, cytotoxic t cells and memory t cells (gerner et al., ; mair et al., ) , and participates in regulating the immune and inflammatory responses (van reeth and nauwynck, ) . in fact, an early increase of nkt cells has been associated with viraemia peak in piglets infected with pr cd and inos are involved in lung inflammation after infection with prrsv (chen et al., ; lee and kleiboeker, ; van gucht et al., yan et al., ) upregulation of cd , as lipopolysaccharides (lps) co-receptor, after infection with prrsv sensitises the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial lps (van gucht et al., ) . for its part, inos is mainly expressed in response to different stimuli, such as cytokines and lps, playing a role in tissue injury upon production of no (chen et al., ; cho and chae, ; vlahos et al., ; yan et al., ) . in this study, an increase in the number of cd + cells after prrsv- infection was observed in association with suppurative j o u r n a l p r e -p r o o f bronchopneumonia, which was more evident in lena-infected piglets at - dpi. this increase was mainly due to cd + monocytes, interstitial and intravascular macrophages infiltrating extensive areas of the interstitium. the influx of cd + monocytes and immature macrophages may be explained by an attempt to replenish the loss of cd + macrophages contributing to clearance of cellular debris and resolution of inflammation, restoring the normal lung function. on the other hand, the increase of cd + cells implies a higher availability of the lps-lbp complex receptor, which is likely to sensitise the lung to future secondary bacterial infections making the onset of prdc easier (van gucht et al., ) . in the case of inos, a significant increase in the number of inos + cells was observed in areas of interstitial pneumonia as well as bronchopneumonia in lena-infected pigs. the induction of inos has been associated with both a direct effect of the viral replication or viral components and an indirect effect mediated by cytokines, such as ifnγ, or by lps (akaike and maeda, ; chen et al., ; lee and kleiboeker, ) . of note, the peak of inos in lena-infected animals appeared in our study just after the peak of prrsv replication in the lung as well as after the peak of serum ifn-γ, being associated with the maximum lung injury and bronchopneumonia lesion. these factors may play a role in the regulation of inos expression along prrsv infection and its role in lung injury development (chen et al., ; lee and kleiboeker, ; yan et al., ) . after a cascade of proinflammatory events, the host is able to trigger off the release of anti-inflammatory or regulatory mediators to restrain the extent of the injury. thus, the role of cd r and foxp was evaluated in the present study. a strong positive correlation was detected among the frequency of cd r + cells and the microscopic score which was mainly associated with a higher severity of typical interstitial pneumonia j o u r n a l p r e -p r o o f and suppurative bronchopneumonia in lena-infected pigs. likewise, an increase in the frequency of foxp + cells between and dpi was triggered by both prrsv- strains when the lung injury was higher. cd r has been involved in reducing the expression of pro-inflammatory cytokines in a wide range of inflammatory diseases (vaine and soberman, ) , nevertheless to the best of the authors' knowledge, the role of cd r in viral diseases of swine is largely unknown. previous studies in a murine model reported that influenza virus infection induced the upregulation of cd r in macrophages, decreasing their responsiveness and increasing the sensitivity to bacterial infection and finally severe lung injury (snelgrove et al., ; vaine and soberman, ) . in contrast, cd /cd r signalling pathway limited type i ifn production during coronavirus infection protecting the host from cytokine storm (vaine and soberman, ) . in addition, foxp has been reported as a potential inhibitor of the cell- the present study dissects the immunopathology of lung injury along an acute infection with prrsv- strains of different virulence, revealing a drop in the number of cd + cells together with an enhancement in the expression of cd and inos as mechanisms involved in the earlier and higher extent of lung lesion in lena-infected piglets. these changes could sensitise the lung to future secondary bacterial infections. in addition, the increase in the number of cd + cells is likely to respond to an attempt to replenish the cd + macrophages subset lost along the infection with both prrsv- strains. on the other hand, the increase in the expression of cd r and foxp represents potential pathways activated to contain the inflammatory response. the authors declare that they have no competing interest. marked microglia activation in the hippocampus and deficits in spatial learning. journal of neuroscience, ( ) temperature (b) for control group (gray circles), -infected group (green triangle) and lena-infected group (red diamonds). "a" indicates a significant difference between the lena and and control groups, "b" a significant difference between the lena and groups and "c" a significant difference between the and control groups. p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . and *** p ≤ . . days post-inoculation, dpi. (c) at necropsy, lungs were scored and processed for histological and immunohistochemical studies. box plots display the macroscopic lung score for each group (control, gray circles; , green triangles; lena, red for all data, a p value lower than . was considered statistically significant and represented as * p ≤ . , ** p ≤ . , *** p ≤ . and ****p ≤ . . inos -ns: not statistically significant or with r < . . correlation coefficients were considered relevant with r > . and p < . . a comparative study of the local cytokine response in the lungs of pigs experimentally infected with different prrsv- strains: upregulation of il- α in highly pathogenic strain induced lesions a distinct function of regulatory t cells in tissue protection genetic diversity of prrsv in central eastern europe in - : origin and evolution of the virus in the region porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus the presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus comparison of asian porcine high fever disease isolates of porcine reproductive and respiratory syndrome virus to united states isolates for their ability to cause disease and secondary bacterial infection in swine precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function phenotypic characterization of a highly pathogenic italian porcine reproductive and respiratory syndrome virus (prrsv) type subtype isolate in experimentally infected pigs upregulation of pro-inflammatory factors by hp-prrsv infection in microglia: implications for hp-prrsv neuropathogenesis immunohistochemical detection and distribution of inducible nitric oxide synthase in pigs naturally infected with actinobacillus pleuropneumoniae changes in macrophage phenotype after infection of pigs with haemophilus parasuis strains with different levels of virulence genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype i strains the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) respiratory viral infection in neonatal piglets causes inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges phenotypic and functional differentiation of porcine αβ t cells: current knowledge and available tools cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs international committee on taxonomy of viruses (ictv) area under the viraemia curve versus absolute viral load: utility for predicting symptomatic cytomegalovirus infections in kidney transplant patients comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate detection of foxp protein expression in porcine t lymphocytes porcine arterivirus activates the nf-κb pathway through iκb degradation f injury: cytokines, uncontrolled inflammation, and therapeutic implications dynamic changes in inflammatory cytokines in pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus attenuation and immunogenicity of a live high pathogenic prrsv vaccine candidate with a -amino acid deletion in the nsp interleukin- , interleukin- beta, and interferon-gamma levels are linked to prrs virus clearance the porcine innate immune system: an update detection of mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the s rrna gene soluble cd pathology and virus distribution in the lung and lymphoid tissues of pigs experimentally inoculated with three distinct type prrs virus isolates of varying pathogenicity increased pathogenicity of european porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance induction of porcine reproductive and respiratory syndrome virus (prrsv)-specific regulatory t lymphocytes (treg) in the lungs and tracheobronchial lymph nodes of prrsvinfected pigs impact of prrsv strains of different in vivo virulence on the macrophage population of the j o u r n a l p r e -p r o o f thymus development and application of a porcine specific elisa for the quantification of soluble cd analysis of the expression of porcine cd r and cd r l by using newly developed monoclonal antibodies dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype prrsv strain downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virusinfected pigs virulent lena strain induced an earlier and stronger downregulation of cd in bronchoalveolar lavage cells the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- activation of the extrinsic apoptotic pathway in the thymus of piglets infected with prrsv- strains of different virulence kinetics of the expression of cd and cd a in the lung and tonsil of pigs after infection with prrsv- strains of different virulence the porcine a antigen is homologous to human cd and related to macrophage differentiation the pd- /pd-l axis and virus infections: a delicate balance molecular epidemiology of prrsv: a phylogenetic perspective use of a polymerase chain reaction assay and an elisa to monitor porcine circovirus type infection in pigs from farms with and without postweaning multisystemic wasting syndrome european genotype of porcine reproductive and respiratory syndrome ( prrsv ) infects monocyte-derived dendritic cells but does not induce treg cells induction of t helper regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus regulatory t cells in respiratory health and diseases. pulmonary medicine emergence of a virulent porcine reproductive and respiratory syndrome virus (prrsv) strain in lower austria a critical function for cd in lung immune homeostasis and the severity of influenza infection pathogenicity of three genetically diverse strains of prrsv type in specific pathogen free pigs the role of pulmonary intravascular macrophages in porcine reproductive and respiratory syndrome virus infection the cd -cd r inhibitory signaling pathway. immune regulation and host-pathogen interactions sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus the combination of prrs virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs porcine reproductive and respiratory syndrome virus infection increases cd expression and lipopolysaccharide-binding protein in the lungs of pigs proinflammatory cytokines and viral respiratory disease in pigs inhibition of nox oxidase activity ameliorates influenza a virus-induced lung inflammation recovery from acute lung injury can be regulated via modulation of regulatory t cells and th cells comparative analysis of immune responses following experimental infection of pigs with european porcine reproductive and respiratory syndrome virus strains of differing virulence lung pathogenicity of european genotype strain porcine reproductive and respiratory syndrome virus (prrsv) differs from that of subtype strains gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus regulation of inos-derived ros generation by hsp and cav- in porcine reproductive and respiratory syndrome virus-infected swine lung injury porcine reproductive and respiratory syndrome virus; cd r , cd receptor ; inos, inducible nitric oxide synthase; foxp , forkhead box protein ; mab, monoclonal antibody; pab citrate ph , microwave heat treatment at w for minutes enzymatic digestion with protease type xiv (sigma-aldrich) at º c for minutes; citrate ph *, autoclave treatment at º c for min we express our appreciation to gema muñoz, alberto alcántara and esmeralda cano for their technical assistance and dr. hans nauwynck for providing us the prrsv- subtype key: cord- - ydh jev authors: meng, x.j title: heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ydh jev porcine reproductive and respiratory syndrome virus (prrsv) continues to be a major problem to the pork industry worldwide. increasing data indicate that prrsv strains differ in virulence in infected pigs and are biologically, antigenically, and genetically heterogeneous. it is evident that the current vaccines, based on a single prrsv strain, are not effective in protecting against infections with the genetically diverse field strains of prrsv. the recent outbreaks of atypical or acute prrs in vaccinated pigs have raised a serious concern about the efficacy of the current vaccines and provided the impetus for developing more effective vaccines. special attention in this review is given to published work on antigenic, pathogenic and genetic variations of prrsv and its potential implications for vaccine efficacy and development. although there are ample data documenting the heterogeneous nature of prrsv strains, information regarding how the heterogeneity is generated and what clinical impact it may have is very scarce. the observed heterogeneity will likely pose a major obstacle for effective prevention and control of prrs. there remains an urgent need for fundamental research on this virus to understand the basic biology and the mechanism of heterogeneity and pathogenesis of prrsv. porcine reproductive and respiratory syndrome (prrs), characterized by severe reproductive failure in sows and respiratory diseases in young pigs, was ®rst recognized in the united states (u.s.) in (keffaber, ; hill, ) . since its appearance, prrs has devastated the swine industry with tremendous economic losses (polson et al., ) . the causative agent of prrs, porcine reproductive and respiratory syndrome virus (prrsv), was ®rst isolated by wensvoort et al. ( ) in the netherlands using porcine alveolar macrophages (pam) and was designated as lelystad virus (lv). in the u.s., prrsv was ®rst isolated and characterized in a continuous cell line atcc cl collins et al., ) . prrsv is a small, enveloped, single positive-stranded rna virus (wensvoort et al., ; ben®eld et al., ; meulenberg et al., a, b) . prrs has now been recognized worldwide (plana et al., ; saito et al., ; valicek et al., ; madsen et al., ; chueh et al., ) and is considered to be an economically important global disease. although prrsv strains identi®ed from around the world cause similar diseases in pigs, increasing data indicate that prrsv strains differ in virulence in infected animals and are antigenically and genetically heterogeneous. more recently, swine herds in the u.s. have experienced outbreaks of a severe form of prrs characterized by abortion and high mortality in pregnant sows (botner et al., a; bell, ; mengeling et al., ; lager et al., ; osorio et al., ) . this form of prrs has been referred to as sow abortion and mortality syndrome, atypical prrs, severe prrs and acute prrs. surprisingly, many of the affected herds were vaccinated, suggesting that the current prrs vaccines do not confer % protection and that a new generation of vaccines is needed. this review will summarize published data describing the heterogeneity of prrsv and discuss the potential implications for current vaccine ef®cacy and future vaccine development. recent reviews on other aspects of prrsv (rossow, ; botner, b; molitor et al., ; meulenberg et al., a; zimmerman et al., ; van reeth, ) also discuss some of the work cited here. meulenberg et al. ( a) ®rst cloned and sequenced the genome of lv, a european strain of prrsv. subsequently, partial sequences of another european strain (conzelmann et al., ) and two north american strains of prrsv (mardassi et al., ; meng et al., ) were reported. more recently, the complete genomes of two north american strains of prrsv have been determined (allende et al., ; nelsen et al., ) . the genome of prrsv is an kb positive strand rna molecule that encodes eight overlapping open reading frames (orfs) organized similarly to the orfs of coronaviruses (lai and cavanagh, ; meulenberg et al., a; allende et al., ; nelsen et al., ) . the overlaps between the orfs of lv range from bp (between the orfs and ) to bp (between the orfs and ). the u.s. strains of prrsv have a bp noncoding region separating orfs and morozov et al., ) . orfs a and b comprise about % of the viral genome and are predicted to encode the viral rna polymerase (meulenberg et al., a; allende et al., ; nelsen et al., ) . the c-terminus of orf a overlaps the n-terminus of orf b by nucleotides. a heptanucleotide slippery sequence, uuuaaac, located just upstream of the uag stop codon of orf a, and a pseudo-knot structure downstream of the slippery sequence are believed to be essential for the expression of orf b of prrsv via a mechanism of ribosomal frame-shifting (meulenberg et al., a; allende et al., ; nelsen et al., ) . orfs , and of prrsv encode virion-associated proteins designated as gp , gp and gp , respectively (meulenberg et al., , b meulenberg and petersen-den besten, ; van nieuwstadt et al., ) . however, the gp protein of a canadian prrsv isolate encodes a nonvirion-associated, soluble protein (mardassi et al., ) , which is similar to the orf protein encoded by lactate dehydrogenase-elevating virus (ldv) (faaberg and plagemann, ) . the reason for the discrepancy in whether gp protein of prrsv is a structural protein is not clear, but genetic variation of the orf gene might be responsible. therefore, the gp protein of other diverse strains of prrsv should also be evaluated. orfs , and of prrsv encode envelope (gp ), membrane (m) and nucleocapsid (n) proteins, respectively (mardassi et al., ; meulenberg et al., meulenberg et al., , a . monoclonal antibodies directed against gp and gp proteins are neutralizing (meulenberg et al., b; pirzadeh and dea, ) . the m protein is an unglycosylated protein of kda which has the same hydrophobicity pro®le as the m proteins of equine arteritis virus (eav) (de vries et al., ) and ldv (godeny et al., ) . the n protein is not n-glycosylated, although it contains or potential n-glycosylation sites . the order of prrsv genes, -viral polymerase (orfs a/ b)-virion-associated proteins gp (orf )-gp (orf )-gp (orf )-gp (orf )-m (orf )-n (orf )- , is the same as in eav (den boon et al., ) , ldv (plagemann and moennig, ) and simian hemorrhagic fever virus (shfv) . therefore, prrsv, along with eav, ldv and shfv, is now classi®ed within a single genus arterivirus in the family arteriviridae in the order nidovirales (cavanagh, ) . the expression and replication of prrsv requires the production of at least six subgenomic mrnas (sg mrnas) (conzelmann et al., ; meulenberg et al., a meulenberg et al., , b, a meng et al., meng et al., , b snijder and meulenberg, ) . these sg mrnas, together with the genomic virion rna, form a -coterminal nested set. each of these sg mrnas contains a common leader sequence of about bp in size (meulenberg et al., a, b; morozov et al., ; nelsen et al., ; allende et al., ; oleksiewicz et al., ) . the leader-mrna junction sequence of prrsv, in which the leader joins to the body of the sg mrnas, is a conserved sequence motif of six nucleotides (ucaacc) or a highly similar sequence (meulenberg et al., a, b; meng et al., b meng et al., , b nelsen et al., ) . the sg mrnas of prrsv are not packaged into the virions , suggesting that the encapsidation signal of prrsvis likely localized within the orf region that is unique to the viral genome, but not present in the sg mrnas. northern blot analysis with orf-speci®c probes indicates that sg mrnas are polycistronic . it is generally believed that only the orf at the end of each sg mrna is translationally active and, thus, each of the sg mrnas is functionally monocistronic. the precise mechanism of transcription and translation of prrsv is not understood, although it is believed to be similar to that of coronaviruses (lai and cavanagh, ) . the recently constructed infectious cdna clone of prrsv should facilitate investigation of the mechanism of prrsv replication. biological variations among prrsv isolates have been reported. the european prrsv isolates were preferentially propagated in pam cultures (wensvoort et al., ; wensvoort, ) , whereas the north american isolates were grown in pam cultures as well as in three continuous cell lines, cl , marc- and crl collins et al., ; kim et al., ; meng et al., meng et al., , a . swine testis (st) cells were also reported to support prrsv replication (plana et al., ) . however, the st cells cannot propagate a high virulence prrsv isolate vr . variations in the susceptibilities of cl cells and pam to prrsv infection were reported. not all prrsv isolates growing in cl cells replicated in pam, and vice versa (bautista et al., a) . failure to propagate some strains of prrsv in certain cell cultures indicates the existence of prrsv variants and, thus, both pam and other cell lines should be used when attempting virus isolation from clinical samples. it has been reported that ®eld strains of prrsv vary in their susceptibility to antibodydependent enhancement (ade) of infection . therefore, the altered ability to infect may be due to the selection of variants that can facilitate infection of macrophages through ade. antigenic variations among prrsv isolates have been well documented. wensvoort et al. ( ) and wensvoort ( ) reported that, antigenically, four european isolates resembled each other closely, but differed from the u.s. isolates, and that three u.s. isolates differed from each other. serologic survey of the ®eld samples by immunouorescence assay indicated that about % of the samples were positive for european lv, but negative for u.s. isolate vr , and that about % of the samples were positive for vr , but negative for lv (bautista et al., b) . in another study, north american isolates were found to be more closely related serologically to each other than to the european isolates (frey et al., ) . of canadian swine sera tested, samples were positive for vr antibody, but only samples were positive for lv. when swine sera from the netherlands were tested, samples were positive for lv antibody, but only samples were positive for vr . western blot analysis indicated that the orf protein of mn- isolate reacted with only % of prrsv-infected pig sera tested (kwang et al., ) . differential reactivity of monoclonal antibodies (mabs) with different prrsv isolates was also reported. two mabs to n protein recognized a conserved epitope in u.s. and european prrsv isolates, but four other mabs to n protein reacted with u.s. isolates only (nelson et al., ) . six mabs raised against a british isolate of prrsv did not react with u.s. isolates tested (drew et al., ) . five mabs against gp protein of a canadian isolate did not react with lv (pirzadeh and dea, ; pirzadeh et al., ). an mab to m protein reacted with all north american prrsv isolates tested, but failed to react with any of the european isolates . the reactivity of mabs against gp , gp and n proteins with european and american prrsv isolates also revealed antigenic differences not only between the u.s. and european isolates, but among different european or u.s. isolates as well (katz et al., ; wieczorek-krohmer et al., ) . in addition, antigenic variation was demonstrated between an isolate and its progeny recovered after in vivo passages (le gall et al., ) , suggesting that a relatively high rate of mutations occurs during prrsv replication in its natural host. given the degree of antigenic diversity observed among prrsv strains, it is unlikely that a vaccine based on one strain of prrsv will effectively protect against antigenically different enzootic ®eld strains of prrsv. in fact, lager et al. ( ) recently showed that gilts inoculated with one strain of prrsv did not completely protect against heterologous challenge with an antigenically distinct prrsv strain. therefore, the effectiveness of a vaccine against heterologous enzootic ®eld strains of prrsv will largely depend on the antigenic relatedness of the virus strain to which the vaccinated animals were exposed. the design of future vaccines will have to take into consideration the antigenic diversity. the author believes that a multivalent vaccine consisting of multiple antigenically distinct strains of prrsv is the most promising candidate for the next generation of vaccines. the mechanism of prrsv pathogenesis is poorly understood. it is generally believed that prrsv initiates an infection in pigs via entry through nasal epithelial, tonsillar, and pulmonary macrophages. prrsv replicates in these cells, causes viremia and, subsequently, results in pneumonia, myocarditis, encephalitis, rhinitis, vasculitis, lymphadenopathy, etc. in target organs (rossow et al., (rossow et al., , . it has been well documented that prrsv causes persistent infections in pigs (albina et al., ; christopher-hennings et al., a, b; wills et al., a, b) . in experimentally infected boars, prrsv can be detected by pcr in semen samples at days postinoculation (dpi) (christopher-hennings et al., a, b) . pigs persistently infected with prrsv can transmit the virus to naive pigs by direct or indirect contact, and the transmission by direct contact occurs up to weeks after infection (albina et al., ; wills et al., a, b) . bilodeau et al. ( ) showed that when speci®c-pathogen-free (spf) pigs were introduced into a barn that had housed prrsv-infected pigs as much as months after clinical signs of infection had disappeared, the newly introduced spf pigs became infected. this study indicates that subclinical prrsv infection can persist in the animals. wills et al. ( a, b) demonstrated that prrsv can be isolated from oropharyngeal samples for up to dpi. pigs persistently infected with prrsv may appear clinically normal, but can still transmit virus to pigs in naive swine herds. therefore, persistent infection of prrsv plays an important role in prrsv survival and transmission, and will likely pose a major obstacle in prrs control programs. despite the ample data documenting prrsv persistence, little has been done to understand the mechanism of persistent infection or what clinical impact it may have. clearly, more studies are needed in the future to determine the host and virus factors that lead to the persistent state and to fully elucidate the mechanism of prrsv pathogenesis. marked differences in virulence among prrsv strains have been observed in experimentally-infected pigs . signi®cant differences in severity of clinical respiratory disease, rectal temperatures, gross lung lesions and microscopic lung lesions were observed among nine different u.s. isolates of prrsv. the european lv and the low-virulence u.s. prrsv isolate vr (isu ) induced mild transient pyrexia, dyspnea and tachypnea, but several high virulence u.s. isolates induced labored respiration, pyrexia, lethargy, anorexia and patchy dermal cyanosis. at dpi, mean lung lesion scores estimating the percentage of lungs affected by pneumonia ranged from . % for lv, . % for vr (isu ), . % for vr , to . % for isu- . despite the observed difference in virulence among prrsv isolates, tissue tropism and distribution of prrsv antigen or nucleic acid within tissues and organs were very similar in pigs inoculated with different strains of prrsv haynes et al., ) . strains of prrsv also vary in virulence for their ability to cause reproductive failure (mengeling et al., ) . mengeling et al. ( ) reported that the effects of prrsv on reproductive performance are strain-dependent. in addition, apathogenic ®eld isolates of prrsv have been reported (ohlinger et al., ; van alstine, ) , indicating that ®eld isolates of prrsv differ in virulence. the recent outbreaks of severe atypical or acute prrs further indicate that the recent atypical prrsv strains circulating in the u.s. swine herds are more virulent than those strains isolated earlier (botner et al., a; mengeling et al., ; bell, ; lager et al., ; osorio et al., ) . mengeling et al. ( ) demonstrated that a ®eld strain of atypical prrsv produced especially severe clinical signs of disease and reproductive failure in experimentally infected gilts. rossow et al. ( ) reported that marked neurovirulence in neonatal pigs was found to be associated with infection by some ®eld isolates of prrsv. prrsv was identi®ed in macrophages or microglia of brain lesions by immunohistochemical staining of brain sections. the replication of the virus in the brain was veri®ed by in situ hybridization. the mechanism for the observed prrsv neurovirulence in neonatal pigs is not known, but genetic changes in prrsv genome may alter the tissue tropism of prrsv. the mechanism for pathogenic variation observed among prrsv strains remains unknown, but the genetic make-up of a particular virus strain will likely determine the virulence of the virus in animals. therefore, it is important to genetically characterize ®eld strains of prrsv with differing virulence. one recent breakthrough in prrsv research is the construction of an infectious cdna clone of prrsv . using this infectious clone, one should be able to construct viruses that are chimeras of low and high virulence strains of prrsv or to mutant genes of interest to study the structural and functional relationship of prrsv genes. the availability of this infectious cdna clone will eventually aid prrsv researchers in identifying the genetic virulence determinant(s) of prrsv. prrsv is genetically heterogeneous. extensive sequence variation was found between the european and the u.s. isolates (mardassi et al., ; meng et al., meng et al., , a meng et al., , b, b morozov et al., ; murtaugh et al., ; nelsen et al., ; allende et al., ) . the nucleotide sequence identity between lv and the u.s. isolates is ± % in orf , ± % in orf , ± % in orf , and ± % in orf (meng et al., a, b) . orfs and genes are relatively conserved among the u.s. isolates or among the european isolates, but extensive genetic variation was observed in the orfs and genes between european and u.s. isolates. it has been shown that the nucleotide sequence identity was ± % in orf , ± % in orf , ± % in orf , and ± % in orf among six u.s. isolates . interestingly, the least virulent u.s. isolate, isu (atcc vr ), has the most divergent sequence compared to the other ®ve u.s. isolates. the nucleotide sequence identity between isu and the other u.s. isolates was ± % in orf , ± % in orf , and ± % in orf . the orf of isu has a three-nucleotide deletion and shares ± % nucleotide sequence identity compared to that of the other u.s. isolates. the sequence variation between the least virulent strain isu and other u.s. isolates appears to be randomly distributed throughout the genome , thus it is dif®cult to speculate regarding any correlation between prrsv virulence and a particular gene or sequence. kapur et al. ( ) analyzed the nucleotide sequence of orfs ± of u.s. prrsv isolates, and found that the genetic distance ranges from . ± . % among these u.s. isolates and is about % between lv and the u.s. isolates. simple accumulation of random neutral mutations cannot explain the substantial nucleotide differences among prrsv isolates, and the mechanism for generating the genetic heterogeneity remains unknown. the leader sequence of prrsv strains also varies signi®cantly. the bp leader sequence of vr strain is bp shorter than that of lv, and has a sequence identity of % with that of lv . the leader sequence of another north american strain, b, is bp in length and also differs considerably in nucleotide sequence with that of lv (allende et al., ) . like the leader sequence, the orf gene sequence also differs extensively between the u.s. and the european strains (allende et al., ; nelsen et al., ) . the orf a of vr strain shares only about % nucleotide sequence identity with that of the lv strain. orf b is more conserved than orf a and shares about % nucleotide sequence identity with that of lv. however, a stretch of amino acids at the carboxyl terminus of orf b of vr has only % similarity with that of lv . allende et al. ( ) also showed that north american strain b shares only about % amino acid identity in the orf a polyprotein region with that of lv. the greatest divergence is found in the nonstructural protein (nsp ), which shares only about % amino acid identity with the corresponding region of lv. surprisingly, the nsp of strain b is amino acids longer than that of lv (allende et al., ) . like strain vr , strain b also exhibits greater divergence in the carboxyl terminal region of orf b (cp protein), with only % identity with the corresponding region of lv. considering the striking differences in the leader sequence and in all orfs between european and north american strains, it is surprising that both european and north american strains cause a similar disease. although the origin of prrsv remains unknown, these data strongly suggest that the european strains and the north american strains of prrsv have undergone divergent evolution on separate continents from a common ancestor. based on the sequence and evolution analyses of u.s. isolates, kapur et al. ( ) estimated that the time for generating the amount of nucleotide variation in the midwestern u.s. isolates takes about ± years of virus evolution, suggesting that prrsv probably emerged as a swine pathogen approximately a decade ago. it has been speculated that european and north american prrsv strains may have evolved from an ldv-like ancestor (plagemann, ; nelsen et al., ) . however, the almost simultaneous emergence of prrs in swine on two continents makes the theory of divergent evolution dif®cult to believe. more likely, the simultaneous emergence of prrs on two continents might relate to global changes in commercial swine management and husbandry . the sg mrnas of prrsv are heterogeneous (meulenberg et al., b; meng et al., b; snijder and meulenberg, ; faaberg et al., ; nelsen et al., ) . meng et al. ( b) reported that, in addition to the genomic rna, a nested set of six or seven sg mrnas is present in cells infected with different isolates of u.s. prrsv that differ in virulence. prrsv isolates isu , isu and isu produce seven sg mrnas, whereas isolates isu and isu produce only six sg mrnas. the additional species of sg mrna (designated as sg mrna - ) is located between sg mrnas and , and is generated from the sequence upstream of orf . however, there is no apparent correlation between virus virulence and the additional sg mrna - . interestingly, a small orf with a coding capacity of amino acid residues at the -end of the sg mrna - was identi®ed. thus, the sg mrna - in some isolates is potentially bicistronic. however, whether this small orf - is actually translated or has any biological functions remains to be studied. morozov et al. ( ) reported that there are two leader-body junction sites for sg mrnas and . nelsen et al. ( ) also demonstrated that vr strain of prrsv utilizes two leader-body junction sites for sg mrna transcription: one site at bp upstream of the orf start codon for most mrna transcripts, and the other site at bp upstream of the orf start codon for a minority of transcripts. in contrast, lv only utilizes the site at bp upstream of the orf start codon. faaberg et al. ( ) has also shown that the sg mrna is transcribed with different leader-body junction sites. the differences in sg mrnas and leader-body junction sites among prrsv isolates further re¯ect the heterogeneous nature of the virus. future studies are needed to elucidate the biological signi®cance of the heterogeneity. at least two distinct genotypes of prrsv have been reported: the european and the north american . recently, an impressive amount of sequence data on prrsv isolates has been generated. to gain a better understanding of the genetic relationship and evolution of prrsv, phylogenetic analyses were performed based on the sequences of orf (fig. a) and orf (fig. b) genes of strains of prrsv worldwide. these sequences were either published (andreyev et al., ; casal et al., ; chueh et al., ; conzelmann et al., ; drew et al., ; gagnon and dea, ; kapur et al., ; le gall et al., ; madsen et al., ; mardassi et al., ; meulenberg et al., a; murtaugh et al., ; nelsen et al., ; pirzadeh et al., ; plana et al., ; rodriguez et al., ; saito et al., ; suarez et al., ; sur et al., ; valicek et al., ; wesley et al., ; wootton et al., ; yang et al., ) or are available in genbank (af , af , af , af , af , af , x , af , u , u ). phylogenetic analysis was also performed on the basis of the complete sequences of the structural genes (orfs ± ), which are available for isolates of prrsv (fig. c) . all three phylogenetic trees, based on different regions of the genome, indicate that the north american and european isolates of prrsv represent two distinct genotypes as reported previously. however, within each of the two major genotypes, several minor genotypes (or variants) of prrsv were also identi®ed ( fig. a and c) . the n gene of prrsv is relatively conserved within each of the two major genotypes (fig. b) . in contrast, the major envelope protein gene (gp ) of prrsv exhibits greater genetic diversity within each major genotype (fig. a) . similarly, the phylogenetic tree based on the complete sequence of the structural genes ( fig. c ) displays greater genetic diversity than the n gene within each major genotype. interestingly, the strains from japan, china, taiwan, guatemala and three danish strains of prrsv are all clustered within the north american genotype. the three danish strains, the chinese strain (s ), a canadian strain (pa ), and a strain from nebraska (ne b) are all found to be closely related to the modi®ed-live vaccine (mlv) virus and the vaccine strain vr . the three danish strains of prrsv are believed to originate from the mlv vaccine virus (vr ) that was used in danish swine herds (madsen et al., ) . phylogenetic trees (fig. a and c) con®rm that these three danish strains are most closely related to the mlv respprrs vaccine virus, but are less related to other north american strains, further indicating that the introduction of north american type of prrsv in denmark was due to the spread of vaccine virus vr (madsen et al., ) . the origin of other strains that are closely related to the mlv and strain vr is not known. it is possible that these strains isolated from different geographic regions also evolved from the mlv vaccine virus vr as a result of largescale vaccination programs in swine herds around the world. quasispecies is de®ned as a population of closely related, yet heterogeneous sequences that are variants of one dominant sequence (bukh et al., ) . viral quasispecies are closely related mutant and recombinant viral genomes subjected to continuous genetic variation, competition and selection (domingo et al., ) . within a single infected animal, many rna viruses have been found to exist as quasispecies. this heterogeneity can cause persistent infection resulting from selection of mutants that escape neutralizing antibody or cytotoxic t lymphocytes (ctl) or as a result of the presence of defective particles (duarte et al., ; ahmed et al., ; domingo et al., ) . extensive genetic variations have been observed among different strains of prrsv; however, little is known about the mechanism of generating genetic diversity. recently, rowland et al. ( ) reported evidence for quasispecies evolution and emergence of a virus subpopulation during in utero infection of pigs with a prrsv isolate. a single nucleotide change in the ectodomain of gp protein was identi®ed during infection of pigs with prrsv strain vr . this ®nding suggests that the genetic variability in the ectodomain of the orf may be due to positive or negative selection forces, such as selection by antibody or other host defenses. other factors such as rna secondary structure, especially in orf , the diverse leader-body junction sites, and the size and sequence difference of the leader sequence among prrsv strains should also be considered as potential driving forces for heterogeneity and quasispecies evolution of prrsv. quasispecies has been well documented in other viruses that cause persistent infections including ldv, an arterivirus closely related to prrsv (duarte et al., ; plagemann et al., ; plagemann, ; bukh et al., ; ahmed et al., ; domingo et al., ) . the extent and nature of prrsv quasispecies evolution and whether quasispecies evolution are related to prrsv persistency remain to be determined. in ldv, quasispecies have been identi®ed and biologically characterized (chen et al., (chen et al., , a (chen et al., , b, . the ldv quasispecies were found to differ in neuropathogenicity, and the neuropathogenic ldvs are incapable of establishing persistent infection in mice. the existence of a quasispecies population during virus infection will affect vaccine ef®cacy and may lead to vaccine failure (domingo and holland, ; duarte et al., ; domingo et al., ) . the ®rst report of prrsv quasispecies evolution by rowland et al. ( ) should stimulate further study of the nature of prrsv quasispecies evolution and its clinical implications. rna recombination can provide a powerful and effective mechanism for evolution of an rna virus. the ability to exchange genetic information may allow rna viruses to adapt a changing environment and to escape a selection pressure (such as neutralizing antibodies), thus providing the recombinant virus with an evolutionary advantage (lai and cavanagh, ) . recently, yuan et al. ( ) provided evidence for homologous rna recombination between prrsv isolates propagated in cell culture. recombinant viral particles containing chimeric orf and orf proteins were identi®ed in ma- cells co-infected with two prrsv isolates. nucleotide sequence analyses con®rmed independent recombination events. the frequency of recombination was estimated from < % up to % within the -bp fragment analyzed. sequence analyses of ®eld isolates of prrsv suggest that rna recombination of prrsv may also occur in nature (yuan et al., ) . by analyzing ®eld isolates of u.s. prrsv, kapur et al. ( ) also provided evidence for intragenic recombination in orfs ± and in orf among prrsv isolates. whether rna recombination plays any roles in generating genetic heterogeneity of prrsv is not known. direct experimental evidence for in vivo rna recombination of prrsv is still lacking. most rna virus recombination studies have been performed in cell cultures, although rna recombination has also been demonstrated in the natural host of picornaviruses, coronavirus and ldv (lai and cavanagh, ; minor et al., ; li et al., ) . for example, a case of poliovirus vaccine-associated poliomyelitis in a human was caused by recombination between two poliovirus vaccine strains (minor et al., ) . high frequency homologous genetic recombination was reported in mice dually infected with two strains of ldv . therefore, future studies are warranted to determine whether rna recombination of prrsv is a phenomenon unique to cell culture or whether this actually occurs in vivo. the frequency of recombination, crossover sites and the clinical implications of prrsv rna recombination also need to be studied. the sg mrna species may also be involved in rna recombination events during infection. thus, future studies are needed to examine rna recombination not only at genomic rna level, but at the sg mrna level as well. several prrs vaccines are currently available; however, there are mixed results regarding the ef®cacy of these vaccines against the genetically diverse ®eld strains of prrsv (lager and mengeling, ; plana-duran et al., ; christopher-hennings et al., ; van woensel et al., ; osorio et al., ; madsen et al., ; mengeling et al., a, b, c; wesley et al., ) . respprrs/repro (boehringer ingelheim), an mlv, is recommended for use in ± week-old pigs and in nonpregnant females (lager and mengeling, ; dee and joo, ) . the prime pac prrs vaccine (schering plough animal health corporation) (hesse et al., ) is also an mlv which has been shown to reduce the severity and duration of disease following challenge. however, it did not prevent infection of vaccinated pigs by a virulent heterologous strain. a live vaccine based on a european isolate of prrsv (porcilis prrs) was found to protect fattening pigs against the respiratory manifestations of prrs (mavromatis et al., ) . osorio et al. ( ) compared three commercial vaccines in their ability to induce protection against prrsv strains of high virulence, and found that these vaccines confer protection against clinical disease, but not against infection. the use of mlvs in boars resulted in vaccine virus shedding in semen and reduced semen quality, but there was reduced or no shedding of wild-type prrsv after challenge (christopher-hennings et al., ) . in another study, vaccination with a mlv in boars resulted in marked reduction in viremia and shedding of virus in semen, but vaccination with inactivated vaccine did not change the onset, duration or level of viremia or shedding of virus in semen (nielsen et al., ) . by using a restriction-site marker that is present in the vaccine virus (vr ), mengeling et al. ( b) demonstrated that the marker was not detected in any of the ®eld strains of prrsv isolated before use of the vaccine. however, the restriction-site marker was detected in of ®eld strains isolated after the introduction of the vaccine, and these ®eld strains were believed to be direct-line descendants of the vaccine virus (mengeling et al., b) . more importantly, these putative vaccine-related strains produced more pronounced pathological changes than did the vaccine virus alone (mengeling et al., b) . wesley et al. ( ) also showed that the restriction fragment length polymorphism (rflp) patterns change as the vaccine virus spreads among a swine population. a glycine marker in the orf gene of the vaccine virus is rapidly lost and replaced with arginine. the use of mlvs in herds may lessen the clinical signs of prrs following infection. however, the potential risk for reversion of mlvs to virulent phenotypes cannot be overlooked. in the absence of a new generation of vaccines, more studies are needed to fully evaluate the safety and ef®cacy of the current mlvs. the emergence and re-emergence of viral infectious diseases is often in¯uenced by the genetics of the viruses (domingo and holland, ; duarte et al., ; domingo et al., ) . genetic heterogeneity of prrsv, due to quasispecies evolution and rna recombination, could lead to the selection of virulent viruses and to the emergence or reemergence of new forms of prrs. quasispecies evolution of prrsv in response to positive or negative selection pressures may signi®cantly change the genomic sequence of mlvs over time as the vaccine virus spreads among swine herds, and ultimately, these genetic changes may revert mlvs to virulent phenotypes. it is also possible that virulent strains of prrsv could be generated through rna recombination between mlvs used in the vaccination programs and enzootic ®eld strains of prrsv. the recent outbreaks of the atypical or acute prrs re¯ect the need to further study this virus to better understand its biology and develop more effective vaccines. most of the herds affected by the atypical prrs had been vaccinated with the current vaccines (bell, ; lager et al., ) . it is possible that a mutant strain(s) of prrsv may be responsible for the recent outbreaks of acute prrs. the heterogeneous nature of prrsv suggests that complete elimination of the virus from the environment is unlikely. the observed genetic diversity among ®eld isolates of prrsv will continue to be the major obstacle for prrs control. therefore, the design for the next generation of vaccines will have to take into consideration the genetic heterogeneity of prrsv, or prrs will remain dif®cult to control. intensive research is required to answer the many questions that remain. the recently constructed infectious cdna clone of prrsv should enable us to study the basic biology of prrsv. using the infectious cdna clone, one can monitor the nature of quasispecies evolution of prrsv in pigs infected by a homogeneous virus derived from the infectious clone. one can also genetically engineer the virus to produce a modi®ed avirulent strain that could be used as an mlv. the immunogenic gene(s) of the genetically-engineered avirulent strain of prrsv can be replaced with that of other antigenically and genetically distinct prrsv strains to produce avirulent virus strains that can be used as a multivalent mlv. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions persistence of viruses genetic variation and phylogenetic relationships of porcine reproductive and respiratory syndrome virus (prrsv) ®eld strains based on sequence analysis of open reading frame comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody serologic survey for lelystad and vr- strains of porcine respiratory and reproductive syndrome (prrs) virus in u.s. swine herds characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation appearance of acute prrs-like symptoms in sow herds after vaccination with a modi®ed live prrs vaccine diagnosis of prrs genetic heterogeneity of hepatitis c virus: quasispecies and genotypes identi®cation of a common antigenic site in the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus nidovirales: a new order comprising coronaviridae and arteriviridae coexistence in lactate dehydrogenase-elevating virus pools of variants that differ in neuropathogenicity and ability to establish a persistent infection neuropathogenicity and susceptibility to immune response are interdependent properties of lactate dehydrogenase-elevating virus (ldv) and correlate with the number of n-linked polylactosaminoglycan chains on the ectodomain of the primary envelope glycoprotein lactate dehydrogenase elevating virus variants: cosegregation of neuropathogenicity and impaired capability for high viremic persistent infection selective antibody neutralization prevents neuropathogenic ldv from causing paralytic disease in immunocompetent mice sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus taiwan md- strain persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars detection of porcine reproductive and respiratory syndrome virus in boar semen by pcr effects of a modi®ed-live virus vaccine against porcine reproductive and respiratory syndrome in boars isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group strategies to control prrs: a summary of ®eld and research experiences structural proteins of equine arteritis virus equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily complications of rna heterogeneity for the engineering of virus vaccines and antiviral agents quasispecies structure and persistence of rna virus production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus rna virus quasispecies: signi®cance for viral disease and epidemiology orf of lactate dehydrogenase-elevating virus encodes a soluble, nonstructural, highly glycosylated, and antigenic protein subgenomic rna is transcribed with different leader-body junction sites in prrsv (strain vr ) infection of cl cells diagnostic testing for sirs virus at the national veterinary service laboratory (nvsl) differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their orfs and genes complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) development of a streptavidin-biotin immunoperoxidase procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung immunohistochemical identi®cation of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two u.s. porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparison of the antigen distribution of two u.s. porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine u.s. porcine reproductive and respiratory syndrome virus (prrsv) isolates in a ®ve-week-old cesarean-derived, colostrum-deprived pig model temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (prrsv) by in situ hybridization in pigs infected with isolates of prrsv that differ in virulence ef®cacy of prime pac prrs in controlling prrs respiratory disease: homologous and heterologous challenge overview and history of mystery swine disease (swine infertility and respiratory syndrome) genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states antigenic differences between european and american isolates of porcine reproductive and respiratory syndrome virus (prrsv) are encoded by the carboxyterminal portion of viral open reading frame reproductive failure of unknown etiology enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line cloning, expression and sequence analysis of the orf gene of porcine reproductive and respiratory syndrome virus mn- b current status of vaccines and vaccination for porcine reproductive and respiratory syndrome acute prrs evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challengeexposed with an antigenically distinct prrsv isolate the molecular biology of coronaviruses antigenic variability of porcine reproductive and respiratory syndrome (prrs) virus isolates: in¯uence of virus passage in pig molecular variation in the nucleoprotein gene (orf ) of the porcine reproductive and respiratory syndrome virus (prrsv) high-frequency homologous genetic recombination of an arterivirus, lactate dehydrogenase-elevating virus, in mice and evolution of neuropathogenic variants sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus identi®cation of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus a subset of porcine reproductive and respiratory syndrome virus gp glycoprotein is released into the culture medium of cells as a non-virionassociated and membrane-free (soluble) form field evaluation of a live vaccine against porcine reproductive and respiratory syndrome in fattening pigs molecular cloning and nucleotide sequencing of the terminal genomic rna of porcine reproductive and respiratory syndrome virus phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the usa and europe sequence comparison of open reading frames to of low and high virulence united states isolates of porcine reproductive and respiratory syndrome virus characterization of a high-virulence u.s. isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from ®eld cases of atypical prrs safety and ef®cacy of vaccination of pregnant gilts against porcine reproductive and respiratory syndrome identi®cation and clinical assessment of suspected vaccine-related ®eld strains of porcine reproductive and respiratory syndrome virus diagnostic implications of concurrent inoculation with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav subgenomic rnas of lelystad virus contain a conserved leader±body junction sequence characterization of proteins encoded by orfs to of lelystad virus identi®cation and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp encoded by orf is incorporated in virus particles molecular characterization of lelystad virus posttranslational processing and identi®cation of a neutralization domain of the gp protein encoded by orf of lelystad virus infectious transcripts from cloned genome-length cdna of porcine reproductive and respiratory syndrome virus antigenic and molecular evolution of the vaccine strain of type poliovirus during the period of excretion by a primary vaccine immunity to prrsv: double-edged sword sequence analysis of open reading frames (orfs) to of a u.s. isolate of porcine reproductive and respiratory syndrome virus characterization of leader-body junction sites in subgenomic mrnas of a u.s. prrsv isolate comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies examination of virus shedding in semen from vaccinated and from previously infected boars after experimental challenge with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents some aspects of the virus causing prrs in germany determination of -leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs prrsv: comparison of commercial vaccines in their ability to induce protection against current prrsv strains of high virulence monoclonal antibodies to the orf product of porcine reproductive and respiratory syndrome virus de®ne linear neutralizing determinants genetic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope gp glycoprotein lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus, a new group of positive strand rna virus lactate dehydrogenase-elevating virus: an ideal persistent virus? lactate dehydrogenase-elevating virus and related viruses porcine epidemic abortion and respiratory syndrome (mystery swine disease). isolation in spain of the causative agent and experimental reproduction of the disease ef®cacy of an inactivated vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus financial evaluation and decision making in the swine breeding herd epitope mapping of the nucleocapsid protein of european and north american isolates of porcine reproductive and respiratory syndrome virus pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- characteristics of major structural protein coding gene and leader-body sequence in subgenomic mrna of porcine reproductive and respiratory syndrome virus isolated in japan phylogenetic relationships of european strains of porcine reproductive and respiratory syndrome virus (prrsv) inferred from dna sequences of putative orf- and orf- genes porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis isolation and identi®ca-tion of porcine reproductive and respiratory syndrome virus in cell cultures isolation of sirs virus from nursery pigs of two herds without current reproductive failure proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection european serotype prrsv vaccine protects against european serotype challenge whereas an american serotype vaccine does not organization of the simian hemorrhagic fever virus genome and identi®cation of the sgrna junction sequences mystery swine disease in the netherlands: the isolation of lelystad virus antigenic comparison of lelystad virus and swine infertility and respiratory syndrome virus lelystad virus and the porcine epidemic abortion and respiratory syndrome differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from north american ®eld strains by restriction fragment length polymorphism analysis of orf evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): monoclonal antibodies detect common epitopes on two viral proteins of porcine reproductive and respiratory syndrome virus: a persistent infection antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus comparative sequence analysis of open reading frames to of the modi®ed live vaccine virus and other north american isolates of the porcine reproductive and respiratory syndrome virus antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs field isolates of porcine reproductive and respiratory syndrome virus (prrsv) vary in their susceptibility to antibody-dependent enhancement (ade) of infection recombination between north american strains of porcine reproductive and respiratory syndrome virus general overview of prrsv: a perspective from the united states acknowledgements i wish to thank drs. roger avery and thomas toth of virginia±maryland regional college of veterinary medicine, and dr. jill sible of department of biology at virginia tech for their critic reviews of the manuscript; drs. prem paul and patrick halbur of iowa state university's college of veterinary medicine for their continuous support and collaboration, and mr. denis guenette for editorial assistance. i apologize to the authors of important papers not cited here because of the narrow scope of this review and of space limitations. key: cord- - f xe zc authors: rowland, r. r. r.; robinson, b.; stefanick, j.; kim, t. s.; guanghua, l.; lawson, s. r.; benfield, d. a. title: inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with -aminopurine date: journal: arch virol doi: . /s sha: doc_id: cord_uid: f xe zc porcine reproductive and respiratory syndrome virus (prrsv) belongs to a group of rna viruses that establish persistent infections. a proposed strategy for evading immunity during persistent prrsv infection is by preventing the induction of ifn activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. ifn-γ mrna expression was observed in the lymph nodes and lungs of pigs infected with wild-type prrsv strain sdsu- . pretreatment of marc- cells with ifn-γ inhibited wild-type (sdsu- p ) and culture-adapted (sdsu- p ) prrs viruses in a dose-dependent manner and at relatively low concentrations. the effect of ifn-γ on virus replication included reductions in the number of infected cells, virus yield, and rna content in single cells. virus replication was partially restored by the addition of -aminopurine ( -ap), an inhibitor of dsrna inducible protein kinase (pkr). the addition of -ap also restored the viral rna content per cell to near normal levels, suggesting that inhibition of viral rna synthesis was through pkr. the principal difference between p and p isolates was the recovery of p replication with lower concentrations of -ap. immunostaining with anti-pkr antibody showed a redistribution of pkr from the cytoplasm into nucleoli of infected cells. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus placed in the family arteriviridae, order nidovirales [ , , , ] . other members in this family include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv). the arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested -coterminal set of subgenomic mrnas possessing a common leader and a poly-a tail [ ] . the outcome following prrsv infection depends on the age and reproductive status of the pig [ ] . infection of adult pigs generally produces a nonfatal disease characterized by flu-like symptoms, including mild interstitial pneumonia, elevated temperature, and inappetance. in sharp contrast, the infection of pregnant gilts and sows results in abortion and the birth of dead and weak-born piglets. surviving piglets exhibit the severest form of respiratory distress with mortality often approaching % within weeks after farrowing. surviving pigs continue to suffer the negative effect of prrsv by exhibiting increased susceptibility to secondary infection. during acute infection, prrsv replication is found in all organs and tissues, which is consistent with the macrophage as the principal cell population supporting virus replication [ , ] . non-macrophage cells, including type ii pneumocytes [ ] , bronchiolar epithelial cells and arteriolar endothelial cells [ ] may provide additional sources of virus in infected pigs. in culture, prrsv replicates in porcine alveolar macrophages, blood monocytes, monocyte-derived macrophages and monkey kidney lines derived from ma- cells [ , , , ] . pigs surviving acute prrs support a long-term asymptomatic infection, which has contributed to the worldwide spread of the disease [ , , , , ] . even though prrsv-specific humoral and cell-mediated immune responses appear early [ , ] , persistently infected pigs continue to shed virus for several months [ , ] . the mechanistic basis for prrsv persistence is not known. similar to ldv, prrsv may sustain replication in a subpopulation of renewable macrophages, while avoiding host-defenses [ ] or restrict the localization of virus replication within "immunoprivileged" anatomical sites, such as testes [ , , ] . the amino acid variability among prrsv field isolates within the ectodomain of the orf envelope protein suggests another strategy; antigenic drift [ ] . persistent viruses frequently incorporate evasion strategies that block the production or inhibit the activation of proteins up-regulated by interferon (ifn; [ , , , , ] ). studies suggest that prrsv may avoid ifn by preventing its induction in macrophages. both type-i and type-ii ifns efficiently inhibit prrsv replication in macrophages; however, small amounts of ifn are found in prrsv-infected pigs and infected macrophages in culture [ , , ] . since prrsv-specific cell-mediated immunity is present during infection, it is assumed that ifn-␥, a product of activated t cells and nk cells, is present in infected pigs [ , ] . however, nothing has been reported regarding the induction of ifn-␥ in prrsv-infected pigs or the mechanism of ifn-␥-inhibition of virus replication. the purpose of this study was to evaluate the production of ifn-␥ in pigs following infection with prrsv and to characterize the mechanism of ifn-␥ action following infection of marc- cells with wild-type and culture adapted prrsv isolates. the results show that ifn-␥ efficiently inhibits prrsv replication in marc- cells, and at least part of the antiviral activity may be through pkr. the north american macrophage-tropic strain, sdsu- -p , is a low-passage isolate possessing attributes similar to other north american field strains. the marc- celladapted strain, sdsu- -p , was obtained by passaging p an additional times on marc- cells. adaptation was performed by infecting marc- cells on -well plates with serial : dilutions of virus. medium from the last well showing cpe was retained, diluted : , and ml added to a new t- flask of marc- cells. after incubation at • c for hr, the monolayer was washed with pbs, and fresh medium added. after days the flask was freeze/thawed at − • c, and medium was again end-point diluted on a new well plate. this process repeated until the total number of passages reached . marc- cells [ ] were maintained in mem supplemented with % heat-inactivated fetal bovine serum (fbs) and antibiotics at • c in % co . the indiana strain of vesicular stomatitis virus (vsv) was obtained from christopher chase at south dakota state university. unless otherwise indicated, confluent marc- cells were infected at an moi of approximately . tcid /cell. increasing the moi of sdsu- beyond . does not produce a further increase in the number of cells infected during the first round of infection or the virus yield (personal observation). recombinant human ifn-␥ (boehringer mannheim) and ifn-␣ (prepo) were stored at − • c. a mm solution of the nitrate salt of -aminopurine ( -ap; sigma) was prepared in mem and ph adjusted by adding m tris until the cell culture medium changed to pink. ifn-␥ and -ap were added at approximately h and h, respectively, prior to infection with virus. after h the medium was removed and cells washed twice with pbs. incubation was continued in fresh medium, containing freshly prepared ifn-␥ and -ap. cytopathic effect (cpe) was observed by phase contrast microscopy of live cells and after staining acetone-fixed monolayers with % crystal violet. cytokine mrna expression in lung and lymph nodes was evaluated in pigs that were exposed to prrsv in utero. this model is based on observations that infected neonates exhibit the severest form of disease [ , ] . seronegative sows were infected at days gestation with wild-type p or attenuated p . sows were allowed to farrow and live-born piglets sacrificed at days after birth. samples of lung and lymph node tissues were immediately frozen in liquid nitrogen and stored at − • c. total rna was extracted from tissues according to christopher-hennings et al. [ ] and stored at − • c. rt-pcr of porcine cytokine and ␤ -microglobulin mrnas was performed according to reddy et al. [ ] . cdna was prepared from ug of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase and random hexamers as primers. pcr amplification of ifn-␥, il- and ␤ -microglobulin cdnas was performed using the primers described in table . pcr amplification consisted of cycles ( sec at • c, sec at • c, and sec at • c) and dna products electrophoresed on a . % agarose gel and visualized using ethidium bromide. the identity of each amplified product was confirmed by cloning and sequencing the gel-purified fragment. [ ] . serial -fold dilutions of virus were placed on -well tissue culture plates containing marc- cells. after days, plates were fixed in % acetone then stained with sdow- . results were reported as tcid /ml. the intracellular localization of pkr was determined by staining with anti-pkr mab (anti-p from transduction laboratories). acetone-fixed cells were incubated for h at room temp with a : dilution of anti-pkr antibody, followed by a h incubation with biotinylated anti-mouse igg (sigma), diluted : . a final one hr incubation included texas redstrepavidin (molecular probes), diluted : and fitc-labeled sdow- , diluted : . antibodies and stepavidin were diluted in pbs-fbs and slides washed twice for min in pbs between the addition of each reagent. fitc-labeled sdow- and texas red-labeled anti-pkr antibodies were visualized under a fluorescence microscope using nm and nm (rhodamine) excitation filters, respectively. for northern blots total rna was extracted from cells at h after infection using the acid phenol guanidinium technique of chomcynski and sachi [ ] . rna was electrophoresed on % agarose following denaturation with glyoxal and dimethyl sulfoxide [ ] , then transferred to a nylon membrane. the amount of rna added to each well was adjusted to reflect the viral rna from the same number of infected cells (determined by the percentage of sdow- -postive cells in a small sample of cells removed from culture prior to rna extraction). the [ p] dctp-labeled prrsv-specific probe, designed to detect genomic and subgenomic rnas, was prepared by random-primed labeling a bp cdna template derived from rt-pcr amplification of the orf region [ ] . nylon membranes were hybridized with the probe overnight at • c, using a hybridization buffer prepared according to church and gilbert [ ] . nylon membranes were washed in × ssc at • c followed by a high stringency wash in . × ssc at • c for min, then exposed to x-ray film. the relative viral rna content in single cells was determined using a modification of the in situ hybridization technique described by peng et al. [ ] . approximately cells in ul of tissue culture medium were spotted onto denhardt's-treated slides. fixation, pre-treatment, hybridization, and post-hybridization procedures were performed according to lawson et al., [ ] using a modification of the original method described by anderson et al. [ ] . the [ s] dctp-labeled random-primed probe was prepared from the same bp orf template used in northern blot hybridization. after the last post-hybridization wash, slides were dipped in nbt- autoradiography emulsion (kodak), exposed for h in the dark, developed, then h and e stained with mayer's hematoxylin and % eosin y in ethanol. prrsv-infected cells were identified as having a silver grain content above that of mock-infected cells. histogram distributions were constructed by counting the number of silver grains over the cytoplasmic and nuclear regions of infected cells. statistical analysis was performed using a two-tailed student's t test. previous studies [ , ] identified the reduced ability of pigs and pig macrophages to produce ifn-␣. the purpose of this experiment was to characterize ifn-␥ mrna expression in acutely infected pigs. sows, at days gestation, were infected with wild-type p or attenuated p isolates. all p -infected piglets (n = ) were positive for prrsv at the time of necropsy ( days after birth) and exhibited typical congenital prrsv pathology, including microscopic lesions in lungs and lymph nodes. the piglets born to the p -infected sow (n = ) showed no signs of prrs and were negative for virus at the time of necropsy, confirming that this virus was rapidly cleared from the mother and did not cross the placenta. three pigs from each of the p and p groups were randomly chosen for analysis of cytokine mrna expression in lungs and mandibular lymph nodes. ifn-␥ mrna expression was detected in the lymph nodes from two of the pigs infected with the wild-type p virus (fig. ) . a third p pig showed no ifn-␥ mrna in lymph node; however, ifn-␥ mrna was detected in the lungs. there was no evidence of ifn-␥ expression in the lymph nodes or lungs of the p detected by rt-pcr amplification of total rna from three infected (p virus) and three control pigs (p virus). infection of pregant sows with sdsu- wildtype p and attenuated p isolates is described in materials and methods. n lymph node; l lung pigs. ␤ microgloblin mrna was amplified to nearly equal levels in all tissues. il- mrna was also amplified from all tissues, a cytokine mrna we frequently find it in lymphocytes and lymphoid tissues of un-infected pigs. the effect of ifn-␥ on the growth of prrsv in marc- was studied by incubating -well plates of confluent marc- cells with human ifn-␥ overnight followed by infection with prrsv p or p isolates. the results of a representative experiment, presented in fig. a , showed loss of crystal violet staining in p and p cultures not treated with ifn-␥. the effect of ifn-␥ on cpe was apparent at concentrations as low as u/ml ifn-␥. intact cell monolayers were observed in wells incubated with or , u/ml of ifn-␥. phase-contrast microscopy, performed prior to crystal violet staining, confirmed the absence of cpe. for the purpose of comparison, ifn-␥-treated marc- cells were also infected with vsv, a virus sensitive to interferon [ , ] . the experimental conditions were the same as those used for prrsv infection. intact cell monolayers were only observed in vsv-infected cultures treated with , u/ml ifn-␥ ( fig. a) . the effect of human ifn-␣ on prrsv replication was also investigated. the experimental conditions were the same as described for the ifn-␥ experiments. representative results, presented in fig. b , showed ifn-␣ inhibition of p and p isolates. similar to ifn-␥, ifn-␣ activity was apparent at concentrations as low as u/ml. unlike ifn-␥, the p isolate was more sensitive to ifn-␣ than p (compare wells treated with and ug/ml ifn-␣). this difference was confirmed in subsequent experiments. further assessments of ifn-␥ inhibition of virus replication were made by counting the number of infected cells and by measuring virus yields in cultures incubated with ifn-␥ overnight. ifn-␥ treatment produced dramatic decreases in the percent-infected cells and virus yield for both p and p viruses (fig. ) . similar to the cpe data the effect was evident at concentrations as low u/ml. ifn-␥, reduced virus yields at all time points and consistently reduced peak yields of p and p by greater than to logtcid /ml. ifn-␥ also reduced the number of infected cells. the growth curve experiments also revealed differences between p ad p isolates. improved adaptation of p to marc- cells was evident by increased virus yields and percent infected cells. another difference was the timing of peak virus yield for p , which was observed at h after infection versus viruses, respectively. ifn-␥ treatment, infection, and measurement of virus yields were performed in the same manner as described in fig. h for p . the experiment presented in fig. also revealed what appeared to be an increased sensitivity of p to ifn-␥. however, due to the different growth characteristics of p and p isolates, a differential effect of ifn-␥ was difficult to assess. there are several approaches that can be used to study the activation of pkr. a role for activated pkr in the ifn-inhibition of prrsv replication was studied by following the recovery of virus replication after the addition of -ap, an inhibitor of pkr phosphorylation [ ] . even though we did not evaluate the phosphorylation state of pkr, we assumed that reversal of ifn inhibition of virus replication following addition of -ap could only occur by preventing pkr phosphorylation. in these experiments -ap was added h prior to infection and after overnight incubation with u/ml ifn-␥. inhibition of ifn-␥ activity by -ap was dosedependent and evident at concentrations as low as mm for p (fig. ) . the optimal concentration of -ap required for maximum virus recovery of p and p infection were and mm, respectively. following several experiments, it was noted that -ap never recovered virus replication to the same levels obtained in infected cultures not treated with ifn-␥ ( fig. ; compare treatments and for p and treatments and for p ). and as shown in fig. further increasing -ap to mm in p -infected cultures actually inhibited virus yield. a similar effect was observed for the p virus incubated mm -ap (data not shown). pkr is typically found in both cytoplasmic and nuclear compartments [ ] . in uninfected marc- cells pkr was distributed throughout the cytoplasm with increased anti-pkr fluorescence in the perinuclear region (fig. d ). we did not observe a significant increase or redistribution in pkr immunofluorescence in cells following overnight incubation with u/ml ifn-␥. however, the intracellular distribution of pkr was dramatically different in prrsv-infected cells. in a representative experiment (fig. e ) pkr immunofluorescence was still present in the cytoplasm, but with increased fluorescence around the perinuclear region and within the nucleoli of p -infected cells (fig. e ). similar results were observed in cells infected with p . incubation with mm -ap prior to infection with p did not alter the pattern of pkr staining, suggesting that pkr activation was not required for nucleolar localization. interestingly, the intracellular distributions of pkr and prrsv nucleocapsid (n) proteins were almost identical (compare fig. b and e) . to determine a possible association between pkr and n protein in the absence of infection, we performed a similar experiment that expressed the n protein fused to enhanced green fluorescent protein (egfp). a representative result, presented in fig. c and f, showed n-egfp fluorescence in the nucleolus and cytoplasm; whereas, pkr remained in the cytoplasm. since the n-egfp fusion construct may not be representative the native n protein, we repeated the experiment using a construct that expressed only the n protein, which was detected using fitc-sdow- . under these conditions the n and pkr proteins did not co-localize to the nucleolus (data not shown). the inability of -ap to completely recover virus replication following ifn-␥treatment suggested that other anti-viral pathways, up-regulated by ifn-␥, were activated during prrsv infection. for example, the , oligoadenylate synthetase ( - a synthetase) pathway, activated by dsrna, inhibits translation by degrading mrna [ ] . northern blots were used to evaluate the integrity of p subgenomic mrnas. the results of a single experiment, presented in fig. a , showed decreased expression of all prrsv rnas following treatment with u/ml ifn-␥. we did not observe degraded rna in the ifn-treated cultures. viral rna expression was also reduced in the presence of mm -ap alone; however, the addition of -ap to ifn-␥ treated cells increased the level of viral rna expression above the ifn-␥-treated culture. to further confirm that ifn-␥ treatment decreased viral rna expression, relative changes in viral rna content in single cells were measured using a modified in situ hybridization technique [ ] . histograms showing the number of p -infected cells versus number of silver grains/cell in infected cells at hr r are presented in fig. b . the mean silver grain content in infected cultures was . grains/cell (fig. b, panel ) . pre-treatment with u/ml of ifn-␥ shifted the distribution towards cells containing significantly less viral rna (mean = . silver grains/cell, p = . , fig. b panel vs panel ) . the addition of -ap restored the mean rna content per cell to near normal levels (fig. b, panel vs panel ). this study confirms previous reports describing ifn inhibition of prrsv [ , , ] . furthermore, we extend these observations by demonstrating that ifn inhibits the replication of both wild-type and tissue culture-adapted isolates derived from a north american prrsv isolate. inhibition of prrsv was observed following pre-incubation with either type-i or type-ii ifns. furthermore, the sensitivity of prrsv to ifn-␥ was greater than vsv ( fig. a) , a virus frequently used for the detection of ifn activity [ ] . consistent with other reports describing the presence of cell-mediated immunity during prrsv infection [ , ] , we observed ifn-␥ mrna expression in lymph nodes and lungs of prrsv-infected pigs (fig. ) . whether or not this amount of expression represents a biologically relevant quantity of ifn-␥ remains to be determined. even though we demonstrated the effectiveness of ifn in inhibiting prrsv replication in marc- cells, little is known regarding the properties of the macrophages supporting prrsv replication in vivo. in infected pigs, prrsv replication is initially found in all organs and tissues [ ] . during the long asymptomatic stage of infection, virus replication is restricted to a subpopulation of cells located in tonsil and lymph nodes (personal observation), suggesting that these cells may be more resistant to ifn-␥, even when exposed to local elevations of cytokines from activated t cells. this observation is similar to studies of ldv infection of mice. ldv normally replicates in a subpopulation of macrophages, but in some strains of mice infects motor neurons causing paralysis. injection of mice with ifn-␥ prior to infection inhibits virus replication in motor neurons without significantly affecting the overall level of viremia [ ] . we propose a model in which the induction of ifn-␥ production during prrsv infection is sufficient to suppress virus replication in permissive cells in non-lymphoid organs, but does not affect replication in a small subpopulation of permissive cells in lymph nodes and tonsil. the identity of this prrsv-permissive population remains to be determined. anti-viral proteins, including pkr, - a synthetase and mx are up-regulated in response to ifn [ , , ] . pkr, after binding viral rna, is autophosphorylated on serine and threonine residues and inhibits translation by phosphorylating the alpha subunit of eif . phosphorylation of pkr is inhibited by -ap, an atp analog. the addition of -ap to cells pre-incubated with u/ml ifn-␥ partially recovered prrsv replication in marc- cells (fig. ) . these results demonstrate that at least, in part, the interferon-induced anti-prrsv activity may occur through the pkr pathway. the ability of -ap to partially restore viral rna synthesis in ifn-treated cells supports the work of deboon et al. [ ] who concluded that ongoing protein synthesis is required for the production of arterivirus rnas. one explanation for the failure of -ap to completely restore virus replication is the activation of other antiviral pathways not regulated by pkr. for example the activation of the - a synthetase pathway can be identified by the presence of degraded forms of both viral and ribosomal rnas [ ] . degraded rna was not detected in northern blots (see fig. a ) in total rna from prrsv infected cultures (personal observation). recently, zhang et al. [ ] reported increased expression of mx in cultured macrophages infected with prrsv. a role for mx in the inhibition of prrsv replication has not been demonstrated. another explanation for the failure of -ap to restore prrsv replication is the inhibition of -ap sensitive phosphorylation event required for virus replication. for example, mm -ap prevents the phosphorylation of the pe /e envelope glycoprotein, which is required for the production of infectious progeny [ ] . pkr is normally distributed between the cytoplasm and nucleus, which is consistent with its roles in regulation of translation and transcription. in marc- cells, we found pkr primarily concentrated in the cytoplasm and perinuclear regions (fig. d ). an unexpected observation was the localization of pkr to the nucleoli of prrsv-infected cells (fig. e ). nucleolar localization was not affected by -ap, suggesting that pkr activation is not necessary for nucleolar localization. this observation confirms the result of besse et al. [ ] who identified a non-phosphorylated form of pkr in the nuclei of hela cells. furthermore, pkr nucleolar localization during prrsv replication is not the result of a physical association with the prrsv nucleocapsid protein ( fig. c and f). from these results we can conclude that nucleolar localization of pkr in marc- cells is dependent on prrsv infection, but is independent of activation or an association with the nucleocapsid protein. prrsv infection of pigs continues to represent a worldwide disease problem. this study, combined with the results of others, clearly demonstrates an important role for ifn-␥ in the control of prrsv infection and suggests that the induction of ifn activity should be considered in the design of new vaccines. however, the mechanism for evasion of host defenses during persistent infection remains to be determined. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus lactate dehydrogenase-elevating virus persists in liver, spleen, lymph nodes and testis and results in accumulation of viral rna in germinal centers concomitant with the polyclonal activation of b cells cell-mediated immunity to porcine reproductive and respiratory syndrome virus ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages indiscriminate degradation in interferon-treated, vaccinia-infected mouse l cells characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) ultrastructural localization of interferon-inducible double-stranded rna-activated enzymes in human cells cellular responses to interferon-gamma in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) inhibition of virus-induced age-dependent poliomyelitis by interferon-gamma identification of interferon-resistant subpopulations in several strains of measles virus: positive selection by growth of the virus in brain tissue nidovirales: a new order comprising coronaviridae and arteriviridae single-step method of rna isolation by acid guanidinium thyocyanate-phenol-chloroform extraction detection of prrsv in boar semen using the polymerase chain reaction persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds genomic sequencing isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs equine arteritis virus rna transcription: uv inactivation and translation inhibition studies the interferon sensitivity of selected porcine viruses chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence neonatal infection of mice with lactate dehydrogenase-elevating virus results in suppression of humoral antiviral immune response but does not alter the course of viraemia or the polyclonal activation of b cells and immune complex formation the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- convenient assay for interferons molecular cloning: a laboratory manual antiviral actions of interferon: interferon-regulated proteins and their surprisingly selective antiviral activities rrna cleavage as an index of ppp(a 'p)na activity in interferon-treated encephalomyocarditis virusinfected cells in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection detection of intracellular porcine reproductive and respiratory syndrome virus nucleocapsid protein in porcine macrophages by flow cytometry antiviral defense in mice lacking both alpha/beta and gamma interferon receptors differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity porcine reproductive and respiratory syndrome virus: a persistent infection characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection key: cord- -uijc qdo authors: molitor, t. w.; bautista, e. m.; choi, c. s. title: immunity to prrsv: double-edged sword date: - - journal: veterinary microbiology doi: . /s - ( ) - sha: doc_id: cord_uid: uijc qdo abstract the immune system is a double-edged sword for porcine reproductive and respiratory syndrome virus (prrsv) infection. on one edge prrsv has a predilection for immune cells and the disease manifestations can be linked directly to changes in the immune system. prrsv appears to replicate extensively, if not exclusively, in cells of the immune lineage, notably macrophages; the direct replication of which may lead to immunosuppression, precipitate secondary infection and/or mediate disease. on the other edge, the virus stimulates immunity post-infection that protects an animal from re-infection. a vast array of structural and functionally distinct antibody specific to prrsv are generated following infection or vaccination. discrete populations of functional antibodies appear at different times and possibly reflect reactivity to different prrsv polypeptides. cell-mediated immune responses specific to prrsv can be detected in various exposed pigs as well. thus, the immune system appears to be intimately involved in both the disease process and protection from disease. it is unclear at this state of understanding what immune compartment provides protective immunity. is it humoral (i.e. antibodies), selective functionally distinct populations of antibodies specific for selected prrsv polypeptides or is cellular immunity essential for protection, or both. this review will attempt to summarize the current state of knowledge of the complex interaction of the immune system and prrsv. the immune system is a double-edged sword for porcine reproductive and respiratory syndrome virus (prrsv) infection. on one edge prrsv has a predilection for immune cells and the disease manifestations can be linked directly to changes in the immune system. prrsv appears to replicate extensively. if not exclusively, in cells of the immune lineage, notably macrophages; the direct replication of which may lead to immunosuppression, precipitate secondary infection and/or mediate disease. on the other edge. the virus stimulates immunity post-infection that protects an animal from re-infection. thus. the immune system appears to be intimately involved in both the disease process and protection from disease. it is unclear at this state of understanding what immune compartment provides protective immunity. is it humoral (i.e. antibodies), even selective antibodies of functionally distinct populations or antibodies specific for selected prrsv polypeptides or is cellular immunity essential for protection. this review will attempt to summarize the current state of knowledge of the complex interaction of the immune system and prrsv. prrs virus was first isolated on swine alveolar macrophages (am) (wensvoort et al., ; pol et al., ) . am represent a highly permissive cell for infection yielding progeny virus titers of greater than io' tcid,,/ml. am serve as sensitive cells for isolation of virus from diagnostic samples and antibody detection using indirect fa or ipt methods. am are primary targets for virus replication in the infected animal, yet prrsv is a multisystem disease characterized by profound viremia and virus distribution and replication in multiple organs causing interstitial pneumonia, vasculitis. lymphadenopathy, myocarditis and encephalitis (rossow et al., ) . thus, replication solely in am does not fully explain the pathogenesis of prrsv in the infected animal. furthermore, in vitro studies with am and collections from prrsv-infected animals reveal that not all am become infected. also. in vitro studies with am show that not % of cells are affected and that there is great variability in susceptibility of am from various donor pigs. in an attempt to elucidate the susceptibility of various cell types and variability within cell types, we examined the susceptibility of various macrophage populations (i.e. microglia, monocytes) and the heterogeneity of alveolar macrophages to infection by prrsv. to examine the heterogeneity of am. a procedure was devised to separate am into subpopulations by subjecting am lavaged from lungs to a discontinued gradient centrifugation and each fraction was evaluated for morphology, expression of cell surface markers. macrophage functions and susceptibility to infection by prrsv. all fractions consisted of greater than % macrophages using a panel of monoclonal antibodies against porcine macrophages. the largest cells were found in the lowest density fraction (i) and the smallest cells were in the highest density fraction (vi, showing an inverse relationship between cell size and density. fractions in the lower densities (i and ii> appeared to have more cytoplasmic vacuoles and lower nuclear to cytoplasmic ratios relative to fractions iii, iv and v and appear to represent mature, differentiated macrophages. functional differences existed between the fractions of pam. pam from the higher density fractions (iii. iv and v) showed increased phagocytosis of pasteurella multocida, superoxide anion production and tnf-a production relative to pam from lower density fractions (i and ii). in contrast, binding of opsonized sheep red blood cells through fc receptors was greatest in the lowest density cells (i). increased prrs virus replication was observed in pam from the higher density fractions (iii, iv and v> representing more immature cells (choi et al., ) . further studies examined the susceptibility of populations of macrophages including brain microglia and peripheral blood monocytes. swine microglia were highly permissive to infection with prrsv yielding progeny titers of greater than ' tcidjml. in contrast, monocytes directly collected and exposed to prrsv yielded low progeny titers of less than lo* tcid,,/ml. yet when monocytes were induced to mature through treatment with m-csf or activated via the adherence to porcine endothelial cells, monocytes became permissive to prrsv infection. voicu et al. ( ) demonstrated that peripheral blood monocytes collected via adherence to plastic were permissive to prrsv infection yielding virus titers of ' tcid,/ml, a ,ooo-fold reduction compared to am yet nonetheless significant titers. although there is an apparent paradox in the two findings, it appears that maturation and activation of monocytes is necessary for productive infection. the adherence to plastic could in fact activate the cells. collectively, these results indicate that heterogenous pam populations exist and that these cells showed different morphological and functional properties. furthermore, the activation/maturation stage of macrophages modulate the susceptibility to virus infection. the susceptiblity of various immune cells to prrsv is summarized in table . clinical and experimental studies suggest that prrsv modulates host responses based on two observations: ( ) secondary infections, e.g. pneumonia, arthritis, eye infections, meningitis and infections with prv, ppv and siv are common following prrs virus infection (collins and rossow, ; zeman et al., ; done and paton, t. w. molitor pt al. / veterinu? microbiology clyy ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and ( ) experimental infection with prrsv precipitates clinical disease in piglets challenged with streprococcus suis (calina et al., ) or with either of two respiratory viruses, porcine respiratory coronavirus (prcv) or swine influenza (si, van reeth et al., ) . prrsv infections are commonly followed by severe, secondary bacterial diseases which greatly reduce the performance of growing pigs (collins and rossow, ) . in a few selected herds. a thorough pathological and microbiological work-up has been performed. a variety of infectious agents have been isolated in concert with prrsv: actinobacillus pleuropneumonia, myoplasma hyymeumoniae, haemoplilus parasuis, actinomyes pygenes. streptococcus suis and swine influenza (zeman et al., ) . the conclusion from this field investigation was that prrsv can increase the incidence of other common diseases. one of the agents identified, h. parasuis, has been diagnosed increasingly in the upper midwest. since the appearance of prrsv. iowa state diagnostic laboratory identified isolations in . compared to in . south dakota state laboratory identified cases in . compared to in . minnesota state laboratory identified i in , compared to in . indirect evidence of the role of prrsv in precipitating secondary infections comes from successfully improved growth and decreased mortality on farms that have eliminated prrsv through nursery depopulation (dee and joo, ) . with the elimination of prrsv on farms, production improved and the isolation and association of disease with secondary agents dramatically decreased. clinically, there is little doubt that concurrent prrsv frequently results in devastating secondary infectious diseases in pigs; however, although clinical and diagnostic laboratory data support the hypothesis of an interaction between prrsv and other agents, yet attempts to demonstrate prrsv as a predisposing factor in respiratory disease under experimental conditions. with the exceptions of streptococcus suis and two viruses, porcine respiratory coronavirus and swine influenza, have been unsuccessful. streptococcus suis (s. suis) serotype infection causes septicemia, meningitis, arthritis and death, usually in young weaned pigs. interactions of s. suis serotype with other microorganisms such as bordetella bronchiseptica (vecht et al.. ) and pseudorabies virus (prv) (iglesias et al., ) have been demonstrated experimentally. however, it has been difficult to study clinical disease caused by s. suis infection because clinical disease is difficult to reproduce using natural routes of exposure when challenging with s. suis alone. the objectives of experimental studies performed by galina et al. ( ) were to characterize the interaction of prrsv and s. suis under controlled conditions and to determine if prrsv pre-disposes pigs to s. suis disease. pigs inoculated with prrsv (vr- ) followed by challenge with a virulent strain ( ) of s. suis serotype developed clinical signs, suppurative meningitis and abundant growth of s. suis from tissues, including brain-meninges. pigs inoculated with prrsv alone, s. suis alone, prrsv and the dhs strain of s. suis serotype (lacking a protein associated with virulence) or control piglets did not have clinical signs, lesions or large amounts of bacteria in their tissues. results of this study suggest that prrsv pre-disposes spf pigs to infection and disease caused by virulent s. suis serotype . in a study examining the effect of concurrent infection of prrsv with porcine respiratory coronavirus (prcv) and swine influenza (si) van reeth et al. ( ) found that prrsv by itself was clinically inapparent in feeder pigs while severe respiratory disease and production losses occur when prcv and si were superimposed on prrsv infection. studies with sequential infection of prrsv followed by h. parasuis, p. multocidia or a. pleuropneumoniae have failed to demonstrate increased severity of disease (solano et al., ) . if indeed pigs are more susceptible to secondary infections following prrsv, a presumed mechanism underlying the increased susceptibility could be suppression of the immune system. two studies have attempted to define putative immunosuppression, following experimental infections with prrsv in pigs and measuring immune response parameters including immune cell profiles and antibody-mediated and cellular immunity to foreign antigens albina et al., ) . in the study of , cell distribution in the lung of infected animals changed dramatically by day post-infection, evidenced by a marked decrease in the percentage of alveolar macrophages and an increase in the percentage of lymphocytes and neutrophils. associated with the altered lung cell dynamics was a decrease in the functional ability of alveolar macrophages to release superoxide anion. in sharp contrast was the finding of profound enhancement of humoral and cell-mediated function in the systemic circulation of prrsv infected swine. pseudorabies virus (prv) and brucella abortus antibody titers and delayed type hypersensitivity responses to the antigen dnfb were significantly enhanced in infected pigs of all age groups. a separate study by albina et al. ( ) demonstrated that prrsv did not impair the immune response following prv vaccination. on the contrary, immune response and disease resistance of pigs previously infected with prrsv and then challenged with virulent prv were increased. in addition, immune parameters such as killer-cell activity and lymphocyte response to antigenic stimulation were not affected following prrsv infection, leaving to question the role of prrsv in causing systemic immunosuppression. the effects of prrsv on non-specific and antigen-specific immune responses are summarized in table . local virus-mediated destruction of alveolar macrophages may account for the extensive respiratory infection in prrsv infected pigs. enhanced systemic responses to exogenous antigens may be due, in part, to polyclonal activation of immune components, which has been described in ldv infection. upon exposure of pigs to prrsv, pigs immunologically respond with a heterogeneous array of responses specific to prrsv virus. included are structural (i.e. class of immunoglobulins) and functional distinct antibodies and cell-mediated responses. experimental data showing that previous infection prevented animals from developing clinical signs after re-exposure (gorcyca et al., ) suggest that protective immune mechanisms are developed in swine upon infection. at present, it is unclear which immune mechanisms are involved in protection against prrsv in swine. serological assays, including immunoperoxidase monolayer assay (ipma, wensvoort et al., ) , indirect fluorescent antibodies (ifa, yoon et al., , serum neutralization (sn, hill et al., depression in response compared to noninfected control t f stimulation over that of noninfected control. --: no effect. ), western blot (nelson et al., ; nelson et al., , indirect enzyme-linked immunosorbent assay (elisa, albina et al., ) and blocking elisa (houben et al.. ) have been used to identify antibody responses to prrsv (table ) . following exposure to virus, swine synthesize prrsv-specific antibodies detected by ifa and ipma which appear at one to two weeks post-infection and can persist for up to one year. antibodies detected by serum neutralization test snt appear later (as much as six weeks post-infection) and disappear sooner (morrison et al., . a modified sn test has been reported (yoon et al., ; yoon et al., ) that detects sn antibody responses at -l days post-inoculation. the antigenic specificity of the snt is more definitive than that of the ipma as well. antibodies can also be shown that recognize virus-specific polypeptides by western blot (nelson et al., ) and immunoprecipitation. an indirect elisa was first described for detecting antibodies to prrsv in sera (albina et al., ) . subsequently, a blocking elisa technique was developed by houben et al. ( ) . the blocking elisa appears to have the advantage of detecting low antibody titers. thus, there exist discrete populations of functional yoon et al., ; nelson et al.. wensvoort et al., nelson et al., yoon et al., ; yoon et al., nelson et al., yoon et al., ; yoon et al., albina et al., houben et al., nelson et al., choi et al.. : yoon et al., yoon et al.. bautista et al., bautista et al.. antibodies that display different kinetics post-infection and possibly reflect reactivity to different prrs virus polypeptides. pigs vaccinated with modified-live virus similar to infected pig vaccine immunologically respond and synthesize antibodies of various structure and functional heterogeneity. pigs born from prrsv-infected dams maintain maternal antibody titers to prrsv until weeks of age, as determined by ifa (dee et al., ) , - weeks of age in a study using the indirect elisa (albina et al., ) and up to weeks of age using the blocking elisa (houben et al., ) . these three studies were performed on pigs from infected herds, representing potential differences in pigs, source, virus strain and farm management factors. the role of the humoral immune response in protection from challenge remains questionable, based on the observations that viremia is detected albeit in the presence of antibodies (rossow et al., ) and that a population of antibodies enhance virus replication in alveolar macrophages in vitro (choi et al., ; yoon et al., ) and in vivo in infected fetuses (christianson et al., ) and feeder pigs (yoon et al., ; yoon et al., ) . it is postulated that the mechanisms of enhancement of replication is through fc receptors. protection from re-infection has been documented in field conditions and experimentally described in pregnant sows . sows that were infected with the prototype usa isolate of prrsv, vr- and that recovered from infection were protected from manifestations of clinical signs and showed no viremia upon re-infection with the homologous strain. this protection could be transferred through colostrum to susceptible newborn pigs . to determine whether this protection was provided by antibodies an experiment was performed by passively transferring high titered anti-prrsv antibodies to l-week-old pigs from non-immune dams and challenging the pigs with prrs virus h later. pigs from immune dams were protected from experimental challenge, but pigs that received anti-prrsv sera failed to be protected from challenge as were pigs receiving non-immune sera or pbs as controls. these results document that immunity can be transferred via colostrum, but antibodies by themselves fail to totally protect, thereby suggesting a role of cellular immunity in protection from disease. it is well recognized that in viral diseases an important role of cellular immunity is the clearance of virus and protection against disease. the data available suggest that cellular immune mechanisms might have an important role (choi et al., ; rossow et al., ) and that antigenic diversity among prrsv isolates may originate strain-specific immune responses (wensvoort et al., ; nelson et al., ; bautista et al., ) . in an attempt to determine whether prrsv induced a cell-mediated immune response, studies were undertaken to establish methods for detecting antigen-specific cell-mediated immune responses to prrs virus. the purpose was to develop methods to detect cm responses to prrsv, both ex-vivo, in the form of t-cell proliferation, and in vivo as delayed-type hypersensitivity (dth) responses. to demonstrate proliferation responses, peripheral blood mononuclear cells from prrsv-infected and control pigs were stimulated in vitro with virus antigens for various incubation periods and t-cell proliferation determined by the uptake of h-thymidine. t-cell proliferation to prrsv virus was robustly detected in virus-exposed animals. the lymphocyte proliferation was prrsv specific and virus concentration dependent. the proliferation was mediated primarily by cd + t-cells since antibodies to cd blocked the response. the kinetics of t-cell proliferation response were evaluated in virus-infected pigs before and every other week post-infection (pi) and compared to viremia and antibody response (sn and ifa). the secondary response was analyzed in the same pigs after re-exposure at weeks pi. virus-infected, but not control pigs, developed viremia detected at and weeks post-infection and antibody titers (fig. la) . the ifa titers developed rapidly and were detected in all infected animals with the highest response at weeks post-infection. the sn titers developed slowly with lower titers detected first at weeks post-infection and the highest response at weeks post-infection. antigen-specific lymphocyte proliferation response to prrsv was first detected in virus-infected animals at weeks post-infection, peaked at weeks post-infection and appeared to decline after weeks post-infection (fig. lb) . the response and decrease in lag time of the same animals to a secondary exposure to virus fig. . kinetics of humoral and cell-mediated immune responses to prrsv. the primary immune responses were determined in infected pigs as compared to control animals. data for the first weeks are shown for antibody response (panel a) and lymphocyte proliferation (panel b). the secondary response is shown in panel c. antibodies were detected in infected pigs by sn and ifa tests at weeks post-infection (pi) and persisted through the week period. proliferation responses were also detected at weeks pi, peaked at weeks pi and declined after weeks pi. no antibody or proliferation responses were detected in control pigs. the proliferation response increased after secondary exposure. the results showed that pigs develop both humoral and cell-mediated primary immune responses specific to prrsv infection. furthermore a memory t-cell response was evident upon secondary exposure. resulted in a t-cell proliferation response which increased in magnitude (fig. ~ ). although there was some variability in the response among the infected animals, the proliferative response was significantly different from the control non-infected pigs ( p < . ) as determined statistically by the method of repeated measure analysis of variance and orthogonal polynomial contrast to test the difference in time effect. in an in vivo measure of a cellular immune response in delayed-type hypersensitivity (dth), pigs either infected or vaccinated with resp prrsv"' responded with a dth response, specific to virus antigen. thus, it is clear that cellular immune response in addition to humoral immunal responses are induced following exposure to virus vaccination. yet, the question left unresolved is the role of cm in protection. it is abundantly clear that a vast array of antibody populations are generated following exposure to virus. functions of antibody populations differ in their onset and duration. in addition, cell-mediated responses are generated following infection with prrsv and following vaccination. notably, pigs previously exposed to virus are protected from re-exposure to at least the homologous virus challenge. the immune response can be both beneficial and detrimental to the host following infection. these are probably critical components of immunological defenses, critical for protection, yet our knowledge to date is restricted to the documentation of the heterologous response. there remains a lengthy list of questions relating to immune mechanism of protection. the application of a vaccine enhances an awareness of the immunological mechanism of protection. attempts to answer these questions are currently underway. an enzyme-linked immunosorbent assay for the detection of antibodies to the porcine reproductive and respiratory syndrome (prrs) virus immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units porcine reproductive and respiratory syndrome virus (prrsv) does not reduce the immune response and diaease susceptibility of pigs comparison of porcine alveolar macrophages and cl- for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody cell mediated immune responses to porcine reproductive and respiratory syndrome virus antibody-dependent enhancement of prrs virus replication heterogeneity of porcine alveolar macrophages: functional properties and susceptibility to virus infection pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid-gestation sows and fetuses pathogen&s of prrs. proc. a.d. leman conf prevention of spread of porcine reproductive and respiratory syndrome (prrs) virus spread in endemically infected swine herds by nursery depopulation eradicating porcine reproductive and respiratory syndrome (prss) virus using two-site production and nursery depopulation porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression interaction between streprococcus suis serotype and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets the effect of porcine reproductive and respiratory syndrome (prrs) virus challenge in pregnant sows after subsequent exposure to virulent virus. th ann". meeting, american association of swine practitioners. missouri a comparison of the indirect fluorescent antibody test and the serum virus neutralization test for the detection of antibodies against prrs virus comparative study of a blocking enzyme-linked immunosorbent assay and the immunoperoxidase monolayer assay for the detection of antibodies to the porcine reproductive and respiratory syndrome virus in pigs inoculation of pigs with streptococcus suis type alone or in combination with pseudorabies virus immunomodulation of host immune responses following porcine reproductive and respiratory syndrome virus infection serologic evidence incriminating a recently isolated virus (atcc vr- ) as the cause of swine infertility and respiratory syndrome (sirs) differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus pathological, ultrastructural and immunohistochemical changes caused by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome pdthogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs porcine reproductive and respiratory syndrome virus (prrsv) interaction with haen ophitusparasui.r clinical effects of dual infections with porcine epidemic abortion and respiratory syndrome virus, porcine respiratory coroma virus and swine influenza virus virulence of streptococcm suis type strains in newborn germfree pigs depends on phenotype interaction of porcine reproductive and respiratory syndrome virus with swine monocytes antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mystery swine disease in the netherlands: the isolation of the lelystad virus an indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera a modified serum neutralization test for the detection of antibodies to porcine reproductive and respiratory syndrome virus in swine sera antibody dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs laboratory investigation of prrs virus infection in three swine herds key: cord- -zu ubwfq authors: mateu, e.; diaz, i. title: the challenge of prrs immunology date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: zu ubwfq porcine reproductive and respiratory syndrome (prrs) is one of the most challenging subjects of research in veterinary viral immunology, and the immune response against prrs virus (prrsv) still is poorly understood. infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an antibody-dependent enhancement of disease. in contrast, development of neutralising antibodies (nas) is delayed and generation of cell-mediated immune responses, such as prrsv-specific interferon (ifn)-γ secreting cells, is initially erratic. in spite of this, induction of strong and rapid nas and ifn-γ responses seem to be required for effective vaccination. prrsv strongly modulates the host’s immune responses. the virus inhibits key cytokines, such as ifn-α, and may induce regulatory cytokines, such as interleukin (il)- . development of nas seems to be impaired by the existence of a decoy epitope close to the main neutralisation epitope in glycoprotein . this ability to modulate the host immune response probably varies among strains or isolates. the genetic diversity of the virus is very high and it has been shown that this diversity can have serious implications for the development of vaccines, since the immunity induced by one strain may be only partial against a different strain, even within the same genotype. with this panorama, the development of newer and universally efficacious prrsv vaccines is challenging, but the present state of knowledge allows optimism if collaborative efforts are undertaken in the scientific community. more than years after the emergence of porcine reproductive and respiratory syndrome (prrs), our understanding of the disease still is far from complete. the clinical features of prrs are well known, a global picture of the epidemiology has been drawn, although some gaps remain to be filled, management and husbandry procedures have been devised for controlling the disease and vaccines are available. however, infection by prrs virus (prrsv) is still widespread and the virus is frequently reintroduced to farms after eradication. what are the reasons for such failures? the answer is not simple, but examination of the scientific literature and problems reported by pig veterinarians indicates that vaccines can be a useful tool, although their efficacy is far from being universal and complete. the approach of ''let's take a strain, let's attenuate or inactivate it and, voilà ! the vaccine will generate sufficient immunity to protect against the disease'' is not valid for prrsv. the present review discusses what is known and what gaps remain in our understanding of prrsv immunity and its immunopathogenesis. it concludes with the positive message that currently available information should allow us to identify the crucial points that need to be studied in order to understand the disease and overcome the challenge of prrsv immunology. adaptive immune response to prrsv the development of adaptive immunity against prrsv is a tale of unusual features in both the humoral and cellular components of the immune response. circulating antibodies against prrsv are detectable in some pigs by days - post-infection (pi) and all animals have seroconverted by day pi (yoon et al., (yoon et al., , . prrsv-specific immunoglobulin m (igm) reaches a peak at day pi and then declines to undetectable levels by days pi. concentrations of igg reach a maximum at - days pi (vezina et al., ; loemba et al., ) . however, this rapid igm and igg response does not correspond to neutralising antibodies (nas) (yoon et al., ) . nelson et al. ( ) studied the kinetics of the humoral response of pigs against an american strain of prrsv. the earliest antibodies were directed against the kda nucleoprotein, followed by the kda m protein, then the kda glycoprotein (gp ). other studies showed that non-structural protein (nsp ) contains a cluster of non-neutralising b-epitopes and probably is the immunodominant protein of prrsv (oleksiewicz et al., ; de lima et al., ) . most diagnostic tests detect antibodies mainly against the n protein. these antibodies appear around the first week pi and persist for several months, but do not correlate with protection. nas are not detected by conventional virus neutralisation tests (vnts) in the first weeks pi. addition of fresh complement and prolonged incubation of virus-serum mixtures increases the sensitivity of the vnt and allows detection of nas earlier (days - pi) (takikawa et al., ) . other authors showed that complement may increase vnt titres by one dilution (diaz et al., (diaz et al., , . since the addition of -mercaptoethanol significantly reduced the sensitivity of the modified vnt, it is likely that low levels of neutralising igm appear in the early phases of the infection (takikawa et al., ) . however, even with this modified vnt, na titres were still relatively low ( / - / ) by day pi. nas are consistently detected by day pi or later for both european and american-type strains of prrsv (yoon et al., ; meier et al., ; diaz et al., ) . these nas are mainly directed against gp , which contains the major neutralisation epitope (nelson et al., ; dea, , ; gonin et al., ) . it has been claimed that gp and m proteins also contain neutralising epitopes (meulenberg et al., ; gonin et al., ; weiland et al., ; yang et al., ; cancel-tirado et al., ) and one report suggested that gp also contains a neutralising epitope (cancel-tirado et al., ) . however, these proteins seem to be of minor biological significance compared to gp . the early development of non-nas may have a significant effect on the development of prrs. it has been shown that non-nas enhance viral replication in alveolar macrophages, a phenomenon known as antibodydependent enhancement (ade) (yoon et al., (yoon et al., , . targets for these antibodies are gp and n proteins (yoon et al., ; cancel-tirado et al., ) . the non-neutralising humoral response may act as a trojan horse for prrsv by coating the virus and enhancing the internalisation of viral particles into macrophages. the question is: why nas do not develop as early as non-nas? nas may play an important role, although their importance may be different in natural infection compared to vaccination. vezina et al. ( ) reported the isolation of prrsv from the blood of pigs with nas. following experimental infection, viraemia may be resolved without detectable levels of neutralising antibodies (diaz et al., ) . similar to the related virus lactate dehydrogenase virus (ldv), the dynamics of prrsv-susceptible macrophages may govern the levels of viraemia (diaz et al., ) . if cytolysis or apoptosis exhaust most susceptible macrophages, infection will be confined to macrophage-rich organs, such as lymph nodes. in this hypothesis, nas may be required for resolution of viraemia, if not infection. a different picture arises when protection before infection is considered. nas block prrsv infectivity for macrophages in vitro (delputte et al., ) . transfer of nas to pregnant sows (na titres / ) protects them against reproductive failure and blocks transplacental infection . using the same antibody transfer system, a titre of / or higher protected piglets against the development of viraemia, whereas sterilising immunity was attained at na titres of / (lopez et al., ) . these results suggest that a vaccine capable of inducing na titres of / should prevent clinical disease and be a key tool in eradication of prrsv. cell-mediated immunity (cmi) is also extremely important in prrs. early studies showed that pigs recovering from experimental prrsv infection had strong lymphocyte proliferative responses, although these responses were not detected until four weeks pi and paralleled the na response (bautista and molitor, ; lopez fuertes et al., ) . cytokine responses were mainly interferon (ifn)-c and, to a lesser extent, il- (lopez fuertes et al., ) . after vaccination with a modified live vaccine using an american strain of prrsv, virus-specific ifn-c secreting cells first appeared in the third week post-vaccination, fluctuated erratically from - per million peripheral blood mononuclear cells (pbmcs) for the next ten weeks, then increased to - per million pbmcs at weeks post-vaccination (meier et al., ) . ifn-c secreting cells were mainly cd + cd + cells, with a small proportion of cd À /cd ab + cytotoxic t cells. a similar delayed development of prrsv-specific ifn-c secreting cells was evident after infection or vaccination with european strains of prrsv (diaz et al., (diaz et al., , . in contrast, - ifn-c secreting cells per million pbmcs were evident by weeks after vaccination against aujeszky's disease virus (meier et al., ) . the unusual characteristics of the adaptive immune response to prrsv suggest that the virus strongly modulates the immune response. early studies showed that prrsv is highly susceptible to the action of type i ifns and suggested that the virus was able to inhibit ifn-a responses, since this cytokine could not be detected in the lungs of pigs in which prrsv was actively replicating (albina et al., ; buddaert et al., ) . ifn-a levels in the lungs of prrsv-infected pigs were much lower than in the lungs of pigs infected with porcine coronavirus or swine influenza virus (van reeth et al., ) . frequencies of virus-specific ifn-c secreting cells were correlated with the frequencies of ifn-a secreting cells in pigs infected with prrsv (royaee et al., ) . although the exact mechanism by which prrsv inhibits ifn-a is unknown, it does not involve inhibition of the nuclear factor (nf)-j b pathway (lee and kleiboeker, ) . different prrsv isolates and different plaque clones of the same strain have different abilities to induce or inhibit ifn-a (lee et al., ) . preliminary results indicate that different european prrsv isolates have different abilities to induce not only ifn-a, but also tnf-a, il- and il- , in alveolar macrophages and dendritic cells (unpublished observations). impairment of ifn-a secretion would be expected to affect the development of an effective t helper type (th ) immune response. cytokines il- may have an important role in the regulation of the immune response to prrsv. after infection with either european or american strains of prrsv, levels of il- mrna were increased in porcine pbmcs and concentrations of il- were increased in bronchoalveolar lavage (bal) fluid (thanawongnuwech et al., ) . some european strains of prrsv induce strong il- responses in pbmcs from naïve pigs, suggesting that this is a not a memory feature (diaz et al., ) . pigs vaccinated with il- -inducing strains had lower frequencies of prrsvspecific ifn-c secreting cells than animals vaccinated with a non-il- inducing strain (diaz et al., ) . monocytes appear to be the major source of il- in prrsv infection (charerntantanakul et al., ) . prrsv also appears to induce il- (asai et al., ; sipos et al., ) , whereas the role of transforming growth factor-b in prrsv infection is unclear (royaee et al., ) . prrsv may interfere with correct antigen presentation and activation of t lymphocytes. prrsv down-regulated expression of major histocompatibility complex (mhc)-i in dendritic cells (dcs), although this was not correlated with impaired proliferative responses in the mixed leucocyte reaction (loving et al., ) . expression of mhc-i and mhc-ii, as well as cd , was down-regulated in monocyte-derived dcs stimulated by infectious but not inactivated prrsv (wang et al., ) . in this study, proliferative responses were decreased when infected dcs were used with syngeneic or allogeneic lymphocytes, suggesting that infected dcs present antigens less efficiently (wang et al., ) . prrsv may down-regulate the innate immune response by altering the cytokine patterns of macrophages and dendritic cells, as well as by modifying the expression of molecules involved in antigen presentation. as discussed previously, one of the intriguing features of prrsv infection is the delayed development of nas. the main neutralisation epitope of prrsv, designated epitope b, is located in the n-terminal ectodomain of gp (amino acids - ) in both american and european strains (ostrowski et al., ; plagemann et al., ; wissink et al., ; plagemann, ) . this neutralisation epitope is flanked by glycosylation sites. an additional immunodominant epitope, designated epitope a, is located in the n-terminal ectodomain of gp (amino acids and ) and has the characteristics of a decoy epitope, similar to that in human immunodeficiency virus type (ostrowski et al., ) . the decoy epitope may interfere with the immune response to the main neutralisation epitope b, resulting in a delay in the na response. insertion of a pan-dr helper t cell epitope between the epitope b and the decoy epitope increased the immunogenicity of epitope b in mice (fang et al., ) , suggesting that the proximity of epitopes a and b is important in delaying the na response. the decoy epitope is not the only way by which prrsv can evade the humoral immune response. gp , the main target for nas, contains up to four glycosylation sites, located in or close to the neutralising epitope. american field strains of prrsv lacking glycosylation sites in the upstream hypervariable region induced nas more rapidly and more strongly in infected pigs than strains lacking the downstream glycosylation site at position (n- ), even though all strains were equally susceptible to nas (faaberg et al., ) . as spanish prrsv strains have evolved from - , there has been a trend to lose the glycosylation site at n- (equivalent to n- of american strains) and to maintain or gain glycosylations in the flanking regions (n- and n- ), consistent with selection of strains inducing weaker na responses (mateu et al., ) . prrsv is divided into european (type i) and american (type ii) genotypes; four subtypes have been identified within the european genotype (stadejek et al., ) . diversity within a genotype or subtype can be high (forsberg et al., ; stadejek et al., ; larochelle et al., ; mateu et al., ; stadejek et al., ) . furthermore, prrsv also demonstrates the phenomenon of quasispecies generation (rowland et al., ; goldberg et al., ) . what is the impact of this genetic diversity of prrsv upon the immune response and protection afforded by vaccination? prrs emerged in europe and america almost simultaneously. the first vaccine against prrsv marketed internationally was a modified live vaccine derived from the prototypic american strain vr- . at that time, it was obvious that the genetic diversity of prrsv could pose problems regarding the efficacy of the vaccine, particularly with infections by the european genotype. pregnant gilts infected with the american isolate nadc- and challenged with the european isolate lelystad virus (lv) late in gestation had only partial protection against transplacental infection (virus crossed the placenta in / gilts), whereas all gilts challenged with the homologous virus were fully protected (lager et al., ) . these results showed that heterologous protection existed, but was only partial, and suggested that common epitopes are likely to be involved in protection in both european and american-type strains. furthermore, monoclonal antibodies against the neutralising epitope of the related virus ldv are able to neutralise both vr- and lv, indicating that the neutralising epitope in gp is shared to some extent by diverse arteriviruses (plagemann et al., ) although partial heterologous protection might be beneficial under some circumstances, a genotype-based vaccine is insufficient to produce immunising sterility. given the genetic diversity of the virus within one genotype, the question then was whether this phenomenon might influence the efficacy of a homologous vaccine. piglets vaccinated with attenuated versions of american strains nadc- , or were challenged days later with a mixture of the virulent versions of the same strains (mengeling et al., ) . a given virulent strain was not present after challenge if the piglets had previously received the attenuated version of that strain, whereas infections with the other virulent strains were established, indicating that immunity to prrsv may be strain-related. after vaccination with a european-type modified live vaccine, pigs were mostly negative for virus in serum or bal fluid after challenge with lv (labarque et al., ) in contrast, vaccinated pigs developed viraemia over days and were positive for virus in bal fluid when challenged with an italian variant strain that had % similarity in orf to the vaccine strain. protection against prrsv infection by a strain different to the one used as a vaccine is somewhat more complex than a matter of genetic similarity. pigs were vaccinated with two different european-type vaccines (v and v ), then challenged with a strain similar to one of the vaccines and slightly different to the other ( - % similarity) (diaz et al., ) . surprisingly, the ''heterologous'' v vaccine afforded sterilising immunity, while the homologous v vaccine did not. the v vaccine induced higher levels of ifn-c secreting cells, whereas v induced il- release by pmbcs. the ability of each strain to induce a strong cell-mediated immune response was more important that the genetic similarity inducing protection. the complexity of the immune response to prrsv and the ability of the virus to escape or modulate the host's immune system make it difficult to develop a vaccine that can be used to eradicate the disease. such a vaccine should accomplish at least four requirements: efficacy, universality, safety and ability to differentiate vaccinated from infected animals. the first line of investigation is the detailed investigation of b and t cell epitopes involved in the development of protective immunity. neutralising epitopes have been established definitively. little is known regarding t cell epitopes, although t cell responses to individual prrsv polypeptides have been reported in virusinfected animals (bautista et al., ) . common critical epitopes in both european and american strains of prrsv have to be clearly identified to support the development of universal vaccines. the second line of research is to determine which components of the virion or viral genome are involved in the down-regulation or modulation of the host's immune system and the mechanisms by which this occurs. this is critical for development of a live attenuated vaccine. the efficacy of a given vaccine is not only related to its immunological properties, but also to the characteristics of the challenging strain. therefore, studies on the relationship between genetic diversity and the immunopathological properties of different strains are needed. reverse genetics and characterisation of the modulating properties of an extensive set of strains are necessary; this can only be achieved through a serious international collaborative effort. thirdly, the developed vaccine should be safe. this means that any possibility of reversion to virulence should be eliminated and transmission of the vaccine strain between pigs should be minimal or non-existent. one obvious way to gain in safety is by using non-replicating vaccines. however, it is unclear if non-replicating vaccines are able to induce nas and adequate cell-mediated immune responses (zuckermann et al., ) . research on subunit or vector-based vaccines and adjuvants should be undertaken. fourthly, the development of a differential vaccine is highly desirable. since prrsv is a virus with a relatively small genome, it is difficult to find targets for deletion, although, to our knowledge, an extensive study of essential and non-essential parts of the viral genome has not been performed. the occurrence of natural variants with small deletions in nsp indicates that non-structural proteins could be a target for constructing differential vaccines (fang et al., ) . it is necessary to demonstrate that a vaccine protects against infection, not only in challenge experiments against one strain of the same or a different genotype, but also against different strains within a given genotype. table summarises some of the knowledge already known or required for developing newer vaccines. the number of important questions that remain to be solved in prrs immunology is considerable. for example, the development of the adaptive immune response after infection with prrsv or vaccination is anomalous. ifnc secreting cells appear late and evolve erratically during the first weeks after infection; na responses are also delayed. nas may protect against disease if present in sufficient quantities before infection, but they do not seem to be essential for clearing virus in blood during the course of the infection. prrsv is able to modulate innate responses, probably through the regulation of ifn-a and il- responses. two different prrsv genotypes exist that have evolved in parallel. cross protection afforded by each genotype is only partial and genetic diversity within each genotype can be high enough to allow a vaccinated animal to be re-infected by a different strain of the same genotype. these circumstances create difficulty in understanding how the immune system and the virus interact. it is possible that different prrsv strains are able to modulate or regulate the immune system in different ways. therefore, published experiments should be always interpreted with caution, particularly when trying to extract general principles from a particular experiment. collaboration between researchers is the best way to enhance our understanding of prrsv immunology. to all those that have contributed to the better understanding of prrs. structural proteins seem to be mainly essential some non-structural proteins might be non-essential exact map of essential and nonessential proteins some of the non-structural proteins may suffer natural deletions structural proteins of a north american strain of porcine reproductive and respiratory syndrome virus. virology , - . delputte, p.l., meerts, p., costers, s., nauwynck, h.j., . effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages. veterinary immunology and immunopathology , - . diaz, i., darwich, l., pappaterra, g., pujols, j., mateu, e., in pigs recovered from porcine respiratory and reproductive syndrome infection differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus genetic diversity and phylogenetic analysis of glycoprotein of european-type porcine reproductive and respiratory virus strains in spain evolution of orf of spanish porcine reproductive and respiratory syndrome virus strains from gradual development of the interferon-(response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus molecular characterization of lelystad virus serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp fragment of the replicase polyprotein contains a cluster of b-cell epitopes passive transfer of virusspecific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain antibodies to the orf product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants immune response in pigs vaccinated with plasmid dna encoding orf of porcine reproductive and respiratory syndrome virus the primary gp neutralization epitope of north american isolates of porcine reproductive and respiratory syndrome virus the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr- deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination parameters of humoral and cellular immunity following vaccination of pigs with a european modified-live strain of porcine reproductive and respiratory syndrome virus (prrsv) identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes upregulation of interleukin- gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus upregulation of il- gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus detection of antibodies against porcine reproductive and respiratory syndrome (prrs) virus in swine sera by enzyme-linked immunosorbent assay increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability monoclonal antibodies to the gp of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the gp the major envelope protein, gp , of a european porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its n-terminal ectodomain categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp and gp proteins and susceptibility to neutralization an indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera antibody-dependent enhancement (ade) of porcine reproductive and respiratory syndrome virus (prrsv) infection in pigs field isolates of porcine reproductive and respiratory syndrome virus (prrsv) vary in their susceptibility to antibody dependent enhancement (ade) of infection characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of c-ifn-producing cells and virological parameters of protection upon challenge key: cord- -tsfxpaj authors: chai, weidong; wang, zhenya; janczyk, pawel; twardziok, sven; blohm, ulrike; osterrieder, nikolaus; burwinkel, michael title: elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (pprsv) vaccination and infection date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: tsfxpaj background: porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important infectious agents for the swine industry worldwide. zinc (zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. the purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with prrsv. findings: the clinical course of prrs and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet ( ppm zn, control group) and diets supplemented with zn oxide (zno) at final zn concentrations of or , ppm. pigs receiving higher dietary zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary zn levels did not substantially influence the number of antiviral ifn-gamma secreting cells (ifn-gamma-sc) or percentages of blood immune cell subsets after infection. finally, feeding higher dietary zn levels reduced neither clinical symptoms nor viral loads. conclusions: our results suggest that higher levels of dietary zno do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous prrsv infection to an extent that could improve the clinical and virological outcome. electronic supplementary material: the online version of this article (doi: . / - x- - ) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome (prrs) is one of the most significant swine diseases worldwide [ ] . efficient prrs virus (prrsv) vaccines would be invaluable in minimizing the clinical and economic impact of prrsv infections, but currently safe and effective vaccines which protect against a wide variety of strains are not available [ ] . zinc (zn) ion salts exhibit a broad-spectrum antiviral activity against a variety of viruses in vitro, including the animal viruses equine arteritis virus and transmissible gastroenteritis virus [ , ] . in the european union standard dietary zn levels in feedingstuffs are limited to ppm due to environmental reasons. however, in other countries high levels of in-feed zn oxide (zno, , - , ppm) may be added to the diet of pigs during a restricted period following weaning to prevent post-weaning diarrhea [ ] as high levels of zno have been proven to conserve the intestinal flora during the critical period following the change of diet that place at weaning [ ] . despite this effect, the exact mechanisms of zno action remain uncertain, and the local or systemic effects of zno against specific viral pathogens also remain largely unknown. we evaluated the systemic effects of different zn levels added to a conventional diet containing ppm zn (zn low , control group) against prrsv. two other groups were fed the diet supplemented with zno at final concentrations of ppm zn (zn med ), or , ppm zn (zn high ). half of the animals received a single vaccination with an experimental uv-inactivated type i prrsv (lelystad virus; lv), since it was shown that a similar vaccination with such a virus in combination with a suitable adjuvant could strongly prime the virusneutralizing (vn) response and reduce duration of viremia after homologous challenge [ ] . in contrast to vanhee et al., we chose a single-vaccination approach and challenge-infected the animals with a heterologous type i prrsv ( , % sequence identity for the envelope glycoprotein gp , which bears a major neutralizing epitope) in order test the influence of elevated zn levels on an suboptimal antigen stimulus and on crossprotection. the study was approved by the local animal welfare authority (landesamt für gesundheit und soziales, berlin, germany) under the registration number g / . german landrace piglets (n = ) of both sexes from a prrsv-free herd were weaned at the age of days, moved to a biosafety level experimental facility (bundesinstitut für risikobewertung, berlin, germany), and randomly allocated to six pens (n = per pen). piglets were assigned to three different diets ( pens per diet). at the age of days, the animals receiving the zn high diet were switched to the zn med diet, in order to avoid toxic effects of zn. one week after commencing the different diets, one pen per diet was chosen randomly and the animals were vaccinated intramuscularly with inactivated lv (accession m ; kindly provided by prof. h. nauwynck (ghent university, ghent, belgium)). four weeks after vaccination, at the age of days, all pigs were challenged with prrsv field strain cresa (accession jf ; kindly provided by prof. j. segalés and prof. e. mateu (cresa, barcelona, spain). animals were infected by intranasal application of ml of virus suspension with a titer of × tcid /ml to each nostril using a spray nebulizer. pigs were monitored daily for the presence of clinical signs and body weights were recorded weekly. blood samples were collected weekly after vaccination and at , , , , , , and days post infection (dpi). nasal swabs were taken on the same dpi as blood samples for quantification of virus shedding. all pigs were necropsied on day pi. lung and lymphoid tissues were evaluated by visual inspection for macroscopic lesions, and samples from lungs, lymph nodes, tonsils, and spleen were taken and immediately frozen in liquid nitrogen and stored at − °c. for virological analysis, serum samples ( , , , , dpi), nasal swabs ( , , dpi), and tonsil, lung and tracheobronchial lymph node samples ( dpi) were examined by qpcr to determine prrsv copy numbers. rna extraction was performed using a viral rna/dna purification kit (stratec) applying μl of serum or mg of tissue each. rna yields and quality were determined with a nanodrop® nd- spectrophotometer (nanodrop technologies). reverse transcription (rt) was performed using the dy-namo™ cdna synthesis kit (thermo fisher scientific). viral loads were quantified using a taqman fluorescent probe-based real-time qpcr assay in an icycler iq™ detection system (bio-rad) with primers described elsewhere [ ] . prrsv-specific igm and igg antibodies were measured by elisa (ingezim prrs dr, ingenasa) according to the manufacturer's instructions. vn antibodies against prrsv were quantified by a viral neutralization test as previously described [ ] . neutralization of prrsv strain cresa was examined using prrsv gp specific monoclonal antibody h (ingenasa) and alexa fluor™ conjugated anti-mouse igg (h + l) secondary antibody (invitrogen). peripheral blood mononuclear cells (pbmc) were isolated using density centrifugation through a ficoll gradient (lsm , paa laboratories). samples were treated with erythrocyte lysis buffer for min on ice, pbmc were washed with ml of pbs with . % bsa and centrifuged for min at × g at °c. in all samples, pbmc viability was confirmed using standard procedures. the cell-mediated prrsv-specific immune response was measured by using elispot for the enumeration of ifn-gamma-sc in pbmc (mabtech). in order to compare homologous and heterologous responses, pbmc were stimulated in parallel ( . × pbmc/well, wells per pig and stimulus) with cresa or lv at a multiplicity of infection of . . unstimulated and phastimulated cells ( μg/ml) were used as negative and positive controls, respectively. ifn-gamma-sc numbers were counted using an elispot reader system (a.el. vis gmbh). flow cytometry analysis was performed as described before [ ] using a bd facscanto™ flow cytometer (bd biosciences). data were analyzed with flowjo™ software (treestar). almost all infected pigs showed clinical symptoms typical for prrsv infection and similar to a previous study with the same prrsv strain [ ] . increased rectal temperatures were detected for more than days, and edema of the eyelids and conjunctivitis for more than days. there was no significant dietary effect on fever magnitude and duration (figure a,b) . other clinical signs such as cough were observed only sporadically and lasted only for - days. regarding the weight gain, we could only analyze the effect of the zn med diet for the vaccinated groups, given that time points were missing for the pigs receiving the zn high pigs. for the remaining time points as well as for the non-vaccinated groups we found no benefits of feeding higher dietary zn levels (figure c,d) . mean prrsv load in serum as determined by qpcr peaked at dpi and gradually reduced later on. neither vaccination nor elevated zn levels showed an influence on prrsv viremia at any time during the observation period (figure a,b) . the same was observed regarding virus shedding as determined by analysis of nasal swabs (figure c,d) . persistent virus was found at dpi in the majority of tonsil samples, regardless of diet, while all lung and tracheobronchial lymph node samples were tested negative for the presence of prrsv genomes. prrsv-specific igg and igm antibodies were not detected by elisa in randomly chosen samples ( vaccinated/ non-vaccinated) before infection on day before infection. on day , only one vaccinated animal was positive for prrsv antibodies (figure a,b) . this is in contrast with previous results showing earlier seroconversion after vaccination with uv-inactivated lv [ ] and might be due to the fact that we used a cell culture-adapted non-purified lv with possibly decreased immunogenicity compared to purified lv grown on porcine alveolar macrophages used in the cited study. at dpi, piglets from all groups were seropositive, except for pig each in the vaccinated and non-vaccinated zn low groups. higher (p ≤ . ) antibody levels were detected in vaccinated groups than in non-vaccinated groups at and dpi after prrsv challenge, while the diet had no influence on antibody levels. the generation of neutralizing antibodies is delayed in prrsv infection and usually appears three to four weeks after infection [ ] . accordingly, in our study virus neutralizing vn antibodies were not detectable until dpi in the serum. a single-vaccination with an inactivated lv did not lead to an earlier induction of vn antibodies, but vaccinated groups developed higher (p = . ) vn antibody titers than their non-vaccinated counterparts at dpi ( figure c, d) . a tendency towards higher vn antibody levels was evident in pigs receiving higher levels of zn (zn med ) at dpi. this tendency continued to dpi in both zn med and zn high groups. thus, the possibility remains that animals receiving higher dietary zn levels might be better protected against reinfection with prrsv. the number of ifn-gamma-sc at dpi revealed no differences after homologous or heterologous re-stimulation (additional file ). flow cytometry analysis of pbmc phenotypes was performed weekly from day to day post infection. single vaccination only delayed but not prevented the prrsv-induced decrease of cd + , cd + and cd + cd + t cell populations, which are important for viral clearance [ ] . we found no sustained effect of dietary zn levels on any of the analysed cell subsets (additional file ). necropsies at dpi revealed no gross lung lesions and lymphoid hyperplasia in tonsils, lymph nodes or spleen in any of the pigs. overall, the study shows that challenge infection with a wild-type prrsv without additional environmental and social stress and the impact of secondary infections results in relatively mild clinical signs. under these conditions, elevated dietary zn levels could not provide enhanced protection against infection with a type i prrsv field strain and could not improve efficacy after a singlevaccination with a heterologous inactivated vaccine. the data sets supporting the results of this article are included within the article (and its additional files). additional file : figure s . prrsv-specific numbers of ifn-gamma-sc determined by elispot. pbmc collected at dpi were restimulated with either of the prrsv strains (lv or cresa ) used in the study. results are shown as average frequencies of virus-specific ifn-gamma-sc per . × pbmc. filled symbols indicate results obtained after in vitro restimulation with the same prrsv used for infections (homologous) while empty symbols show the results of in vitro restimulation with lv (heterologous). additional file : figure s . modulation of pbmc immune cells frequencies determined by flow cytometry analysis. a and b, cytotoxic lymphocytes (cd + cd − cd α high ); c and d, naïve t h cells (cd + cd + cd − ); e and f, cd + γδ t cells (cd + cd + cd + ); g and h, antibody forming and/ or memory b cells (cd − cd + cd − ); i and j, nk cells. asterisks indicate statistically significant differences (p < . ) between averages at each dpi. immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology. vaccine zn( +) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture antiviral activity of zinc salts against transmissible gastroenteritis virus in vitro effect and interaction between wheat bran and zinc oxide on productive performance and intestinal health in post-weaning piglets dietary zinc oxide in weaned pigs-effects on perfor-mance, tissue concentrations, morphology, neutrophil functions and fecal microflora development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant dietary enterococcus faecium ncimb and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection the challenge of prrs immunology evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (pprsv) vaccination and infection the authors would like to acknowledge the animal welfare officer m. ladwig and all animal technicians supervised by dr. s. banneke at bundesinstitut für risikobewertung for their engagement. we further acknowledge e. luge for his excellent technical assistance. we thank dr. s. kreuzer, züchtungsbiologie und molekulare genetik, humboldt-universität zu berlin, for helping with the flow cytometry data analysis and prof. m. schmidt, institut für immunologie, freie universität berlin, for helpful comments about the experimental design. we also thank b. esch, institut für virologie, freie universität berlin for expert technical assistance. the study was funded by the deutsche forschungsgemeinschaft through grant sfb / . the authors declare that they have no competing interests. key: cord- -i fw afj authors: lau, susanna k. p. title: molecular research on emerging viruses: evolution, diagnostics, pathogenesis, and therapeutics date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: i fw afj viruses are increasingly recognized as emerging infectious disease agents in both humans and animals.[...]. viruses are increasingly recognized as emerging infectious disease agents in both humans and animals. zoonotic viruses, in particular, often evolve rapidly to adapt to new hosts with enhanced virulence and emerge or re-emerge to cause epidemics. factors such as urbanization, global warming, and dense human and animal populations may have contributed to the emergence of some viruses. the recent epidemics caused by zika virus and middle east respiratory syndrome coronavirus (mers-cov) clearly illustrate the ability of emerging viruses to pose huge public health problems within a short time. much more research effort is needed to understand the evolution and pathogenesis of these emerging viruses, as well as the development of diagnostics and therapeutics to combat existing and future epidemics. recent advances in molecular techniques, such as sequencing and metagenomics, have accelerated our understanding of genetic and host diversity of emerging viruses, and hence their evolutionary pathways. nevertheless, many questions remain unanswered. for example, while severe acute respiratory syndrome coronavirus (sars-cov) was originated from horseshoe bats as the primary reservoir [ , ] , the ancestral origin of mers-cov has not been ascertained yet despite its close relationship with some bat coronaviruses [ ] [ ] [ ] . molecular assays play a crucial role in the diagnosis of emerging viruses such as zika virus, which may overcome the limitations of viral cultures and serological tests, allowing more convenient, rapid, and accurate diagnosis. immense research efforts have also been recently made to reveal the diverse virulence mechanisms and host-pathogen interactions of emerging viruses. on the other hand, with only a few exceptions such as influenza virus, treatment of emerging viruses is mostly supportive, as a result of the lack of effective antivirals for most of these viruses. discovery of novel antiviral agents is eagerly awaited for many emerging viruses, though identification of antiviral targets which spare the host replication machinery can be difficult. therefore, new research directions are crucial to predict, prevent, and combat virus diseases. in this special issue, "molecular research on emerging viruses: evolution, diagnostics, pathogenesis, and therapeutics", insights into advances and discoveries in understanding the different aspects of various emerging viruses are given by eight original studies and four review articles. three articles focus on arthropod-borne viruses (arboviruses) which are important emerging pathogens having caused various epidemics in recent years. in the systematic review and meta-analysis study by coelho et al., the prevalence of microcephaly in infants born to zika virus-infected women among all pregnancies was estimated [ ] , which may contribute to the understanding of the public health impact of this emerging arbovirus. the article by le coupanec et al., studies the viral replication during co-infection of chikungunya and dengue viruses which has been observed in some patients [ ] . co-infection with both viruses in aedes aegypti mosquitoes was found to facilitate viral replication, suggesting the importance of pathogen-pathogen interactions. in another article, lu et al. investigated the antiviral activity of histone deacetylase (hdac) inhibitors as host-targeting agents against japanese encephalitis virus (jev) [ ] . tubacin, a selective hdac inhibitor was found to be a potential host-targeting agent, demonstrating preventive and therapeutic activities against jev infection. zoonotic influenza viruses remain a significant concern to both human health and food industry, with their tendency to re-assort and mutate to generate novel strains capable of interspecies transmission. yet, it remains difficult to predict the emergence potential of new strains. in the article by eng et al., a machine learning approach was taken to build a zoonotic strain prediction model which could classify avian, human, or zoonotic strains with an estimated zoonotic risk [ ] . in another article, zhang et al. investigated the role of swine cellular micrornas in regulating swine pandemic h n / influenza a virus (siv-h n / ) replication [ ] . two micrornas, ssc-mir- and ssc-mir- , were found to target viral haemagglutinin (ha) and non-structural protein (ns), respectively, and inhibit viral replication, providing insights into virus-host interaction and control of the virus in swine population. hepatitis viruses pose significant disease burdens worldwide and some have emerged or re-emerged in different populations. in the review article by sridhar et al., the genotypic diversity and evolution of existing hepatitis e virus strains are reviewed, with a special focus on the emergence of camel hepatitis e variants [ ] . in another article, lee et al. reported novel hepatitis b virus intergenotypic recombinants from a patient co-infected with genotype a and c [ ] . the results may prompt further studies on the clinical implications of such novel recombinant virus strains. coronaviruses have continued to emerge or re-emerge in the last two decades to cause epidemics in humans and animals. in the review article by lin et al., current knowledge on the molecular evolution, pathogenicity, and epidemiology of infectious bronchitis virus, which poses huge economic threats to poultry farms worldwide, was summarized [ ] . enteroviruses are another group of emerging viruses that can cause severe sporadic infections or epidemics especially in young children. in particular, enterovirus-d has emerged in recent years in various countries and is increasingly recognized as an important respiratory pathogen in the young and immunocompromised. in the article by yip et al., the first fatal case of ev-d infection and genetic diversity of ev-d strains in hong kong were described [ ] . the study also found a newly emerged subclade b and an interclade recombination between clade a and d strains in china. two articles report on the virus-host interaction during porcine reproductive and respiratory syndrome virus (prrsv) infection which causes severe losses in the swine industry worldwide. in one article, ji et al. describes the role of porcine interferon stimulated gene a (isg a) in restricting prrsv replication [ ] . isg a was found to be upregulated in cells or tissues of pigs and could suppress prrsv replication in infected marc- cells, supporting its role in host immune response to prrsv. in another article, liang et al. describes the transcriptomic differences in porcine alveolar macrophages from two different pig breeds, tongcheng and large white pigs, in response to prrsv infections [ ] . transcriptomics profiling of infected macrophages suggested that tongcheng pigs, being more resistant to prrsv infection, may promote the extravasation and migration of leukocytes from the capillaries to the surrounding tissues to defend against prrsv and suppress apoptosis of macrophages in order to enhance antigen presentation. last but not least, some mammalian arenaviruses are emerging viruses that may infect humans to cause lethal hemorrhagic fever. in the article by ly, current knowledge on the differential immune responses to arenaviruses were summarized, which may help understand their pathogenesis and contribute to the development of vaccines [ ] . severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace receptor isolation of a novel coronavirus from a man with pneumonia in saudi arabia comparative analysis of genomes of three novel group c and group d coronaviruses reveals unique group and subgroup features genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus microcephaly prevalence in infants born to zika virus-infected women: a systematic review and meta-analysis co-infection of mosquitoes with chikungunya and dengue viruses reveals modulation of the replication of both viruses in midguts and salivary glands of aedes aegypti mosquitoes reduces the replication of the japanese encephalitis virus via the decrease of viral rna synthesis predicting zoonotic risk of influenza a viruses from host tropism protein signature using random forest sus scrofa mir- and mir- negatively regulate swine h n / influenza a virus replication by targeting viral ha and ns, respectively hepatitis e virus genotypes and evolution: emergence of camel hepatitis e variants identification of novel a /c inter-genotype recombinants of hepatitis b virus from a korean chronic patient co-infected with both genotype a and c infectious bronchitis virus variants: molecular analysis and pathogenicity investigation first report of a fatal case associated with ev-d infection in hong kong and emergence of an interclade recombinant in china revealed by genome analysis porcine interferon stimulated gene a restricts porcine reproductive and respiratory syndrome virus replication in marc- cells transcriptome differences in porcine alveolar macrophages from tongcheng and large white pigs in response to highly pathogenic porcine reproductive and respiratory syndrome virus (prrsv) infection differential immune responses to new world and old world mammalian arenaviruses the author declares no conflict of interest. int. j. mol. sci. , , key: cord- -zolwjl u authors: xiao, shuqi; jia, jianyu; mo, delin; wang, qiwei; qin, limei; he, zuyong; zhao, xiao; huang, yuankai; li, anning; yu, jingwei; niu, yuna; liu, xiaohong; chen, yaosheng title: understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: zolwjl u porcine reproductive and respiratory syndrome (prrs) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. prrs virus (prrsv) replicates mainly in porcine alveolar macrophages (pams) and dendritic cells (dcs) and develops persistent infections, antibody-dependent enhancement (ade), interstitial pneumonia and immunosuppression. but the molecular mechanisms of prrsv infection still are poorly understood. here we report on the first genome-wide host transcriptional responses to classical north american type prrsv (n-prrsv) strain ch a infection using solexa/illumina's digital gene expression (dge) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology. our results suggest that n-prrsv appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during n-prrsv infection processes. n-prrsv-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. porcine reproductive and respiratory syndrome (prrs), also called ''blue ear'' disease due to a typical, but not often observed hallmark of ''blue ears'', is widely accepted as being one of the most economically important diseases affecting swine industry. since its first appearance in the late s in the us and europe, prrs has spread worldwide [ , , ] . prrs is characterized with high mortality in piglets, reproductive failure (late-term abortions and stillbirths, premature farrowing, mummified pigs) in pregnant sows and respiratory disease (interstitial pneumonia, respiratory difficulties) in nursery and grower/finishing pigs, causing highly significant economic losses to the swine industry worldwide, resulting in .$ . million losses each year in the us alone [ ] . the etiologic agent of prrs is prrs virus (prrsv), a small enveloped, linear, single, positive-stranded rna virus, which is a member of the family arteriviridae which includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus and enters in the newly established order delayed and their levels remain low, which can not eliminate effectively prrsv-infected cells [ , ] . because of these features of prrsv infection, prrs has been one of the most challenging subjects of research in veterinary viral immunology [ ] . regulation of immune responses and genetic resistance to infectious viral diseases is an area of concern for human and swine [ ] . prrsv strongly modulates the host's immune responses, and changes the host's gene expression. studies showed that prrsv inhibits type i interferons (ifn-a/b, spi ifn), especially ifn-a [ ] , and induces interleukin- (il ) [ , ] . because the primary cellular target of prrsv is the porcine alveolar macrophages (pams) of the lung, several studies have analysed the immune responses of pams to prrsv infection. one group [ ] used differential display reverse-transcription pcr to identify molecular genetic changes within prrsv-infected pams over a h pi period. their results suggest that myxovirus resistance (mx ) and ubiquitin specific proteases (usp) genes may play important roles in clinical disease during prrsv infection. notably, one recent paper on genome-wide transcriptional response of pams following infection with the lelystad prrsv strain (european type, eu prrsv) using affymetrix microarrays has been published during the preparation of our manuscript [ ] . they found that the expression of beta interferon (ifn-b) was strongly upregulated while the expression of il- and tnf-a was weakly upregulated. almost in the same time, the other group employed serial analysis of gene expression (sage) to examine the global expression of genes in vr- prrsv strain (north american type, na prrsv)-infected pams. they identified over unique tags with significantly altered expression levels [ ] . in vitro studies will be useful for investigating how prrsv modifies genes expression in primary target cells, such as pams. however, many of the outstanding issues will be answered only in the context of prrsv-infected animals. hence, the characterization of host immune response under in vivo environment to prrsv is still an area in urgent need of investigation. lung pathogenesis is a major feature of prrsv infection. moreover, in addition to serving as a source of protein in the human diet, the pig is also an excellent biomedical model for humans because of the similarity in size and physiology, and in organ development and disease progression [ ] . thus, understanding the host's immune response to prrsv infection is important not only for swine production but also for human consumption. however, to date, the immune response to prrsv in porcine lung has not been analyzed by transcriptome profiling. next generation high-throughput sequencing technology has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data [ , , , ] . the technology developed by illumina (formerly solexa sequencing) [ ] , which is also referred to as digital gene expression (dge) tag profiling, allows identification of millions of short rnas in a sample and of differentially expressed genes without the need for prior annotations. here we employed the illumina genome analyzer platform to perform a digital gene expression analysis of the porcine lung transcriptome response to n-prrsv infection, and used histopathology examination to analyze the pulmonary pathological changes of the infected-porcine lungs. the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology was systematically analyzed. the comprehensive analysis of the global host response induced by n-prrsv suggested an inflammatory response, mediated by multiple inflammatory molecules early during infection that induced tissue injury, an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. our study had been approved by animal care and use committee of guangdong province, china. all animal procedures were performed according to guidelines developed by the china council on animal care and protocol approved by animal care and use committee of guangdong province, china. nine conventionally-reared, healthy -week-old, crossbred weaned pigs (landrace yorkshire) were selected from a highhealth commercial farm that has historically been free of all major pig diseases, such as prrsv, porcine circovirus type , classical swine fever virus, porcine parvovirus, pseudorabies virus, swine influenza virus and mycoplasma hyopneumoniae infections. all pigs were prrsv-seronegative determined by elisa (herdchek prrs xr; idexx laboratories) and absence of prrsv tested by rt-pcr. pigs were randomly assigned to two groups in the experiment and raised in isolation rooms. six pigs were inoculated with ml viral suspension ( ml intranasally and ml intramuscularly) of classical north american type prrsv (n-prrsv) strain ch a, isolated from china in , gifted by dr. zhang guihong, south china agricultural university) at a dose of . tcid ml on day . three uninfected negative control (unc) pigs were treated similarly with an identical volume of dmem culture media from uninfected marc- cells day prior to experimental infection, and were immediately necropsied. n-prrsv-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days to pi. viral reisolates were performed after the pigs were killed. the infected group showed positive, and the unc group was negative. tissue homogenates and serum were examined by n-prrsv-specific quantitative pcr (qpcr). the oligonucleotide primers used were nsp f( -gtgggtcggcaccagtt- ) and nsp r , designed in the gene segment encoding for nsp . the taqman probe, fam-cacagttctacgcggtgcagg -tamra , was synthesized. three infected pigs randomly chosen were necropsied at each time point of h pi and h pi. lung samples were collected from unc group (c), three pigs at h pi (n ), three pigs at h pi (n ) and immediately frozen in liquid nitrogen for rna isolation or fixed in % neutralized buffered formalin for histological processing. lungs of unc and experimentally infected pigs were processed routinely for haematoxylin and eosin (h&e) and immunohistochemistry staining, as described previously [ ] . total rna was extracted from frozen lungs using standard protocols (trizol) and then treated with dnase to remove potential genomic dna contamination according to the manufactures's protocols. rna integrity and concentration were evaluated by agilent bioanalyzer (agilent technologies). for rna library construction and deep sequencing, equal quantities of rna samples from three unc individual lungs were pooled, rna samples from the three infected pig lungs (n ) were pooled, and rna samples from the three infected individual lungs (n ) were pooled. approximately mg of rna representing each group were submitted to solexa (now illumina inc.) for sequencing. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol. in brief, mrna was isolated from mg total rna by binding the mrna to a magnetic oligo bead. first-and secondstrand cdna were synthesized while the mrna was attached to the beads. the double stranded cdnas were digested with nlaiii to wash away all fragmens other than the catg fragment attached to the oligo bead. then gex nlaiii adapter was ligated at the site of nlaiii cleavage. in addition, gex nlaiii adapter contains the sequence for the restriction enzyme mmei, subsequently, we applied the restriction enzyme mmei to create the bp tag. the gex adapter was ligated at the site of mmei cleavage. a pcr with cycles was performed with two primers that anneal to the ends of the adapters to enrich the adapterligated cdna construct. the resulting bp fragments were purified from % novex tbe page gel. subsequently, the purified cdna tags were sequenced on the illumina cluster station and genome analyzer. image recognition and base calling were performed using the illumina pipeline. all data is miame compliant. the raw data (tag sequences and counts) has been submitted to gene expression omnibus (geo) under series gse . for the raw data, we filtered adaptor tags, low quality tags and tags of copy number = to get clean tags. subsequently, we classified the clean tags according their copy number in the library and show their percentage in the total clean tags and analyzed saturation of the library. the preprocessed database of all possible catg + -nt tag sequences was created, using sus scrofa unigene (http://www.ncbi. nlm.nih.gov/unigene/ugorg.cgi?taxid = , unigene build # sus scrofa, nov, th, ) from ncbi. for monitoring the mapping events on both strands, both the sense and the complementary antisense sequences were included in the data collection. information on the position of polyadenylation signals was also collected from the transcript dababase. then we aligned all clean tags to the reference sequences, and unambiguous tags were annotated. we counted the clean tag number corresponding to each gene. to compare the de of gene across samples (n /c, n /c, n /n ), the number of raw clean tags in each library was normalized to tags per million (tpm) to obtain normalized gene expression level. de detection of gene or tag across samples was performed according to the previous description [ ] . genes were deemed significantly differentially expressed with a p-value , . , a false discovery rate (fdr) , . and an estimated absolute log -fold change . . in sequence counts across libraries. in order to verify the dge results, we used qpcr analysis. the rna samples used for the qpcr assays were both the same as for the dge experiments and independent rna extractions from biological replicates. qpcrs were done on the lightcycler (roche), with sybr-green detection (sybr primescript rt-pcr kit, takara biotechnology co., ltd.), according to the manufacture's instruction. each cdna was analyzed in triplicate, after which the average threshold cycle (ct) was calculated per sample. the relative expression levels were calculated with the ddct method. the results were normalized to the expression level of hprt and relative to the c sample. through browsing all health traits of pig quantitative trait locus (qtl) database (pigqtldb, http://www.animalgenome. org/qtldb/pig.html) by trait classes, we obtained mapping details of qtl on the corresponding pig chromosome. then pig affymetrix elements corresponding to health trait qtl regions were downloaded to an excel file. by matching the id of de genes to all genes in the qtl regions, we obtained de genes of the corresponding qtl region. pathway analysis was mainly based on the kyoto encyclopedia of genes and genomes (kegg) database. two-side fisher's exact test with a multiple testing and x test were used to classify the pathway category. the false discovery rate (fdr) was used to correct the p-value. we chose only pathway categories that had a p, . . within the significant category, the enrichment re was , where n f is the number of flagged proteins within the particular category, n is the total number of proteins within the same category, nf is the number of flagged proteins in the protein reference database list, n is the total number of proteins in the gene reference database list. stc is implemented entirely in java. the clustering algorithm first selects a set of distinct and representative temporal expression profiles. these model profiles are selected independent of the data. the clustering algorithm then assigns each gene passing the filtering criteria to the model profile that most closely matches the gene's expression profile as determined by the correlation coefficient. since the model profiles were selected independent of the data, the algorithm can then determine which profiles have a statistically significant higher numberthan genes assigned using a permutation test. this test determines an assignment of genes to model profiles using a large number of permutations of the time points. it then uses standard hypothesis testing to determine which model profiles have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs. significant model profiles can either be analyzed independently, or grouped together based on similarity to form clusters of significant profiles [ , ] . stc-go supports gene ontology enrichment analyses for sets of genes having the same significant temporal expression pattern. we select random samples of s a (s a is the number of genes assigned to the same model temporal expression profile r.) genes at each iteration and compute fisher's exact test p-values for the selected genes in all go biological categories [ ] . the two-sided fisher's exact test p-value for a category reflects a test of the null hypothesis that the category is enriched in genes assigned to profile r with respect to what would have been expected by chance alone. to decide whether or not to follow up a category that appears enriched in these genes, we would know the statistical reliability of the apparent enrichment. to assess the significance of a particular category, we need to know the distribution of p-values that would occur by random chance. the percentage of false positives to be tolerated will generally depend on the relative costs of false positives and false negatives in whatever follow-up study is to be done. this way of framing the question leads us to specify the false discovery rate (fdr) for a set of categories, rather than significance level (p-value) for each category. with the significance at the . level, for a given category, the enrichment r e is given by r e~( i=m)=(s a =n) where i is the number of genes assigned to profile r within the go category of interest, m is the total number of genes within the go category of interest, and n is total number of unique genes in the gene reference database list. after n-prrsv infection, the affected pigs exhibited the following clinical symptoms within - days: fever of . - . uc, depression, anorexia, rough hair coats, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, mild diarrhoea, shivering. those unc pigs did not show any obvious changes in body temperature and clinical signs. qpcr assay showed that n-prrsv virus was present in each of the infected pigs. but n-prrsv nsp gene was not differentially expressed at h pi and h pi (table s ). histopathology examination of n-prrsv-affected pigs showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells ( figure b ). most viral antigen was detected in alveolar cells and bronchiolar epithelial cells in lesions ( figure c ). to investigate the regulation of the host response to the n-prrsv virus, we considered the global gene expression profiles in lungs using solexa/illumina's dge system, a tag-based transcriptome sequencing method. we sequenced three porcine lung dge libraries from c, n , n using massively parallel sequencing on the illumina platform. major characteristics of these three libraries were summarized table . we obtained approximately . million total sequence tags per library with distinct tag sequences. prior to mapping these tag sequences to reference sequences, we filtered adaptor tags, low quality tags and tags of copy number = , producing approximately . million total clean sequence tags per library with distinct clean tag sequences. the c library had the highest number of both total sequence tags and distinct sequence tags; this was followed by the n , n libraries. moreover, the c library had the highest ratio of number of distinct tags to total tags and the lowest percentage of distinct clean high copy number tags. the data showed that more genes were detected in the c library than other two libraries and more transcripts were expressed at lower levels in the c library. saturation analysis of capacity of libraries showed that new emerging distinct tags were gradually reduced with increasing of total sequence tags when the number of sequencing tags was big enough. when the number of sequencing tags reached million, library capacity approached saturation ( figure s ). for tag mapping, we preprocessed one reference tag database that included sequences from sus scrofa unigene. to get the reference tags, we used nlaiii to digest all the samples and took all the catg+ tags in the gene as the gene's reference tags, not only the most one. we obtained total reference tag sequences with unambigous tag sequences. considering polymorphism across samples, tolerances were set to allow one mismatch in each alignment. by the criteria, . %, . % of distinct clean tags mapped to the unigene virtual tag database, . %, . % of the distinct clean tags mapped unambiguously to the unigene, and . %, . % of the distinct clean tags didn't map to the unigene virtual tag database ( table ). the occurrence of unknown tags was probably due to the incompleteness of pig genome sequencing. most solexa experimental tags matched to the st or nd catg site in high-confidence transcripts ( figure s ). for solexa sequencing can distinguish between transcripts originating from both dna strands, employing the strand-specific nature of the sequencing tags obtained, we found evidence for bidirectional transcription in to of all detectable unigen clusters and to antisense-stand specific transcripts (table s ). by comparison, the ratio of sense to antisense strand of the transcripts was approximately . : for all libraries. this suggests that in spite of the high number of antisense mapping events detected, the transcriptional regulation in the n-prrsv-induced immune response acts most strongly on the sense strand. to analyze the depth of transcriptome sampling in the dge libraries, we studied the rate of increase of the number of genes (sense+antisense strand) identified as the size of the corresponding library increases. when the library size reached one million, we could identify % and % all genes and genes identified by unambigous tags, respectively ( figure s ). at this time, library capacity approached saturation. to gain the global transcriptional changes in n-prrsv infected porcine lungs, we applied the method described previously [ ] to identify de genes from the normalized dge data by pairwise comparisons between all differential time points (n /c, n /c, n /n ) during infection. results showed that genes had p values , . , false discovery rate (fdr) , . and estimated absolute log -fold change . . in at least one of the pairwise comparisons, which were declared to be differentially expressed during infection course (table s ) . to characterize the functional consequences of gene expression changes associated with infection with n-prrsv, we performed pathway analysis of de genes based on the kegg database by two-side fisher's exact test. we chose only significant pathway categories that had a p-value of , . and an fdr of , . . as shown in figure s , the significant signaling pathways include cell adhesion molecules (cams), t cell receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, biosynthesis of unsaturated fatty acids, pantothenate and coa biosynthesis, etc (table s ) . to validate de genes identified by solexa sequencing, we selected genes for qpcr confirmation. the set included two down-regulated genes (epithelial chloride channel protein (aecc) and hyaluronan and proteoglycan link protein (hapln )) and six up-regulated genes (inflammatory response protein (irg ), dead (asp-glu-ala-asp) box polypeptide (ddx ), usp , cxcl , cytochrome p (cyp a ), and cd ). data were presented as fold changes in gene expression normalized to the hprt gene and relative to the c sample. pearson's correlation coefficient (r) showed that both the dge and qpcr data (pooling samples) were highly correlated, for the genes modulated by n-prrsv had a high consistency and r values ranging from . (cyp a ) to . (aecc) between the two methods ( figure ). qpcr analysis (both pooling samples and independent rna extractions from biological replicates) confirmed the direction of change detected by dge analysis. this correlation indicated the reliability of dge results. qtl play a central role in linking genomic information with phenotypes. the ultimate goal of qtl studies is to identificate the actual gene(s) that are responsible for the phenotypic variation observed in a particular trait [ ] . in the present paper, we mapped the de genes to pig qtl regions of health traits in pig qtldatabase (pigqtldb). our search found that de genes were distributed in different known qtl regions related to pig health traits ( figure s ; table s ). among the de genes, and were located in qtl regions of the cd -positive leukocytes and cd -positive leukocytes, respectively; were distributed in the qtl region of the band-formed neutrophils and cd -positive leukocytes. immune responses against pathogens depend in part on the generation of fully differentiated 'killer' (or effector) and memory cd + t cell. effective priming and maintenance of cd + t cell responses to viral infection require 'help' from cd + t cells, the latter play also a critical role in programming cd + t cell memory development [ ] . moreover, recent study showed that cd + t cells guide effector cytotoxic t lymphocytes (ctls) to virally infected tissues where they can destroy infected cells [ , ] . in order to profile gene expression time series and search for the most probable set of clusters generating the observed time series, we used stc algorithm of gene expression dynamics, which explicitly took into account the dynamic nature of temporal gene expression profiles during clustering and identified the number of figure s ; table s ) with (profile , , , ) significant cluster profiles which have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs ( figure s ). then gene ontology (go) based on biological process (bp) enrichment analyses for sets of de genes having significant cluster profiles was performed by two-side fisher's exact test (table s and table s ; figures s , s , s s ). we chose only significant go categories that had a p-value of , . . the most prominently overrepresented go terms of significant cluster profile ( , , ) and profile ( , , ) , which are down-regulated genes, involved in regulation of lipid, cholesterol biosynthetic and metabolic process; regulation of skeletal muscle development, muscle cell differentiation; digestion; negative regulation of neuron apoptosis and neurological system process (table s ; figures s and s ) . the most prominently overrepresented go terms of significant cluster profile ( , , ) and profile ( , , ), which are up-regulated genes, included negative regulation of fibroblast proliferation; natural killer cell, macrophage, lymphocyte, mononuclear cell, leukocyte and t cell proliferation, differentiation and activation; complement activation, immune response, inflammatory response, defense response, and apoptosis; response to stimulus(stress); lipid and fatty acid metabolic process and oxidation; positive regulation of ubiquitinprotein ligase activity and protein proteolysis, protein targeting to mitochondrion (table s ; figure s and s ). these results are consistent with these genes and their associated processes playing important roles in n-prrsv replication and pathogenesis. viral infection of host leads to the initiation of antiviral innate immune responses, which results in the induction of expression of the type i interferons [ ] . meanwhile, many viruses have also developed strategies to evade and subvert the immune response. as shown in figure a , transcripts of the ifn c was significantly induced in n-prrsv-infected pigs at days through pi, but short type i interferon (spi ifn) gene expression was suppressed, and interferon alpha (ifna ) gene expression was markedly down-regulated. lipid rafts, lipid microdomains of the cell membrane enriched in sphingolipids, cholesterol and associated proteins, play critical roles in the life cycle of many viruses [ ] . some viruses enhance their replication by modulating host cell lipid metabolism [ ] . dge analysis of pigs infected with n-prrsv showed significant increase of transcript abundance in many genes involved in lipid metabolism, including those for apolipoprotein b receptor (apob r), apolipoprotein-e (apoe), low density lipoprotein b (ldlb), phosphatidylinositol -kinase catalytic subunit type (pik c ) ( figure b ). perhaps n-prrsv alters hosts' lipid metabolism to create a lipid-rich intracellular environment to facilitate its own multiplication. moreover, we also observed that n-prrsv induced upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including myeloid cell leukemia sequence (bcl -related) (mcl ), nuclear factor kappa-b (nfkb ), nfkb , adrenomedullin (adm), and interleukin (il ), and downregulation expression of pro-apoptotic genes, including bak protein (bak), (apoptosis-related protein ) (apr ) ( figure d ) to inhibit apoptosis, which might prolong cell life and increase the yield of progeny virions. n-prrsv infection caused anorexia and subsequent slow growth. accordingly, we observed that transcript abundance of genes involved in digestion, such as gastric mucin (muc ac) and cytochrome p (cyp a ), was significantly decreased ( figure e ). simultaneously, transcript abundance of the genes associated with cell, muscle and cartilage development was markedly decreased ( figure f ). these genes include insulin-like growth factor binding protein (igfbp ), collagen, type ii, alpha isoform (col a ), connective tissue growth factor (ctgf), epidermal growth factor (egf). fever and heat shock fever is frequently the host's initial response to infection [ ] . after viral infection, pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids were recognized by host pathogen-recognition receptors (prrs), such as toll-like receptors (tlrs), which trigger gene expression and synthesis of the il- b precursor. active caspase- (casp ) cleaves the il- b precursor into mature, bioactive il- b, which is an inflammatory cytokine most responsible for fever [ , , ] . as shown in figure a , transcript abundance of tlr , , , , il- b and casp was significantly increased in n-prrsv infected porcine lungs. moreover, transcript abundance of genes involved in the activation of casp and il- b secretion including apoptosisassociated speck-like protein containing a card (asc), prostaglandin e synthase (pge ) and phospholipase a , group vii (pla g ) was significantly increased ( figure a ). the expression of heat shock proteins (hsps), known as stress proteins, can be markedly upregulated by all cells under conditions of stress, such as increased temperature (fever) and viral infection [ ] . transcript abundance for most of these heat shock genes, including -kda hsp (hsp ), hsp , and heat shock protein beta- (hsp ) was significantly elevated in n-prrsv infected lungs relative to unc lungs ( figure b ). viral infection results in an inflammatory response, which is an essential component of the antiviral innate immune response [ ] . after recognizing the pamps, either surface or intracellular prrs trigger intracellular signaling cascades that results in the activation of transcription factors, including nuclear factor-kb (nf-kb), interferon-regulatory factors (irfs), and signal transducer and activator of transcription (stats). as shown in figure a , transcripts of the toll-like prrs tlr , tlr , tlr , tlr , were significantly increased in n-prrsv-infected pigs at days through pi, but no change in tlr which specializes in the recognition of viral dsrna was detected. cytoplasmic prrs ( figure a ), retinoic-acid-inducible protein i (rig-i, ddx ) and melanoma differentiation-associated gene (mda ), the two most relevant for defense against viruses, were expressed at a high level after n-prrsv infection. cell surface prrs such as cd , md- protein (md ) and cd (which is probably involved in prrsv entry during uncoating [ ] ) were likewise up-regulated expression after n-prrsv infection ( figure a ). after binding to n-prrsv viral pamps, prrs initiate intracellular signaling cascades that activate transcription factors, including irf , irf , irf , but not irf and stat , stat , stat ( figure b ). activated transcription factors and stats in turn induce the transcription of specific sets of interferon-stimulated genes (isgs) [ , ] , and expression of multiple inflammatory genes [ ] , which induce a pro-inflammatory response and attract cells, such as neutrophils and macrophages, to sites of infection. accordingly, we observed significant increase of transcript abundance in many genes involved in isgs ( figure c ), pro-inflammatory cytokines (such as il b, il ) ( figure d ), chemokines (ccl , cxcl , cxcl ) ( figure e ), adhesion molecules (vcam, icam , sell), and other inflammatory molecules (such as mmp- ,) ( figure f ). moreover, immunoglobulin (such as igg b, igg ) ( figure g ), three categories of fc receptors and mannose receptor c (mrc ) ( figure h ), and complement proteins ( figure i ) were also significantly induced in the n-prrsv-infected lungs. however, several complement inhibitors that possess inhibitory and/or decay-accelerating acitivity, such as decay-accelerating factor cd , complement component binding protein, alpha (c bpa), c bpb, were significantly repressed in the n-prrsvinfected lungs ( figure i ). cytotoxic t lymphocytes (ctls) detect cells infected with a virus and destroy them through perforin-mediated apoptosis [ ] . cd + t cells activation require t cell receptors (tcrs) to recognize cognate antigenic peptides for presentation on mhc class i molecules displayed on the surface of antigen presenting cells (apcs) [ ] . accordingly, we observed that transcript abundance of ubiquitin specific peptidase (usp) and ubiquitin enzyme ( figure a ), proteasomes, and aminopeptidases ( figure b ) was significantly increased in n-prrsv-infected lungs. the transcript abundance of b m, mhc class i antigen (sla- ), sla- , tap , and chaperones (such as grp ) was markedly increased after infection with n-prrsv while the transcript abundance of sla-b was significantly decreased ( figure c ). in addition to recognization of cognate peptides presented by mhc class i molecules, cd + t cells activation needs also to receive 'costimulatory' signals and help by helper cd t + cells [ ] . as shown in figure d and e, cathepsins and mhc class ii antigens were significantly induced in n-prrsv-infected lungs. the transcript abundance of costimulatory molecules (such as cd , icos), cams, and tcrs/cd complex as well as co-receptor molecules (such as cd a, table s for full gene names. doi: . /journal.pone. .g cd b) was remarkably increased after infection with n-prrsv ( figure f and g) . activated ctls release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. as shown in figure h , prf and granzyme b (gzmb) transcript abundance was significantly elevated in n-prrsv infected lungs relative to unc lungs. in addition to cytotoxins released from ctls, the transcript abundance of other pro-apoptotic members ( figure i ), such as nfkbia, growth arrest and dna-damage-inducible protein alpha (gadd a), bh interacting domain death agonist (bid), xiap-associated factor (xaf ), cytochrome c (cycs), casp , was also significantly increased after infection with n-prrsv, which can induce apoptosis of virus-infected cells. in addition, we also identified the upregulated expression of cytochrome b heavy chain (gp -phox), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), and the downregulated expression of heme oxygenase (hmox ) during n-prrsv infections, which might result in the oxidative stress response and subsequent oxidative damage of tissues ( figure j ). from the data presented in the paper, a model for the relationship between pulmonary gene expression profiles and infection pathology can be surmised in figure , n-prrsv virus replicates and spreads by subverting host innate immune response and hijacking host lipid metabolism as well as inducing an antiapoptotic and anti-inflammatory state, as indicated by suppression expression of spi ifn, ifn-a, down-regulation expression of proapoptotic genes for bak, apr- , sarp , high levels expression of genes involved in lipid metabolism, such as apoe, ldlb, pik c , anti-apoptotic genes for mcl , bcl a , chfr, adm, nfkb, il , and anti-inflammatory molecule pge as well as cd . infections of n-prrsv viruses resulted in fever and inflammatory response, as indicated by high expression of proinflammatory cytokines and chemokines, adhesion molecules, inflammatory enzymes and receptors, such as il- b, il , sell, icam, ccl , cxcl , cxcl , b m, proteasomes, cathepsins. this was compounded by cell death and elevated expression of nfkbia, xaf , gadd a, perforin, granzymes, and cytochrome c, coupled with increased ros-mediated oxidative stress, as indicated by by up-regulation expression of cytochrome b . taken together, the n-prrsv infections may have resulted in an excessively immune and inflammatory response that contributed to tissue damage. infection of pigs with n-prrsv caused fever, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, depression, anorexia, mild diarrhoea. histopathology examination showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells [ ] (figure ). although great efforts have been made by many researchers, the molecular basis of n-prrsv infection is largely unknown. here we report on the first genome-wide host transcriptional response to n-prrsv infection using solexa/illumina's digital gene expression (dge) system, a tag-based novel high-throughput transcriptome deep sequencing method. given the nature of the methodology of illumina's dge system, we have pooled biological replicates from three pigs for each group to make representative samples for deep sequencing analysis. we could reach a sequencing depth of . - . million tags per library (table ) and found over genes to be differentially expressed during n-prrsv infection processes (table s ) . although others studies have also pooled biological replicates for library construction and deep sequencing [ , ] , resulting in the lack of biological replicate, one may blur the impact of variations in pooling samples. because of the variations of pigs in response to prrsv infection, it is possible that one pig could significantly affect results without independent libraries. but we performed the qpcr validation both on the same pooled material that was used for deep sequencing and on independent rna extractions from each pig, which all confirmed the direction of change detected by dge analysis (figure ). our dge analysis showed massive changes in the transcript abundace of known immune response genes and of genes that have been implicated in prrsv infection [ , , ] . we also identified many interesting genes that had not been linked to prrsv infection in previous studies. for example, transcript abundace of lipid metabolism-related genes including apob r, apoe, pik c, was significantly increased during n-prrsv infection processes. alterations in lipid metabolism, perhaps including those with significant upregulation in this study, have been observed in response to infection by a range of viruses including sars-cov, hcv, influenza a virus, or dengue virus [ , , , ] . although in vitro studies have investigated how prrsv modifying genes expression in pams [ , , ] , many of the outstanding issues will be answered only in the context of prrsv-infected animals [ ] . hence, we characterized the genome-wide transcriptome response to prrsv infection in porcine lung by deeping sequencing. but studies of transcript abundance in lung tissues have also their intrinsic limitations. for example, the transcriptome of lung tissues is actually a merging transcriptional responses of a wide range of cell types, some of which are infected, some of which are responding directly to the infectious process and others of which are bystanders. moreover, increased cellularity of tissues may be confused as biologically important increased transcript abundance. despite such limitations, our dge study offers a broad, system-wide window into molecular processes that regulate gene expression and also provides new leads for functional studies of candidate genes involved in host-virus interaction, as illustrated in this paper. the induction of expression of type i interferons (ifns; including ifn-a and ifn-b) is a well-known innate antiviral immune reaction in the virus-infected cells [ , ] . however, n-prrsv infection suppressed spi ifn gene expression and decreased the transcript abundance of ifn-a ( figure a ). previous studies [ , , ] , both in vitro and in vivo, have also showed that prrsv elicited only a minimal ifn-a production or even suppressed it's expression. the suppression of spi ifn, in particular of ifn-a, is probably a crucial step in the pathogenesis, because ifn-a has been shown to inhibit prrsv replication [ ] . other viruses infection, such as the influenza virus [ ] , hepatitis c virus (hcv) [ ] , ebola virus [ ] , also suppressed type i ifn gene expression which led to extensive viral replication and increased pathogenesis. irf plays an important role for type i ifn gene expression. the transcript abundance of irf was decreased intensively in n-prrsv-infected pigs by h pi ( figure b ). one study [ ] indicated that prrsv nsp b inhibited irf , and then down-regulated ifn-b gene expression. it is worth mentioning that the nsp of the influenza a can also suppress innate immunity by inhibiting irf activation, and subsequently disrupting the induction of a/b-interferon [ ] . research has indicated that the expression of cd , a prrsv receptor [ ] , on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenecity of prrsv [ ] . transcript abundance of cd was significantly increased after n-prrsv infection ( figure a ). the internalization of prrsv via cd in the target cells may induce the expression of il , and in turn induce the expression of cd on neighboring undifferentiated monocytes and increased overall prrsv susceptibility [ ] . moreover, infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an ade [ ] . these antibodies enhance prrsv viral replication by coating the virus and enhancing the internalization of viral particles into macrophages [ ] . as shown in figure g and h, igg and fcc receptors were significantly induced during n-prrsv infection processes. interestingly, the presence of antibodies during feline enteric coronaviruses (fecvs) infection does not also provide sterilizing immunity, instead, these antibodies opsonize virus particles and facilitate their entry to monocytes and/or macrophages through fcc receptors, resulting in disease enhancement [ ] . the activation of pro-inflammatory transcription factor nf-kb induces robust activation of the casp inflammasome and subsequent release of il- b that cause fever and inflammation [ , , , ] . accordingly, we identified upregulation expression of casp , nf-kb, and il- b genes during n-prrsv infection processes ( figure a ). nf-kb activation also enhanced the expression of matrix metalloproteinases (mmp ) and mmp , two cytotoxic substances in prrsv-infected cells [ , ] . similarly, transcript abundance of mmp and ngal ( kda alpha- -microglobulin-related subunit of mmp- ) was significantly increased in the lungs of n-prrsv-infected pigs ( figure f ). upregulation expression of mmps would likely facilitate infiltration of inflammatory cells and increase inflammation. upregulation expression of il (also known as cxcl ), which is an attractant for neutrophils and other polymorphonuclear leukocytes produced after acute infection, in prrsv-infected pams [ , ] and lungs of n-prrsv-infected pigs ( figure d ), was observed. other chemokines such as ccl (also known as mcp ), cxcl , cxcl (also known as ip ), which were significantly increased ( figure d ), may be also crucial for lymphocyte and macrophage infiltration into the sites of n-prrsv infection. ccl , il and ip expression were upregulated during sars-cov [ , ] , and murine coronavirus [ ] infections process, which may recruit monocytes and/or macrophages to sites of infection and be a major cause of lung pathology. although the present study indicates that upregulation expression of pro-inflammatory molecules contributes to the pathogenesis of n-prrsv, increased transcript abundance of anti-inflammatory molecules, such as il , pge , was also detected in the study. upregulation of il gene expression was found previously in prrsv-infected porcine leukocytes, pams, dcs, and in vivo in prrsv infected pigs [ , , , ] . perhaps an increase in pro-inflammatory molecules followed by increased anti-inflammatory molecules is the normal progression of events in inflammation [ ] . the upregulation expression of il might skew the immune response away from a protective th -cell response towards a non-protective th -cell response, therefore impairing clearance of virus, which benefits viral infections [ ] . upregulation expression of anti-inflammatory molecules and proinflammatory molecules occurring concurrently was also observed after sars-cov and fipv infection [ , ] . antibodies might also contribute to immunopathogenesis through increasing the uptake of virus by macrophages, resulting in activation of these macrophages and secretion of pro-inflammatory cytokines and chemokines. antigen-antibody complexes might increase transcript abundance of complement ( figure i ), which leads to generation of the classical inflammatory response through the production of potent proinflammatory molecules [ ] . furthermore, complement activation might also contribute to the development of pulmonary edema and oedema of the eyelids. further understanding the roles complement plays in the hostpathogen interactions may help to develop more effective pharmacological agents against infection. moreover, damage to the lungs of n-prrsv-infected pigs seems to occur directly by viral destruction of alveolar and bronchial epithelial cells and macrophages ( figure c) , as well as indirectly through production of immune mediators. activated ctls and nk cells release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. transcript abundance of pfr and granzymes increased in the lungs of n-prrsv infected pigs ( figure h ). pro-apoptotic molecules xaf , bid, cyto c, casp , aifm , were significantly up-regulated after infection with n-prrsv, which may induce apoptosis of virus-infected cells ( figure i ). simultaneously, we also observed upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including bcl a , mcl , chfr, nfkb, adm, il etc ( figure d ). upregulation expression of anti-apoptotic genes and pro-apoptotic genes occurring concurrently after n-prrsv infection seems in contradiction of each other. however, this may reflect a balance between apoptotic and anti-apoptotic mechanisms. perhaps prrsv actively induces an anti-apoptotic state to complete its virus replication cycle through delaying cell death while induces apoptosis of virus-infected cells after completion of virus replication to increase rate of spread of virus [ , , ] . anti-apoptotic and pro-apoptotic activaties were also observed in prrsv-infected marc- cells, in which prrsv stimulated anti-apoptotic pathways early in infection while caused apoptosis of prrsv-infected cells late in infection [ , ] . infection with n-prrsv also increased transcript abundance of nfkbia ( figure i ), an inhibitor of the tnf receptor activated transcription factor nf-kb. loss of nf-kb activity has been shown to increase the cytotoxic effects of tnf which resulted in increased cell death [ ] . an increase of transcript abundance in proapoptotic genes might result in disruption of the mitochondria transmembrane potential, thereby inducing release of cyto c from mitochondrial membranes to induce apoptosis and secondary necrosis [ ] . the production of ros, especially superoxide radicals, and the subsequent oxidative damage of cells and tissues are recognized as key contributors to the viral pathogenesis [ , ] . ros-mediated oxidative stress might also be involved in inducing apoptosis by prrsv [ ] . accordingly, we identified the remarkable upregulation of cytochrome b heavy chain (gp -phox) ( figure j ), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), after infection with n-prrsv, that generated superoxide radicals that killed both infected and normal cells at sites of infection, which would further exacerbate the immunopathological response. infection of macrophages, monocytes and dcs that are essential for immune function, is likely to be a key component in n-prrsv-induced pathogenesis [ , , , ] . apoptosis of infected cells causes immune suppression by two mechanism: apoptosis either induces a decrease in the numbers of immune cells that compromises both the innate and adaptive immune response in which they are unable to eradicate the primary infection, or impairs immunity by inducing immunosuppressive effects in the surviving cells [ ] . for example, uptake of apoptotic cells by normal macrophages and dcs stimulates immune tolerance by inducing the release of anti-inflammatory cytokines, such as il , and suppressing the release of pro-inflammatory cytokines [ ] . histopathological analysis of the lymphnodes of pigs infection with n-prrsv revealed a profound depletion of immune cells compared with those of unc (data not shown). in summary, the data presented in this study suggest that n-prrsv appears to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. after infection of macrophages and possibly dcs, prr-pamp interactions triggered signaling cascades that increased the transcript abundance of multiple inflammatory molecules, including cytokines, chemokines, adhesion molecules and inflammatory enzymes that induce a proinflammatory response, activate and recruit immune cells, such as macrophages and neutrophils, to sites of infection for virus elimination and thereby produce the clinical symptoms of viral infection, such as fever, dyspnoea, interstitial pneumonia in lungs. further, antibodies and complement activation might exacerbate inflammatory response. n-prrsv might induce an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. figure s signaling pathways of de genes. pathway analysis was mainly based on the kegg database. a p-value of , . and an fdr of , . in the two-side fisher's exact test were selected as the significant criteria. the vertical axis is the pathway category and the horizontal axis is the log (p value) of these significant pathways. found at: doi: . /journal.pone. .s ( . mb tif) figure s genes that distributed in the known pig qtls of health traits. the x axis represents the qtl symbol, and the y axis indicates the number of genes associated with health traits. see table s for full qtl names. figure s biological process go terms of profile . functional classification of the de genes was performed according to go biological processes. a p-value of , . in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi: . /journal.pone. .s ( . mb tif) figure s biological process go terms of profile . functional classification of the de genes was performed according to go biological processes. a p-value of , . in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi: . /journal.pone. .s ( . mb tif) figure s biological process go terms of profile . functional classification of the de genes was performed according to go biological processes. a p-value of , . in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview isolation and identification of porcine reproductory and respiratory syndrome(prrs) virus from aborted fetuses suspected of prrs emergence of porcine reproductive and respiratory syndrome in sweden: detection, response and eradication assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states nidovirales: a new order comprising coronaviridae and arteriviridae rapid differential detection of classical and highly pathogenic north american porcine reproductive and respiratory syndrome virus in china by a duplex real-time rt-pcr construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china highly virulent porcine reproductive and respiratory syndrome virus emerged in china challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology the challenge of prrs immunology porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis advances in swine biomedical model genomics interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus upregulation of il- gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus upregulation of interleukin- gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection genomewide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (sage) gene expression analysis goes digital rna processing and its regulation: global insights into biological networks prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity sequencing technologies -the next generation toward the , dollars human genome protective immunity induced by a recombinant pseudorabies virus expressing the gp of porcine reproductive and respiratory syndrome virus in piglets the significance of digital gene expression profiles cluster analysis of gene expression dynamics optimal gene expression analysis by microarrays gene ontology: tool for the unification of biology. the gene ontology consortium a qtl resource and comparison tool for pigs: pigqtldb mobilizing forces-cd + helper t cells script adaptive immunity cd (+) t lymphocyte mobilization to virus-infected tissue requires cd (+) t-cell help viral evasion and subversion of patternrecognition receptor signalling lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle hepatitis c virus hijacks host lipid metabolism common and divergent immune response signaling pathways discovered in peripheral blood mononuclear cell gene expression patterns in presymptomatic and clinically apparent malaria the caspase- inflammasome: a pilot of innate immune responses recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production heat-shock proteins induce tcell regulation of chronic inflammation post-transcriptional regulons coordinate the initiation and resolution of inflammation sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus cytokine determinants of viral tropism innate immune modulation by rna viruses: emerging insights from functional genomics perforin-mediated target-cell death and immune homeostasis a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus deep sequencing of the zebrafish transcriptome response to mycobacterium infection a microrna catalog of the developing chicken embryo identified by a deep sequencing approach lipid raft disruption by cholesterol depletion enhances influenza a virus budding from mdck cells jnk phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol immunopathogenesis of coronavirus infections: implications for sars interferon-inducible antiviral effectors interferons and viral infections in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity aberrant innate immune response in lethal infection of macaques with the influenza virus interferon regulatory factor- activation, hepatic interferon-stimulated gene expression, and immune cell infiltration in hepatitis c virus patients global suppression of the host antiviral response by ebola-and marburgviruses: increased antagonism of the type i interferon response is associated with enhanced virulence porcine reproductive and respiratory syndrome virus non structural protein {beta} modulates host innate immune response by antagonizing irf activation inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses modulation of cd receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages blocking il- in systemic inflammation infection, fever, and exogenous and endogenous pyrogens: some concepts have changed caterpillers, pyrin and hereditary immunological disorders inflammasome adaptors and sensors: intracellular regulators of infection and inflammation increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated swine alveolar macrophages severe acute respiratory syndrome: clinical outcome and prognostic correlates sars coronavirus and innate immunity the t cell chemoattractant ifn-inducible protein is essential in host defense against viral-induced neurologic disease porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin- production host gene expression profiling in pathogen-host interactions altered p mitogen-activated protein kinase expression in different leukocytes with increment of immunosuppressive mediators in patients with severe acute respiratory syndrome in vivo cytokine response to experimental feline infectious peritonitis virus infection complement and its role in innate and adaptive immune responses induced apoptosis supports spread of adenovirus vectors in tumors viral appropriation of apoptotic and nf-kappab signaling pathways porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the pandemic virus mitochondria and apoptosis pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals ifn-alpha treatment enhances porcine arterivirus infection of monocytes via upregulation of the porcine arterivirus receptor sialoadhesin differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability apoptosis and caspases regulate death and inflammation in sepsis death-defying immunity: do apoptotic cells influence antigen processing and presentation? we thank the beijing genomics institute (bgi) shenzhen and genminix informatics ltd.,co for their providing us with technical assistance in dge and bioinformatics analysis. key: cord- -w ukokl authors: tian, xinsheng; lu, guangwen; gao, feng; peng, hao; feng, youjun; ma, guangpeng; bartlam, mark; tian, kegong; yan, jinghua; hilgenfeld, rolf; gao, george f. title: structure and cleavage specificity of the chymotrypsin-like serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: journal of molecular biology doi: . /j.jmb. . . sha: doc_id: cord_uid: w ukokl summary biogenesis and replication of the porcine reproductive and respiratory syndrome virus (prrsv) include the crucial step of replicative polyprotein processing by self-encoded proteases. whole genome bioinformatics analysis suggests that nonstructural protein (nsp ) is a c-like serine protease ( clsp), responsible for most of the nonstructural protein processing. the gene encoding this protease was cloned and expressed in escherichia coli in order to confirm this prediction. the purified protein was crystallized, and the structure was solved at . Å resolution. in addition, the crystal structure of the ser ala mutant was determined at . Å resolution. the monomeric enzyme folds into three domains, similar to that of the homologous protease of equine arteritis virus, which, like prrsv, is a member of the family arteriviridae in the order of nidovirales. the active site of the prrsv clsp is located between domains i and ii and harbors a canonical catalytic triad comprising ser , his , and asp . the structure also shows an atypical oxyanion hole and a partially collapsed s specificity pocket. the proteolytic activity of the purified protein was assessed in vitro. three sites joining nonstructural protein domains in the prrsv replicative polyprotein are confirmed to be processed by the enzyme. two of them, the nsp /nsp and nsp /nsp junctions, are shown to be cleaved in trans, while cis cleavage is demonstrated for the nsp /nsp linker. thus, we provide structural evidence as well as enzymatic proof of the nsp protein being a functional clsp. we also show that the enzyme has a strong preference for glutamic acid at the p position of the substrate. porcine reproductive and respiratory syndrome virus (prrsv) was first discovered in north america in , and almost simultaneously (in ) in europe. it manifests itself clinically with severe reproductive failure in sows and respiratory distress in neonatal pigs. in recent years, the virus has become endemic throughout the world, leading to significant economic losses in pig production. in the autumn of , unparalleled outbreaks of atypical prrs occurred in more than provinces (or autonomous cities) of china, affecting more than million pigs with nearly , fatal cases. adult and growing pigs were also affected, which was different from classical prrs. in , an almost identical atypical prrsv was found to be epidemic in vietnam and china. presently, only inactivated or attenuated vaccines are available to combat prrs, but their efficacy is less than satisfactory due to adaptive evolution of prrsv, , especially in the cases of the atypical prrs outbreaks in china and vietnam. , this urgent issue prompted us to exploit the possibility of drug discovery for treatment of the highly pathogenic prrsv infection. like other rna viruses in the order nidovirales, such as equine arteritis virus (eav), human coronavirus e, and severe acute respiratory syndrome coronavirus (sars-cov), prrsv has a singlestranded positive-sense rna genome that is about . kb in size, containing a large ′-terminal replicase gene and a downstream set of structural protein genes. the replicase gene is translated into two large polyprotein precursors, a and ab (with molecular masses of about . and . kda for pp a and pp ab, respectively), with the latter being synthesized following a ribosomal frame-shifting event. after the rapid autocatalytic release of nonstructural protein (nsp ) and nsp , the remainder of the polyproteins are cleaved into mature nonstructural proteins (nsp -nsp ) by the c-like serine protease ( clsp), which is also often simply called c-like proteinase ( cl pro ). , the functional importance of the cl proteinase in the viral life cycle makes it an attractive target for the development of drugs directed against prrsv. here, we confirm that prrsv nsp , which we demonstrate to extend from residue g to e in the viral polyprotein of the virus strain jxa , is a clsp from both the structural and enzymatic points of view. the structure of the enzyme reveals two chymotrypsin-like β-barrel domains and an extra c-terminal α/β domain similar to that observed in eav nsp . a canonical catalytic triad that is composed of ser , his , and asp is located in the open cleft between the two β-barrel domains. an s subsite consisting of his and very likely ser and an oxyanion hole without the signature helix-like turn are seen in our structure. our results also clearly show that the recombinant enzyme displays proteolytic activity in vitro. besides, based on the results of the cleavage assay, three nsp junction sites in the prrsv replicase precursor are confirmed, thereby shedding light on the processing pathway of pp a and pp ab in the virus. overall description of the recombinant prrsv clsp used in this study as a member of the arteriviridae, prrsv shows substantial similarities in genome organization to eav-the prototype virus of this family. [ ] [ ] [ ] accordingly, whole genome bioinformatics analysis predicts that the prrsv nsp domain should be the main protease responsible for processing most of polyproteins pp a and pp ab. previously, we reported on the successful expression of a gene construct encoding this putative serine protease in escherichia coli as well as the crystallization of the protein. as shown in fig. a , after removal of the glutathione s-transferase (gst) tag, nine additional amino acid residues were still left at the n-terminus of the protein. the in vitro peptide cleavage assay in this study was performed using this recombinant enzyme with the nine extra residues present, while for the cis cleavage assay, even more additional nterminal residues were present (fig. b) . in both cases, the recombinant protein displayed proteolytic activity in vitro, thereby confirming the identity of nsp as the main protease in prrsv replication (see results below). structure determination and quality of the refined structure although the sequence identity between prrsv and eav nsp s is more than %, we failed to solve the structure of prrsv clsp by molecular replacement (mr) using the structure of eav nsp as a search model. the presence of two methionine residues in the recombinant prrsv clsp suggested that selenomethionine (semet)-based multiple-wavelength anomalous dispersion could be used to solve the phase problem. the unit-cell dimensions of the crystals (a = . Å, b = . Å, c = . Å, β = . °; space group c ) indicate that there is only one molecule per asymmetric unit, and we successfully located both of the two crystallographically independent selenium sites. based on the obtained phases, a final model with good stereochemistry was built (for details, see materials and methods). as shown in table , the structure was refined to . Å resolution, with r c r y s t = . % and r free = . %; . % of the residues lie in the most favored regions of the ramachandran plot and . % lie in the additionally allowed regions. the quality of the electron density map of the recombinant protease at . Å is excellent, with an overall temperature factor of . Å , and allowed us to model the positions of the main chain, most sidechain atoms, and solvent molecules unambiguously. at the polypeptide chain termini, electron density is visible starting from residue and ending at residue , but loop - is not seen in the maps due to disorder. the overall structure of prrsv clsp and comparison with eav nsp the prrsv clsp is folded into three domains, including two antiparallel β-barrels and an extra cterminal α/β domain ( fig. a and b) . the nterminal β-barrel (domain i) consists of six β-strands (ai to fi) and a short α-helix (helix a) that closes the barrel like a lid, whereas the middle β-barrel (domain ii) comprises seven β-strands (aii to gii) and is connected to domain i via a very long loop (amino acids - ) (fig. a) . the catalytic site is located at the opening of the cleft between domains i and ii and consists of residues ser , his , and asp (fig. a) , which are totally conserved among members of the arteriviridae (fig. c) . the extra cterminal domain (domain iii) comprises residues - and consists of two pairs of short antiparallel β-sheets (strands aiii-ciii and biii-diii) and two α-helices. the crossing angle is ∼ °between helix b and helix c (fig. a) . a long n-terminal loop extends over domain ii to the vicinity of domain iii. the orientation of this loop is stabilized by the three hydrogen bonds arg n…glu oe , thr n… val o, and thr og …val n (fig. a) . the phenyl ring of phe is directed into the hydrophobic interface between domains ii and iii and, therefore, also helps to stabilize the orientation of the nterminal loop (fig. a, left image) . (a) overview of the proteolytic processing of prrsv replicase. the rapid autocatalytic release of nsp α, nsp β, and nsp after translation of the orf a or orf ab is indicated. the rest of the junction sites are processed by nsp . all the predicted cleavage sites are listed above the schematic boxes representing nsp to nsp . three of the putative cleavage sites were confirmed by our work. the rest of them were not evidenced to be processed in our study and therefore are labeled with a question mark on the top. the numerals indicated in each cleavage site are based on the genomic information of prrsv strain jxa (genbank accession no. ef ). the recombinant protein for crystallization trials and in vitro peptide cleavage assay was produced using the gst fusion expression system (ge healthcare). nine extra n-terminal amino acid residues remained after removal of the gst tag. (b) the translational fusion proteins used in the cis cleavage assay. the red-colored portion indicates the additional residues introduced by the pet- b vector. the eight n-terminal amino acid residues of nsp are colored pink and the myc epitope is highlighted in blue. a dali search of the protein data bank (pdb) database gave a variety of proteins with significant structural similarities to our structure, among which the eav nsp expectedly had the highest z score ( . ). as the prototype structure of nsp s in arteriviridae, the eav clsp also folds into three domains. superposing domains i and ii of prrsv clsp onto those of the eav nsp (copy a of the four copies in the asymmetric unit) yielded an r.m.s.d. of . Å for equivalent (out of compared) c α pairs (fig. a) , while domain iii alone exhibited an r. m.s.d. of . Å for (out of ) c α pairs (fig. d) , indicating that the two enzymes share great structural similarity at the tertiary level. however, the steric arrangement of the domains in the two proteases is quite different. an ∼ Å shift of domain iii relative to domains i and ii was observed in our structure, when compared to eav nsp . this shift causes the whole domain iii to move away from the cleft where the catalytic triad is located (fig. a) . clear electron density for the loop asn -ile that connects domains ii and iii, as shown in fig. c , leaves no doubt about the chain tracing of the loop and verifies the -Å shift of domain iii, compared to eav nsp . the shift may also account for our failure to solve the structure by mr using eav nsp as the search model. further discrepancies were also observed in two loop regions. one is the loop connecting strands cii and dii (cys -pro in prrsv nsp ). in eav nsp , this loop is much like a tadpole with its head lying next to the catalytic nucleophile ser (fig. b ). however, the corresponding loop in our structure is approximately "⨼" shaped with a protuberance pointing to domain iii ( fig. a and b ) on which the hydrophobic side chain of phe resides. this residue is conserved in prrsv and lactate dehydrogenase-elevating virus clsp but not in eav nsp , where it is substituted by trp (fig. c) . the phenyl ring of phe protrudes into the hydrophobic interface between domains ii and iii (fig. a , left image) and thereby causes the "⨼"shaped conformation of the cii-dii loop. another loop of great variance is the one connecting strands eii and fii. in prrsv clsp, this loop is eight residues in length, from gly to gly , and is highly flexible as proved by the total lack of electron density for residues - . however, in eav nsp , the corresponding region is mainly a βstrand with a much shorter loop, indicating that the conformation of this part is relatively rigid (fig. b) . interestingly these two regions showing significant variance are actually believed to cover, respectively, the s specificity pocket and the oxyanion hole in eav nsp , two highly important features involved in substrate recognition and catalysis by cl proteases. the s specificity pocket in cl proteases, the s specificity pocket accommodates the side chain of the substrate p residue and therefore determines the enzyme's where i i is the observed intensity and 〈i〉 is the average intensity from multiple measurements. where f o and f c are the structure-factor amplitudes from the data and the model, respectively. r free is the r-factor for a subset ( %) of reflections that was selected prior to refinement calculations and was not included in the refinement. d ramachandran plots were generated by using the program procheck. the catalytic triad is indicated by red arrows, and the three residues that are believed to constitute the s specificity pocket in eav nsp are marked with blue stars. the phe residue, which is thought to lead to the atypical oxyanion hole in prrsv clsp, is labeled by a dark triangle. cys and cys , which have been referred to in the text, are marked with black squares. ldvc and ldvp, lactate dehydrogenase-elevating virus neurovirulent type c and strain plagemann, respectively; prrsvas and prrsves, prrsv american strain and european strain, respectively. preference for specific amino acid residues at this position. to identify the possible residues that may be involved in the formation of the s pocket in prrsv clsp, we used the structure of streptomyces griseus proteinase e (sgpe) in complex with a tetrapeptide as the reference structure for superposition. sgpe is another chymotrypsin-like proteinase that has a substrate preference for glu at the p position. both the eav nsp (copy a) and our structure were aligned. as shown in fig. a , the s specificity pocket of both the sgpe and eav nsp is composed of three residues, with a conserved his (his in eav nsp and his in sgpe) lying at the bottom of the pocket and one ser (eav nsp : ser ; sgpe: ser ) lining one side and another thr/ser (eav nsp : thr ; sgpe: ser ) located on the opposite side. however, only the histidine (his ) could be found in our structure. the first serine involved in the s subsite is conserved in prrsv clsp (ser ) (fig. c ), but we cannot define its position due to the absence of electron density for the loop - as mentioned before. nevertheless, there is no residue in prrsv nsp to match the thr/ser (thr in eav nsp , ser in sgpe) on the other side of the pocket. thr of prrsv nsp , which is also highly conserved among members of arteriviridae (fig. c) , is too far away to contact the substrate p residue (fig. a ). an atypical oxyanion hole without the signature helix-like turn both eav nsp and sgpe form a canonical oxyanion hole that is characterized by a helix-like turn preceding the nucleophilic serine. as shown in fig. b , the backbone amides of ser and gly in sgpe can form hydrogen bonds with and therefore stabilize the carbonyl oxyanion in the tetrahedral intermediate during catalysis. although, in eav nsp , only copy b has the normal active oxyanion hole geometry while the other three copies only form collapsed ones, this difference is mainly caused by flipping of the peptide bond between ser and gly rather than a big change of the orientation of the signature turn. however, in prrsv nsp , this oxyanion-specific turn is missing. instead, a much more stretched loop leaves only the amide group of the catalytic nucleophile (ser ) in contact with the oxyanion. we term this rare property an atypical oxyanion hole. compared to that of the eav nsp , an even stronger hydrophobic interaction between domains ii and iii is observed in our structure. with phe , val , phe , phe , phe , ile , leu , phe , phe , ile , l , and l , the interface harbors hydrophobic residues, whereas in eav nsp , this interface only includes eight such residues (fig. a ). this hydrophobic core holds domains ii and iii together and likely forces the loop connecting the two domains to adopt its observed orientation, leading to the -Å shift as described above. another unexpected feature of the extra domain is the presence of two patches of solvent-exposed hydrophobic residues, implying possible interactions of prrsv nsp with other proteins. one patch, which is also present in eav nsp (fig. b , right image), consists of leu , ile , and ile (fig. b , left image). another patch, comprising val , val , ala , ala , and ala , is even more extended and locates on a surface that is almost perpendicular to the former one (fig. c , left image, and b, left image). it is noteworthy that this second hydrophobic patch is totally absent in eav nsp (fig. c, right image) . peptides corresponding to eight predicted junctions in prrsv pp a or pp ab ( fig. a ; table ) were synthesized, tested for trans cleavage, and further analyzed by high-performance liquid chromatography (hplc) in order to see whether the recombinant nsp is proteolytically active in vitro. in our assay, we showed that peptides corresponding to two junction sites can be cleaved by the enzyme. for each predicted cleavage site, the scissile peptide bond was between glu and gly. just like other reports on peptide-based cleavage assays, , our enzyme also shows a remarkable difference in efficiency towards different peptides. of the nine potential substrates investigated, only three can be cleaved to the extent of being detectable on an hplc system (see table ). peptide sp - , derived from the junction sequence between nsp and , is most susceptible to this enzyme. after incubation with the recombinant enzyme, two new peaks with retention times of . and . min, respectively, were observed after analyzing a reaction volume of μl on a c column (fig. b) . the uncleaved peptide gave a peak at . min, consistent with that for the peptide-alone control (fig. a) . the two product peaks were collected and analyzed by mass spectrometry (ms). the results showed that the calculated molecular masses corresponding to the peaks were . and . , respectively, matching their theoretical monoisotopic molecular weight (mw) perfectly (the theoretical mw for fragment kdktayfqle is . , and for grhftw, it is . ). a typical representation of the ms result is shown in fig. f . when the catalytic serine residue was replaced by alanine (ser ala mutant), no detectable peaks of cleaved peptides were observed in the hplc profile (fig. c) . this confirmed the crucial function of ser as the nucleophile in catalysis. another peptide that was shown to be processed is sp , which corresponds to the nsp /nsp junction. however, the proteolytic efficiency for sp was much lower than that for sp - . only a small portion (b %) of the peptide was cleaved after a -h incubation (data not shown). the kinetic parameters of the protein were determined in vitro using a fluorescent assay system to quantitatively test the proteolytic activity of the recombinant enzyme. the results showed that the enzyme exhibits a k m of about . mm and a k cat of about . min − for the substrate dabsyl-ktayfqle↓grhfe-edans. the glutamic acid at the p position of the peptide substrate is crucial for effective cleavage and cannot be replaced by glutamine c-like proteases of rna viruses are specific for either gln or glu at the p position of the substrate. among the nidoviruses, coronavirus cl pro cleavage sites always have a gln at the p position. , , however, in the same order nidovirales, the clsps of arteriviridae, which are represented by the prototype eav nsp , show a clear preference for glutamic acid over glutamine at the p position of the substrate. in eav, of the eight cleavage sites of the replicative polyprotein processed by the main protease, only one junction contains a glutamine at the p position (q /s joining nsp / ), while all other cleavage sites have glutamic acid in this position. , , based on the similarity in genome organization between prrsv and eav, the putative cleavage sites processed by clsp were predicted in prrsv, all of which harbor a glu at the p position. therefore, it is a reasonable assumption that the clsp of prrsv also shows preference for glu over gln at the p position of the substrate. the peptide sp -m was synthesized and tested for cleavage by clsp to confirm this prediction. sp -m is identical in sequence to sp - except that the glu in p was replaced by gln. as shown in fig. d , the retention time for this peptide is . min. as expected, after incubation of the peptide with clsp for h at °c, only tiny amounts of cleavage products can be detected (fig. e) , indicating that the glutamic acid at the p position in the substrate is crucial for effective cleavage by clsp and cannot be replaced by glutamine. the nsp /nsp junction site is processed by clsp via cis cleavage whereas the linker between nsp and nsp was shown to be processed in vitro, the sp peptide that harbors the junction of nsp and nsp was not observed to be cleaved in trans in our in vitro assay (table ). however, processing of the nsp /nsp junction is crucial for the release of the enzyme to free itself from the polyprotein precursor. therefore, a cis cleavage assay was designed in order to investigate processing of this site. as shown in fig. b , two constructs bearing the nsp /nsp junction fused to a myc epitope at the c-terminus of wildtype clsp or its s a mutant were made. extra n-terminal and c-terminal hexa-histidine tags were introduced into the fusion proteins by the pet- b vector to facilitate detection, resulting in the wildtype protein his-nsp -[nsp /nsp ]-myc-his and the mutant product his-nsp (s a)-[nsp /nsp ]-mychis. after expression of the two gene constructs in e. coli, an apparent molecular mass difference between the two protein products was observed in sds-page, where the mass of the wild-type protein is smaller than that of the mutant by about kda, indicating the occurrence of cis cleavage at the cterminal junction site of his-nsp -[nsp /nsp ]-mychis (fig. a) . both the wild-type and the mutant proteins were purified and subjected to western blot analysis for detection of the myc epitope as well as the hexa-histidine tag to confirm this conclusion. two different amounts ( and μg) of both proteins were loaded. at both amounts, the wild-type protein showed no signal for the myc epitope, in contrast to a strong myc signal given by the mutant protein (fig. c) . both proteins displayed an unambiguous his-tag signal, which was much stronger in the case of the mutant product, consistent with the fact that it contains a hexa-his tag at both termini while the wild-type protein only has the n-terminal tag left (fig. b) . these results clearly showed that his-nsp -[nsp /nsp ]-myc-his removed its c-terminal tail via cis cleavage after expression. the purified wild-type protein was sent to the national center of biochemical analysis, academy synthetic -mer peptides that represent the eight predicted junction sequences in the replicative polyprotein were incubated with μm recombinant prrsv nsp as described in materials and methods and analyzed by hplc for the extent of cleavage. after incubation at °c for h, if more than % of the peptide was found to be cleaved as is determined by the decrease in area of the peak representing the parental peptide, "+++" is given; if - % of the parental peptide is cleaved, we perceive it as "+"; if less than % is cleaved but still detectable amounts of products are observed as marked by the appearance of new peaks in the hplc profile, we mark it "+/−". "−" means no visible product peaks in the hplc profile could be observed at all. all the cleaved products were verified by ms and/or comparison with hplc traces of peptide standards. sp - and sp correspond to the same junction site in pp b. sp - was synthesized and analyzed for the purpose of separating the two product peaks from each other. of military medical sciences, for c-terminal sequencing to further ascertain the exact peptide bond cut by the enzyme. after digestion of the protein using trypsin, a peptide fragment with observed mass of . was found and selected for further sequencing. as shown in fig. d , the amino acid sequence determined for this fragment is % identical with that of the c-terminal portion of the wild-type protein after cleavage at the e /g junction, which, in turn, confirms this site to be the nsp / nsp junction in pp a or pp ab. the structure of the s a mutant of clsp the . -Å structure of the s a mutant protein was solved by mr using the wild-type structure as the search model and refined to r cryst = . % and r free = . (see table ). the overall structure of the mutant protein is almost identical with that of the native clsp, with an r.m.s.d. of only . Å. the structure contains residues - but, like the wildtype structure, lacks electron density for the loop - . as shown in fig. a , there is clear electron density for the catalytic center, demonstrating that substitution of ser by ala was successful. at the same time, the substitution did not change the microenvironment surrounding residue , as seen from the unchanged conformations of the (truncated) catalytic triad, the s subsite residue his , and the oxyanion hole (see fig. b ). the consistent conformations of these parts between the native and the mutant proteins make the mutant structure an appropriate model for enzyme-substrate interaction studies. prrsv nsp has been predicted and accepted as the main proteinase, with a pivotal role in the viral life cycle, but experimental proof had been lacking. our work, for the first time, reports the crystal structure and in vitro proteolytic activity of this enzyme. the s specificity pocket that accommodates the side chain of the substrate's p residue plays a vital role in substrate recognition and catalysis. in all the reported cl pro s with known structures, a conserved histidine is found sitting at the bottom of the pocket. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] mutational analysis found that any replacement of this residue both in human and feline coronavirus m pro completely abolished their proteolytic activities. , as a member of the c-like protease family, it is not surprising that in prrsv nsp , his is localized appropriately to fulfill this function. however, besides this conserved his, the s subsite of both sgpe and eav nsp includes two additional ser/thr residues as shown in fig. a , , for which no counterparts can be found in our structure. we suspect that ser must also be involved in p side-chain recognition based on two facts. first, this ser is absolutely conserved in arterivirus nsp sequences. in eav nsp , the same ser has been proposed to contact the side chain of the p glutamic acid (fig. a) . furthermore, although we cannot define the position of ser due to total lack of electron density, the two residues directly preceding it are clearly defined. therefore, ser is confined to a limited steric space close to the modeled p residue. a hydrogen bond between this disordered residue and the p amino acid could help stabilize both residues upon substrate binding. we also note that loop - lines one wall of the s subsite and is highly flexible. it may function like a lid that can partially cover the pocket after the entry of the p side chain and therefore help clinch the substrate to allow the occurrence of nucleophilic attack onto the scissile bond. this may also, to some extent, compensate the lack of a third partner in the s specificity pocket of prrsv nsp (see results). snijder et al. reported that mutation of this third residue, thr in eav nsp , to other amino acids can have various effects on catalysis, indicating its important role as part of the s subsite. it is noteworthy that our enzyme also shows weak proteolytic activity against sp -m with a gln at the p position ( fig. e; table ). this might reflect the flexibility of the substrate binding of the enzyme that can also accommodate gln in addition to glu. the catalytic triad is shown as sticks. his is also shown because of its crucial role in substrate recognition. the missing loop and the oxyanion hole region are also highlighted. the consistent conformations of these parts between the native and the mutant proteins make the mutant structure an appropriate model for enzyme-substrate interaction studies. recognition of both residues has been reported in other c proteinases, for example, c from members of the picornaviridae. , taking the similarity of the side chains of glu and gln into account, this might not be surprising. furthermore, the flexibility of ser might also be attributed to this partial compatibility. we also report a rare form of oxyanion hole in our structure, which is totally lacking the signature helixlike turn. in serine proteinases, the catalytic cleavage of the substrate peptide bond is assumed to undergo a transformation from the ground planar state to a transition state that is characterized by the formation of a tetrahedral intermediate. the backbone nh groups (in the case of subtilisin also including the side chain of asn ) can hydrogen-bond with the resultant carbonyl oxyanion of the substrate p residue during cleavage at the scissile bond and are therefore very important in catalysis. [ ] [ ] [ ] [ ] both in eav nsp and sars-cov m pro , two forms of the oxyanion hole were observed, an active form and a collapsed one. , nevertheless, in both forms, the signature turn remains almost the same. in eav nsp , the different forms of the oxyanion hole are believed to arise mainly from the flipping of the peptide bond between ser and gly , which is common in pairs of homologous protein structures. , however, as shown in fig. b , the stretched form observed in our structure (lacking the helix-like turn) can only support the formation of one hydrogen bond with the oxyanion to stabilize the negative charge. we suspect that the hydrophobic strength of phe might be the main cause for this atypical oxyanion hole. this is supported by our recent mutational work of phe ala. the mutant enzyme shows some higher enzymatic activity, implying the role that phe played in the formation of the atypical oxyanion hole, which might be attributed to the observed low enzymatic activity (data not shown). this needs to be verified by a clear crystal structure and further mutational work in the future. the unusual properties of both the s subsite and the oxyanion hole observed in our structure, as we discussed above, might well explain the low in vitro catalytic activity of prrsv nsp compared to that of other c-like proteases in similar experiments. , another interesting point is that in the neighborhood of the substrate-binding site, cys is located in close proximity to cys but has its side chain directed away from that of the latter, so that a disulfide bond cannot be formed. disulfide bonds are rare in proteins that function in the cytosol, but for instance in coronavirus nonstructural proteins, several cases have been observed. [ ] [ ] [ ] we presume that the side chain of cys can adopt a different conformation and form a disulfide bond with cys , perhaps after substrate binding to facilitate the proteolysis. this should be addressed in the future with a substrate-enzyme complex structure. with great efforts, we failed to obtain the complex crystals so far. as the main proteinase in arteriviridae, nsp must first release itself from the replicative polyprotein to fulfill its proteolytic function of processing the downstream domains into functional replicative subunits. unlike eav, whose nsp /nsp cleavage site is reported to be cleaved in the major processing pathway with the presence of nsp as the cofactor, we have shown in our cis cleavage assay that a quick and thorough cleavage at the c-terminus of prrsv nsp occurs automatically in vitro. therefore, based on the results obtained in this study, we propose a possible nsp release model for prrsv: translation of orf a is followed by a rapid autocatalytic release of nsp α, nsp β, and nsp ; , subsequently, the nsp /nsp junction site is quickly processed via cis cleavage by the clsp to yield nsp -nsp and nsp -nsp . then, a trans cleavage of the nsp /nsp joining sequence occurs to generate nsp and free nsp , which can then fulfill its role as the main proteinase. nevertheless, taking into account that the trans processing of the nsp /nsp junction is very slow as shown by the peptide cleavage assay (see results), we believe that cis cleavage at this site might also happen, especially in the early stage of translation of orf a. in support of this, a very long n-terminal loop was observed in our structure (fig. a) . this terminus can be brought into an appropriate position for cis cleavage by a conformational rearrangement upon breaking of the three hydrogen bonds mentioned above. refinement of this cleavage model will require further studies in the future. for coronaviral cl pro s such as in sars-cov, there is plenty of evidence showing that the nterminus of the protease is crucial for its proteolytic activity. either addition of extra residues to the nterminus or deletion of the n-terminal residues is deleterious to enzyme activity. [ ] [ ] [ ] there are also many reports showing that the homodimeric form of the sars-cov cl pro is the catalytically active form and the unique c-terminal extra domain plays a key role in controlling the dimer-monomer equilibrium. [ ] [ ] [ ] [ ] in our previous report, we showed that the recombinant prrsv nsp with nine extra amino acid residues at the n-terminus is monomeric in solution. these extra residues might also affect the enzymatic activity of prrsv nsp and might be attributed to the high k m observed for the enzyme. nevertheless, we confirm here that the recombinant enzyme displays obvious catalytic activity in vitro. furthermore, the enzyme is actually active in vitro even with extra n-terminal residues added as in the cis cleavage assay. thus, the c-like proteases of nidovirales likely evolved different proteolytic mechanisms for coronaviridae such as sars-cov and arteriviridae such as prrsv. these aspects merit further study in the future. the construction of the recombinant plasmid encoding wild-type clsp has been described previously. briefly, the coding sequence of the prrsv nsp was amplified by reverse transcription-pcr reaction from the genomic rna extracts of prrsv isolate (genbank accession no. ef ) and then inserted into smai and xhoi sites of pgex- p- to yield plasmid pgex- cl. the s a (ser substituted by ala) mutant construct was achieved by overlapping extension pcr. using pgex- cl as template, with primer combinations of cl-f / clm-r or clm-f / cl-r , two segments were obtained first and then they were mixed together at equal molar ratio as new templates in a second round of pcr by primer pairs cl-f / cl-r . the resultant fragment that carries the s a substitution was inserted into pgex- p- to generate pgex- clm-s a. with primer pairs cl-ea-f / cl-ea-r , using either pgex- cl or pgex- clm-s a as template, dna fragments encoding translational fusion proteins his-nsp -[nsp /nsp ]-myc-his and his-nsp (s a)-[nsp / nsp ]-myc-his were amplified respectively. the fragments were introduced into the pet- b plasmid to create pet- cl-ea and pet- cl-s a-ea (fig. b) . all the constructs were verified by direct dna sequencing, and the sequences of the primers used in this study are listed in table . the detailed production and purification procedure for the wild-type clsp have been described previously. for the mutant enzyme, the same method was adopted. in brief, μl of the plasmid was transformed into e. coli bl (de ) competent cells for protein expression. the resultant gst fusion protein was purified by glutathione-sepharose chromatography, cleaved with prescission protease, and the recombinant protein was further purified by sizeexclusion chromatography. semet-labeled clsp was produced in bl (de ) e. coli grown in semet minimal medium ( . % ynb, % glucose, and mm mgso in m medium) supplemented with l-semet at mg l − ; lysine, threonine, and phenylalanine at mg l − ; and leucine, isoleucine, and valine at mg l − . the expressed protein was purified as described above. the purified proteins were concentrated, and their concentration was determined using bca™ protein assay kit (pierce) according to the manufacturer's instructions. crystallization conditions were optimized, and the best crystals for both the wild-type and semet-labeled clsp were obtained by vapor diffusion in hanging drops consisting of μl of reservoir solution ( . m sodium citrate, ph . , and . m monobasic ammonium phosphate) and μl of concentrated protein solution ( mg ml − in mm tris-hcl and mm nacl, ph . ), followed by incubation at °c for days. for the s a mutant protein, the crystals were obtained under conditions similar to those for the wildtype clsp, except that the ph value was elevated to . and the concentration of the mutant protein was mg/ ml. crystals appeared after days at °c. crystals were flash-cooled in liquid nitrogen in cryoprotectant solution containing % glycerol and % reservoir solution. multiwavelength x-ray diffraction data to . Å were collected from crystals of semetlabeled protein on beamline w b of the beijing synchrotron radiation facility and processed with the hkl suite of programs. two selenium sites were located from data collected at peak and remote wavelengths using solve. an initial model was built using resolve followed by model refinement using refmac (ccp suite). x-ray diffraction data collected from crystals of the native protein on an in-house rigaku micromax rotating-anode x-ray generator were phased using mr procedures in molrep as implemented in ccp version . . the initially determined structure was used as a search model. a series of iterative cycles of manual rebuilding were carried out in coot and refinement with refmac (ccp suite). the s a mutant structure was solved by mr method using the wild-type structure as the search model and refined to . Å. the final statistics are summarized in table . during the course of model building and refinement, the stereochemistry of the structures was checked by procheck. all figures were generated using pymol and espript. in vitro peptide trans cleavage assay the -mer substrate peptides (representing eight putative cleavage sites in prrsv pp a or pp ab) for the in vitro cl protease peptide cleavage assay were purchased from beijing scilight biotechnology ltd. co. each peptide was dissolved in % dimethyl sulfoxide at a concentration of mm and stored at − °c. the nsp activity assay was performed in a reaction volume of μl, using μl nsp (or s a mutant nsp ) ( mg/ml) and μl peptide ( mm) in the reaction buffer ( mm tris, ph . , mm nacl, mm ethylenediaminetetraacetic acid, mm dtt, and % glycerol) to yield a final concentration of μm enzyme and μm peptide in the assay. cleavage reactions were routinely incubated at °c for h. the reactions were terminated by the addition of an equal volume of % trifluoroacetic acid, and samples were frozen in liquid nitrogen immediately before being stored at − °c. the samples were centrifuged for min at , rpm before analysis by reverse-phase hplc on a c column ( . mm × mm). cleavage products were resolved by using a -min, - % linear gradient of acetonitrile in . % trifluoroacetic acid. the absorbance was determined at nm, and peak areas were calculated by integration. the identities of the product peptides were verified by quadrupole fourier transform-ms analysis and/or comparison with hplc traces of peptide standards. a fluorescence-based assay with the fluorogenic peptide substrate dabsyl-ktayfqle↓grhfe-edans ( % purity, biosyntan gmbh, berlin, germany) was used to assess the activity of the prrsv clsp. this peptide contains the nsp /nsp cleavage site (indicated by the arrow). the enhanced fluorescence due to the cleavage of this substrate as catalyzed by the enzyme was monitored at nm with excitation at nm, using a cary eclipse fluorescence spectrophotometer. the experiments were performed in a buffer consisting of mm tris-hcl (ph . ), mm nacl, mm ethylenediaminetetraacetic acid, and mm dtt. kinetic parameters k m and k cat were determined by initial-rate measurements at °c. the reaction was initiated by adding proteinase (final concentration, . μm) to a solution containing different final concentrations of the fluorogenic peptide ( - μm). the initial rates were converted to enzyme activities (micromole substrate cleaved per second). kinetic constants were derived by fitting the data to the michaelis-menten equation with the nonlinear regression analysis program sigma plot. to investigate the cis cleavage activity of clsp, we designed two constructs bearing the nsp /nsp scissile bond fused to a myc epitope at the c-terminus of clsp or clsp (s a), resulting in the expression vectors pet- cl-ea and pet- cl-s a-ea. the n-terminal and c-terminal hexa-his tags were introduced into the recombinant proteins via vector pet- b (fig. b) . the recombinant proteins were expressed and purified by using a ni-nta affinity chromatography column and a superdex hiload / column (ge healthcare). the uninduced cell sample, cell lysate after induction, and the purified protein were separated by % sds-page for coomassie brilliant blue staining. for western blot analysis, or μg of the purified his-nsp -[nsp /nsp ]-myc-his and his-nsp (s a)-[nsp / nsp ]-myc-his were loaded onto % sds-page for separation and then transferred onto hybond nitrocellulose membranes (amersham biosciences). membranes were blocked overnight in % milk powder dissolved in tris-buffered saline. blotted proteins were detected with the rabbit polyclonal anti-his primary antibody (santa cruz) or the mouse monoclonal anti-myc antibody (santa cruz). a horseradish-peroxidase-labeled anti-rabbit or anti-mouse secondary antibody (santa cruz) was used. after each incubation step, membranes were washed four times with . % tween- in tris-buffered saline. detection was performed by using supersignal west pico chemiluminescent substrate solution (pierce biotechnology). the protein was prepared in mm nh hco at a concentration of mg/ml followed by digestion using trypsin (roche) at °c for h with the mass ratio of trypsin to the protein set at : . the digested products were further reduced by the addition of dtt to the digestion volume at mm followed by incubation at °c for h. the resultant products were analyzed by electrospray ionization (esi)-ms/ms. then, a peptide fragment of . da was chosen for sequencing by esi-ms/ms on a q-tof hybrid quadrupole/time-of-flight mass spectrometer (micromass, uk). the coordinates and associated structure factors have been deposited in the research collaboratory for structural bioinformatics pdb (pdb code fan for the native protein; pdb code fao for the s a mutant structure). generation of an infectious clone of vr- , a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus blue ear' disease of pigs porcine reproductive and respiratory syndrome (prrs or blue-eared pig disease) assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark porcine respiratory and reproductive syndrome virus variants, vietnam and china genetic variation of the prevailing porcine respiratory and reproductive syndrome viruses occurring on a pig farm upon vaccination complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains virus-encoded proteinases and proteolytic processing in the nidovirales equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily the arterivirus nsp protease is the prototype of a novel group of chymotrypsin-like enzymes, the c-like serine proteases structure of arterivirus nsp . the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole the "colorful" epidemiology of prrs general overview of prrsv: a perspective from the united states full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate molecular cloning, expression, purification and crystallographic analysis of prrsv cl protease automated mad and mir structure solution procheck: a program to check the stereochemical quality of protein structures mapping the protein universe a glutamic acid specific serine protease utilizes a novel histidine triad in substrate binding expression, purification, and in vitro activity of an arterivirus main proteinase crystal structure of foot-and-mouth disease virus c protease. new insights into catalytic mechanism and cleavage specificity prediction of proteinase cleavage sites in polyproteins of coronaviruses and its applications in analyzing sars-cov genomes mechanisms and enzymes involved in sars coronavirus genome expression proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication alternative proteolytic processing of the arterivirus replicase orf a polyprotein: evidence that nsp acts as a cofactor for the nsp serine protease structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor structure of human rhinovirus c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes refined x-ray crystallographic structure of the poliovirus c gene product the refined crystal structure of the c gene product from hepatitis a virus: specific proteinase activity and rna recognition coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs biosynthesis, purification, and characterization of the human coronavirus e c-like proteinase conservation of substrate specificities among coronavirus main proteases hepatitis a virus c proteinase substrate specificity serine proteases: structure and mechanism of catalysis subtilisin; a stereochemical mechanism involving transition-state stabilization structure of crystalline alphachymotrypsin. iv. the structure of indoleacryloylalpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme structural basis of the activation and action of trypsin peptide-plane flipping in proteins variable oligomerization modes in coronavirus non-structural protein turkey coronavirus non-structure protein nsp -an endoribonuclease the sars-unique domain (sud) of sars coroanvirus contains two macrodomains that bind g-quadruplexes the nsp alpha and nsp papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases production of authentic sars-cov m (pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction severe acute respiratory syndrome coronavirus c-like proteinase n terminus is indispensable for proteolytic activity but not for enzyme dimerization. biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations a structural view of the inactivation of the sars coronavirus main proteinase by benzotriazole esters sars cov main proteinase: the monomerdimer equilibrium dissociation constant critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease the catalysis of the sars c-like protease is under extensive regulation by its extra domain dissection study on the severe acute respiratory syndrome c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors atomic structures of the human immunophilin fkbp- complexes with fk and rapamycin maximum-likelihood density modification the ccp suite: programs for protein crystallography the pymol molecular graphics system espript: analysis of multiple sequence alignments in postscript we thank stefan anemüller for measuring the enzyme kinetics of prrsv nsp . key: cord- -yo libo authors: zhou, yan; zheng, haihong; gao, fei; tian, debin; yuan, shishan title: mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase date: - - journal: sci china life sci doi: . /s - - - sha: doc_id: cord_uid: yo libo the subgenomic mrna transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (prrsv) are directed by the viral replicase. the replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp , containing the viral replicase, has characteristic sequence motifs conserved among the rna-dependent rna polymerases (rdrp) of positive-strand (ps) rna viruses. to test whether the conserved sdd motif can tolerate other conserved motifs of rna viruses and the influence of every residue on rdrp catalytic activity, many amino acids substitutions were introduced into it. only one nsp substitution, of serine by glycine (s g), could rescue mutant viruses. the rescued virus was genetically stable. alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. we also found that the sdd motif was essentially invariant for the signature sequence of prrsv rdrp. it could not accommodate other conserved motifs found in other rna viral polymerases, except the gdd motif, which is conserved in all the other ps rna viruses. these findings indicated that nidoviruses are evolutionarily related to other ps rna viruses. our studies support the idea that the two aspartate residues of the sdd motif are critical and essential for prrsv transcription and represent a sequence variant of the gdd motif in ps rna viruses. rna-dependent rna polymerases (rdrps) function as the catalytic subunit of the viral replicase. rdrps are required for the replication and transcription of all positive-strand (ps) rna viruses, in concert with host proteins and, sometimes, other viral proteins [ , ] . the rdrp domain is essential for rna replication [ , ] . several conserved sequence motifs have been identified in the putative rdrps of several animal and plant ps rna viruses [ ] . currently, eight conserved motifs are known to be present in all classes of polymerases, and four of them reside in the catalytic portion of the "palm" domain [ , , ] . the most conserved palm domain contains the four amino acid sequence motifs found in all classes of polymerases, named a, b, c, and d [ ] , plus a fifth motif e, unique to rdrps and reverse transcriptases [ ] . this structural conservation implies that palm domains of all rdrps may have evolved from a common, ancient ancestor. motif c forms a "-strand, turn, -strand" structure that contains the highly conserved gly-asp-asp (gdd) motif, with the aspartic acid residues exposed in the loop region found in rdrps, an rdrp hallmark [ , [ ] [ ] [ ] . this structure is very similar in all classes of polymerases, and positions the two dd residues close to the conserved d of motif a [ ] . the two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme [ , [ ] [ ] [ ] . the rna-dependent rna polymerases (rdrps) of all positive stranded rna viruses and double stranded rna viruses, such as reoviruses, contain a gdd motif, a highly conserved sequence located at the active site. however, with one exception, the rdrps of nidoviruses have a replacement of the gly residue by a ser residue preceding the two key catalytic asp residues in the gdd motif [ ] . thus, porcine reproductive and respiratory syndrome virus (prrsv) has an sdd signature instead of the canonical gdd in the rdrp (nsp ) [ ] [ ] [ ] (figure , table ). the role of the serine residue in the rdrps of nidovirus has not been studied; however, the corresponding glycine residue is essential for poliovirus rdrp and for other rdrps carrying a gdd signature [ , ] . it is said that the phylogeny of rdrps mainly parallels the taxonomy of rna viruses. the other rna viruses also have a particular conserved motif in the place of the gdd motif, such as the gdn motif of nonsegment-negative stranded rna viruses [ , ] , the sdd motif of segment-negative stranded rna viruses [ , , ] , and the mdd motif of retroviruses [ ] ( table ). the universal occurrence and exceptional conservation of rdrps has led to rdrps, along with a few other replicative proteins, being used for the identification and classification of rna viruses. what then, is the phylogenetic relationship of these different motifs among rna viruses? based on the present studies of the gdd motif, the requirement for the glycine residue of the gdd motif is somewhat flexible for in vitro rna synthesis [ ] . studies on polioviruses, hcv [ ] , rhdv, tombusviruses [ ] , rubella viruses (rv) [ ] , csfv [ ] , and caliciviruses [ ] showed that the first aspartate of the gdd motif appears to be a strict requirement, as any changes to this position are not tolerated for in vivo viral replication and/or in vitro rna synthesis. studies of the second aspartate of the gdd motif showed it was not absolutely conserved in all classes of polymerases, suggesting some flexibility at this position. however, mutational analysis indicates a relatively strict requirement for an aspartate at this position in the rdrps of calicivirus [ ] and encephalomyocarditis virus [ ] . overall, single or multiple substitutions of the equivalent residues in viral rdrps were shown to change or even abolish polymer-ase activity, depending on the assay conditions used [ ] . usually, rdrp domains are readily identifiable by comparative sequence analysis. little is known about the biochemical and structural properties of the replicase [ ] . although many studies suggest nsp is the rdrp of prrsv [ , ] , there is no experimental evidence to demonstrate its catalytic activity. a core palm structure containing the four motifs (a-d) found in all classes of polymerases [ ] is present in the c-terminal region of nsp ; therefore, it is possible to identify rdrp catalytic activity by testing whether the conserved sdd motif (at residues to of nsp ) itself is essential for virus replication (figure ) . this work provides an opportunity to better understand the biology of prrsv, as well as to identify antiviral targets for the prevention of prrsv. cells transfected with an sga mutant of eav did not show detectable rna replication or subgenomic mrna transcription [ , ] . moreover, previous studies have focused on the structure-function analysis of the gdd motif and mainly depended on in vitro biochemical experiments. however, characterization of the sdd sequence motif of prrsv has not yet been reported. given the heterogeneity observed with regard to the gdd motif in rna polymerases of ps rna viruses, we wanted to ascertain whether flexibility exists with respect to amino acid substitutions at this site of the prrsv rna polymerase and the influences of amino acid change on virus replication and transcription. we utilized site-directed mutagenesis to construct several different combinations of mutations in the sdd motif in an infectious clone of prrsv, and investigated the functional role of the motif. marc- cells (atcc, manassas, va, usa) and baby hamster kidney (bhk- ) cells were propagated in eagle's minimum essential medium (emem; gibco-brl, gaithers- figure schematic representation of prrsv genome showing the location of the conserved sdd motif within rdrp. shown is a part of domains in the pp ab replicase polyproteins of prrsv. arrows represent sites in pp ab that are cleaved by chymotrypsin-like ( clpro) proteinase. the proteolytic cleavage products are numbered according to full cleavage site map. within the cleavage products, the location and names of the domains that have been identified as structurally and functionally related are highlighted. these include diverse domains with conserved cys and his residues (c/h), rna-dependent rna polymerase (rdrp), helicase (hel) and uridylate-specific endoribonuclease (n or nendou) activities. the number of amino acid d indicates the location of sdd in pp ab. burg, md, usa) with % fetal bovine serum (fbs; gibco-brl, gaithersburg, md, usa), and maintained in emem with % fetal bovine serum at °c with % co . bhk- cells and marc- cells were used for incubation of prrsv. the viruses were rescued from the type ii prrsv infectious clone paprrs (with cmv promoter) [ ] and used as parental virus in all experiments. standard recombinant dna techniques were used to generate all the constructs. the full-length infectious cdna clone, paprrs [ ] was used in the basis for subcloning. construction of sdd mutants. the full-length mutant plasmids were constructed by site-directed pcr mutagenesis and soe (gene splicing by overlap extension) pcr. a series of mutations was introduced into the nsp sdd motif by making single mutations at amino acid residue , , or . the synthetic deoxyribonucleotides used in the mutagenesis are shown in table . to create plasmids pgdd, psed, psde, and psga, a shuttle plasmid named ptb , representing nucleotide positions - of the viral genome, was constructed to introduce specific modifications to the sdd motif in the full-length genomic clone. the fragment - nt was cloned into the vector pbluescript ii sk (+/) (stratagene, la jolla, ca, usa). ptb plasmid was used as the template in site-directed pcr mutagenesis. pcr-mediated mutagenesis was carried out with the appropriate primers containing the required nucleo-tide changes. to construct plasmids pmdd, psad, psnd, psda, psdn, psgd, and pgdn, soe pcr was used with paprrs as the template and two pairs of primers. the strategy of construction of them was the same with psnd. for mutant psnd, a pair of primers (sf plus sr snd) was used to amplify approximately . kb from nt to , and the downstream . kb fragment from nt to was obtained by a second pcr using primers, sf snd and sr . the . kb fragment containing the desired d→n mutation was generated by the soe pcr with . and . kb fragments as templates, and sf and sr as the primers. the pcr products were purified using the watson gel extraction mini kit (watson, shanghai, china). purified products were digested with avr ii and ecor v and the major fragment was used to replace the avr ii/ecor v fragment of the infectious clone paprrs to form the mutant clones. site-directed pcr mutagenesis was carried out using cycles of °c for s, or °c for s, and °c for min using pfuturbo hotstart dna polymerase (stratagene, la jolla, ca, usa), added to the reaction mixture after an initial denaturation ( °c, min). after cycles, the products were extended at °c for another min. soe pcrs were carried out using cycles of °c for s, °c for s, and °c for min using pfuultraii fusion hs dna polymerase (stratagene, la jolla, ca, usa), added to the reaction mixture after an initial denaturation ( °c, min). after cycles, the products were extended at °c for another min. for detecting replication, pas was constructed by deleting to nt from paprrs using afl ii and spe i as a negative control. bhk- cells were propagated in a -well plate ( × cells/ mm well) and grown for d to approximately %- % confluence. the subconfluent bhk- cells were transfected with equal amounts ( . μg) of full-length mutant plasmid using lipofectamine ltx&plus reagents (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions and incubated at °c with % co . the supernatant of the cell culture was harvested and inoculated onto marc- cells h posttransfection, and was monitored daily for cytopathic effect (cpe). when % cpe was observed, the supernatant of the cell culture was harvested and stored in a  °c freezer as the primary rescued virus stock, designated as vgdd passage (p ). the -fold diluted p was used for inoculating fresh marc- cells, and the supernatant was harvested at dpi, and designated as p . the p -p virus stocks were prepared in the same manner. for rna extraction, viral genomic rna was isolated using a qiaprep viral rna mini kit (qiagen, hilden, germany), and cells were lysed at h posttransfection. total intracellular rnas were isolated using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. rnas were suspended in rnase-free water, aliquoted, and stored at  °c. for the analysis of virus transcription, the subgenomic (sg) mrna s of prrsv was detected by reverse transcription-pcr (rt-pcr) from the total intracellular rnas of transfected cells ( figure ). as an internal control for rt-pcr analysis of sg mrna s, the β-actin gene of bhk- cells was also detected using a pair of primers, actin-f and actin-r. for the sg mrna s rt reaction, primer sr was used. for the sg mrna s pcr, primers sf and sr were used. the pcr conditions comprised cycles of °c denaturation ( s), °c annealing ( s), and °c extension ( s), followed by incubation at °c for min. the cycles were followed by a -min incubation at °c. for the analysis of the virus mutational site, a pair of primers, sf and sr , was used in the synthesis of cdna covering the sdd motif at nt - . the pcr products were gel-purified and sub-cloned into pgem-t vector (promega, madison, usa), and sequenced. for negative-strand-specific rt-pcr, we used the approach figure the influence of aa substitutions in the sdd motif on subgenomic transcription and genomic replication. a, rt-pcr strategy for the detection of genomic negative strands and subgenomic mrna s. for the rt-pcr, primers sf and sr were used to amplify regions specific for plasmid, positiveand negative-stranded genomic rna. primer sr is directed against sequences within the orf a region and sf is directed against the leader sequences of the ′utr; both are used for priming cdna synthesis (dashed line) on the genomic negative strands. primers sf and sr are used for the pcr specific for positive-stranded sg mrna s. primer sr is directed against the body sequence of sg mrna s and is used for priming cdna synthesis (dashed line) on the positive strand of sg mrna s. b, rt-pcr amplification of leader-body junction specific regions of subgenomic mrna s. to study the impact of mutations in the sdd motif on prrsv transcription, rt-pcr analysis of the sg mrna s was performed on the total rnas obtained from mock-transfected bhk- cells and cells transfected with pmdd, psad, psed, psnd, psgd, psda, psde, psdn, psga, pgnd, pgdd, and paprrs, using a forward primer located in the leader region and a reverse primer in orf . the bands representing the specific sg mrna s are shown in the top panel and semiquantitative rt-pcr analysis of β-actin gene of bhk- cells to estimate the amounts sg mrna s is shown in the middle panel. to study the impact of mutations in the sdd motif on prrsv replication, the rnas were isolated from bhk- cells transfected with mock-transfected bhk- cells and cells transfected with pmdd, psad, psed, psnd, psgd, psda, psde, psdn, pgdn, psga, pgdd, and paprrs, using a forward primer located in the leader region and a reverse primer in the orf . rt-pcr analysis of genomic negative strands is shown in the bottom panel. outlined in figure . bhk- cells transfected with full-length mutant plasmid and paprrs were grown on a -well plate at °c. indirect ifas were carried out by standard procedures [ ] , using a : dilution of a prrsv monoclonal antibody directed against the prrsv open reading frame (orf , encoding a nucleocapsid protein), and antibody anti-prrsv nsp (kindly provided by dr. fang ying at south dakota state university) (figure ). after three washes with pbs, cells were incubated at °c for h with goat anti mouse secondary antibody alexa fluor® dyes (invitrogen, carlsbad, ca, usa). the samples were examined with an olympus fluorescence microscope equipped with a digital camera and qfluoro software (leica). to determine the multi-step growth curve of the p vgdd and vaprrs, marc- cells in mm dishes were in-fected with μl ( . moi) of p viruses, repeated three times. an aliquot of the cell culture supernatant was harvested and replenished with an equal volume of emem containing % fbs at the indicated time points ( , , , , , , , , , and h) post-infection and stored at  °c. viral titration was performed by tcid , and a growth curve was determined from the tcid results, as described previously [ ] (figure ). for detection of viral replication, an aliquot of the cell culture supernatant was harvested at h after transfection to bhk- cells with psdd and paprrs. viral titration was performed by tcid as described previously [ ] (table ). vgdd in vitro generated from marc- cells were serially -fold diluted. . ml viruses was inoculated onto marc- monolayers ( × cells/well) in -well plates. after h at °c, cell monolayers were washed once with pbs and overlaid with ml mem containing % (w/v) low melting-point agarose and % fbs. after incubation for further four days at °c, single plaques were selected and dissolved into l % mem, and then inoculated onto marc- cells for producing viruses. for sequencing the whole genome of these viruses, the strategy we adopted is shown in table and figure , and was described previously [ ] . cdna fragments were then cloned to pgem-t vector and sequenced. northern blotting was performed according to the north-ernmax kit (ambion austin, tx, usa) manufacturer's protocol. cultures of marc- cells were infected with p mutant and wt viruses at moi. total intracellular rnas were isolated from infected cells harvested at h postinfection using trizol reagent. the total rnas were separated on % denatured agarose gels using agarose-le (ambion, austin, tx, usa) as described previously [ , ] , blotted onto a nitrocellulose membrane (ambion, austin, tx, usa), and probed with an n-specific probe, ′-ccgttttacagcttttctgccacccaacacgag-gctcttcaacccgggcaccaatgtgcc- ′. this probe could not detect the sg mrna . . to examine the function of the conserved sdd motif in prrsv replication, a series of mutations were introduced into nsp by substituting each of the three residues (positions , , and ) (figure ). pcr-mediated mutagenesis was carried out with primers containing the required nucleotide changes ( table ) . to construct the other mutations, such as psnd, soe pcr was used with pap-rrs dna as the template and two pairs of primers, sf plus sr snd and sf snd plus sr ( table ). the pcr product containing the mutation and paprrs was digested by avr ii and ecor v, used to replace the avr ii-ecor v fragment ( - nt) of paprrs. the intro-duced mutations were verified by sequencing and restriction analysis. mutant plasmids were transfected into bhk- cells, whose actual infectivity was examined. no transcription of sg mrna s was detected when mutants pmdd, psad, psed, psnd, psda, psde, psdn, psgd, pgdn, and psga were transfected into bhk- cells ( figure ) ; therefore, it was important to determine whether these mutants were able to replicate and produce genomic negative strands. in the infected cell, the genomic negative strands of arteriviruses, like those of coronaviruses, are present in double-stranded replicative intermediates (ris). to detect the influences of replicase mutants on replication, we used a more sensitive rt-pcr approach to analyze the presence of negative strands in cells transfected with the above mutants. as outlined in figure , specificity for the antigenomic template was achieved by using primer pair sf and sr . for the detection of genomic negative strands, primer sr matches sequences within the orf a region and sf matches the leader region within the ′utr. bhk- cells were transfected with the above described mutant clones and pas . in the latter clone, the predicted nsp rna polymerase domain was removed, a deletion which has been shown to render prrsv replication incompetent. we used this mutant clone as a negative control for replication detection. rna was isolated h after transfection and analyzed for the presence of genomic negative strands (figure ) . the rt-pcr results for pas were negative, confirming the specificity of our rt-pcr approach. in the cases of pmdd, psad, psed, psnd, psda, psde, psdn, psgd, pgdn, psga, pgdd, and paprrs, the replicase mutants produced detectable amounts of genomic negative strands (figure ). the results indicated that none of the mutations abolished the replication function of rdrp. the rdrp of prrsv is required for prrsv replication; therefore, the effect of sdd motif mutations on the catalytic activity of rdrp can be determined by analysis of rna synthesis and virus production. the wt paprrs and the mutant pgdd could produce viruses after transfection into bhk- cells and inoculation onto marc- cells. the primers used were complementary to the leader sequence (sf ) and the end of orf (sr ), and primed cdna synthesis on all subgenomic positive stranded mrna s of prrsv ( figure ). the mutant prrsv full-length cdna clones pmdd, psad, psed, psnd, psgd, psda, psde, psdn, pgdn, and psga carrying mutations specifying the sdd motif in the viral replicase (table ) , were transfected into bhk- cells and failed to produce sg mrna s (figure ) . no cpe was detected in marc- cells inoculated with undiluted culture medium of bhk- cells transfected with the above-described mutants. moreover, expression of the n protein was not detected by ifas in bhk- cells transfected with the mutant full-length cdna clones, except for wt paprrs and pgdd ( figure ) . however, detection of the nsp protein demonstrated that the transfection and plasmid vector system were both functional. the results indicated that all the mutations destroyed the transcription function of rdrp (figure ) . to analyze the viral rna profiles, total rnas were isolated at the h postinfection from marc- cells infected by the p mutant viruses (vgdd) and p vapprs at an moi of . the rnas were separated on formaldehyde-denatured agarose gel and subjected to northern blotting with a dig-labeled probe complementary to the n protein sequence of type ii prrsv. the northern blot results revealed that the sg mrnas of mutants could be recognized by the specific probe and that the sg mrna was the most abundant rna in the virus transcription process (figure ) . compared with vaprrs, the transcription pattern in the mutant virus vgdd was altered as a result of the change to the motif (figure ). passage experiments were carried out to detect the reversion of the s g mutant harvested from bhk- cells transfected with pgdd. the mutation specifying the sdd motif was sequenced from p to p of vgdd. the substitution of s g was found not revert (table ) , which indicated that vgdd was stable ( figure b ). to investigate the influence of the s g mutation of rdrp on the vgdd genome, marc- cells were infected with vaprrs or vgdd. p viral supernatants were serially passaged through plaque purification five times, named p -p , and stored for further experiments. rna was isolated from p to p virus particles and used for cdna synthesis and subsequent pcr amplification (table , figure a ). the sequence covering the regions of the vgdd genome was determined in at least three isolated cdna clones. in all three sequenced cdna clones, the original substitution had not reverted; however, two synonymous mutations, i.e., c u and u c had occurred ( figure c ). the results indicated that mutant virus s g is stable and did not influence the fidelity of rdrp. in this study, we analyzed some of the properties of the prrsv rdrp by site-directed mutagenesis of the sdd motif, which is associated with the polymerase activity and is particular to nidoviruses. we took two approaches for systematic mutational analysis of the sdd motif: (i) we introduced either conserved or non-conserved amino acid changes in the sdd motif; and (ii) we introduced mutations that resembled the conserved sequence of other viral rdrps (table ) . based on the sequence alignment, sdd motif was predicted to be a variant of the gdd motif of positive-strand rna viruses. our observation that the rdrp of prrsv is active in replication and transcription when the sdd motif is mutated to gdd suggests that, conversely, the polymerase of other positive-strand rna viruses might function with an sdd motif. it is reported that the requirement for a glycine residue in gdd motif is strict in the positive-strand rna viruses [ ] . mutational analysis of the gdd motif of rdrp of rv [ ] and tbsv confirmed this view, indeed the mutant add was found to revert back [ ] . however, our results showed that the mutant amino acid residue s g of vgdd did not revert back and was stable until p . the serine residue of the sdd motif could be substituted by glycine residue. this was the first demonstra-tion of flexibility of the serine residue of sdd motif in prrsv. in terms of genetic stability, the mutant vgdd could be used as a candidate for genetic marker vaccines. however, it is not obvious why nidoviruses have evolved an sdd sequence in place of the gdd motif. from the multi-step virus growth curve, although vgdd maintained a growth pattern similar to that of vaprrs, the peak titers of vgdd were lower than those of vaprrs ( figure ) . in terms of how the mutant gdd motif of rdrp affected the replication and transcription of prrsv, it produced less mrna s than vaprrs, despite producing more sg mrna . than vaprrs ( figure ) . these results were confirmed in three independent experiments. it has been reported that there is a quantitative balance among sg mrnas species [ ] ; thus, we proposed that a quantitative balance between sg mrna . and sg mrna . exists, which was confirmed by our lab (data not shown). rdrp interacts with the promoters of every sg mrna to produce the sg mrnas, and a change in the active site of rdrp may influence this interaction, disturbing the quantitative balance between sg mrna . and sg mrna . . motif c contains highly conserved amino acid residues that represent specific signatures of each major group of rna viruses. in motif c, the sequence gdd is conserved among polymerases of positive-strand rna viruses, mdd belongs to retroviruses, gdn and sdd are conserved signature motifs of nonsegmented-negative stranded and segmented-negative stranded rna viruses, respectively [ ] , whereas sdd is conserved in nidoviruses. high-resolution structures of rdrps of positive-and double-strand rna viruses revealed clear similarities, despite sharing little sequence identity, suggesting an evolutionary link between the rdrps of these rna viruses [ , , ] . to investigate the role of the conserved c motif in the evolutionary process of the polymerase, we mutated amino acid residues of sdd to those conserved amino acids described above. when sdd was mutated to mdd or gdn, rdrp lost transcriptional activity. from the aspect of evolutionary relationship, nidoviruses, which are also positive single-stranded rna viruses, are closely related to the positive stranded rna viruses, but are further from the nonsegmented-negative stranded rna viruses and retroviruses. however, it is unclear why segmented-negative stranded rna viruses also have an sdd motif, but abrogated polymerase activity when was mutated to gdd [ , ] . there appears to be a strict requirement for the first aspartate, because any changes to this position, including a change to a negatively charged glutamate, are almost never tolerated for in vivo viral replication and/or in vitro rna synthesis [ ] . in the present study, all substitutions of the first aspartate residue of the sdd motif resulted in a lethal mutation. in agreement with previous studies [ ] , the first aspartate residue of sdd is integral to the catalytic active site of the molecule and is absolutely conserved in the rdrp of prrsv. however, results obtained from the mutational analysis of the second aspartate residue were different in several viral rdrps, mainly depending on the assay conditions used [ ] . mutational analysis of the gdd motif of rdrp of calicivirus and bvdv obtained results similar to ours, single substitutions of the second aspartate residue were shown to destroy rdrp activity. the second aspartate is also important and cannot tolerate any substitutions. in conclusion, our findings were similar to the results of poliovirus [ ] and rv: substitutions of either aspartate residue were not tolerated and were lethal. it is said that perhaps the gdd motif plays a central role in rna synthesis by binding metal ions [ , , ] , which may be determined by the characteristics of amino acids (table ) . to investigate the effect of different charge and polarity of amino acids substituted in the sdd motif on the activity of rdrp, we introduced various amino acid substitutions (table ) . first, the aspartate residue was substituted by the simplest amino acid, i.e., alanine, which has no polarity; however, it was lethal to rdrp. methionine also has no polarity, but mutant pmdd did not produce virus either. the serine residue has no charge; therefore, we substituted the serine residue with glycine residue, which also has no charge. perhaps the substitution of ser by gly produced a functional mutant because of the less polar no charge similarities between ser and gly. aspartate is negatively charged; therefore, we substituted the aspartate residues with glutamate residues, which are also negative charge. however, neither mutant d e nor d e produced virus, which was similar to a previous study [ ] . replacement of the aspartate residues with uncharged asparagines residues (d n or d n) also did not produce virus. it can be concluded that the strict requirement of dd residues meant that no substitutions could be tolerated, probably because the dd residues are required for metal ion binding [ , ] . the mutants could not produce sg positive strands or sg negative strands (data not show), but could produce genomic negative strands ( figure ). the results demonstrated that mutation of the sdd motif did not completely abolish the function of rdrp, which was different from the results of eav [ ] and other viruses [ , , [ ] [ ] [ ] [ ] . this could be because the primary function of replication is not determined by the sdd motif in prrsv, and the sdd motif is specifically involved in transcription. in fact, viral rdrps function with other factors, such as viral and host factors, in natural infection, in which rdrps combine to differentiate genomic rna replication from sg mrna transcription. for positive stranded rna viruses, replication occurs in membrane-bound, multiprotein complexes termed the replication/transcription complex (rtc). however, the domains of rdrp that interact with the other factors, and the process of transcription and replication in the rtc are still unknown. the fact that conservation of the sdd motif is not required for the replication of rdrp in prrsv, suggested that transcription and replication of prrsv are two independent processes and may be uncoupled. it is reported that nsp of eav is essential for sg mrnas production and is dispensable for genome replication [ ] . in the synthesis of sg mrnas, nsp combines with the viral rdrp complex to engage in transcription; however, the mechanism is still unknown, and we speculated that the sdd motif of rdrp could interact with nsp in this process. taken together, our results largely demonstrate that the sdd motif is a variant of the gdd motif of positive-strand rna viruses. a better knowledge of the biochemical and structural characterization of arterivirus key enzymes, like the rdrp, and the processes of transcription and replication may provide important leads for the development of specific pre-prrsv vaccines and anti-prrsv drugs [ ] . the present study helps to elucidate the biology of prrsv and contributes to the development of antiviral targets for the prevention of prrs. analysis of rna-dependent rna polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure mutations in the gdd motif of rubella virus putative rna-dependent rna polymerase affect virus replication. virology characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis nidovirales: evolving the largest rna virus genome primary structural comparison of rna-dependent polymerases from plant, animal and bacterial viruses mutations in the palm region of a plus-strand rna virus polymerase result in attenuated phenotype permutation of the active site of putative rna-dependent rna polymerase in a newly identified species of plant alpha-like virus effect of mutation in the hepatitis c virus nonstructural b region on hcv replication catalytic core of alphavirus nonstructural protein nsp possesses terminal adenylyltransferase activity rna virus replication complexes mutation of the aspartic acid residues of the gdd sequence motif of poliovirus rna-dependent rna polymerase results in enzymes with altered metal ion requirements for activity structural and functional characterization of sapovirus rna-dependent rna polymerase the molecular biology of arteriviruses porcine reproductive and respiratory syndrome virus: origin hypothesis de novo initiation of rna synthesis by the arterivirus rna-dependent rna polymerase genetic and biochemical evidence for an oligomeric structure of the functional l polymerase of the prototypic arenavirus lymphocytic choriomeningitis virus transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus rna polymerase mutational analysis of the conserved motifs of influenza a virus polymerase basic protein rift valley fever virus l protein forms a biologically active oligomer analysis of tombusvirus revertants to identify essential amino acid residues within rna dependent rna polymerase motifs mutational analysis of the gdd sequence motif of classical swine fever virus rna-dependent rna polymerases mutation analysis of the gdd sequence motif of a calicivirus rna-dependent rna polymerase the rna polymerase activity of sars-coronavirus nsp is primer dependent virus-encoded proteinases and proteolytic processing in the nidovirales the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins identification of dispensable nucleotide sequence in ' untranslated region of porcine reproductive and respiratory syndrome virus arterivirus nsp modulates the accumulation of minus-strand templates to control the relative abundance of viral mrnas nidovirus transcription: how to make sense? poly(a)-and primer-independent rna polymerase of norovirus expression and characterization of rna-dependent rna polymerase of dendrolimus punctatus tetravirus the interplay of rna elements in the dengue virus ′ and ′ ends required for viral rna replication production of infectious hepatitis c virus by using rna polymerase mediated transcription open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited key: cord- -lopcb c authors: tan, feifei; wei, zuzhang; li, yanhua; zhang, rong; zhuang, jinshan; sun, zhi; yuan, shishan title: identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: lopcb c nucleocapsid (n) protein of porcine reproductive and respiratory syndrome virus (prrsv) plays a central role in virus replication. in this study, serial n- and c-terminal truncations of n protein were performed in the context of type prrsv infectious cdna clone, and our results revealed that a stretch of inter-genotypic variable n terminal residues aa – (( )ngkqqkkk( )k) and the last four inter-genotypic variable aa residues (( )sps( )a) at the c terminus of n protein were dispensable for type prrsv infectivity. all the recovered deletion mutant viruses had spontaneous mutations in the n coding region, including substitution, deletion and insertion. we re-engineered the additional internal deletion with or without the original c-terminal deletion back into wild-type aprrs and found that the internal domain spanning the inter-genotypic variable residues – (( )kgp( )g) and conserved residues – (( )knpe( )k), respectively, were dispensable for type prrsv viability. these results demonstrated that n protein contains non-essential regions for virus viability in cell culture. such dispensable regions could be utilized as insertion site for foreign tag expression and the rescued viruses could be the candidates for marker vaccine. porcine reproductive and respiratory syndrome virus (prrsv) is a single-strand, positive-sense rna virus, and is a member of the family arteriviridae, together with equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv). arteriviridae and coronaviridae are classified as members of the order nidovirales, mainly based on their similar replication and transcription strategy (den boon et al., ; gorbalenya et al., ; snijder and meulenberg, ) . two genotypes of prrsv have been classified, (european) type and (north american) type , lelystad virus (lv) (wensvoort et al., ) and vr (benfield et al., ) are the representative strains of two types, respectively. prrsv virion consists of at least seven structural proteins, including glycoprotein (gp ) , gp (de lima et al., ) , gp (van nieuwstadt et al., ) , gp , and unglycosylated envelope (e) protein (wu et al., ) , membrane (m) protein, and nucleocapsid (n) protein (bautista et al., ) . structural proteins of arteriviruses are expressed via a nested set of subgenomic mrnas (sgmrnas) formed by a unique discontinuous transcription process (de vries et al., ; meng et al., ; nelsen et al., ) . the common leader sequence of sgmrnas is derived from the proximal untranslated region (utr). the leader-body junction sequence can be amplified by sgmrna-specific rt-pcr (chen et al., ; den boon et al., ; meulenberg et al., ; nelsen et al., ; zheng et al., ) . n protein is the most abundant component of the virion and plays a crucial role in virus assembly. generally, type and type prrsv n proteins consist of and amino acids (aa), respectively. however, stadejek et al. ( ) reported that n protein of type exhibits size polymorphism, the number of amino acid residues varies from to . although n protein is one of the inter-genotypically conserved proteins, lv and vr share only % identity at the amino acid level (dea et al., ) . n protein is assembled into an icosahedral nucleocapsid in the form of disulfide-linked homodimer , and the cterminal half of prrsv n protein is believed to be the dimerization domain (spilman et al., ) . the n-terminal half of n protein is enriched in basic amino acids and intrinsically disordered, which are common properties of rna-binding proteins (daginakatte and kapil, ; doan and dokland, ; yoo et al., ) . the crystal structure of the c-terminal dimerization domain comprises two antiparallel ␤-sheet floors superposed by two long ␣ helices and flanked by two n-and c-terminal ␣ helices (doan and dokland, ) , as shown in fig. a . the structure is homologous to that of eav (deshpande et al., ) , and surprisingly, has a fold similar to that of severe acute respiratory syndromeassociated coronavirus (yu et al., ) . by replacing each amino (doan and dokland, ) (pdb id: p ). alignment of the n sequence from type strain vr (genbank id: ay ), the wild-type aprrs (genbank id: gq ) in this study and type strain lv (genbank id: m ). (b) sequence alignment of the first nt sequence of orf of lv, aprrs and n-terminal deletion mutants, the identical sequence with lv is denoted as dot while natural sequence deletion compared with lv designated as dash line. the engineered deletion is represented by asterisk. the nt that was documented to be essential for rna replication of lv (verheije et al., ) is indicated in the open box, the nt sequence which had kissing interaction with utr is indicated by shaded box. acid with proline, wootton et al. ( ) demonstrated that the - aa substitution of type prrsv n protein reduces the conformational-dependent monoclonal antibody (mcab) binding significantly. therefore, the c-terminus of n protein is important for maintaining the local and/or the overall configuration of the prrsv n protein. verheije et al. ( ) reported that c-terminal six amino acids of n protein are non-essential for virus infectivity of type prrsv. these authors also indicated that the n-terminal and internal regions of n protein cannot tolerate deletion. as shown in fig. a , n protein is intra-genotypically conserved and only one (d ) out of the aa of type prototypic strain, vr , is different from the aprrs strain (y ) used in this study. several inter-genotypically conserved domains were observed too. in addition, crystal structure of c-terminal dimerization domain except for the last aa of vr n protein was determined (doan and dokland, ) . in this study, to further illustrate the roles of the terminal and internal aa residues of n protein in type prrsv replication, particularly in viral viability. we performed serial deletions at the n-and c-termininal of n protein in the context of full-length cdna clone paprrs. we found that type prrsv contained extensive regions that were dispensable for virus viability in cell culture. these results are of great significance for foreign gene expression and genetic engineered vaccine development. marc- cells were grown in minimal essential medium (mem) (sigma) complemented with % fetal bovine serum (fbs) (invitrogen), and maintained in mem with % fbs at • c with % co . type prrsv infectious cdna clone paprrs (yuan and wei, ) was used as the wild-type control in all experiments. its complete genomic sequence was available as genbank accession number gq . pbsx was a shuttle plasmid that cloned the spe i-xho i region (nt - ) of aprrs genome into the pbluescript sk(+) vector (fermentas). p usc was constructed by inserting asc i immediately after the stop codon of orf . n-terminal deletion was performed from the fifth amino acid to retain the integrity of orf . fragments containing the corresponding deletion region were generated using the mutagenic pcr method as described before (yu et al., ) , pbsx was the template. then the spe i-xho i fragment of paprrs was replaced by the analogous fragments derived from mutagenic pcr products. internal deletion mutants were constructed in the same manner. c-terminal deletion mutants were constructed based on the fulllength clone p usc. the fragments containing the desired deletion were amplified by pcr, asc i was introduced in anti-sense primers (table ) , and the sense primer was spef (table ). the spe i-asc i fragment of p usc was replaced by the pcr fragments carrying the desired deletion. for the construction of plasmids that contained primary engineered deletions and spontaneous mutations, appropriate fragments were prepared by rt-pcr products from the viral cell culture supernatants. the amplified fragments between xba i and xho i were swapped into the corresponding region of paprrs. all full-length clones of mutants were verified by sma i mapping and nucleotide sequencing. full-length plasmids were prepared with qiaprep spin miniprep kit (qiagen). marc- cells were seeded in six-well plates and grown to % confluence. the monolayer cells were transfected with g plasmid using l fugene hd regent (roche) according to the manufacturer's instructions. after visible cpe was observed (about h posttransfection for wild-type aprrs) and % of the monolayer cells were detached, the culture supernatants were collected and labeled as passage (p) viruses. the wildtype and recovered mutant viruses were passaged in marc- cells five times to gain p -p virus stocks. at least three further passages were performed if no visible cpe was observed after transfection. marc- cells were transfected with plasmids of wild-type or mutants. intracellular expressions of nsp and n were visualized by immunofluorescence staining at hpt and hpt, respectively, the protocol was same with that described before (yu et al., ). the mcabs against nsp and n protein (mr ) (wootton et al., ) were kindly donated by dr. ying fang at south dakota state university. alexa fluor goat anti-mouse igg (h + l) (invitrogen) was used as a secondary antibody. viral rna was extracted from cell supernatants using qiaamp viral rna kit (qiagen), and treated with turbo dnase (ambion) to eliminate input genomic dna according to the manufacturers' instructions. first-strand cdna was synthesized using reverse transcriptase xl (amv) (takara) and anti-sense primer qst (table ). the fragment containing the n gene was amplified by taq dna polymerase (takara) using primer pairs sf and sr . the pcr products were purified using tiangel mini purification kit (tiangen) and sequenced directly. the full-length genome of the recovered viruses was amplified by five primer pairs as described previously (yuan and wei, ) , and the primer sequences were available upon request. the leader-body junction sites of prrsv were detected by sgmrna-specific rt-pcr (nelsen et al., ; zheng et al., ) . as shown in fig. b , sf (nt - ) and sr (nt - ) are the primer pairs of sgmrna amplification of aprrs as described before (yu et al., ). for the recovered p viruses that had a mixed population, plaque purification was performed. the transfected cell supernatants of c / - and n - were used to infect marc- cells at a moi of approximately . . after h incubation, cell monolayer was overlaid with × mem (invitrogen) supplemented with % fbs and % low melting agarose (promega). well-isolated plaques were passaged onto fresh marc- cells. rna was extracted and rt-pcr was performed as previously described, then the n gene was sequenced. marc- cells were infected with wild-type and p rescued viruses at ∼ . moi. cell culture supernatants were collected at various times after infection. titers were determined by standard tcid assay. three independent titrations were performed and the mean value was used for determination of the viral multi-step growth-curve. overlapping genes are a dominant feature of arterivirus genome, and there are nucleotides that overlap between orf and orf . therefore, n-terminal deletion mutants were truncated from the fifth residue of orf to ensure the integrity of orf . intergenotypic aa sequence alignment indicated that the lv strain (type ) contains four extra aa residues (tapm) than aprrs (type ) immediately upstream of the inter-genotypically conserved region from g of aprrs (fig. a) . it was therefore possible that this variable region plays little role in type prrsv n functionality. accordingly, we constructed five deletion mutants that comprised residues - , , , and at the n terminus, which were designated as pn / - , pn / - , pn / - , pn / - and pn / - , respectively ( fig. a) . we also wanted to investigate the c-terminal requirements of n protein for type prrsv viability. to this end, a series of c-terminal truncation mutants were constructed. firstly, a recombinant full-length clone p usc containing an asc i immediately after the stop codon of orf was constructed (fig. b, d) . transfection assay demonstrated that the asc i insertion did not affect viral infectivity, and the rescued virus usc had similar growth properties with the wild-type virus aprrs (fig. b ). therefore, p usc was used as the backbone for constructing c-terminal truncations, which contained residues - , - , - , and - deletions, named as pc / - , pc / - , pc / - and pc / - , respectively (fig. b) . to define whether the n-terminal deletions affect virus replication, full-length clones pn / - , pn / - , pn / - , pn / - , pn / - , and the wild-type paprrs, were transfected into marc- cells. intracellular expression of nonstructural protein (nsp ) and n protein was determined by immunofluorescence assay (ifa) at and days post-transfection (dpt), respectively, which indicated the genomic rna replication and sgmrna transcription properties of the mutants. as shown in fig. a , all but pn / - displayed nsp and n protein expression, but the fluorescence intensity was different. it was noteworthy that there were only several single cells stained for mutant pn / - , suggesting virus spreading between neighboring cells was affected. moreover, no fluorescent signal was detected in the case of pn / - (even after prolonged incubation at dpt; data not shown), indicating that deletion of residues - ( ngkqqkkkkgdgqpv n) might have blocked both virus replication and transcription. in the parallel transfection experiments, visible cytopathic effect (cpe) appeared for the wild-type paprrs as well as for mutants pn / - , pn / - and pn / - . the longer deletion mutants pn / - and pn / - , however, produced no infectious particles even after three further passages. these results demonstrated that the nterminal residues - of n protein were non-essential for virus infectivity in cultured cells. we next investigated the possible blockage for the n-terminal deletion mutants that failed to produce progeny viruses. two sgm-rnas (sgmrna . and sgmrna . ) of orf have been defined in type prrsv infected cells (nelsen et al., ; zheng et al., ) . as outlined in fig. b , total rnas of cells transfected with pn / - , pn / - , pn / - and paprrs, respectively, were extracted and sgmrna -specific rt-pcr was performed as previously described (yu et al., ; zheng et al., ) . as shown in fig. c , the two expected bands representing sgm-rna . and sgmrna . were amplified from cells transfected with wild-type paprrs, pn / - and non-viable pn / - , but the band of pn / - was weaker than others, indicating the lower transcription level than others. this was consistent with the ifa result and further demonstrated that sgmrna transcription of pn / - was down-regulated. however, pn / - showed no sign of sgmrna transcription (fig. c ) which was coincident with the ifa results against n protein expression (fig. a) . to assess their genetic stability, the rescued viruses n / - , n / - and n / - were serially passaged five times to establish virus stocks of p - virus. the supernatants of each passage were collected, and the cdna fragment [nucleotides (nt) - ] containing orf was amplified by rt-pcr. nucleotide sequencing analysis confirmed that the engineered deletions were retained in all of the passaged viruses. however, several spontaneous mutations appeared when compared with wild-type virus aprrs. another independent transfection experiment was carried out to confirm the occurrence of spontaneous mutations. the spontaneous mutations of the n-terminal deletion mutant viruses at p in two independent experiments were summarized in fig. a . almost all of the spontaneous mutations were different in two experiments. the spontaneous mutation nt t c (aa s p) existed in n / - was identical with that in mutant virus n / - in experiment . most of the spontaneous mutations emerged at p could be stably passaged to p . given that n plays multiple functions in virus replication process, it was necessary to investigate whether additional genetic alterations were present in other genomic regions. in doing so, a total of five overlapping cdna fragments were amplified by rt-pcr, followed by direct nucleotide sequencing. the consensus sequence of the full-length genome of mutant virus n / - (p ) was assembled and showed no other detectable mutations besides the detected s p mutation in n coding region. we also determined the sequence of utr, utr and m coding region of other mutant viruses, and found no additional genetic alterations. to further quantitatively assess the growth behavior of the recovered n-terminal deletion mutant viruses in cultured cells, multiple-step growth curves were determined for p of rescued viruses n / - , n / - and n / - . as depicted in fig. d , virus n / - and n / - had similar growth kinetics with the parental aprrs. the mutant virus n / - reached the peak titer at h post-infection (hpi), which was h delayed compared with aprrs, and the titer was nearly -fold lower than that of the wild type aprrs (fig. d) . this suggested that a larger deletion at the n-terminal of the n protein adversely affected virus replication process, and the spontaneous mutations in the rescued viruses may account for the growth difference. we next attempted to define the role of the c-terminal unstructured residues for virus replication. upon transfection of c-terminal deletion mutants, the intracellular expression of nsp and n protein were ifa positive for all mutants (fig. a ), though only a few positive cells were detected for pc / - and pc / - , indicating the rna synthesis level was reduced. moreover, the marc- cells transfected with mutants pc / - and pc / - developed typical prrsv cpe, albeit it was delayed by h compared with the parental p usc. no cpe was observed for the larger deletion mutants pc / - and pc / - , in spite of three additional passages. this was confirmed by rt-pcr with sf /sr which showed no sign of infectious particles. these results indicated that the last aa ( sps a) at the c terminus of type prrsv n protein were dispensable for virus viability, which was aa upstream of the previous result based on lv strain (verheije et al., ) . the recovered c-terminal deletion viruses were also passaged for assessment of their genetic stability. the presence of the engineered deletions was confirmed by rt-pcr and nucleotide sequencing at all five passages for the recovered viruses c / - and c / - . however, direct nucleotide sequencing of the rt-pcr products of c / - displayed ambiguous sequence at some positions of orf , suggesting that the rt-pcr products contained a mixed population. we then performed plaque purification assay for transfectant virus c / - (p ). a total of plaques were picked and nucleotide sequencing of rt-pcr product (nt - ) displayed no ambiguity for an individual plaque. collectively, we found three different types of spontaneous substitutions including nt t c, a g and a g, which resulted in aa y h, y c and t a substitution, respectively (fig. b) . surprisingly, two types of additional deletions (aa - and - ) were found (fig. b) . in an effort to assess the variety of the spontaneous mutations, we repeated the transfection experiment, plaque purification and rt-pcr, which showed only one type of additional mutation (nt a g (aa y c)) (fig. b) . mutant virus c / - was analyzed in the same manner. the analysis revealed different spontaneous mutations in two independent transfections, including e g substitution and a -aa insertion (fig. b) . the full-length genome sequence of c / - (p ) containing the e g mutation showed no further mutation in the other genomic regions. surprisingly, the -nt sequence (acagtgctt) was inserted between t and t. the fusion sequence rendered the encoded aa i unchanged, and followed by a stretch of aa (qcf) insertion before the last truncated aa ( rvt a) (fig. b) . nucleotide sequence comparison showed that the inserted -nt was a direct repeat of nt - (gq ) of nsp coding region, implying possible non-homologous rna recombination between these two regions. growth kinetics were assessed for two plaque-purified viruses (ppvs) of c / - generated in the experiment (fig. b) , including ppv (c / - containing the y c mutation) that also was present in the repeated experiment, and ppv (c / - containing residues - deletion) that had the maximal internal deletion (fig. b ). for c / - , the p virus containing the e g mutation was analyzed. fig. b depicts the growth curves of these mutant viruses, compared with the parental usc and aprrs. the c / - ppv had a slightly lower titer than usc at each time point. however, the titers of c / - ppv and c / - were approximately -fold lower than that of usc, and their peaks appeared at and hpi, respectively, which was noticeably delayed (fig. b ). this suggested that the absence of the last four residues ( sps a) and the internal region ( knpe k) could adversely affected virus replication. to further investigate whether the emergence of internal aa deletions was the result of the primary engineered deletion, or the internal aa residues per se were not required for n protein functionality, we introduced two internal deletions found in c / - ppvs back into the wild-type paprrs, named as pn - and pn - , respectively (fig. c) . meanwhile, two double mutants containing the originally engineered c-terminal -aa deletion, together with second-site mutation y c and the residue - deletion were reconstructed. the resultant plasmids were desig- fig. . multiple alignment of the first aa of n protein of five different ppvs of recovered n - , the right panel indicates the plaque numbers of each ppv. aprrs is the wild-type, whereas pn - indicates the engineered deletion. n - ppv , ppv , ppv , ppv and ppv represent the different ppvs of the mutant virus n - . the residues that match aprrs are obscured, and differed from the sequence of pn - are labeled. the short line indicates the deleted residues in pn - and recovered viruses. the -aa insertion in ppv is indicated by the brackets. nated as pc /y c and pc /n - , respectively (fig. d) . all of these mutant plasmids displayed nsp and n expression (fig. a) and developed visible cpe after transfection, suggesting that the internal aa residues ( kgp g) and ( knpe k) were not essential for virus viability, regardless of the presence or absence of the primary deletion. in view of the frequent occurrence of the second-site mutations, the genetic stability of these reconstructed mutants was also investigated, but no further additional mutation was identified for recovered viruses n - and c /y c at all passage levels in the two independent transfection assays. however, mutant virus c /n - had yet one additional point mutation, nt a g (aa i v), which was genetically stable for at least five passages. intriguingly, no additional mutation appeared within n in the independent transfection experiment in all five passages. in the case of n - , sequence analysis indicated that the recovered viruses consisted of a mixed population in both independent experiments; viral plaque purification was therefore performed for n - viruses in experiment . eighteen plaques were selected and sequence analysis showed five different second-site mutations, including g r&k e (ppv in fig. ) , and four different substitutions together with / k/k repaired, as summarized in fig. . we further determined the master sequence of the full-length genome sequence of ppv , no detectable genetic alteration was found in the other region of the viral genome besides n gene. it is also worth noting that the five aa (pgpvm) insertion between residues and of the n protein in virus n - ppv resulted in an unusual length of n protein ( aa). sequence comparison showed that the inserted -nt (cccgggccctgtcat) was identical to a region in orf (nt - , gq ), which coded for nsp (ziebuhr et al., ) . whether genomic recombination between these two regions or some other factors plays a role in this process is under investigation. two of the p ppvs of n - (ppv and ppv in fig. ) , together with recovered viruses n - , c /y c and c /n - containing the i v substitution, were used to assess their growth properties. these viruses exhibited indistinguishable growth kinetics from the wild type aprrs (fig. b) , which suggested that the introduced or induced internal deletions did not affect virus replication significantly. the n protein could tolerate the internal deletion per se, which might not be related to the c-terminal engineered deletion. n protein is the most abundant and important structural protein in prrsv virion, and plays a crucial role in virion assembly. in this study, we found that the n-terminal residues - and last four c-terminal residues were non-essential for type prrsv viability. in addition, we also demonstrated that deletion in the middle region of n protein did not block the production of infec-tious virus. our study is believed to be the first report that the inter-genotypically variable n terminal and internal residues of n protein could tolerate deletion without affecting type prrsv viability in cultured cells, while discrete inter-genotypically conserved terminal residues play crucial roles in viral rna synthesis and/or virus growth. the nonessential regions identified here could be further utilized as insertion site for foreign gene tag and the rescued viruses could be the candidates for genetic marker vaccine. the n protein of coronavirus has been shown to participating virus rna synthesis through interacting with the transcription regulatory sequence (grossoehme et al., ) or as the rna chaperone (zuniga et al., (zuniga et al., , . for arterivirus, it was documented that all of the structural proteins are not required for genomic rna replication and sgmrna transcription of eav (molenkamp et al., ) . in this study, we demonstrated that both terminal and internal domains contained aa residues that are nonessential for virus viability. however, the replication and transcription level of some mutants were reduced, indicating prrsv n protein and/or its coding sequence may affect viral rna synthesis. in comparison with the prrsv n amino acid sequence (fig. a) , we found that the inter-genotypically conserved residue g was the only difference between viable pn / - and non-viable pn / - . it was possibly that the g sequence specificity was required for virus viability, which needs further site-directed mutagenesis. we also proved that the internal residues ( kgp g) and ( knpe k), respectively, were not essential for virus viability. interestingly, these regions sandwiched the reported nls ( pgkk (n/s) k k) of n protein (rowland et al., . lee et al. ( ) demonstrated the nls-null mutant clone produced infectious virus, and the rescued virus displayed a titer -fold lower than that of wild-type virus. the mutagenized nls underwent strong selection pressure in pig that resulted in partial or complete reversion and reacquisition of nls function (pei et al., ) . in our study, the rescued virus n - also displayed additional mutation when transfected into the marc- cells. whether the rescued virus n - undergoes selective pressure in cells still needs further study. the maximal deletion mutant virus n / - and c / - had severe defect on virus growth kinetics, as shown in figs. d and b, suggesting these deletions might adversely affect virus growth. it was also worth noting that the clone pc /n - had identical sequence of n protein with ppv of c / - . however, the growth kinetics of ppv was much lower than that of c /n - (figs. b and b); the only difference was that the latter had one more additional mutation i v in n protein. further investigation on whether the i v substitution determines the different growth property is under way. there are at least two experimentally identified rna signals involving in rna synthesis at the -terminus of arterivirus. one is the pseudoknot interaction between two -proximal stem loops (sl and sl ) demonstrated in eav system snijder, , ) . however, the nucleotides involved in the pseudoknot interaction in prrsv are located in utr. another is the kissing loop structure identified in lv strain, verheije et al. ( ) reported that a stretch of nt within lv orf is essential for rna replication because of a kissing interaction between the -nt sequence in orf and its complementary counterpart in utr. in this study, the mutant pn / - deleted part of this corresponding structure, including the putative -nt kissing-loop sequence in aprrs orf (fig. b) . therefore, we deduced that such a kissing-loop interaction might also play a role in type prrsv replication, which was supported by the failure of fluorescent signal and sgmrna detection in transfected cells (fig. a, c) . as a multi-functional protein, viral n protein is known for interacting with other structural protein for virion assembly (kuo and masters, ) , and with rna for genome encapsidation (doan and dokland, ) . therefore, it is necessary to investigate whether genetic alterations exist in other genomic regions. we determined the nucleotide sequences of utr, utr and m region of all mutant viruses, however, no obvious genetic alterations were observed in these regions. therefore, the n protein domains that may be responsible for protein-rna and protein-protein interactions are most likely located outside of the mutated region. intriguingly, we found that a stretch of nt sequence (acagtgctt, nt - ) insertion in n coding region of mutant virus c / - was a direct repeat from nsp coding region. meanwhile, another -nt (cccgggccctgtcat, nt - ) found in virus n - ppv was part of the nsp coding sequences. as the nt insertion was out-of-frame, we speculate that such recombination most likely happened at the rna level via non-homologous rna recombination. we demonstrated that the n protein truncated viruses were accompanied by multiple patterns of spontaneous mutations, whereas few missense mutations were found in other genomic regions of the p mutant viruses n / - , c / - , n - ppv , and wild-type aprrs. we cannot completely rule out the possibility that quasispecies nature may account for some of the additional mutations, as we essentially determined the consensus sequences of the mutant viruses by direct sequencing of the rt-pcr products. another reason for the lack of mutation is that the parental virus, aprrs, is highly adapted on marc- cells, which could limit the number of the quasispecies. more importantly, these spontaneous mutations are often in the forms of deletions and insertions, which could not be simply attributed to the quasispecies nature. our results suggested that the spontaneous mutations in n protein were caused by the introduced deletion rather than replication error. n protein is one of the genetically conserved structural proteins. on the contrary, we found various patterns of spontaneous mutations generated from the initial terminal deletion of n protein. in present study, we did repeat transfection to investigate the properties of the spontaneous mutations. however, almost all of the recovered viruses had different additional mutations in the independent transfection assay. for example, point mutation and out-of-frame insertion were found in the recovered virus c / - in two experiments (fig. b) . also the same spontaneous mutation might respond to different primary deletions, such as s p that arose simultaneously in the recovered viruses n / - and n / - . on the other hand, most of the spontaneous mutations were located at the n-terminal domain of n protein, the structure of which is still unresolved. therefore, it was difficult to illustrate any spatial relationship between the primary deletion and spontaneous mutations described in our study. in addition, we did not find a clear pattern of local charge compensation for the spontaneous mutations. another explanation of the spontaneous mutations would be the role of the underlying rna secondary structure. we also analyzed the local secondary structure of all mutants, but no significantly change was found. except for pn / - , none of the other mutants and the recovered viruses affects the potential kissing loop structure of n protein. therefore, the inherent mechanism still needs further manipulation of n protein and structural analysis of the n-terminal domain of n protein. taken together, we dissected the prrsv structure-function relationship and found that ( ) a stretch of inter-genotypic variable n terminal residues aa - ( ngkqqkkk k) were nonessential for virus viability; ( ) the last four inter-genotypic variable aa residues ( sps a) at the c terminus of n protein were dispensable for type prrsv viability; ( ) the internal aa residues ranging from inter-genotypic variable aa - ( kgp g) and inter-genotypic conserved - ( knpe k), respectively, were dispensable for type prrsv viability. our results indicated the non-essential regions in n protein for prrsv replication in cell culture, which lays a foundation for foreign gene expression and development of genetic tagged vaccine. structural polypeptides of the american (vr- ) strain of porcine reproductive and respiratory syndrome virus rna signals in the terminus of the genome of equine arteritis virus are required for viral rna synthesis an rna pseudoknot in the end of the arterivirus genome has a critical role in regulating viral rna synthesis characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) sequences of end of genome and of end of open reading frame a of lactate dehydrogenase-elevating virus and common junction motifs between leader and bodies of seven subgenomic mrnas mapping of the rna-binding domain of the porcine reproductive and respiratory syndrome virus nucleocapsid protein gp is a structural component of the prrsv type ii (us) virion all subgenomic mrnas of equine arteritis virus contain a common leader sequence current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicativeform rnas structure of the equine arteritis virus nucleocapsid protein reveals a dimer-dimer arrangement structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus nidovirales: evolving the largest rna virus genome coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus identification and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp encoded by orf is incorporated in virus particles subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence the arterivirus replicase is the only viral protein required for genome replication and subgenomic mrna transcription porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus the molecular biology of arteriviruses cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe proteins encoded by open reading frames and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion viable porcine arteriviruses with deletions proximal to the end of the genome kissing interaction between noncoding and coding sequences is essential for porcine arterivirus rna replication lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at lelystad homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein the b protein as a minor structural component of prrsv colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae reverse genetic manipulation of the overlapping coding regions for structural proteins of the type ii porcine reproductive and respiratory syndrome virus construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins recombinant prrsv expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus virus-encoded proteinases and proteolytic processing in the nidovirales coronavirus nucleocapsid protein is an rna chaperone coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this study was co-sponsored by the natural science foundation of china (# ) and the european union (seventh framework program; project no. ) to s.y. key: cord- -mn r x authors: hodgins, douglas c.; chattha, kuldeep; vlasova, anastasia; parreño, viviana; corbeil, lynette b.; renukaradhya, gourapura j.; saif, linda j. title: mucosal veterinary vaccines: comparative vaccinology date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: mn r x infections of mucosal surfaces are major causes of morbidity, mortality, and economic loss in species of veterinary interest, and a concern for animal welfare. vaccines are used extensively in veterinary medicine, and innovative vaccine technologies such as recombinant dna-vectored and distinguishing infected from vaccinated animals (diva) vaccines and automated in ovo vaccination (of embryonated chicken eggs) have been rapidly adopted commercially. immunological research using outbred, nonrodent animal models has contributed to a broader understanding of mucosal defenses, and has provided the initial impetus for investigation of the common mucosal immune system. studies of the potential of novel adjuvants to improve vaccine efficacy against genetically unstable, immune-subverting rna viruses, such as porcine reproductive and respiratory syndrome virus in pigs, should assist in the control of pathogens with similar characteristics in other species. successful development of vaccines to prevent and treat ascending infections of the reproductive tract of cattle set a precedent for applications in other species including humans. studies of mucosal adjuvants and delivery systems continue at the interface between passive and active immunity, with the goal of inducing the earliest possible protection against enteric and respiratory pathogens of neonates. despite advances in nutrition, genetics, housing, and therapeutics, diseases of the respiratory, reproductive, and gastrointestinal tracts of domestic animals and poultry continue to be major causes of morbidity and mortality. although vaccines have been developed and licensed for prevention of many of these diseases, there is a need for improvement in vaccine efficacy and for new vaccines. reasons for low vaccine efficacy include inappropriate, unstable, or outdated antigens in vaccine preparations (e.g., in vitro expressed antigens instead of in vivo expressed antigens), inappropriate immune responses (e.g., systemic instead of mucosal, or th type instead of th type or vise versa), and inappropriate use of otherwise efficacious vaccines (e.g., inappropriate timing of vaccination) (tizard, ). an overview of some of the vaccines currently licensed for vaccination of domesticated animals and wildlife by mucosal routes is provided in table . many of these vaccines consist of pathogens attenuated by traditional methods, but engineered virus-vectored vaccines are now used extensively (by both mucosal and parenteral routes) in veterinary medicine. the majority of current poultry vaccines are attenuated agents delivered in ovo, orally, intranasally (in) or by other mucosal routes, for reasons of ease of administration, economy, and protective efficacy. in comparison, fewer vaccines for domestic mammals are delivered by mucosal routes. management practices for mammals differ from those for poultry; mass vaccination techniques for mucosal delivery have been developed for poultry, but have not been pursued as zealously for mammals. recently however, a number of attenuated live vaccines for in administration have been developed for respiratory tract infections, using traditional methodologies. improved protective efficacy and rapid onset of immunity compared to killed vaccine products have led to widespread acceptance. attenuated live oral vaccines for enteric diseases have also been marketed, but in many cases efficacy has been disappointing due to lack of potency in adults or interference by maternal antibodies in suckling animals. better strategies for induction of immunity in the gastrointestinal tract are needed, especially for neonates in the presence of maternal antibodies. in contrast, effective parenteral vaccines for the most common diseases of the reproductive tract in veterinary species have been available for years, and there has been little motivation to develop mucosal vaccines. many of the diseases of the respiratory and gastrointestinal tracts are most devastating in the neonatal period. for these diseases active immunization may not provide protection before natural exposure to the pathogen. maternal vaccination to enhance passive immunity has been widely used in veterinary medicine, especially for control of enteric diseases. practical difficulties arise, however, with diseases such as parvovirus enteritis in puppies in which a smooth transition must be made from protection by passive maternal antibodies to protection by active immunity, without permitting a window in between of disease susceptibility. this transition is difficult to achieve because induction of active immunity is commonly inhibited by maternal antibodies. various strategies are used to address this problem, but improved vaccines, adjuvants, and antigen delivery systems would improve the reliability of neonatal immunization. although progress is being made in disease prevention in veterinary species, ever changing management practices (e.g., earlier weaning of piglets, larger animal operations) generate new patterns of disease, requiring new control strategies. the emergence of new pathogens (e.g., porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus- ) provides new challenges for vaccine research before some of the older challenges have been met. in this chapter, we focus on mucosal veterinary vaccines and vaccine concepts related to selected pathogens of economic importance. our intent is to highlight progress, to review existing and future vaccination strategies, and to acknowledge the unique contributions of this research to our understanding of mucosal vaccines and immunology. respiratory tract infections are a major cause of morbidity and mortality in farm animals, poultry, and pets. disease conditions are intensified by current management practices such as mixing of recently weaned, stressed beef calves from multiple sources in auction barns. certain disease conditions, such as atrophic rhinitis of pigs, result from the interplay of several pathogens, and multiple agents must be represented in vaccines. some respiratory pathogens such as mycoplasma hyopneumoniae in young pigs are causing new patterns of disease as management practices change (e.g., weaning at an earlier age), requiring changes in vaccine strategies. other pathogens such as prrsv have only recently emerged and improved vaccines await advances in understanding of the agent and of disease pathogenesis. a discussion of respiratory vaccines for even the major pathogens of veterinary species is beyond the scope of the present review. this section will focus on vaccines for prrsv infections in pigs and influenza in horses to illustrate general principles. porcine reproductive and respiratory syndrome (prrs) is a chronic reproductive and respiratory disease of pigs caused by prrsv, a member of the family arteriviridae (benfield et al., ) . signs associated with prrs are anorexia, fever, lethargy, pneumonia, red/blue discoloration of the ears and vulva, delayed return of sows to estrus after weaning, abortion, fetal mummification, stillborn or weak born piglets, and high preweaning mortality (mengeling et al., ) . prrsv is prevalent in swine-producing countries worldwide. annual economic losses to the pork industry in the united states due to prrs have been estimated at $ million (holtkamp and kliebenstein, ) . according to the american animal and plant health inspection service (aphis) report of january , . % of unvaccinated pigs in the united states are seropositive to prrsv. infected pigs excrete prrsv in saliva, nasal secretions, urine, milk, colostrum, and feces at low levels or intermittently, and also in semen of infected boars (rossow et al., ) . in the absence of control measures, prrsv is spread by aerosols, fomites, and personnel. prrsv is divided into two distinct genotypes, type i (european) and type ii (north american). each genotype contains subtypes and strains, which are genetically diverse and vary in virulence and pathogenicity (kim et al., ) . immunity to one genotype of prrsv may provide partial or no protection against reinfection, reflecting the complexity of prrsv genetics and immunological variation among strains (botner et al., ) . swine are the only known species susceptible to prrsv. alveolar macrophages (mΦs) are the primary permissive cells to prrsv; mΦs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta, and umbilical cord may also be infected by the virus (halbur et al., ) . the most efficient and rapid host response against viruses consists of production of type i interferons (ifns) (ifnα and ifnβ). in prrsv-infected pigs, innate ifnα secretion is significantly suppressed (albina et al., ) and the virus dampens innate nk cell-mediated cytotoxicity as early as day postinfection (renukaradhya et al., ; dwivedi et al., ) . in pigs, a significant correlation has been observed between the prrsv infection and an increased expression of il- , associated with reduced expression of ifnα, ifnγ, il- , and tnfα (gómez-laguna et al., ) . induction of il- is mediated by interaction between prrsv proteins and mΦs/dendritic cells (dcs). the prrsv nucleocapsid protein induces il- production by peripheral blood mononuclear cells (pbmcs) and alveolar mΦs (wongyanin et al., ) , delaying the onset of protective cell-mediated immunity (cmi) to prrsv (dwivedi et al., a . dysregulated immune responses in infected pigs affect viral pathogenesis, disease severity, and susceptibility to secondary microbial infections (thanawongnuwech et al., ; renukaradhya et al., ) . immune responses against prrsv are inadequate to completely clear the virus. viremia disappears in - weeks, but the virus persists in the tonsils and lymphoid tissues for several months (moyron-quiroz et al., ) . prrsv infection in both germ-free and conventionally raised pigs is associated with polyclonal b cell activation, hypergammaglobulinemia, lymphoid adenopathy, renal lesions, and lymphoid hyperplasia (cooper et al., ) . polyclonal lymphoplasia also occurs in mice infected with lactate dehydrogenase-elevating virus, a related virus (grossmann et al., ) . primary antibody responses occur promptly after prrsv infection (loemba et al., ) , but the majority of secreted antibodies are autoantibodies or prrsv nonneutralizing antibodies (lemke et al., ) . the virusneutralizing antibody (na) response is weak, and delayed (mateu et al., ) . both killed and modified live virus (mlv) prrsv vaccines are available commercially for intramuscular or intradermal administration (charerntantanakul, ) . mlv vaccines confer protection against genetically homologous prrsv, but incomplete protection against heterologous viruses (mengeling et al., ) . in both prrsv-infected and mlv-prrsv-vaccinated pigs, virus-specific cell-mediated immune responses are either delayed or dampened . reversion to virulence is a major concern with modified live vaccines. there are numerous reports of reversion of prrsv vaccine virus to virulence and of the presence of reverted vaccine strain prrsv in unvaccinated sows and pigs . vaccine-derived virus has been isolated from fetuses, stillborn, and dead piglets, indicating the spread of disease from vaccinated pigs. identification of mutations in multiple vaccine-derived isolates at identical positions of the viral genome suggests a strong selective pressure on critical viral proteins in field situations. available killed prrsv vaccines fail to protect even against homologous virus, do not elicit na, and induce weak cell-mediated immune responses (bassaganya-riera et al., ) . pregnant sows and gilts and breeding boars are not protected from prrsv due to a lack of safe and protective vaccines. control of prrsv in breeding stock is critical in preventing vertical and horizontal transmission of the virus. early attempts to develop killed prrsv vaccines using recombinant prrsv proteins, plasmids expressing viral genes, and inactivated prrsv administered with or without adjuvants have been unsuccessful ( charerntantanakul, ) . however, recent studies show promise. killed prrsv vaccine coadministered in with cpg oligodeoxynucleotide adjuvant (a tlr- ligand) induced antibodies, virus-specific t cells, and secretion of ifnγ and il- (zhang et al., ) . in another study, prrsv inactivated by uv light or binary ethylenimine induced na and protected pigs against homologous viral challenge. the suppressive effect of live prrsv on ifnα production was lost when the virus was inactivated by uv irradiation (vanhee et al., ). an oral immunization strategy using prrsv nucleocapsid protein genetically fused to cholera toxin (ct) stimulated prrsv antibody responses in the intestines, but no detectable response in vaginal secretions (hyland et al., ) . lack of success in developing protective killed prrsv vaccines may reflect an inability to present killed prrsv antigens effectively to the pig immune system, or (like live prrsv) killed virus may induce immunosuppressive effects. in addition, the antigenic mass used in killed vaccines may be insufficient, suggesting the need for potent adjuvants or novel delivery systems. attempts have been made to improve mlv-prrsv vaccines using adjuvants. in one study, pigs were injected with recombinant porcine il- , il- , il- , or ct within week of intramuscular administration of mlv-prrsv. il- and ct enhanced ifnγ and gp antibody responses, respectively (foss et al., ) . however, use of these adjuvants did not reduce the severity of viremia. unfortunately, prrsv (na) responses were not assayed, and protection against heterologous challenge was not assessed in this study. prrsv gains entry through respiratory and reproductive mucosal surfaces and causes disease primarily at mucosal sites. therefore, a mucosal vaccine against prrsv may be an effective strategy for controlling the disease. until recently, attempts to elicit protective mucosal immunity against prrsv have been unsuccessful, probably due to a lack of appropriate adjuvants. because prrsv infection rapidly subverts the host immune responses, an effective adjuvant must overcome immunosuppression caused by vaccine virus and simultaneously potentiate virus-specific adaptive immunity. a panel of bacterial preparations derived from mycobacterium, vibrio, and streptococcus species, which were potent adjuvants in rodent models, were tested in with mlv-prrsv (bonavida et al., ; barral and brenner, ) . based on mucosal and systemic immune responses, three of the preparations, mycobacterium tuberculosis whole cell lysate (m. tb wcl), ct b subunit, and ok- (a product of streptococcus pyogenes), were selected for viral challenge trials. only m. tb wcl significantly dampened prrsv immunosuppressive effects, and enhanced virus-specific adaptive responses (renukaradhya et al., ; dwivedi et al., b) . historically, heat-killed mycobacteria are recognized to have as potent adjuvant effects as components of freund's complete adjuvant (fca) and have been used extensively in experimental animals. the use of fca in humans and food animals is unacceptable due to severe granulomatous inflammatory reactions to toxic cell wall components ( bekierkunst, ). however, a nontoxic water-soluble purified fraction of m. tb wcl (werner et al., ) has adjuvant properties in rodents, guinea pigs, and rabbits. pigs inoculated in with m. tb wcl have no detectable signs of toxicity locally or systemically (dwivedi et al., a,b) . a recombinant vaccine (prrsv gp and m expressed in bacillus calmette-guerin (bcg)) is reported to reduce clinical signs of prrs with decreased viremia and viral load in the tissues (bastos et al., ) . cross-protective immunity against prrsv has been evaluated in pigs receiving mlv-prrsv and m. tb wcl in, by challenging with virulent heterologous prrsv virus (kim et al., ) . reduced clinical disease, viremia, and virus-mediated immunosuppression were noted. in addition, secretion of ifnγ and il- by lung and blood lymphocytes in response to prrsv m and nucleocapsid proteins was enhanced (dwivedi et al., a) . guillonneau et al. ( ) reported similar findings in mice vaccinated in with adjuvanted influenza vaccines. enhanced virus-specific cytotoxic t cell and memory responses to internal viral proteins were noted, as well as cross-protective immunity. studies of passive protection of pigs by prrsv na have indicated that modest na titers (≤ / ) protect pregnant sows against reproductive failure, block placental transmission of infection, prevent viremia in piglets, and provide sterilizing immunity (osorio et al., ) . pigs immunized in with mlv-prrsv alone or with m. tb wcl developed na titers < / and / , respectively, postimmunization (dwivedi et al., b) . in pigs immunized (mlv-prrsv + m. tb wcl) and challenged with prrsv mn , an na titer > / persisted until pid (dwivedi et al., a) . relatively low numbers of circulating virus-specific ifnγ secreting cells have been reported for pigs vaccinated in with mlv-prrsv without adjuvant, ( - cells per million pbmcs) (dwivedi et al., a) . in contrast, meier et al. ( ) have reported - ifnγ secreting cells per million pbmcs in pigs vaccinated against aujeszky's disease virus. pigs immunized in with mlv-prrsv with m. tb wcl and challenged with prrsv had greater than ifnγ secreting cells per million pbmcs, and more than a twofold higher frequency of cd + cd + t cells (dwivedi et al., a) . the literature concerning mucosal immune responses in the pig respiratory tract is limited compared to studies of rodents and humans. recent research highlights the advantages of activating the mucosal immune system using vaccines delivered with potent adjuvants. induction of adequate immune responses in the respiratory and reproductive tract will be essential for control of prrs in swine. in-delivered, potent mucosal vaccines generate better cross-protective immunity against genetically variable prrsv field viruses. efforts to control prrs outbreaks using conventional parenterally delivered vaccines have had limited success; development of an alternative approach of generating immunity in the respiratory tract should be a priority. in particular, mucosal adjuvants based on components of mycobacterial species show promise. respiratory tract disease affects virtually every aspect of equine husbandry, including working, pleasure, and race horses. considerable efforts are expended to prevent epizootics of respiratory disease in stables, fairs, shows, and race tracks. equine influenza virus will be discussed in detail as an example of past, current, and future vaccine approaches. equine influenza virus causes epizootics of upper and lower respiratory tract disease almost worldwide. until equine influenza was excluded from the continent of australia through import restrictions and quarantine. in august of that year, importation of an infected horse led to an explosive epizootic of equine influenza with an estimated , horses infected over a -month period (callinan, ) , demonstrating the potential of the virus to spread in a naïve, unvaccinated population. infection can occur in horses of all ages, but epizootics often involve younger animals (van maanen and cullinane, ) . clinical signs include high fever, a persistent dry cough, nasal discharge, anorexia, and depression. secondary bacterial pneumonia may complicate the clinical picture. equine influenza viruses are classified as type a influenza. antigenic differences in the hemagglutinin (h) and neuraminidase (n) glycoproteins define the two recognized subtypes, a/equine/ (h n ) and a/equine/ (h n ) (wilson, ) . two lineages of a/equine/ , american and european, have been identified; multiple virus strains are included in vaccines since protection against heterologous strains is incomplete (yates and mumford, ) . antigenic drift is sufficient to require regular reappraisal of strains included in vaccines (mumford and wood, ) . natural infection induces iga antibodies in nasal secretions, igga and iggb antibodies in serum nelson et al., ) , and circulating cytotoxic t lymphocytes . protection against reinfection persists for at least a year (hannant et al., ) . vaccination with inactivated virus vaccines induces serum igg (t) antibodies without detectable iga in nasal secretions (nelson et al., ) and without cytotoxic t cell activity (van maanen and cullinane, ) . two or three doses of vaccine are typically administered in the primary series, with booster doses at least once a year thereafter. more frequent vaccination is advised for horses at high risk of infection (wilson, ) . protection is typically incomplete and of limited duration. improved adjuvants can enhance the level and duration of antibody responses to inactivated virus vaccines (mumford et al., c) . suppressive effects of maternal antibodies on responses to inactivated vaccines have led to recommendations not to vaccinate foals before months of age (van oirschot et al., ) . a subunit equine influenza vaccine based on the immune stimulating complex (iscom) adjuvant technology has been licensed and marketed in europe since (newmark, ) . antibody responses to iscom-based vaccines typically are of higher titer and are more persistent than for conventional inactivated vaccines (mumford et al., a) . protection has been demonstrated against experimental challenge, months after a three dose vaccination series (mumford et al., b) . protection may be due in part to the ability of iscom-adjuvanted vaccines to induce cytotoxic t lymphocytes (morein et al., ) . although iscom-based vaccines can induce iga antibody responses following in administration (hu et al., ) , the commercial influenza iscom vaccine is administered parenterally. in administration of inactivated equine influenza virus with ct b has been reported to induce local iga antibodies, and protection against experimental challenge (hannant et al., ) . a cold-adapted, temperature-sensitive live vaccine for in administration is available commercially . protection against experimental challenge has been demonstrated months after a single vaccination . this is a notable improvement in efficacy and practicality over conventional killed vaccines. experimental plasmid vaccines encoding the hemagglutinin gene of equine influenza have been examined in horses. three doses of plasmid administered to the skin and mucosal sites (tongue, conjunctiva, and third eyelid) induced protection against clinical disease and partial protection against viral shedding. protection against clinical disease was reduced if plasmid was administered only to the skin (lunn et al., ) . a canarypox-vectored equine influenza vaccine, expressing hemagglutinins from two strains of h n equine influenza, has been available commercially (intramuscular administration) since (toulemonde et al., ) . in the australian epizootic of , this vaccine was used extensively during the government program to eradicate equine influenza. the vectored vaccine was chosen for this program because of its diva (distinguishing infected from vaccinated animals) characteristics ( kirkland and delbridge, ) . infected horses mount antibody responses to the viral nucleoprotein in addition to the viral hemagglutinin, whereas vaccinated horses respond only to the hemagglutinins expressed by the vaccine vector. a recent report indicates that adequate antibody responses are generated with as little as days between primary and secondary vaccination (el-hage et al., ) . protection against experimental challenge has also been noted weeks after a single dose of vaccine in ponies ( toulemonde et al., ) . for some respiratory pathogens (e.g., m. hyopneumoniae in pigs) the critical antigens associated with protective immune responses have not been identified. for other pathogens (e.g., prrsv) there is also a need to identify the appropriate type of immune response (th or th ) needed for protection. for some complex disease conditions (e.g., bovine respiratory disease complex) there is continuing uncertainty whether all of the relevant contributing pathogens have been identified. although many parenteral vaccines are efficacious in reducing lower respiratory tract disease, there is a need to investigate whether induction of mucosal immunity in the upper airways, in combination with systemic immunity, can further reduce infection rates, transmission of pathogens, and economic losses. finally, there is a need to devise and implement changes in management procedures to reduce disease exposure (by nonimmunological methods), and to optimize immune interventions by improved timing of vaccinations. vaccines to prevent reproductive tract disease have received much emphasis in veterinary medicine. this is especially true of food-producing animals because reproductive failure is a major economic problem. although vaccines to prevent reproduction are of interest for abandoned pets or deer in areas of overpopulation, this section will deal only with vaccines designed to prevent infectious disease of the reproductive mucosa. infections causing adverse pregnancy outcome can be classified by route of infection: hematogenous or ascending. several hematogenous infections have a predilection for the gravid uterus resulting in early to late abortions . these include leptospirosis, chlamydial infection, and brucellosis in several animal species, histophilus somni infection in cattle and sheep and neospora caninum in cattle. although vaccines are available for several of these infections, the vaccine for brucellosis has been available since the s and is the most well known. several brucella species cause abortion or epididymitis/orchitis in the primary hosts ( brucella abortus in cattle, brucella suis in swine, brucella melitensis in goats, brucella ovis in sheep, brucella canis in dogs, and brucella marinum (or brucella delphini) in marine mammals). infection may be acquired via the gut mucosa or the conjunctiva/upper respiratory mucosa and localizes in the reticuloendothelial system and endometrium/placenta by systemic spread. thus, systemic vaccines are effective. a modified live b. abortus vaccine, along with a "test and slaughter" eradication program, has been successful in controlling bovine brucellosis in north america. the modified live vaccine (b. abortus strain ) is very effective in stimulating cmi that is critical for protection against this facultative intracellular pathogen. strain now has been largely replaced by a new attenuated strain, rb , which does not stimulate an antibody response known to interfere with diagnostic serologic assays. there is considerable information on mechanisms of systemic immunity to brucellosis. neospora caninum also causes abortion by a hematogenous route and a vaccine is available (weston et al., ) . histophilus somni can infect either by the hematogenous route or by an ascending genital route to cause abortion or infertility and vaccines are available. since the focus of this volume is mucosal immunity, no more will be said concerning protection against hematogenous infections of the genital tract. ascending local infections of the reproductive tract are usually transmitted sexually. the two best examples of vaccines for sexually transmitted infections of animals are campylobacter fetus subsp. venerealis (formerly vibrio fetus subsp. venerealis) and tritrichomonas foetus. both are host-specific bovine sexually transmitted diseases (stds) that only infect the reproductive mucosa. both are extracellular pathogens that do not invade the mucosa of the reproductive tract but may be found in the placenta and fetus. the localized nature of these infections and transmission limited to coitus suggest that mucosal immunity must be important. because a vaccine has been available for c. fetus subsp. venerealis for several decades and its use has controlled the disease in developed countries, that vaccine will be discussed first. vibriosis (or campylobacteriosis) is a chronic bacterial genital infection with no overt clinical signs other than reproductive failure (corbeil et al., ) . after months of infection, the uterus is cleared first, followed by the vagina. convalescent immunity is partially protective for a limited time. antibody is effective in protection against this extracellular pathogen as demonstrated by systemic passive immunization (berg et al., ) . the antibody response to infection is primarily iga in the vagina and igg in the uterus (corbeil et al., ) . systemic immunization with a whole cell vaccine results in both igg and igg antibody responses to surface antigen in serum, and uterine and vaginal secretions (corbeil et al., ) . this response prevents infection and can rapidly clear infected cows (corbeil and winter, ) . that is, the vaccine can be used prophylactically and even therapeutically. immunization is efficacious even though surface antigenic variation occurs in the face of a local immune response (corbeil and winter, ) . presumably, immune clearance occurs when the dynamic interaction between protection and evasion is shifted in the favor of the host. this appears to occur earlier when the response is primarily igg than when iga predominates (corbeil et al., ) . this may be related to the ability of the igg antibodies to mediate opsonization and intracellular killing of the bacterium, an ability which iga antibodies lack (corbeil and winter, ) . although this work was done many years ago, it sets a precedent for systemic immunization for prophylaxis and therapy of reproductive mucosal infections. trichomoniasis is a similar chronic genital mucosal infection of cattle. it is caused by the protozoan, t. foetus, and results in pregnancy loss. trichomonas vaginalis causes a human std also associated with adverse pregnancy outcome. thus, bovine trichomoniasis serves as a model for immune prevention of both bovine and human reproductive mucosal infections. because of the economic significance of bovine trichomoniasis and because no chemotherapy is approved, investigations have focused on immunoprophylaxis and immunotherapy. t. foetus colonizes the vaginal and uterine or preputial surfaces for months, like c. fetus subsp. venerealis infection. in fact, mature bulls are often infected for life whereas young bulls may clear the infection with time (cobo et al., ) . this is probably related to innate immunity. trichomonads are anaerobic parasites and are found deep in uterine glands and epithelial crypts of the penis and prepuce (rhyan et al., ) where the oxygen tension is probably lowest. older bulls have deeper epithelial crypts, which may have lower oxygen tension. in order to understand protective acquired immune responses, monoclonal antibodies (mabs) with putative protective functions were chosen for immunoaffinity purification of a highly glycosylated surface antigen (bondurant et al., ; corbeil et al., ) . analysis of many isolates of t. foetus indicated that two mabs recognized different epitopes of the same antigen, which was conserved in all isolates tested. this glycosylated surface antigen was later shown to be a lipophosphoglycan (lpg)/protein complex. systemic immunization with the immunoaffinity-purified lpg-containing surface antigen, followed by intravaginal challenge with t. foetus, resulted in significantly earlier clearance of the parasite from vaccinated animals than from controls (bondurant et al., ; corbeil et al., ) . more importantly, clearance of immunized animals most often occurred before weeks of infection. parsonson et al. ( ) showed that significant inflammation accompanied by reproductive failure did not occur until after weeks of infection, so the vaccine should protect against fetal loss. analysis of vaccine-induced antibody responses demonstrated predominantly igg responses in the serum and iga plus igg antibodies in secretions (corbeil et al., , . ige antibodies were also increased during infection. as ige antibodies increased, mast cells degranulated and clearance of t. foetus occurred. these studies raised several questions. first, why is the systemic response to the lpg/protein complex skewed toward igg (a th response in cattle) and not igg (a th response)? this question is under investigation. second, are igg or iga antibodies to the lpg-containing antigen most protective and how can that ig isotype be enriched to enhance protection? to address the latter questions, preliminary studies were done in mice to determine the best routes and adjuvants to enrich for igg or iga in genital secretions . subcutaneous priming with the immunoaffinity-purified lpg-containing surface antigen (called tf . antigen) in quil a adjuvant and subcutaneous boosting with whole cells enriched for igg anti-tf . antibodies in genital secretions whereas subcutaneous priming and intravaginal boosting greatly enriched for iga antibodies in genital secretions. when cattle immunized by these two methods were challenged intravaginally with t. foetus, those with predominantly iga or predominantly igg anti-tf . antibodies in genital secretions were equally protected. later studies with similar in immunizations showed that stimulation of the common mucosal immune system gave results similar to those of intravaginal immunization . this raised the question of inductive sites for local immune responses in the genital tract. others have suggested that the genital tract is not an inductive site because m cells and mucosa-associated lymphoid tissue (malt) are not present. this is true of cattle as well as mice and people. however, even though control cows did not have histologically demonstrable malt in the uterus and vagina, cows experimentally infected with t. foetus did . similar lymphoid nodules and follicles under a modified epithelium were detected in preputial and penial surfaces of bulls infected with t. foetus (rhyan et al., ) . immunostaining of parallel sections with mab to tf . antigen indicated uptake of antigen by epithelial cells and large mΦ or dc under the basement membrane near the lymphoid follicles (rhyan et al., ) . similar antigen uptake has been detected in the infected female uterine and vaginal mucosa (corbeil et al., ) . thus, even though the parasite is noninvasive, released tf . antigen appears to be taken up by epithelial cells. rat uterine epithelial cells can present antigen to t helper cells (wira and rossol, ) . also, mΦ/ dc positive for antigen should be antigen-presenting cells (apcs). detection of igg and iga antibodies in genital secretions of infected bulls (cobo et al., ; rhyan et al., ) and cows and the histologic demonstration of follicles and putative apcs suggests that inductive sites in the genital tract are formed in response to antigenic stimulation. like c. fetus subsp. venerealis, t. foetus has mechanisms for evasion of immune responses. these include coating of the surface with ig nonspecifically , epitope variation (ikeda et al., ) , and cleavage of igg , igg , and complement component by extracellular cysteine proteinase (talbot et al., ; kania et al., ) . studies with cysteine proteinase inhibitors demonstrated decreased cytotoxicity of t. foetus for bovine trophoblast cells and decreased infectivity in a mouse model, confirming the likely role of cysteine proteinases in pathogenesis (cobo et al., ) . even with these mechanisms for immune evasion, as with c. fetus, it is clear that the dynamic interaction between host and parasite can be made to favor the host by systemic or local immunization. the usefulness of whole cell vaccines in preventing t. foetus infection and reproductive failure in cows has been demonstrated in clinical trials (kvasnicka et al., ) . first-generation whole cell vaccines are now commercially available for prevention of trichomoniasis in cows. earlier, clark et al. ( ) demonstrated efficacious immunization of bulls with whole t. foetus cells or crude membrane glycoproteins that probably contained tf . antigen. this study indicated that vaccination of bulls could both prevent infection and clear already established infection. so vaccines for bovine trichomoniasis are both immunoprophylactic and immunotherapeutic. recent studies showed that vaccination of bulls with a commercially available whole cell killed t. foetus vaccine in oil adjuvant resulted in protection against challenge and high levels of igg antibodies in serum and preputial secretions (cobo et al., ) . igg antibodies to whole cell t. foetus antigens predominated but fairly high levels of igg antibodies were also detected. the above studies with c. fetus subsp. venerealis and t. foetus show that: . stds can be prevented or even cured by systemic vaccination of both males and females. . at least for one std, igg and iga of the same antigenic specificity are equally protective at the mucosal surface. either systemic immunization or mucosal (in or intravaginal) immunization with systemic boosting is protective. . inductive sites are formed in the mucosa of infected male and female genital tracts even with noninvasive pathogens. . strong and appropriate immune responses will clear microbial infection from the genital tract even when the microbe has multiple immune evasive mechanisms. . the fact that protection against two stds has been demonstrated in the natural outbred host (cattle) has advantages over murine models of std vaccines. in the latter, the human pathogen is usually inoculated into the unnatural murine host and the disease does not mimic the human infection. furthermore, although inbred mice provide a homogeneous experimental animal, they do not represent the variation in immune responses seen in the outbred human population. the work on bovine vibriosis and trichomoniasis demonstrates protection under field conditions for two stds that cause adverse pregnancy outcome in an outbred host. this is an encouraging precedent for control of human stds and related adverse pregnancy outcomes. future needs include identification of the protective antigens for most stds. for antibody-mediated protection of the genital mucosa, several questions have not yet been addressed. as far as we know, the role of ige in the genital tract is largely unstudied even though it does seem to be involved in clearance of trichomonads from the bovine genital tract. lastly, manipulating genital immune responses to enhance th or th type responses is an unexplored research area. enteric disease is a major cause of mortality and morbidity in animals. agents causing diarrhea in animals include viruses (e.g., adenoviruses, pestiviruses, caliciviruses, coronaviruses, parvoviruses, rotaviruses, toroviruses), bacteria (e.g., campylobacter spp., clostridium spp., diarrheagenic escherichia coli, salmonella spp., yersinia spp.), and parasites (e.g., coccidia spp., cryptosporidium parvum). these infections occur most commonly in suckling animals or in poultry under weeks of age, but may also be common postweaning and in susceptible seronegative or stressed adult animals (saif and jackwood, ) . induction of local secretory iga (s-iga) antibodies (prevent attachment, invasion, and neutralize toxins or infectious agent) and mucosal cellular immune responses (against intracellular bacteria and viruses) by vaccines are essential for protection from enteric pathogens. peyer's patches (pp) and mesenteric lymph nodes are two important organized gut-associated lymphoid tissues (galt) in domestic animals and serve as the induction site for gut immune responses. ileal pp in some domestic animals (sheep, cattle, pigs, dogs) differ from jejunal pp, serving as a primary lymphoid organ similar to the bursa of fabricius in chickens, unlike in humans and rodents where pp are secondary lymphoid organs (chu and liu, ; yasuda et al., ) . cryptopatches or clusters of lymphoid cells in the basal lamina propria occur in mice, but are absent in pigs (pabst et al., ) . mucosal lymphoid tissues and lymphoid cells in domestic animals and humans differ in toll-like receptor (tlr) expression and function (after binding their ligand) when compared to mice (iwasaki and medzhitov, ; tohno et al., ; guzylack-piriou et al., ) . these differences in immune components suggest that vaccine studies carried out in mice may fail to translate to domestic animals or humans. enteric pathogens have different characteristics related to their intestinal tropism and replication, requiring different vaccination strategies. enteric viruses have predilections for replication in distinct vertical and longitudinal regions of the small or large intestines. cytolytic infection of enterocytes leads to varying degrees of villous loss and fusion, reduced small intestinal absorptive capacity, and a malabsorptive, maldigestive diarrhea (saif, a) . secretory diarrhea is also induced by some viruses such as rotavirus (rv), which involves the viral enterotoxin, nonstructural protein (nsp ), and/or stimulation of the enteric nervous system (ball et al., ; lundgren et al., ) . thus, viral diarrheas can be of variable severity and act via multiple mechanisms that differ from those of enteric bacteria, most of which cause secretory diarrhea mediated by enterotoxins (fairbrother and gyles, ) . enteropathogenic viruses can be divided into three types (types i, ii, and iii) according to their preferred site of replication in the intestine (reviewed by saif ( a) ). type i (transmissible gastroenteritis virus (tgev) and rv) and ii virus infections can be prevented by inducing local intestinal immunity, whereas type iii viruses (parvovirus), which infect crypt enterocytes basolaterally, can be prevented by inducing systemic immunity. the enteropathogenicity of bacteria is determined by their virulence factors including adhesion factors (fimbriae or pili) and enterotoxins; therefore, bacterial vaccines generally need to prevent attachment and toxin action within the intestine (fairbrother and gyles, ) . in the following sections, we will review vaccine strategies for these different types of enteric infections. tgev and rv vaccines in pigs will be reviewed to illustrate findings using domestic outbred animals instead of inbred laboratory rodent models. neonates can be protected from enteric infections by providing passive lactogenic immunity, which can be achieved by immunizing mothers preparturition. pioneering work done by bohl and saif in the early s with live oral virulent tgev in pigs was the foundation for the gut-mammary gland-s-iga immunologic axis, which later became the basis for the concept of the common mucosal immune system. their studies showed that in tgev seronegative sows, only oral immunization with virulent tgev induced high rates of protection in suckling neonates, which was associated with high titers of s-iga antibodies in colostrum and milk, whereas systemic immunization induced mainly igg antibodies saif et al., ) . rotavirus is a major cause of dehydrating diarrhea in young livestock, infants, and poultry (saif and fernandez, ) . multiple rv serogroups, based on common inner capsid vp antigens (a-h), and multiple g (vp , glycoprotein) and p (vp , protease-sensitive) serotypes (based on neutralizing epitopes) or genotypes (based on sequence analysis) for group a rvs have been detected in humans, sheep, swine, cattle, horses, dogs, cats, poultry, and wildlife (estes and kapikian, ; martella et al., ) . among the distinct rv serogroups and g/p serotypes/genotypes, cross-protection is limited. the antigenic divergence among different sero/genotypes of rvs presents a challenge for the design of vaccines that are capable of inducing heterotypic protection. commercial modified live and killed rv vaccines for rv diarrhea in livestock and poultry are limited to group a rvs (saif and fernandez, ) . mebus et al. ( ) developed the first oral rv vaccine for calves in ( year prior to the discovery of human rv) using a cell cultureadapted neonatal calf diarrhea rv strain. although a significant reduction in morbidity and mortality was observed in a field trial among vaccinated calves, in the majority of herds (compared to previous years), subsequent field studies revealed variable efficacy. experimental studies suggested that maternal antibodies interfered with live oral vaccine replication and suppressed development of active immunity (saif and fernandez, ) . the neonatal gnotobiotic pig model has been used to investigate immune responses to rv vaccines and infection for nearly three decades (saif et al., , yuan and saif, ; gonzalez et al., ) . gnotobiotic pigs are free of maternal antibodies (placental transfer of igs does not occur in swine), but they are immunocompetent at birth. they are maintained aseptically and are free of exposure to extraneous wildtype rvs, assuring that exposure to a single pathogen can be analyzed. initial studies were conducted to mimic natural rv infection (bohl et al., ) and to study immune correlates of protection gonzalez et al., ; yuan et al., ) . understanding the sequence and kinetics of immune responses stimulated by virulent rvs allows for the determination of markers of protective immunity and pathogenicity, which can then be used to design vaccines that will stimulate protective immunity without inducing pathology. gnotobiotic pigs orally inoculated with virulent porcine or human rvs were completely protected from homotypic but not heterotypic (distinct g/p types) rv challenge (hoshino et al., ; saif et al., ) . significant correlations were observed between the protection to rv-induced diarrhea and shedding and the following immune parameters: the number of intestinal iga rv antibody-secreting cells (ascs), serum and intestinal iga rv antibody titers, and the frequency of intestinal rv-specific ifnγ producing cd + and cd + t cells yuan et al., ; to et al., ; yuan and saif, ; yuan et al., ) . these immune parameters were significantly higher in virulent rv inoculated pigs than in those inoculated with attenuated or inactivated rv (ward et al., b) . pigs inoculated with two or three doses of attenuated rv were moderately protected against diarrhea ( %- doses, %- doses) and virus shedding ( %- doses and %- doses) after homotypic challenge, suggesting a need for multiple doses and suitable mucosal adjuvants to enhance the efficacy of rv vaccines yuan et al., yuan et al., , yuan and saif, ) . gnotobiotic pigs inoculated with virulent rv developed an acute proinflammatory serum cytokine profile (il- , tnfα) coinciding with peak diarrhea and viremia, followed immediately by increased th (il- , ifnγ) cytokines and convalescent th (il ) and tr (il- ) cytokine responses. gnotobiotic pigs inoculated with one dose of attenuated rv showed lower early ifnγ and proinflammatory cytokine responses compared to virulent rv inoculated pigs. both attenuated and virulent rv inoculated pigs developed th /th /tr cytokine-secreting cell (csc) responses at - weeks postinoculation; however, attenuated rv inoculated pigs developed lower intestinal ifnγ and higher intestinal and splenic il- cscs compared to virulent hrv inoculated pigs (azevedo et al., ) . virulent rv inoculated pigs had significantly higher protection rates against rv challenge ( % and % against diarrhea and shedding, respectively) compared to one dose of attenuated rv inoculated pigs ( % and % against diarrhea and shedding, respectively) (azevedo et al., ) . these findings suggest that higher protection rates are associated with early proinflammatory and th cytokine responses, which promote cytotoxic t cell activity and viral clearance, and later th induced cytokines, which are important for protective s-iga antibody responses. thus, protection against rv in pigs requires balanced th /th /treg responses (azevedo et al., ; gonzalez et al., ) . infection of gnotobiotic pigs with virulent rv causes early recruitment of innate apcs (monocytes/mΦ and dcs) and γδ t cells into the ileum, and enhanced tlr , tlr , and tlr expression among apcs in spleen (zhang et al., c; wen et al., wen et al., , . in virulent rv-infected pigs, plasmacytoid dcs are major producers of serum ifnα and, along with other innate immune cells (γδ t cells and apcs) and cytokines (tnfα, ifnγ and il- ), are important for controlling early rv viral replication and subsequent development of protective adaptive immune responses . development of attenuated rv vaccines with mucosal adjuvants that mimic immune responses to virulent rv may improve existing vaccines. immunogenicity and protective efficacy of rv vaccine formulations (attenuated replicating virus, inactivated virus, and recombinant baculovirus-expressed virus-like particles (vlp)), administration routes, and adjuvants have also been evaluated using the gnotobiotic pig model (bohl et al., ; yuan and saif, ; gonzalez et al., ) . inactivated oral or intramuscular rv vaccines failed to protect against virulent rv challenge, despite high igg antibody responses induced in serum and systemic lymphoid tissues. serum igg antibodies did not correlate with protection. however, a recent study showed that an inactivated reassortant rv strain (cdc- strain) formulated with aluminum phosphate and administered systemically in gnotobiotic pigs resulted in induction of serum igg antibody titers, coinciding with partial protection against shedding and diarrhea, suggesting that adjuvant may have stimulated local specific (gut iga antibodies) or nonspecific immune responses, which were not assessed in this study (wang et al., ) . rotavirus subunit vaccines consisting of double-layered vlp composed of rv inner capsid proteins vp and vp ( / -vlps) administered in or orally with mutant heat-labile toxin of e. coli (mlt) or iscoms as adjuvants (yuan and saif, ) induced igg asc responses in systemic lymphoid tissues and low or no iga asc responses in intestinal lymphoid tissues and also failed to mediate protection, contrary to results in adult mouse studies (yuan and saif, ) . the failure of in or oral / -vlp vaccines suggests that protective immunity to rv diarrhea in neonatal pigs requires mainly the presence of systemic or intestinal iga antibodies to the outer capsid rv proteins, vp and vp , each of which induces na. however, when / -vlps adjuvanted with mlt or iscom were used as in or oral booster doses in pigs orally primed with attenuated rv, the protective efficacy increased significantly. an oral attenuated rv prime and in / -vlp-iscom boost regimen (attrv/ / -vlp) induced the highest numbers of intestinal iga ascs and serum and intestinal iga antibody titers, and protection rates were similar to or higher than those induced by three oral doses of attenuated rv (gonzalez et al., azevedo et al., ) . interestingly, priming with two doses of / -vlp (in or oral) followed by live attenuated rv was ineffective for inducing iga antibodies or protection. thus the use of a replicating vaccine to prime at one inductive site (galt) followed by boosting with a nonreplicating vaccine at a second mucosal inductive site (nasal-associated lymphoid tissue, nalt) is an effective strategy for stimulating protective mucosal immune responses, which can be applied to other enteric viral vaccines. furthermore, efficacy of the prime/boost strategy (replicating/nonreplicating vaccine, attrv/ / -vlp) was examined in the presence of high and low titers of passive antibodies to mimic neonatal pigs receiving maternal antibodies (nguyen et al., a,b) . similar to attrv/ / -vlp prime/boost vaccine studies, plasmid dna containing vp was used as a booster subsequent to oral attenuated rv vaccine priming. this regimen showed high protection against shedding, but poor efficacy against rv diarrhea (yuan et al., ) . collectively these findings suggest that mucosal boosters are effective in enhancing iga antibody titers to rvs in orally primed animals . studies have shown that immunogenicity and efficacy of mucosal vaccines can be improved by the use of appropriate strains of probiotics that modulate mucosal and systemic immune responses, by interaction with epithelial cells and the underlying intestinal immune cells (fukushima et al., ; sanz and de palma, ) . certain probiotic strains are reported to enhance immune responses to rv vaccines in children; others reduce rv diarrhea severity, but the mechanisms are not well defined (fang et al., ; holscher et al., ) . supplementation with lactobacillus acidophilus in attenuated rv vaccinated pigs is reported to enhance intestinal ifnγ-producing cd + t cells, intestinal iga and igg rv ascs, and serum iga and igg rv antibody titers (zhang et al., b) . these findings suggest that probiotics are an alternative, cheap, and safe adjuvant for enhancing efficacy of oral attenuated rv vaccines in animals and potentially in humans. colonization of pigs by two different strains of lactic acid bacteria (lab, l. acidophilus and lactobacillus reuteri) and subsequent virulent rv infection resulted in higher and balanced th /th /treg cytokine responses (il- , ifnγ, il- , il- , tgfβ), higher total intestinal iga-secreting cells, total serum igm, and intestinal igm and igg titers, although the numbers of iga rv ascs and serum and intestinal rv antibody titers did not differ compared to virulent rv-only infected pigs. no overall difference in protection rates was noted when compared to virulent rv-only inoculated pigs, which was likely because of the short interval (only days) between lab colonization and virulent rv challenge (zhang et al., a; azevedo et al., ) . dual colonization of the aforementioned lab strains also modulated innate immune components in virulent rv inoculated pigs as follows: ( ) enhanced frequency of γδ t cells in the intestine and their distribution; ( ) enhanced tlr and tlr -expressing cd + apc, and tlr -expressing cd − apcs in the blood, but reduced tlr -and tlr expressing cd − apcs in the spleen, and ( ) reduced frequency of mΦ and cdcs in the spleen. collectively, these findings suggest that probiotics may reduce systemic inflammatory responses induced by virulent rv. effects on tlr expression on apcs in the ileum were not determined (zhang et al., c; wen et al., wen et al., , . probiotics may not only modulate immune responses to rvs or other enteric vaccines, but may also directly ameliorate diarrhea/infection by enhancing gut barrier integrity and maintaining intestinal permeability, by stimulating nonspecific immune responses, by changing gut microbial populations, and by aiding in the regulation and prevention of apoptosis ( madsen et al., ; yan and polk, ; preidis et al., ) . using the tgev model for evaluation of active protection against diarrhea in pigs, researchers also delineated compartmentalization in the common mucosal immune system and its impact on mucosal vaccine strategies and protection (vancott et al., (vancott et al., , . the natural occurrence of a deletion mutant of tgev with exclusive respiratory tropism, referred to as porcine respiratory coronavirus (prcv), provided a unique opportunity to study asc responses and protective immunity to two antigenically related porcine coronaviruses with enteric (tgev) versus respiratory (prcv) tropism. oral immunization of pigs with tgev induced high numbers of iga asc in the intestine and provided complete protection against tgev challenge, whereas in immunization of pigs with prcv induced mainly systemic responses (igg asc) and provided only partial protection against tgev challenge. thus, the in prcv alone failed to elicit sufficient intestinal iga asc to provide full protection against the enteric pathogen, tgev. findings from this study and rv vaccine studies suggest that the use of multiple mucosal inductive sites in a prime/boost vaccination regimen may be an effective approach for overcoming compartmentalization in the common mucosal immune system. canine parvovirus (cpv) infects crypt enterocytes causing hemorrhagic gastroenteritis in pups (saif and jackwood, ) . because cpv is likely disseminated to the basolateral surface of crypts by the hematogenous route, serum na (derived maternally or actively produced) is protective against the disease. pollock and carmichael ( ) demonstrated that pups with hemagglutination inhibition (hi) serum antibody titers of > : were immune to oronasal cpv type challenge. cpv is highly stable in the environment and pups were susceptible to infection as soon as maternal antibodies declined to hi titers of : - : . a maternal hi antibody titer as low as : severely affected the efficacy of a live low titer cpv vaccine ( - th passage in culture) in generating active immune responses (carmichael et al., ) , which resulted in a window of susceptibility for pups. others have shown that an hi titer of less than : in cpv- (low passage, high titer) vaccinated pups did not severely affect active antibody responses (burtonboy et al., ; hoare et al., ) , suggesting that increasing the dose or reducing the attenuation of the virus may help to overcome inhibitory effects of maternal antibodies. studies with modified live variant cpv- b strain (low titer) have shown that these vaccines, when given either parenterally ( th passage) or in ( th tissue culture passage), elicited almost % protection in pups with maternal antibody titers of : to : and even % protection in pups with antibody titers of : . higher efficacy of these vaccines can be attributed to either strong inherent immunogenicity of these new vaccines or antigenic differences among cpv- and cpv- a and cpv- b (pratelli et al., ) . overall, during the past four decades of cpv vaccine development, modified live viruses have proven superior to inactivated intramuscular (im) vaccines (appel, ) . in brief, various strategies such as the use of less attenuated virus (low serial passage), high titer vaccines, multiple doses, or the use of the in route of immunization have been attempted to overcome inhibitory influences of maternal antibodies and to reduce the window of susceptibility in pups. recently new variant cpv- c has emerged, which is highly pathogenic and causes more severe diarrhea in dogs. currently used vaccines (cpv- or cpv- b strains) are effective in protecting dogs against challenge with cpv- c virus under experimental conditions (spibey et al., ) , although efficacy in the field is unknown (reviewed by decaro and buonavoglia ( ) ). antigenic differences between original cpv- and its variants may reduce the efficacy of current cpv vaccines, which is supported by in vitro virus-neutralization tests conducted on vaccinated animals that showed low cross-reactivity between heterologous cpv variants (cavalli et al., ) . however, this may not represent actual cross-protection in clinical cases. there is a need not only to make current vaccines effective in the presence of maternal antibodies, but also to update them based on continuous epidemiological surveillance studies. dna plasmids expressing vp (jiang et al., ) , replicon-based cpv dna vaccine expressing vp (dahiya et al., ) , b cell epitope ( l peptide of vp ) fused to ct b subunits expressed in transgenic tobacco chloroplasts (molina et al., ) , chimeric virus particles expressing cpv peptide (different prime/boost strategies) (nicholas et al., ) , and recombinant vlps formed by baculovirusexpressed vp (casal, ) have been evaluated in dogs or rodents without maternal antibodies and have demonstrated good immunogenicity and/or protective efficacy. further efficacy tests in pups with maternal antibodies are needed to assess their commercial potential. oral vaccines for the induction of active immunity against bacterial diarrhea are not commonly used in livestock, although e. coli diarrhea is an important problem in neonatal and postweaning calves and pigs. f (k ) and f are the major fimbrial adhesins present on swine enterotoxigenic e. coli (etec) and are major targets for e. coli vaccines (fairbrother et al., ) . whole bacteria vaccines are routinely administered parenterally to pregnant cattle, sheep, and swine to protect their suckling neonates against etec infections (moon and bunn, ) . such vaccines are practical and effective because: ( ) fimbriae are required for the adhesion-colonization of bacteria early in the pathogenesis of the disease; ( ) most fatal etec infections in farm animals occur in the neonatal period; ( ) more than % of the etec in farm animals belong to a small family of fimbrial antigen types, ( ) and mothers are seropositive to etec so booster responses are elicited. recent vaccine studies have focused on administration of purified bacterial subunits (transgenically expressed adhesin of f fimbriae in plants) and mucosal adjuvants (verdonck et al., ; joensuu et al., ) and have shown promising results. overall, the vaccine strategy used, parenteral vaccination of field-exposed seropositive mothers, to induce lactogenic immunity is the same as that shown to be effective for parenteral application of rv vaccines in rv seropositive mothers (bohl et al., ; saif and jackwood, ; saif and fernandez, ) . studies of live oral enteric vaccines in animals have clarified the mechanisms of induction of protective immunity against enteric disease and contributed to our understanding of the common mucosal immune system. however, commercial live oral vaccines often have shown inadequate or inconsistent efficacy under field conditions (saif and jackwood, ; saif and fernandez, ) . major obstacles to improved efficacy of oral vaccines include: ( ) maternal antibodies in the intestine of neonates (mainly colostrum and milk antibodies), which interfere with live vaccine replication; ( ) qualitative and quantitative limitations in the neonatal immune system, although neonates are immunocompetent; ( ) the inability of attenuated vaccine strains to adequately infect (too high attenuation) or stimulate s-iga antibodies in the intestine; ( ) the use of inappropriate (or unstable) antigens or route for subunit vaccines; ( ) the lack of oral delivery vehicles or mucosal adjuvants for subunit vaccines; and ( ) infection by pathogens prior to vaccination. studies of neonatal pigs indicate that protection rates against rv diarrhea upon challenge correlate with the magnitude of iga asc and memory b cell responses in intestinal lymphoid tissues gonzalez et al., ; yuan and saif, ) . studies conducted in immunodeficient or specific gene knockout adult mice have shown that neither cd + or cd + t cells nor antibodies were essential for induction of protective immunity to rv infection, but usually one of these effectors (t or b cells) was necessary for elimination of primary rv infection (mcneal et al., (mcneal et al., , . in pigs, recent studies have shown that cd + and cd + ifnγ producing t cells play a role in protection against rv diarrhea and infection (yuan et al., ) , but it is difficult to create genetically modified pigs similar to knockout mice, to assess the contribution of b cells and t cells. the redundant nature of immune responses to rv in mice, the multiple immunologic and possibly nonimmunologic pathways to resolve rv infections (franco and greenberg, ; ward, ) , the age factor and host differences in rv pathogenesis in mice and pigs (saif et al., , ward, ) , and the use of inbred mouse strains contribute to the discrepancies seen between the adult mouse and the neonatal pig models. the majority of pathogens enter and initiate infection at mucosal surfaces, making mucosal sites relevant targets for vaccines to prevent infection. to develop effective mucosal vaccines, it is important to determine correlates of protection against enteric pathogens. generally for localized gut infections, s-iga antibodies and intestinal t cells play an important role. improved vaccines that induce high levels of intestinal iga antibodies against the appropriate microbial antigens can be achieved by choosing the proper vaccine formulation and delivery method. vaccines should also induce: ( ) heterotypic protection; ( ) active immunity in the presence of maternal antibodies; and, ( ) long-lasting immunological memory. novel vaccines (e.g., vlps, transgenic plants), adjuvants (e.g., mlt, iscoms, tlr ligands (e.g., cpg), mycobacterial extracts, α -dihydroxyvitamin d , retinoic acid, probiotics, and cytokines), and vaccine delivery systems (e.g., recombinant plant or animal viruses or bacterial vectors, genetically engineered probiotics, iscoms, liposomes, and nanoparticles) should be explored and evaluated in relevant animal models to further enhance the efficacy of current or new vaccines. recent studies have shown that the targeting of antigens directly to apcs (via surface receptors) is an effective way to tailor immune responses to optimize protection and should be explored further in large animals (trumpfheller et al., ) . the passive transfer of maternal immunity provides essential protection in newborns. although most neonatal immune systems are competent to mount primary immune responses against pathogens, primary responses often do not develop quickly enough to prevent disease. maternal immunologic transfer provides a critical (though temporary) aid to survival for neonates. the enhancement of passive immunity through vaccination of the mother has been a successful disease prevention strategy in domesticated animals. vaccinated mothers develop higher levels of specific antibodies in colostrum and milk and increased levels of immunity in their offspring (glezen, ; saif and fernandez, ) . passive immunity can also be enhanced by oral administration of immune milk or heterologous antibody preparations (e.g., chicken egg yolk igy (ikemori et al., ; kuroki et al., ) or monoclonal antibodies) or by parenteral administration of hyperimmune plasma (becu et al., ) . recent studies using monoclonal, single-chain antibodies (variable heavy domain (vhh) nanoantibodies) of llama origin open new possibilities for providing passive immunity to humans and animals (garaicoechea et al., ) . immunoglobulins of the igg and igg isotypes of camelids consist of heavy chains without associated light chains (hamers-casterman et al., ) . the distinctive biochemical characteristics and binding qualities of vhh antibodies overcome some key limitations of conventional antibodies (see below). unfortunately, passive antibodies often interfere with active immunization of young animals and birds. various vaccination strategies have been developed to minimize the suppressive effects of maternal antibodies, but improved adjuvants and antigen-delivery systems are needed to facilitate efficient induction of active immunity in the presence of maternal antibodies. this section will address past, current, and future approaches for enhancing passive immunity in veterinary species. the transfer of systemic passive immunity from mother to offspring can occur prenatally, via the placenta or yolk sac, or postnasally via ingestion of colostrum and milk, depending on the species. the main ig isotype transferred in most mammalian species is igg. in avian species igy, the functional equivalent of mammalian igg, is transferred to the yolk to passively protect the developing chick (kovacs-nolan and mine, ) . in primates and rabbits, maternal igg is transferred across the placenta to the fetus. in rodents, transplacental transfer occurs in combination with prolonged ( - days) postnatal transfer by means of colostrum and milk (husband, ) . in dogs and cats transfer of igg occurs by a combination of prenatal and postnatal mechanisms, with % to % of total transfer occurring before birth (tizard, ) . in ruminants, horses, and pigs, offspring are born virtually agammaglobulinemic and transmission of ig occurs only via colostrum for a limited time after birth (tizard, ) . after the transition from production of colostrum to milk, ig are no longer absorbed from the intestines and only act locally in the gastrointestinal tract. immunoglobulin absorption in neonates of large domestic species is facilitated by the presence of protease inhibitors in colostrum and its efficiency declines rapidly after birth, with maximal absorption occurring in the first h. the cessation of absorption of intact macromolecules is termed "gut closure," and occurs at different ages in different species. in calves and pigs closure normally occurs by - h after birth. absorption of colostrum ig can be highly effective, supplying the newborn with serum ig at concentrations similar to those of the dam. failure of passive transfer (fpt) is a common problem, however, in newborn calves and foals (besser and gay, ; tyler-mcgowan et al., ) . fpt may occur because of the production of low quantities of colostrum, because of production of colostrum with low concentrations of maternal antibodies, because of ingestion of low quantities of colostrum, or because of inefficient absorption (quigley and drewry, ) . colostrum supplements, colostrum replacers, and plasma products have been developed commercially to address this problem, with variable success. vaccination of the dam in late pregnancy can enhance antibody titers in colostrum and after suckling in the serum of the offspring (saif and fernandez, ) . the benefits of vaccination of the dam for enhancing passive immunity can be lost if colostrum is of low quality or if absorption of colostral ig is inefficient. the half-life of ig varies considerably among species of domestic animals and with the ig isotype. neonatal receptor for fc of igg (fcrn) is involved in homeostasis of serum levels of igg in general, but distinct mechanisms may function in neonates. the main route of clearance of passively acquired maternal igg in calves is transfer from the serum to the intestine (besser et al., ) . approximately % of passively acquired igg is eliminated by this route. if titers of passive circulating antibodies are high enough, the transfer of antibodies from the circulation to the intestinal lumen can mediate short-term protection against rotavirus diarrhea (besser et al., ) . the same mechanism may be functional in piglets (saif and wesley, ; parreño et al., ; ward et al., a) . the persistence of titers of circulating maternal antibodies is generally considered in designing vaccination strategies for young animals because of suppressive effects of maternal antibodies on active immune responses. induction of immune memory can occur in the absence of a detectable serum antibody response (boersema et al., ; parreño et al., ) . the presence of passive antibodies in the intestines can interfere with mucosal immune responses to natural infection as well as to vaccination parreño et al., ; nguyen et al., a,b) . experiments in colostrum-deprived lambs (jones et al., ) and calves (mosier et al., ) have demonstrated the ability of parenterally administered immune antisera to mediate protection following experimental challenge with mannheimia haemolytica. parenteral administration of hyperimmune plasma raised against rhodococcus equi has been shown to protect against pneumonia in young foals in experimental (hooper-mcgrevy et al., ) and field studies (becu et al., ) . hyperimmune plasma is available commercially for use in foals. prepartum vaccination of beef (van donkersgoed et al., ) and dairy (hodgins and shewen, ) cows has been demonstrated to increase antibody titers to m. haemolytica in their colostrum and in the serum of their calves. vaccination of broiler breeder chickens can be used to provide passive protection against respiratory/septicemic disease caused by avian pathogenic e. coli (kariyawasam et al., ) . rodents have been a popular model for the study of passive protection by milk antibodies. however, rats and mice actively transport igg from the gut into the circulation during the first - weeks of life. thus, antibodies in ingested milk contribute to both local and systemic immunity in rodents, in contrast to the strictly local effects occurring in humans and most domestic animals. in pigs, horses, dogs, and cats, igg is the most abundant ig in colostrum but iga predominates in milk. parenteral vaccination, by enhancing serum igg antibody titers, contributes to igg antibodies in colostrum but has limited effects on iga antibodies in milk. milk antibodies provide passive protection to the neonatal intestinal tract by immune exclusion preventing the attachment of viruses, bacteria, and parasites and by neutralizing viruses or enterotoxins. s-iga antibodies, because of their resistance to cleavage by digestive enzymes, and higher levels in milk are more efficient in mediating protection in the gut of pigs and other monogastrics (saif and jackwood, ) , but high persisting levels of passive igg antibodies are also protective. in ruminants igg antibodies, relatively resistant to proteolytic enzymes (brock et al., ) and predominant in milk, have functions similar to those of s-iga. numerous vaccines are marketed for vaccination of cows and sows to provide lactogenic immunity to rotavirus, coronavirus, and e. coli in suckling offspring. vaccine efficacy has been variable. ideally, suckling animals become subclinically infected with enteric pathogens while receiving adequate passive antibodies to prevent disease, and develop active immunity to prevent subsequent diarrhea. this balance between passive immunity and disease can be disrupted in intensive animal production systems by exposing animals to pathogens in confined, contaminated environments. earlier weaning practices reduce intake of milk antibodies. maternal enteric vaccines are commonly used in two populations of pregnant animals. to control epidemic infections, they are used in naïve, seronegative animals to induce primary immune responses. to control endemic infections (such as rotavirus and e. coli), booster vaccines are used in seropositive, field-exposed animals to stimulate anamnestic memory responses. virulent tgev given to pregnant sows stimulates high levels of iga antibodies in milk and passive protection (saif and jackwood, ) . oral attenuated tgev vaccines, which replicate poorly in sows, induce lower iga milk antibody titers and low or variable efficacy in the field (moxley and olson, ) . parenteral killed tgev vaccines induce only low igg milk antibody titers and have the lowest protection rates. attempts to develop maternal tgev recombinant subunit vaccines based on the surface tgev spike (s) protein that induces na, or live vector vaccines expressing the s protein, have also been of limited success in tgev seronegative swine (saif and wesley, ) . however, prime/boost strategies such as im administration of tgev s protein following oral/in priming with attenuated tgev have shown promise as a means of enhancing iga milk antibody titers (park et al., ) . booster vaccination strategies are required to enhance lactogenic immunity to endemic enteric pathogens, such as rotavirus and e. coli, because antibody titers in milk decline dramatically during lactation. breast milk iga antibodies are increased in women endemically exposed to cholera following parenteral boosting with a cholera vaccine (svennerholm et al., ) . in pigs infected with rotavirus, iga memory b cells initially reside in the ileal pps but are subsequently present in substantial numbers in spleen (yuan et al., ) . systemic stimulation of such iga memory b cells by parenteral booster vaccines can yield dimeric iga antibodies in serum for transport to mucosal secretions via the polymeric ig receptor. under field conditions, antibodies to endemic intestinal pathogens are also common in bovine colostrum and milk, but without the boosting effect of highly immunogenic vaccines, antibody titers are often too low to protect calves (besser and gay, ; saif and fernandez, ) . thus vaccines are marketed for prepartum vaccination of cows against rotavirus, coronavirus, and e. coli to enhance passive immunity in their calves, but the field efficacy of these vaccines has been questioned (waltner-toews et al., ) . vaccination of pregnant dairy cows with modified live or binary ethyleneamine inactivated rotavirus or recombinant / / / vlps has been shown to increase igg and virus na titers to rotavirus in colostrum and milk and mediate passive protection in calves against oral rotavirus challenge (saif and fernandez, ; kim et al., ) . prepartum vaccination of cows and sows with bacterins prepared from enteropathogenic (epec) e. coli for prevention of diarrhea in their offspring is also commonly practiced. under modern farming practices, dairy and veal calves rarely are fed whole milk from their dams for more than or days. thus vaccine efficacy is based on antibodies absorbed from colostrum or retained temporarily in the gut, rather than on a continuing supply of immune milk. piglets, in contrast, continue to receive immune milk until weaning at - weeks of age. the importance of a continuous supply of passive antibodies for protection against tgev has been demonstrated (saif and wesley, ) . numerous commercial ig preparations with antibody activity against specific enteric pathogens have been marketed. products intended for prevention of e. coli enteritis in calves include dried bovine colostrum and whey, hyperimmune sera raised in horses, and mouse monoclonal antibodies to the k (f ) antigen of e. coli. these products are administered orally in the first h of life to prevent adhesion of epec e. coli. orally administered bovine colostral whey containing rotavirus antibodies also passively protects piglets against rotavirus (schaller et al., ) . immunization of chickens shows promise as an efficient method for producing polyclonal antibodies for passive protection. specific antibodies of the igy isotype are induced by vaccination and are concentrated in egg yolk. laying hens can produce about g of igy per year. yolk antibodies with virus na provide calves with partial protection against diarrhea caused by rotavirus (kuroki et al., ; vega et al., ) , and etec e. coli (ikemori et al., ) . in a recent study, supplementation of the milk ration of neonatal calves with egg yolk containing igy antibodies to rotavirus for days provided % protection against rotavirus diarrhea after challenge, and also enhanced mucosal asc numbers in the duodenum (vega et al., ) . egg yolk lacking rotavirus specific igy did not provide clinical protection, but did enhance asc responses, suggesting the presence of immune modulators in egg yolk. protective effects of yolk antibodies are dependent on antibody titers in oral preparations (marquardt, ) . development of better means to protect yolk antibodies from digestive processes will improve both the efficacy and the economic viability of yolk antibodies for clinical applications (kovacs-nolan and mine, ). for many diseases of newborns and neonates, passive immunity is the only practical means of providing timely protection. unfortunately, it is well documented that maternal antibodies can suppress active immune responses following vaccination. this effect has been observed with both live and nonreplicating vaccines, and for both systemic and mucosal responses (siegrist et al., ; hodgins et al., ; parreño et al., ; nguyen et al., a,b) . antibody responses especially are affected; t-lymphocyte responses may not be suppressed (siegrist et al., ) . titers of maternal antibodies are maximal for most species of interest in the first week of life and then decline gradually over the next few months, but variability of titers among individuals is high. with many vaccines, a "window of disease susceptibility" of variable duration occurs when titers of maternal antibodies are too low to mediate protection, but are too high to permit effective vaccination. a number of strategies are used to cope with this problem. some veterinary vaccines for cattle are sold with the disclaimer that "animals vaccinated before months of age should receive a booster dose of vaccine at months of age." this provides little solace for the many diseases of cattle occurring in the first weeks or months of life. a common strategy for vaccines of dogs and cats is to administer a series of doses of vaccine from an early age (at which time only a few individuals will be responsive) and to continue vaccinating until an age at which virtually all can respond to vaccination. this strategy has economic disadvantages for the pet owner. some manufacturers produce low passage, high virus titer vaccines especially for use in situations where high titers of maternal antibodies and high pathogen exposure are anticipated. this is similar to a strategy once (but no longer) approved by the world health organization for vaccination of children in developing countries against measles (gellin and katz, ) . preliminary evidence suggests that incorporation of vaccine antigens in highly structured iscoms or in application of vaccines can enhance immune responses in the presence of maternal antibodies (van binnendijk et al., ; brockmeier et al., ) . immunoglobulin g in most mammalian species is composed of two heavy chains, covalently linked by disulfide bonds, and two light chains. "conventional" heavy chains consist of one variable domain and three constant domains (ch , ch , and ch ); light chains are composed of a variable and constant domain. in contrast camelid species produce "heavy chain immunoglobulins" that lack light chains and the first constant heavy domain (hamers-casterman et al., ) . the antigen-binding site of these unusual heavy chain antibodies is formed by a single domain, designated vhh in camelids. vhh are easily produced as recombinant proteins, designated single domain nanoantibodies or nanobodies ® and represent the smallest molecule in nature capable of binding a specific antigen. the cdr region of these nanobodies has the capacity to form long loops that can extend into cavities on antigens, e.g., the active site crevice of enzymes. other advantageous features of nanobodies include their high solubility, thermal stability (resist pasteurization), refolding capacity, and optimal tissue penetration in vivo (reviewed by vanlandschoot et al. ( ) ). nanobodies have demonstrated efficacy as agents of passive immunity for infectious diseases. vhh specific for rotavirus inner capsid protein vp are able to broadly neutralize rotaviruses independently of serotype, and in mouse experiments provide passive protection against challenge with human rotavirus (pant et al., ; garaicoechea et al., ) . nanobodies against other viral diseases with veterinary impact have been developed (foot-and-mouth disease, influenza, rabies (vanlandschoot et al., ) ) and represent a promising next-generation biologic platform for passive immunity. maternal vaccination to enhance passive immunity is already widely used in veterinary medicine. some of these vaccines, especially vaccines against enteric viruses, have limited efficacy. new approaches to enhance immunogenicity are promising, but await commercial development. a clearer understanding of protective mechanisms and immune modulation mediated by passive antibodies would contribute to more effective interventions. there is an urgent need for adjuvants and delivery systems capable of reliably inducing active immunity in neonates despite the presence of maternal antibodies. the ability to provide continuity of immune protection from birth, by combining passive immunity with active immunization, would have a major impact on neonatal morbidity and mortality. the rapid expansion of commercial fish farming (aquaculture) in many countries in recent decades has been accompanied by an urgent need to develop vaccines to prevent (infectious) fish diseases that were previously unknown or obscure. added to the difficulties involved in identifying the pathogens responsible, there has been the challenge of delivering vaccine efficiently with minimal stress to very large numbers of fish. some commercial vaccines for fish are administered by intraperitoneal or im injection, but mucosal vaccines are also widely used (reviewed by brudeseth et al. ( ) ). fish are routinely vaccinated by immersion in tanks of diluted bacterin, modified live bacteria or viruses, with exposure times ranging from s up to min, depending on the vaccine and age of the fish. it is unclear whether the main route of antigen uptake is oral or via the mucosal surface of the gills. several commercial vaccines are now available that consist of recombinant viral proteins for mixing into the feed (evensen and leong, ) ; days of feeding is recommended by the manufacturer. research on mucosal veterinary vaccines has contributed new concepts to the field of mucosal immunity. investigations of pathogen-host interactions in outbred animals have illustrated the complexity of these interactions. early studies of an enteric coronavirus infection of swine (tgev) led to the concept of the gut-mammary gland s-iga immunologic axis and provided part of the basic tenet for a common mucosal immune system. later studies of tgev and a deletion mutant of tgev with respiratory tropism (prcv) revealed functional compartmentalization within the common mucosal immune system whereby in inoculation of pigs with prcv failed to elicit sufficient intestinal iga antibody to fully protect against the enteric pathogen tgev (vancott et al., (vancott et al., , . subsequent studies have explored new prime/boost mucosal immunization strategies to elicit intestinal immunity to rotavirus in naïve pigs (saif, b; yuan and saif, ; gonzalez et al., ) . in these studies only oral priming with attenuated virus led to successful in booster responses using nonreplicating (vlp) vaccines combined with mucosal adjuvants such as iscom or mlt. thus use of a replicating vaccine to prime lymphocytes at a major mucosal inductive site (galt) followed by boosting with a nonreplicating vaccine at a second inductive site (nalt) effectively stimulated intestinal iga antibodies and induced active protection against rotavirus diarrhea. although there is progress in developing safe and effective nonreplicating vaccines to boost mucosal immune responses (saif and fernandez, ) , the dilemma remains to develop effective vaccines to prime for mucosal immunity. mucosal adjuvants (mlt, iscom, cpg, cytokines) and new delivery systems (replicating vectors, microparticles) have shown promise in animal studies reviewed in this chapter. however, their economical production and final evaluation under field conditions, including in the presence of maternal antibodies as relevant, are needed. considerable research effort has been devoted to development of vaccines for respiratory diseases of domestic animals. in some instances attenuated organisms delivered by mucosal routes have demonstrated improved efficacy over nonreplicating antigens given by systemic routes. for many respiratory diseases, however, further progress in the development of mucosal vaccines will have to await advances in understanding of disease pathogenesis and identification of protective antigens. in contrast, studies of ascending infections of the reproductive tract in cattle have demonstrated the efficacy of systemic vaccination to clear established infections, and highlight the possibility of therapeutic vaccines. finally it is important to realize that there are considerable species differences in mucosal immunity. for example, the primary ig in mammary secretions of ruminants is igg which is actively transported to the mammary gland from serum and provides effective passive immunity to the nursing offspring against enteric pathogens. thus parenteral immunization of the dam stimulates passive immunity in ruminants against enteric pathogens. in contrast, in monogastrics, iga predominates in milk and iga lymphoblasts that traffic to the mammary gland originate in the intestine. therefore oral vaccines in monogastrics may provide a more effective vaccine strategy to induce iga antibodies in milk (saif and fernandez, ) . by applying the aforementioned vaccine concepts with new and effective mucosal adjuvants, delivery systems, and bioengineered vectors expressing appropriate microbial antigens, it is likely that a new generation of veterinary vaccines will emerge to better cope with existing and emerging mucosal pathogens. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus forty years of canine vaccination an oral versus in prime/boost regimen using attenuated human rotavirus or vp and vp virus-like particles with immunostimulating complexes influences protection and antibody-secreting cell responses to rotavirus in a neonatal gnotobiotic pig model cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus lactobacillus acidophilus and lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus agedependent diarrhea induced by a rotaviral nonstructural glycoprotein cd antigen presentation: how it works impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of cd + t cells immune response of pigs inoculated with mycobacterium bovis bcg expressing a truncated form of gp and m protein of porcine reproductive and respiratory syndrome virus immunoprophylaxis of rhodococcus equi pneumonia in foals acute granulomatous response produced in mice by trehalose- , -dimycolate characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) effects of passively and actively acquired antibody on bovine campylobacteriosis (vibriosis) the importance of colostrum to the health of the neonatal calf passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen the transfer of serum igg antibody into the gastrointestinal tract in newborn calves silent memory induction in maternal immune young animals antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses mechanism of nk activation by ok- (streptococcus pyogenes). i. spontaneous release of nkcf and augmentation of nkcf production following stimulation with nk target cells immunization of virgin cows with surface antigen tf . of tritrichomonas foetus appearance of acute prrs-symptoms in sow herds after vaccination with a modified live prrs vaccine the effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral igg successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines status and future perspectives of vaccines for industrialized fin-fish farming performance of high titre attenuated canine parvovirus vaccine in pups with maternally derived antibody equine influenza -the a modified live canine parvovirus vaccine. ii. immune response use of parvovirus-like particles for vaccination and induction of multiple immune responses evaluation of the antigenic relationships among canine parvovirus type variants a new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge adjuvants for porcine reproductive and respiratory syndrome virus vaccines porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy, and safety aspects morphological and functional comparisons of peyer's patches in different parts of the swine small intestine therapeutic immunization of bulls with the membranes and glycoproteins of tritrichomonas foetus var. brisbane immunity to infections in the lower genital tract of bulls preputial cellular and antibody responses of bulls vaccinated and/or challenged with tritrichomonas foetus effect of vinyl sulfone inhibitors of cysteine proteinases on tritrichomonas foetus infection renal lesions associated with experimental porcine reproductive and respiratory syndrome virus (prrsv) infection animal model for the study of genital immune mechanisms: venereal vibrosis of cattle immunoglobulin binding by tritrichomonas foetus uterine mast cells and immunoglobulin-e antibody responses during clearance of tritrichomonas foetus bovine trichomoniasis as a model for development of vaccines against sexually-transmitted disease immunity in the female bovine reproductive tract based on the response to campylobacter fetus an oral sindbis virus replicon-based dna vaccine containing vp gene of canine parvovirus delivered by escherichia coli elicits immune responses in dogs canine parvovirus -a review of epidemiological and diagnostic aspects, with emphasis on type c evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions crossprotective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs accelerated vaccination schedule provides protective levels of antibody and complete herd immunity to equine influenza rotaviruses dna vaccines against viral diseases of farmed fish escherichia coli infections escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies dosedependent effect of lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus immunity to homologous rotavirus infection in adult mice effect of a probiotic formula on intestinal immunoglobulin a production in healthy children llama-derived single-chain antibody fragments directed to rotavirus vp protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice measles: state of the art and future directions maternal vaccines cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model intestinal and systemic immunity to rotaviruses in animal models and humans antibody responses to human rotavirus (hrv) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated hrv priming and intranasal vp / rotavirus-like particle (vlp) boosting with iscom autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against golgi antigens and other subcellular elements combined nkt cell activation and influenza virus vaccination boosts memory ctl generation and protective immunity type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-α, tumour necrosis factor-α and interleukin- immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs naturally occurring antibodies devoid of light chains cell mediated immune responses in ponies following infection with equine influenza virus (h n ): the influence of induction culture conditions on the properties of cytotoxic effector cells equine mucosal immune system: intranasal vaccination with inactivated equine influenza virus protects from infection duration of circulating antibody and immunity following infection with equine influenza virus antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (h n ) immunogenicity of a lowpassage, high-titer modified live canine parvovirus vaccine in pups with maternally derived antibodies preparturient vaccination to enhance passive immunity to the capsular polysaccharide of pasteurella haemolytica a effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs bifidobacterium lactis bb enhances intestinal antibody response in formula-fed infants: a randomized, double-blind, controlled trial pork checkoff study: prrs costs industry $ million annually evaluation of equine immunoglobulin specific for rhodococcus equi virulence-associated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia infection immunity of piglets to either vp or vp outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen immunostimulating complexes (iscoms) for nasal vaccination passive transfer of immunity oral immunization induces local and distant mucosal immunity in swine conservation of a protective surface antigen of tritrichomonas foetus protection of neonatal calves against fatal enteric colibacillosis by administration of egg yolk powder from hens immunized with k -pilated enterotoxigenic escherichia coli toll-like receptor control of the adaptive immune responses nucleic acid immunization protects dogs against challenge with virulent canine parvovirus f (k ) fimbrial adhesin faeg expressed in alfalfa reduces f + enterotoxigenic escherichia coli excretion in weaned piglets protection of lambs against experimental pneumonic pasteurellosis by transfer of immune serum degradation of bovine complement c by trichomonad extracellular proteinase resistance of broiler chickens to escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (vlps) of two serotypes or inactivated bovine rotavirus vaccines use of a blocking elisa for antibodies to equine influenza virus as a test to distinguish between naturally infected and vaccinated horses: proof of concept egg yolk antibodies for passive immunity passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk clinical evaluation of the efficacy of inoculating cattle with a vaccine containing tritrichomonas foetus lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea antibody responses to dna vaccination of horses using the influenza virus hemagglutinin gene equine influenza virus infections: an update probiotic bacteria enhance murine and human intestinal epithelial barrier function control of intestinal diseases in pigs by feeding specific chicken egg antibodies zoonotic aspects of rotaviruses evolution of orf of spanish porcine reproductive and respiratory syndrome virus strains from to effector functions of antibody and cd + cells in resolution of rotavirus infection and protection against reinfection in mice cd t cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric vp protein and the adjuvant lt(r g) neonatal calf diarrhea: results of a field trial using a reo-like virus vaccine gradual development of the interferon-γ response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus induction of na by a tobacco chloroplast-derived vaccine based on a b cell epitope from canine parvovirus vaccines for preventing enterotoxigenic escherichia coli infections in farm animals iscom: a delivery system for neonates and for mucosal administration passive protection of calves with pasteurella haemolytica antiserum clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows the function of local lymphoid tissues in pulmonary immune responses who/oie meeting: consultation on newly emerging strains of equine influenza antigenicity and immunogenicity of experimental equine influenza iscom vaccines duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccines antigenicity and immunogenicity of equine influenza vaccines containing a carbomer adjuvant bovine herpes virus infections in cattle local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination bark extract amplifies vaccine high titers of circulating maternal antibodies suppress effector and memory b-cell responses induced by an attenuated rotavirus priming and rotavirus-like particle-immunostimulating complex boosting vaccine regimen low titer maternal antibodies can both enhance and suppress b cell responses to a combined live attenuated human rotavirus and vlp-iscom vaccine effect of priming/booster immunisation protocols on immune response to canine parvovirus peptide induced by vaccination with a chimaeric plant virus construct experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccinederived virus passive transfer of virusspecific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus serum and intestinal isotype antibody responses to wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies early pathogenesis and pathology of tritrichomonas foetus infection in virgin heifers maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination immunization of pups with maternally derived antibodies to canine parvovirus (cpv) using a modified-live variant (cpv- b) probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine nutrient and immunity transfer from cow to calf pre-and postcalving porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally-infected bulls experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and -week-old pigs comparative pathogenesis of enteric viral infections of swine enteric viral infections of pigs and strategies for induction of mucosal immunity group a rotavirus veterinary vaccines enteric virus vaccines: theoretical considerations, current status, and future approaches transmissible gastroenteritis and porcine respiratory coronavirus isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses comparative studies of the pathogenesis, antibody immune responses, and homologous protection to porcine and human rotaviruses in gnotobiotic piglets gut microbiota and probiotics in modulation of epithelium and gut-associated lymphoid tissue function prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody influence of maternal antibodies on vaccine responses: inhibition of antibody but not t cell responses allows successful early prime-boost strategies in mice canine parvovirus type vaccine protects against virulent challenge with type c virus boosting of secretory iga antibody responses in man by parenteral cholera vaccination cleavage of proteins of reproductive secretions by extracellular proteinases of tritrichomonas foetus differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model veterinary immunology, an introduction serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease toll-like receptor and are expressed and functional in gut-associated lymphoid tissues of presuckling newborn swine efficacy of a recombinant equine influenza vaccine against challenge with an american lineage h n influenza virus responsible for the outbreak in the united kingdom efficacy of a cold-adapted, intranasal equine influenza vaccine: challenge trials dendritic cell-targeted protein vaccines: a novel approach to induce t-cell immunity failure of passive transfer in foals: incidence and outcome on four studs in new south wales protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs effects of various vaccination protocols on passive and active immunity to pasteurella haemolytica and haemophilus somnus in beef calves development of an experimental inactivated prrsv vaccine that induces virus-neutralizing antibodies nanobodies ® : new ammunition to battle viruses maternal antibodies against equine influenza virus in foals and their interference with vaccination egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves cholera toxin improves the f (k )-specific immune response following oral immunization of pigs with recombinant faeg a field trial to evaluate the efficacy of a combined rotaviruscoronavirus/escherichia coli vaccine in dairy cattle inactivated rotavirus vaccine induces protective immunity in gnotobiotic piglets possible mechanisms of protection elicited by candidate rotavirus vaccines as determined with the adult mouse model rotavirus vaccines: how they work or don't work role of maternally derived circulating antibodies in protection of neonatal swine against porcine group a rotavirus pathogenesis of an attenuated and a virulent strain of group a human rotavirus in neonatal gnotobiotic pigs toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs development of γδ t cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) efficacy of a neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle comparative field evaluations of in ovo applied technology equine influenza antigen-presenting cells in the female reproductive tract: influence of estrous cycle on antigen presentation by uterine epithelial and stromal cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin- and regulatory t-lymphocytes (treg) probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells the sheep and cattle peyer's patch as a site of b-cell development equine influenza vaccine efficacy: the significance of antigenic variation induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-dna vaccine in a gnotobiotic pig model short-term immunoglobulin a b-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with wa human rotavirus systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-γ producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs intranasal administration of cpg oligonucleotides induces mucosal and systemic type immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo influence of probiotic lactobacilli colonization on neonatal b cell responses in a gnotobiotic pig model of human rotavirus infection and disease probiotic lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs key: cord- -gm nx a authors: franzo, giovanni; cecchinato, mattia; martini, marco; ceglie, letizia; gigli, alessandra; drigo, michele title: observation of high recombination occurrence of porcine reproductive and respiratory syndrome virus in field condition date: - - journal: virus research doi: . /j.virusres. . . sha: doc_id: cord_uid: gm nx a abstract recombination in porcine reproductive and respiratory syndrome virus (prrsv) is a well-documented phenomenon. a high recombination frequency has been reported in experimental conditions both in vitro and in vivo, and its role in driving viral evolution has been postulated by several authors. however field evidences are rare, mainly obtained from large-scale sampling and typically represented by single sequences rather than by groups of circulating “recombinant progenies”. the present work was aimed to investigate the gray area between experimental studies and large-scale epidemiological investigations. the study was performed on orf , orf and concatenated sequences obtained in our laboratory or available in genbank collected between and in northern italy. six independent recombinant strains out of concatenated sequences (∼ %) were found, demonstrating a high recombination frequency respect to previous field studies but comparable to in vitro experiments. in silico analysis let speculate that this new strain displayed physicochemical features diverse enough to potentially alter its immunological properties. taken altogether, the results of our study support previous experimental evidences that depict prrsv to be extremely prone to recombination. the limited temporal and geographical spread of recombinant strains however states in favor of a limited fitness of the recombinant progeny compared to parental strains and the marginal role of this phenomenon in prrsv evolution. porcine reproductive and respiratory syndrome (prrs) was first recognized quite contemporaneously in the u.s. and in europe between the end of s and the early s. since then prrs has emerged as the most prevalent disease of swine in the world, causing remarkable economic losses (neumann et al., ; nieuwenhuis et al., ) . the agent of the disease, porcine reproductive and respiratory syndrome virus (prrsv), classified in the order nidovirales, family arteriviridae, genus arterivirus, is an enveloped, single-stranded positive-sense rna virus. the viral genome is approximately kb in length and contains nine open reading frames (orfs) (firth et al., ; meulenberg, ) . two main genotypes, type i (european-like) and type ii (north american-like) have been identified sharing - % nucleotides and - % amino acids (shi et al., a) . a great and progressively increasing (mateu et al., ; pesch et al., ) genetic variability has been observed: mean nucleotide diversity within european and american genotypes has been estimated to be about % and . %, respectively (cho and dee, ; shi et al., a,b) . genetic distance, calculated on orf , has reached a maximum of about % in type i and % in type ii (murtaugh et al., ) . rna virus evolution is assumed to result primarily from rna polymerase infidelity. indeed the prrsv nucleotide substitution rate has been estimated to vary between . × − and . × − (murtaugh et al., ; yoon et al., ) . although the role of recombination in evolution of rna viruses is still debated (simon-loriere and holmes, ) , several authors assert that recombination is an important mechanism of genetic diversity generation in prrsv (liu et al., ; mengeling, ; murtaugh et al., ) , playing a potential role in conditioning virulence, antigenic escape and diagnostic failure. several studies have demonstrated recombination in both in vitro (van vugt et al., ; yuan et al., ) and in vivo, in experimental (liu et al., ) and field conditions (fang et al., ; forsberg et al., ; li et al., ; shi et al., a; stadejek http://dx.doi.org/ . stadejek http://dx.doi.org/ . /j.virusres. . . - /© elsevier b.v. all rights reserved. et al., ) . in the latter case, results where typically obtained comparing sequences obtained from large-scale (i.e. country level) sampling. the aim of this study was to investigate recombination on a smaller scale in terms of geographic distance and time window (forsberg et al., ) . the samples used in this study were drawn from the istituto zooprofilattico sperimentale delle venezie's historical archive, a regional public veterinary laboratory collecting passive field samples brought by practitioners for diagnostic purposes. all of the samples (serum and lung), coming from pig farms among provinces in northeastern italy (enclosing a geographic area of about , km ), found positive at routine rt-pcr to prrsv between and and stored at − • c, were analyzed. rna had been extracted from l of serum or l of lung homogenate using the high pure viral rna kit and high pure rna tissue kit, respectively (roche diagnostics, monza, italy). each sample had been routinely tested using a classical two step rt-pcr targeting a genomic fragment within the orf region and allowing the differentiation between the type i and type ii strains through electrophoresis on acrylamide gels (persia et al., ) . orf and orf of each sample were amplified using a onestep rt-pcr as described by oleksiewicz et al. ( ) . briefly, orf sequence was amplified using the primer orf f ( caa tga ggt ggg cia caa cc ) and orf r ( tat gti atg cta aag gct agc ac ) while orf was amplified using the primer pair orf f ( gcc cct gcc cai cac g ) and orf r ( tcg ccc taa ttg aat agg tga ), obtaining an amplicon of bp and bp respectively. amplification and band specificity were visualized using a sybr safe stained % agarose gel, after electrophoresis. amplicons were sequenced with the same primers, in both senses, using the bygdye terminator v. . cycle sequencing kit (applied biosystem ® , monza, italy). sequences were obtained using abi prism ® genetic analyzer (applied biosystem ® , monza, italy). chromatograms were evaluated by finchtv (http://www.geospiza.com) and consensus sequences were reconstructed using cromaspro (cromaspro version . ). when both orfs were available, concatenated sequences were constructed using mesquite (maddison and maddison, ) . sequences obtained plus those (i.e. orf and orf ) derived from pesente et al. ( ) were aligned by guidance (using prank as alignment method) (penn et al., ) and score evaluated. for clarification purposes, all orf sequences published by pesente et al. ( ) were renamed with the accession number assigned to orf . jmodeltest . . (darriba et al., ) was used to select the model of evolution according to akaike information criterion (aic). phylogenetic trees based on orf and orf were reconstructed applying the maximum likelihood method implemented in phyml . (guindon et al., ) assuming the gtr + + i nucleotide substitution model. phylogenetic tree reliability was evaluated using a fast nonparametric version of the alrt (shimodaira-hasegawa [sh]-alrt), which was developed and implemented in the phyml . (anisimova et al., ) . orf , orf and concatenated sequence alignments were tested for evidence of recombination using rdp (martin et al., ) . in order to obtain a conservative estimate, a recombination event was accepted only when detected by two or more methods implemented in the program with a p-value lower than × − . a collection of partitions without recombination was obtained dividing the original alignment at the recombination breakpoint. phylogenetic trees were reconstructed for each partition using raxml (silvestro and michalak, ) and used to calculate per site log likelihoods for each alignment partition. statistical significance of topological incongruence between segments separated by recombination breakpoints were assessed through sh, kh, elw and au tests implemented in consel (shimodaira and hasegawa, ) . a p-value < . was assumed to indicate statistical significance. a discrete states phylogeographic reconstruction of prrsv strains migration pattern was performed using beast . . (drummond et al., ) as described by lemey et al. ( ) . the provinces where the samples had been collected were considered to be discrete states. an asymmetric substitution model, coupled with the bayesian stochastic search variable selection (bssvs), was implemented. non-recombinant, concatenated orf -orf sequences, for which sampling data was known, were analyzed for this purpose. bayesian factor (bf) was calculated in order to define well supported diffusion rates using spread (bielejec et al., ) . rates yielding a bf > were considered adequately supported (kass and raftery, ) . the same software was used to generate the kml file compatible with google earth displaying migration history. structural consequences of recombination on gp were considered for a recombinant cluster that demonstrated circulation over time in a farm. nucleotide and amino acid p-distance of recombinant strains from their parents were calculated using mega (tamura et al., ) . hydrophobicity profile was calculated using protscale (wilkins et al., ) assuming the kyte & doolittle scale. secondary structure and transmembrane topology of gp were predicted using psipred (http://bioinf.cs.ucl.ac.uk/psipred/). n-linked glycosylation sites were estimated using netnglyc . server (gupta et al., ) . the possible role of recombination in generating strains with different immunological properties was evaluated through in silico prediction of t-and b-epitopes. linear bcell epitopes were predicted using the bepipred . server (larsen et al., ) . for cytotoxic t lymphocytes epitopes, netctlpan . server (stranzl et al., ) a pan-specific major histocompatibility complex class i epitope predictor, integrating prediction of proteasomal cleavage, antigen transport efficiency and mhc-i binding affinity, was used. all swine mhc-i alleles deposited in the program database were selected to predict -, -, -, -mer peptides. mhc-ii binders were predicted using netmhcii . server (nielsen and lund, ) , searching -mer peptides that bound the collection of human mhc-ii (loci dr and dq). considering that the stronger the binding, the more likely the peptide to become t-cell epitopes (gustiananda, ), highly stringent cut-offs were applied. peptides were accepted as possible epitopes when their rank score was < % and ic nm < (including high and intermediate affinity binder of sla-i). to limit the presence of false positive results using hla-ii based software, only strong binder (ic nm < ) predicted by netmhcii were accepted, according to díaz et al. ( ) and gustiananda ( ). a total of orf and orf were obtained, including those achieved from pesente et al. ( ) . the accession numbers table strains detected as recombinants with rdp . when identified parental viruses are also reported. recombination breakpoints are defined by nucleotide position assuming the beginning of orf as position . major of sequences obtained in our study are provided in supplementary data . all of the strains were collected from a restricted geographic area of < , km , with a maximum distance between farms of km. for samples both orf and orf were available allowing the construction of a third database, based on the concatenation (i.e. the joining of two character strings end-to-end) of the respective orf and orf sequences of each strain. all the strains belonged to the type i subtype i, according to stadejek's classification (stadejek et al., (stadejek et al., , . phylogenetic analysis revealed some incongruences between trees obtained from orf and orf (fig. ) . the recombination scan on concatenated sequences coupled with topology comparison revealed statistically significant recombination events (table and supplementary data ). four recombinants (i.e. strains , / , and ) displayed a single breakpoint between the end of orf and the beginning of orf . unfortunately, a more precise localization could not be performed because the segment spanning these orfs was not sequenced. besides, only for strain both parental viruses were clearly identifiable. only donors of the orf segment were clearly detected for both / and while no closely related sequences were identified in orf . however, the analysis of the wider orf database displays a certain relatedness of / with strains / . another recombination event between orf and orf was detected (strain ay ), although only one parent could be evidently identified (ay ). analysis refinement using the orf dataset confirmed the presence of recombination breakpoints in position and and identifies ay and ay as major and minor parents. the orf tree reconstruction revealed a close relationship between ay and ay . the last recombinant strain was identified to be the result of two recombination events. strains and were classified as recombinants between / (orf ) and / (orf ). at the same time strain and the closely related were predicted as donors in a recombination event within orf (segment - ): strains / , / , / and / were detected as recombinants between (minor parent) and / (major parent). remarkably, both parental and recombinants strains were collected from the same farm at different time periods (see supplementary data ). particularly, recombinant strains were found twice about months apart while their co-circulation with / . on the contrary, the minor parent (strain ), was recorded only two years later. predicted recombination events involved both relatively neighbor (< km) and distant (more than km) farms. remarkably, the same strain (i.e. / ) was implied in two different recombination events (i.e. events and ) ( table ) that took place in the same geographic area. the parental strains involved in these events were sampled from the same farm (i.e. / and ) or in nearby farms (i.e. origin farms of / and / were about km apart), supporting the short-range spread of prrsv strains. at the same time, discrete state phylogeographic analysis evidenced the presence of well-supported migration rates among provinces of northeastern italy and an extensive prrsv circulation over time (supplementary data ). nucleotide and amino acid p-distance in the segment internal and external to recombination breakpoints. distance is calculated as mean distance between recombinants' group and parental viruses. strain gp of recombinant strains / , / , / and / , demonstrating a prolonged circulation, was further analyzed to explore the presence of structural differences from parental strains which may affect viral fitness. strains and / , assumed as representative of minor and major parents, respectively, were also included in the analysis. the calculation of the p-distance demonstrated a relevant amino acidic difference between the two parents: . % in the internal region to recombination breakpoints (aa - ) and about . % in the external. comparable results were obtained considering nucleotides (table ) . hydrophobicity profile and secondary structure were also affected and recombinants displayed some different secondary structures from both major and minor parents (supplementary data ). however, the transmembrane regions' prediction revealed no differences among strains even though recombination spanned part of the first ectodomain, the three transmembrane segments and the first part of the main endodomain. some differences were observed in glycosylation pattern: the strain / , / , / and / were predicted to be glycosylated in position , and . a similar pattern was displayed by strain with the only exception that n-linked glycosylation was present in position instead of . certain dissimilarity was displayed by strain / which lost the glycosylation in position . consequences of recombination on gp antigenicity were also estimated in silico. for computational easiness and clarification, considering the high percentage of identity purposes (p-distance = . and . at amino acid and nucleotide level, respectively), / was assumed as representative of all recombinant strains. linear b-cell epitopes, although slightly different in extension, were substantially unchanged in position: a region around the first glycosylation site and two sequences within the c-terminal endodomain were identified as possible epitopes in all strains (fig. ) . the first one, which claimed to be a neutralizing epitope (aa - ) (mateu and diaz, ; plagemann, ) , spanned the region where the initial fig. . phylogenetic trees of orf (on the left) and orf (on the right) reconstructed using raxml. only strains for which both orf and orf were available were included in this picture. recombinants are marked with black circles to emphasize the different topology between the two orfs. recombination breakpoint was estimated. one amino acidic difference between parental viruses was identifiable at amino acid (g-n), with the recombinants sharing the major parent sequence. aa was different in both parental and recombinant strains with the latter sharing the same residue. the analysis of t-cell epitope evidenced that, comparing major, minor and recombinant strains, approximately the same sla-i alleles recognized the common region of gp . two main differences were estimated between major and minor parents in predicted ligands of sla-i (region - and - ) (fig. ) . accordingly, the recombinant epitopic profile can be described as a combination of parental patterns. a similar picture was obtained with regard to hla-ii. parents differed in region - , - and - . recombinants resulted to be approximately a combination of parental strains. however, epitopes in position aa - and aa - were lost while one peculiar epitope (aa - ) was predicted (fig. ) . rna viruses represent both a fascinating opportunity and a challenge in the study of evolutionary processes (holmes, ) . their populations usually harbor abundant genetic variability, due in large part to the combination of high mutation rate and large population size. although recombination had been thought to be rare in these viruses, recent studies disavowed this theory, demonstrating not only that it is a frequent phenomenon in some virus families, especially retroviruses and positive-sense single stranded rna viruses, but also that it can sometimes have a major impact on their emergence, evolution, and epidemiology (simon-loriere and holmes, ) . a similar picture can be drawn for prrsv, a highly variable rna virus, recently emerged as agent of devastating impact on pig production. although the remarkable genetic distance is mainly attributable to the high substitution rate (yoon , ) and to the abrupt increase in population size, consequent to the advent of high density confinement management practice in europe and north america coupled with increasing pig transportation (murtaugh et al., ) , the role of recombination is nowadays increasingly studied. a high recombination frequency was demonstrated both in vitro (van vugt et al., ; yuan et al., ) and in experimentally infected pigs (liu et al., ) . nevertheless, the frequency of naturally occurring recombination was believed to be much lower. in fact, most of the recombination events identified so far in the field were only represented by single sequences rather than by circulating "recombinant progenies" (fang et al., ; forsberg et al., ; li et al., ; murtaugh et al., ; shi et al., a; stadejek et al., ; yoshii et al., ; yuan et al., ) . however the vast majority of field evidences were based on sequences obtained from strains collected at different time points and from distant locations. an underestimate of the recombination rate in the field due to sampling limitations (shi et al., a) is plausible. our study attempted to deal with this inadequacy, performing an intensive sampling based on a narrow spatial and temporal scale. six recombination events were detected in concatenated orf -orf , affecting orf and the region between orf and orf . according to previous studies (shi et al., a) , no recombination events were displayed within orf . six independent recombinant strains out of were found. this proportion of recombinant strains was comparable with those reported in vitro by yuan and van vugt for prrsv (van vugt et al., ; yuan et al., ) and approximate the range reported for some coronavirus (lai, ) . several hypotheses can be proposed to explain the high frequency of recombination events observed. firstly, it should be accounted that sequences considered, originating from a small area, are relatively similar (mean p-distance: orf = . ; orf = . ; concatenated = . ). van vugt et al. ( ) demonstrated that recombination occurs preferentially between genome regions with a high percentage of similarity, which is compatible with the transcription strategy of arterivirus (pasternak et al., ) . liu et al. ( ) demonstrated that prrsv is really prone to recombine in experimentally co-infected pigs. the occurrence of transmission of different strains between farms therefore plays a key role in the likelihood of recombination (forsberg et al., ) . analysis of geographic relation between parental strains revealed that viruses originating from both distant and neighboring farms participate to this phenomenon. accordingly, phylogeographic analysis revealed the presence of well-supported migration rates among provinces of northeastern italy. these data support the field evidence that multiple strains co-infection is relatively frequent in italian pig farms. high farm density, intense animal movements and ineffective biosecurity measures are the most probable cause of this situation. a further hypothesis is that, collecting numerous samples on a narrow spatial and temporal scale makes it more likely to find recombinants with low fitness, whose survival and spreading among farms is unlikely, and which otherwise would not have been collected performing a less intensive sampling. the evidence that quite all recombinants were sampled only once and did not demonstrate any persistence or geographical spread supports this hypothesis. a partial exception was represented by strains / , / , / and / . these recombinants were collected from the same farm approximately months apart demonstrating a certain fitness. in silico analysis revealed a noteworthy amino acid difference from parental strains resulting in different hydrophobicity and secondary structure. recombinants secondary structure was not a simple combination of the parental ones but presented some peculiar characters, probably as a consequence of interaction between distant amino acidic regions or minor aa substitutions occurred after recombination took place. both b-cell and t-cell epitopes are constrained by sequence specificity, and mutations within epitopes can result in immune escape. obviously, mutations within an epitope can directly affect antibody-antigen interactions or epitope-mhc and t-cell receptor interactions. additionally, mutations outside of the epitope can inhibit antibody binding through conformational changes, or inhibit proper cleavage and processing of t-cell epitopes (korber et al., ) . for these reasons, the role of recombination in generating strains with potentially different immunological features was investigated. two linear b-cell epitopes were predicted within major endodomain and one within the major ectodomain, in substantial agreement with antigenic region identified in vitro by other authors (vanhee et al., ) . the first two are unlikely involved in viral neutralization due to their localization (vanhee et al., ) . on the contrary, the epitope identified within the gp main ectodomain is usually regarded as one of the major neutralization epitopes (mateu and diaz, ) . interestingly the beginning recombination breakpoint was predicted within this region. all the predicted antigenic regions displayed some amino acidic differences between parental viruses affecting also the sequences of recombinants strains (fig. ) . it has been demonstrated that different prrsv isolates differ in susceptibility to neutralization (martínez-lobo et al., ) . even if a clear correlation with protein sequence was not identified for genotype i, kim et al. demonstrated that amino acid substitution in specific positions of ectodomain can affect neutralization in genotype ii strains (kim et al., ) . as a consequence, it is possible to suppose that the recombination events discovered could have influenced viral susceptibility to humoral immunity through changes in epitope sequence, possibly in association with minor conformational changes that might have modified epitope accessibility or immunogenicity (martínez-lobo et al., ) . glycosylation pattern showed some differences among parental and recombinant strains. it is well established that glycosylation plays a major role in protecting against humoral response. it has been reported that removal of n-glycosylation site renders the virus more susceptible to neutralization and elicits a significantly greater neutralizing antibodies response (ansari et al., ; darwich et al., ; vu et al., ) . however an actual correlation between glycosylation number and neutralization phenotype has not been found yet (martínez-lobo et al., ) . loss of glycosylation in position , predicted in strain / , is usually regarded as deleterious, being it strongly required for both assembly and infectivity in lv (dokland, ) . however, although not frequently, field strains without this glycosylation site have been reported (mateu et al., ; stadejek et al., ) . besides, balka reported a case of reversion to virulence of a prrsv vaccine strain in which the new virulent strain was characterized by the loss of glycosylation in position associated with glycosylation in position (while vaccine has n- ) (balka et al., ) . it is also possible that apparently minor changes in glycosylation profile could significantly affect strain virulence. also, considering potential t-cell epitopes, as expected, recombinant strains presented a combination of different parental epitopes, besides the one additional mhc-ii ligand that was predicted. despite our conservative settings, in silico prediction of epitopes cannot be considered an accurate tool, even if used with some success also in veterinary medicine (díaz et al., ) . however it should be stressed that the main purpose of this analysis was to verify if recombination between parental strains may generate an amino acidic difference relevant enough to affect their original physicochemical and antigenic properties. the results provide strong evidence that through recombination a new strain was generated which displayed characters different enough to potentially alter the development of acquired immunity or decrease the effectiveness of recall response against parental strains. nevertheless the definition of actual b and t-cell epitopes of these strains, their effect on cross-protection or the effects of recombination on virulence are beyond the scope of this study. this study, conducted in a restricted geographical area ( , km ) in a limited time frame, evidenced a frequent occurrence of prrsv recombination in pig farms, compatible with that reported in vitro and in vivo in experimental conditions. these results provide further confirmation that prrsv, as other nidovirales family members, is really prone to undergo recombination events, probably due to their peculiar replicative strategy (pasternak et al., ; simon-loriere and holmes, ) . on the other hand, the lower frequency of recombinant strains detected on a broader scale in other studies, suggests that recombinants usually have low fitness and rarely gain an evolutionary advantage over their parents. in the present study the prolonged circulation of recombinant viruses, displaying a combination of parental physicochemical and antigenic profiles, was demonstrated in a farm. their temporal survival and spatial spread remained limited, suggesting a marginal role of recombination in driving prrsv evolution. anyhow, recent evidences demonstrated the emergence of virulent type ii strains through recombination (chen et al., ; shi et al., ) . considering the frequency of recombination and its ability to generate strains of unexpected behavior (li et al., ; liu et al., ) , further efforts should be deserved to study the consequences of this phenomenon on various aspects as infectivity, virulence, immunogenicity and diagnosability providing more extensive knowledge on the evolutionary driving forces of prrsv. survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies genetic diversity of porcine reproductive and respiratory syndrome virus strains circulating in hungarian swine herds spread: spatial phylogenetic reconstruction of evolutionary dynamics two natural recombinant highly pathogenic porcine reproductive and respiratory syndrome viruses with different pathogenicities porcine reproductive and respiratory syndrome virus jmodeltest : more models, new heuristics and parallel computing certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology in silico prediction and ex vivo evaluation of potential t-cell epitopes in glycoproteins and and nucleocapsid protein of genotype-i (european) of porcine reproductive and respiratory syndrome virus the structural biology of prrsv bayesian phylogenetics with beauti and the beast . diversity and evolution of a newly emerged north american type porcine arterivirus: analysis of isolates collected between discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . immunoinformatics analysis of h n proteome for designing an epitope-derived vaccine and predicting the prevalence of pre-existing cellular-mediated immunity toward bird flu virus in indonesian population the evolution and emergence of rna viruses bayes factors significance of genetic variation of prrsv orf in virus neutralization and molecular determinants corresponding to cross neutralization among prrs viruses immunoinformatics comes of age recombination in large rna viruses: coronaviruses improved method for predicting linear b-cell epitopes bayesian phylogeography finds its roots recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo mesquite: a modular system for evolutionary analysis rdp : a flexible and fast computer program for analyzing recombination porcine reproductive and respiratory syndrome virus isolates differ in their susceptibility to neutralization the challenge of prrs immunology evolution of orf of spanish porcine reproductive and respiratory syndrome virus strains from the potential role of genetic recombination in the evolution of new strains of porcine reproductive and respiratory syndrome virus (prrsv) prrsv, the virus the everexpanding diversity of porcine reproductive and respiratory syndrome virus appearance of novel prrsv isolates by recombination in the natural environment assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds sensitive detection and typing of porcine reproductive and respiratory syndrome virus by rt-pcr amplification of whole viral genes nidovirus transcription: how to make sense guid-ance: a web server for assessing alignment confidence scores evaluation of three rt-pcr assays for the detection of porcine and respiratory syndrome virus (prrsv) in diagnostic samples new insights into the genetic diversity of european porcine reproductive and respiratory syndrome virus (prrsv) phylogenetic analysis of orf and orf sequences of porcine reproductive and respiratory syndrome virus (prrsv) from prrs-positive italian farms: a showcase for prrsv epidemiology and its consequences on farm management gp ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain lelystad virus recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china molecular epidemiology of prrsv: a phylogenetic perspective phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses consel: for assessing the confidence of phylogenetic tree selection raxmlgui: a graphical front-end for raxml why do rna viruses recombine? porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe molecular evolution of prrsv in europe: current state of play netctlpan: pan-specific mhc class i pathway epitope predictions mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein protein identification and analysis tools in the expasy server tracing the genetic history of porcine reproductive and respiratory syndrome viruses derived from the complete orf - sequences: a bayesian coalescent approach genetic polymorphism of the nsp gene in north american type-porcine reproductive and respiratory syndrome virus recombination between north american strains of porcine reproductive and respiratory syndrome virus the work here described was supported by the university of padua grant, year ( a - / ). supplementary material related to this article can be found, in the online version, at http://dx.doi.org/ . / j.virusres. . . . key: cord- -gev xq a authors: zhu, liqian; yang, shen; tong, wu; zhu, jianping; yu, hai; zhou, yanjun; morrison, robert b.; tong, guangzhi title: control of the pi k/akt pathway by porcine reproductive and respiratory syndrome virus date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: gev xq a phosphatidylinositol- -kinase (pi k)/akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. in the present study, we compared the phosphorylation status of akt during infection of marc- cells and porcine alveolar macrophages (pams) with highly pathogenic prrsv (hp-prrsv) strain hun . we observed that biphasic activation of akt was induced in at both the early stage ( , and min postinfection) and the late stage ( and h postinfection) of hp-prrsv infection of marc- cells, while an early-phase activation of akt was found exclusively in virus-infected pams in vitro. analysis with the pi k-specific inhibitor ly confirmed that pi k acted as the upstream activator for the virus-induced activation of akt. uv-irradiation-inactivated virus still induced the early event in pams but not in marc- cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated akt within different cell cultures. we further demonstrated that foxo and bad, which serve as downstream targets of akt, were phosphorylated in virus-infected marc- cells. moreover, the suppression of phosphorylated akt with ly significantly inhibited the virus-induced cytopathic effect (cpe) on marc- cells, but it had a negligible effect on virus propagation. collectively, our data provide new evidence of a novel role for the pi k/akt pathway in prrsv infection of marc- cells. porcine reproductive and respiratory syndrome virus (prrsv) is a small, enveloped, positive-strand rna virus belonging to the family arteriviridae [ , ] . since it emerged in late s in europe and north america, it has caused significant economic losses to the pork industry worldwide [ , ] . in , an outbreak of hp-prrsv described as ''pig high fever disease'' occurred in china and caused disastrous loses to the farmers [ , ] . this disease is currently a major concern for the swine industry worldwide. phosphatidylinositol- -kinase (pi k)/akt is a key signaling transduction pathway in the regulation of cell survival, cell proliferation and differentiation, and cell apoptosis [ ] [ ] [ ] ] . the activation of pi k-akt pathway promotes cell survival by the phosphorylation of numerous substrates such as glycogen synthase kinase- (gsk- ), foxo , bad and mtor [ , , , , ] . many viruses are known to manipulate this pathway in favor of their replication, such as influenza a virus [ ] , hepatitis b virus [ ] , hepatitis c virus [ ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] , junín virus [ ] , human immunodeficiency virus type [ ] , bovine herpesvirus type [ ] and infectious bursal disease virus [ ] . previous studies have shown that during prrsv infection of porcine monocyte-derived dendritic cells (mo-dcs), the pi k/akt pathway is activated at min and h postinfection (h p.i.), and it is inhibited at h p.i. [ ] . in vivo, the virus shows a very narrow cell tropism and infects a specific subpopulation of porcine macrophages [ , ] . in vitro, efficient prrsv replication is only observed in primary pig macrophages (e.g., alveolar macrophages), differentiated monocytes [ ] or cells derived from african green monkey kidney, such as marc- [ ] . for easy manipulation, marc- cells provide an important tool for the study of prrsv replication. previously, it was reported that the pi k/akt pathway is regulated by prrsv in a complex manner in mo-dcs [ ] , but whether this is a universal phenomenon in all permissive cells is unknown. a number of studies have shown that the pi k/akt signaling pathway is functionally dependent on downstream substrates of akt such as foxo , bad and mtor for regulation of cell survival [ , , , ] . the downstream targets of akt controlled by prrsv have not been identified. the objective of this study was to address how the pi k/akt pathway is modulated in infection of marc- cells and pams with highly pathogenic prrsv. we demonstrated that the pi k/ akt pathway was activated by hp-prrsv in a cell-culturedependent manner and that the downstream targets foxo and bad were regulated through this pathway. in addition, we found that activated pi k was required for the development of cpe in marc- cells, but not for production of infectious progeny. the hp-prrsv strain hun used in the present study was isolated from a pig showing signs of ''high fever syndrome'' originating from a swine farm in the region of hunan province, china, in [ , ] . a stock of hun virus at passage was prepared in marc- cells, titrated, and stored at - °c until use. marc- cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco brl) supplemented with % fetal bovine serum (gibco brl) in an incubator at °c containing % co . the pam cultures were obtained by broncho-alveolar lavage of -week-old domestic piglets from a prrsv-negative herd affiliated with shanghai agricultural science institute. all animal experiments were conducted according to the guidelines and approved protocol of the shanghai veterinary research institutional animal care committee. the pams were cultured with rpmi- medium supplemented with % fetal bovine serum. all of our experiments were conducted at veterinary laboratory biosafety level . rabbit monoclonal antibodies (mab) recognizing phospho-akt (ser ), phospho-bad (ser ), phospho-foxo (ser ) and phospho-mtor (ser ) were purchased from cell signaling technology inc. mouse monoclonal b-actin antibody was obtained from santa cruz biotechnology, inc., santa cruz, calif. horseradish peroxidase (hrp)-conjugated anti-rabbit igg and anti-mouse igg were purchased from zhongshan golden bridge biotechnology co., ltd. pi k-specific inhibitor ly was supplied by cell signaling technology, and a stock of this inhibitor was prepared with dmso. marc- cells in -mm dishes were grown to %- % confluence, whereupon they were subjected to serum starvation for h in serum-free dmem. subsequently, growth-arrested cells were infected at a multiplicity of infection (moi) of with hun virus or were mock infected with the same medium. one hour postinfection (p.i.), the cells were washed with phosphate-buffered saline (pbs, ph . ) and then cultured in fresh dmem for various times as indicated. for inhibitor experiments, marc- cells were pretreated with the pi k inhibitor ly for h. cells were then infected with the virus at an moi of for h, washed with pbs, placed in serum-free medium containing fresh inhibitor, and sustained for h. dmem containing . % dmso (v/v) was used for the mock treatment unless otherwise specified. growth-arrested pam cells cultured in -mm dishes were infected at an moi of with hun or were mock infected with medium and then incubated for various times as indicated. the cells were then collected for western blotting analysis. to inactivate hp-prrsv by uv irradiation, the virus stocks were dispersed in -cm tissue culture dishes and placed directly under a uv lamp ( w) for min. complete inactivation of the virus was confirmed by the titration on marc- cells. both marc- cells and pams were exposed to uv-irradiation-inactivated virus to analyze the variation of phosphorylated akt within h p.i. as indicated. after hp-prrsv infection for the lengths of time indicated, cells were washed with pbs and lysed with lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . lg/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). the cell lysates were cleared by centrifugation for min at , g prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) under reducing conditions. the separated proteins were transferred onto nitrocellulose membranes (millipore). the membranes were blocked for h at room temperature with skim milk solution ( % in tris-buffered saline containing . % tween ). blots were incubated overnight at °c with primary antibodies followed by incubation for h with the secondary antibody (conjugated horseradish peroxidase). after extensive washing, the immunoreactive bands were detected by enhanced chemiluminescence (ecl) (pierce) and subsequently reprobed for total protein loading using anti-b-actin antibody. to analyze whether inhibition of pi k by ly would affect virus replication, serum-starved marc- cells seeded in -well plates were pre-incubated with the inhibitors or mock pretreated with dmem containing . % dmso for h at °c and then infected with hun virus for h together with or without the inhibitor. after extensive washing with pbs, the cells were placed in dmem with or without fresh inhibitor for or h. after two rounds of freezing and thawing, the infectious progeny was titrated with marc- cells using a tcid assay. we first used the marc- cell model to examine the kinetics of akt phosphorylation at ser , which is required for akt activation, to determine whether hp-prrsv infection modulates the pi k/akt pathway. cells that were mock infected with dmem medium or infected with hp-prrsv strain hun were harvested at min, min, min, min, h, h, h, h, h and h postinfection for western blot analysis. it was found that akt was activated in a biphasic manner after infection of marc- cells with hp-prrsv (fig. a, top panel) . the first phase of akt activation occurred early ( and control of the pi k/akt pathway by prrsv min p.i.) in virus infection (fig. a) . at h p.i., the phosphorylation of akt had returned to the basal level, and after a certain interval, akt phosporylation increased again in the late stage of infection. the amount of phosphorylated akt was evidently increased at h p.i., and this level was sustained for the reminder of the investigation ( h p.i.) (fig. a, upper panel) . the changes in akt phosphorylation were not due to variation in the total amount of protein loaded (fig. a, bottom panel) . since akt can be activated by both pi k-dependent and pi k-independent pathways [ ] , the role of activated pi k in the virus-induced akt activation was subsequently investigated. here, the chemical ly , a potent and specific inhibitor of pi k, was used. as demonstrated by immunoblot assay, lm of ly could efficiently inhibit the phosporylation of akt induced by the virus at the late stage of infection (fig. b) . this indicated that the activated pi k accounted for the virus-triggered akt activation. taken together, the data indicate that the pi k/akt pathway was activated in infection of marc- cells by prrsv. since pams and monocytes are the major natural target cells during both acute and persistent prrsv infection [ ] , it deserved to be investigated whether the virus controls the pi k/akt pathway during infection of pams. here, the kinetics of phosphorylated akt was determined in hp-prrsv-infected pams in vitro. serum-starved pams that were mock infected with rpmi- medium or infected with hun at min, min, min, min, h, h, h, h, h and h postinfection were collected for western blotting. as illustrated in fig. , the amount of phosphorylated akt increased dramatically from to min p.i. however, this activation was transient and quickly returned to the basal level at min postinfection. in addition, the enhanced phosphorylation of akt was not observed at the later stage. this suggested that akt was only activated by hp-prrsv at the early phase in the infection of pams, which is different from what was observed in marc- cells. since increased phosphorylation of akt induced by hp-prrsv was observed at min and min postinfection in virus-infected marc- cells, and at , and min postinfection in pams, we hypothesized that the virus entry process accounted for this early-stage activation. thus the uv-irradiated hp-prrsv virus was employed to address this issue. under the conditions described above, the virus could be completely inactivated (data not shown). such inactivated virus fails to express viral proteins due to the formation of thymidine dimers, which prevent the transcription of viral genes but do not interfere with the capacity of the virus for receptor binding and entry into host cells by endocytosis [ ] . as showed in fig. , the exposure of cells to uv-irradiated virus led to phosphorylation of akt at and min postinfection in pams (fig. b ), but not in marc- cells (fig. a) . this indicates that virus-cell interactions during the binding or entry process are probably responsible for this event in pams, whereas in marc- cells, a different mechanism may account for this activation. by western blotting using rabbit monoclonal antibodies against phospho-bad (ser ), phospho-foxo (ser ) and phospho-mtor (ser ). compared to the mockinfected control, virus replication resulted in significantly increased phosphorylation of both foxo and bad, while no change in mtor was observed (fig. a) . phosphorylation with both foxo and bad were most evident at h p.i., which corresponds in time to the change in phosphorylated akt. to address whether the change in phosphorylated foxo and bad was induced by the virus through the pi k/akt pathway, the pi k-specific inhibitor ly was employed for further investigation. as shown in fig. b) (fig. a) . unexpectedly, the virus-induced activation of the pi k/akt pathway was not essential for production of infectious prrsv progeny virus. the cytopathic effect (cpe) in hun -infected marc- cells is characterized by cell congregation and contraction [ ] . interestingly, in infected marc- cells treated with ly , the cpe was significantly alleviated. under normal conditions, prrsv-infected marc- cells showed considerable cpe at h p.i. (fig. b, b) , but no cpe was observed in hun -infected marc- cells when lm of ly was used to treat the cells (fig. b, d) . in addition, the inhibitor at a concentration of lm could not induce cpe, and it was apparently not cytotoxic to marc- cells, (fig. b, c) , which was confirmed by mtt assay (data not shown). thus, these findings suggest that the pathway may be linked to hp-prrsv-induced cpe formation in marc- cells. many viruses interfere with signaling pathways in their infected host cells to favor productive infection, which can also impact on the physiology of the host cell as well as pathogenesis. therefore, the identification of cellular factors involved in virus replication is important for the design of novel antiviral strategies. in this regard, the manipulation of cellular pathways regulating cell apoptosis or survival affords great advantages to virus replication [ ] , and many viruses have been demonstrated to regulate cell survival via the regulation of the pi k/akt pathway [ , ] . a previous report has indicated that the pi k/akt pathway is activated in the early phase and subsequently inhibited at h p.i. during prrsv infection of mo-dcs [ ] . here, hp-prrsv strain hun was used to further investigate how and why this pathway is controlled by the virus. here, we report that the activation of the pi k/akt pathway was significantly enhanced by the hun virus at both the early and late stage of infection of marc- cells, while a distinct pattern was adopted during infection of pams in which the phosphorylation of akt occurred exclusively in the early stage, as early as min p.i., and then returned to the basal level at min p.i. (figs. a and ). when the cells were exposed to the uv-irradiationinactivated virus, significantly increased phosphorylation of akt was observed in pams, but not in marc- cells. entry of macrophages by prrsv occurs via a few similar but also different mechanisms compared to entry into marc- cells [ ] . it could be inferred that the virus entry process in pams was enough to activate the pi k/ akt pathway, while a different mechanism of activation was used in marc- cells. it is possible that a different entry mechanism results in this difference in the regulation of akt. together with previous data reported by zhang et al. [ ] , we generalize that prrsv regulates the pi k/ akt pathway by different mechanisms in mo-dcs, pams and marc- cells. various pro-apoptotic proteins have been identified as downstream targets of akt, including the bcl- family members bad, gsk- a/b, and mtor and the foxo transcription factor family members (fkhr) [ , , , , ] . they are normally regulated in a pi k/akt-dependent manner. for example, the phosphorylation of bad (ser- ) was decreased but the phosphorylation of mtor (ser ) and foxo (ser ) was increased by infection with varicella-zoster virus [ ] . similarly, it has been shown that the pi k/akt/mtor cascade contributes to the establishment of persistent hcv infection [ ] . in the present study, we demonstrated that both foxo and bad were regulated by prrsv through the control of the pi k/ akt pathway in the late stage of infection in marc- cells (fig. ) , and therefore the, pi k/akt/foxo and pi k/akt/bad cascades may be manipulated by prrsv to control host-cell survival. the pi k/akt pathway has been shown to play important roles in different steps of the life cycles of a variety of viruses [ ] . for example, this pathway has been shown to be involved in the regulation of ebola virus entry of host cells [ ] , to contribute to arenavirus budding [ ] , and to be involved in the control of synthesis of viral rna of nonsegmented negative-stranded rna viruses [ ] . unexpectedly, our investigation with the pi k-specific inhibitor ly showed that the pi k/akt pathway has a negligible effect on the titer of infectious prrsv progeny in marc- cells (p [ . ), as determined at both and h p.i. (fig. a ), but inhibition of this pathway significantly blocked virus-induced cpe formation. to our knowledge, similar results have not been documented for other viruses. therefore, it will be worthwhile in the future to investigate how the pi k/akt pathway regulates cpe formation during prrsv infection. in summary, our findings, together with a previous report of prrsv regulating akt in mo-dcs, suggest that pi k/akt signaling is modulated by prrsv in different ways in different cell types. foxo and bad were employed by the virus as downstream targets of the pi k/ akt pathway. unlike other viruses for which the control of this pathway contributes significantly to virus replication, for prrsv, it is not important for generation of infectious progeny, but this modulation changes the physiology of the host cell, as revealed by the significant alleviation of cpe. this finding may offer a new and important insight into the role of pi k/akt in virus infection, or even in viral pathogenesis. porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection secondary enterovirus infection in the murine model of myocarditis. pathologic and immunologic aspects akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor the torrid affairs of viruses: effects of mammalian dna viruses on the pi k-akt-mtor signalling pathway cell cycle and death control: long live forkheads phosphoinositide -kinase signalling pathways akt/pkb and other d phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation involvement of pi k/akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group the pivotal role of phosphatidylinositol -kinase-akt signal transduction in virus survival inhibition of glycogen synthase kinase- by insulin mediated by protein kinase b susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and cd effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) hijakt: the pi k/akt pathway in virus replication and pathogenesis apoptosis. a bad kinase makes good regulation of hepatitis b virus replication by the phosphatidylinositol -kinase-akt signal transduction pathway control of the pi k/akt pathway by prrsv alterations in microrna expression profile in hcv-infected hepatoma cells: involvement of mir- in regulation of hcv replication via the pi kinase/akt pathway enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line participation of the phosphatidylinositol -kinase/akt pathway in junin virus replication in vitro targeting the pi k/akt cell survival pathway to induce cell death of hiv- infected macrophages with alkylphospholipid compounds activation of the n-ras-pi k-akt-mtor pathway by hepatitis c virus: control of cell survival and viral replication lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav jnk and pi k/akt signaling pathways are required for establishing persistent sars-cov infection in vero e cells the forkhead transcription factor foxo regulates adipocyte differentiation mammalian target of rapamycin is a direct target for protein kinase b: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states varicella-zoster virus requires a functional pi k/akt/gsk- alpha/beta signaling cascade for efficient replication phosphoinositide- kinase-akt pathway controls cellular entry of ebola virus effect of the phosphatidylinositol -kinase/akt pathway on influenza a virus propagation akt plays a critical role in replication of nonsegmented negative-stranded rna viruses highly pathogenic porcine reproductive and respiratory syndrome the pi k/akt pathway contributes to arenavirus budding porcine reproductive and respiratory syndrome virus entry into the porcine macrophage infectious bursal disease virus activates the phosphatidylinositol -kinase (pi k)/akt signaling pathway by interaction of vp protein with the p alpha subunit of pi k activation of the pi k/akt signaling pathway during porcine circovirus type -infection facilitates cell survival and viral replication serine phosphorylation of death agonist bad in response to survival factor results in binding to - - not bcl-x(l) a dual effect of porcine reproductive and respiratory syndrome virus replication on the phosphatidylinositol- -kinase-dependent akt pathway cell biology. gsk- beta and microtubule assembly in axons highly virulent porcine reproductive and respiratory syndrome virus emerged in china biphasic activation of pi k/akt and mapk/erk / signaling pathways in bovine herpesvirus type infection of mdbk cells key: cord- -gtg hiu authors: prather, randall s.; wells, kevin d.; whitworth, kristin m.; kerrigan, maureen a.; samuel, melissa s.; mileham, alan; popescu, luca n.; rowland, raymond r. r. title: knockout of maternal cd protects fetuses from infection with porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: gtg hiu after infection of the porcine dam at about days of gestation, porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta and begins to infect fetuses. outcomes of include abortion, fetal death and respiratory disease in newborn piglets. cd is the receptor for the virus. in this study, cd -positive fetuses, recovered between days of gestation or days after maternal infection, were completely protected from prrsv in dams possessing a complete knockout of the cd receptor. the results demonstrate a practical means to eliminate prrsv-associated reproductive disease, a major source of economic hardship to agriculture. porcine reproductive and respiratory syndrome (prrs) is the most economically important disease of swine in north america, europe and asia, costing north american producers approximately $ million annually . losses are the result of respiratory disease in young pigs, poor growth performance, reproductive failure, and in utero infection . the reproductive form of the disease accounts for an estimated % of losses, the result of abortions, dead fetuses, and respiratory disease in newborns. in its severest form, reproductive prrs can result in % mortality of fetuses/neonates, along with increased mortality for the dams. pigs that survive in utero infection become continuous sources of virus in downstream production phases, resulting in endemically infected herds . the severest form of reproductive disease is associated with a group of highly virulent isolates referred to as atypical prrsv , . interestingly, many of the atypical prrsv isolates emerged from prrs-vaccinated farms . in , an atypical virus, called high pathogenic prrsv (hp-prrsv), appeared in china and continues to decimate pig populations in that country . since the standard commercial breeding facility contains about , sows, an outbreak of high mortality reproductive prrs can have a devastating impact. to ensure sustainability of pork production and food security, solutions for the control of reproductive prrs remain a priority. vaccines have been unable to control the disease, largely because of genetic diversity within the structural proteins of the virus . in practice, intensive biosecurity measures provide the only means of protecting the reproductive herd. along with lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv), prrsv belongs to the family, arterviridae. structurally, the arteriviruses resemble togaviruses, but similar to coronaviruses, replicate via a nested ′-co-terminal set of subgenomic mrnas, which possess a common leader and a poly-a tail. arteriviruses exhibit a tropism for macrophages and possess the capacity to establish subclinical persistent infections, as well as cause severe and fatal disease . the reproductive form of prrs occurs following the infection of pregnant gilts or sows at about days of the day gestation period , . after an initial phase of replication in maternal macrophages, the virus crosses the placenta and begins to productively infect fetuses. the virus initially infects only a small number of fetuses, followed by horizontal transmission of virus from fetus to fetus . the exact mechanism of how the virus crosses the placenta remains unknown, but could be similar to the infected "trojan horse" macrophage, previously described for ldv . unlike the alveolar macrophages in adult animals, the primary site of prrsv replication in the fetus is the thymus . since the pig fetus becomes immunocompetent at about days of gestation, prrsv infection occurs in a fetal immune environment containing functional b and t cells , . cd is a kda type membrane protein composed of nine scavenger receptor cysteine-rich (srcr) domains and two spacer domains along with a transmembrane domain and a short cytoplasmic tail. in addition to functioning as a virus receptor, cd exhibits several important functions related to maintaining normal homeostasis. for instance, following infection or tissue damage, cd functions as a scavenger molecule, removing haptoglobin-hemoglobin complexes from the blood . the resulting heme degradation products regulate the associated inflammatory response . cd as a receptor for prrsv was first described by calvert et al. . transfection of non-permissive cell lines with cd cdna from a variety of species, including simian, human, canine, and mouse can make cells permissive for infection. we recently showed that pigs with a complete knockout (ko) of the cd gene lack cd expression on macrophages and fail to support prrsv infection , . since cd expression is a dominant trait and inherited in a classic mendelian fashion, offspring possessing normal cd expression and function can be derived by crossing a ko cd −/− female pig with a wildtype (wt) cd +/+ male. for this study, cd ko gilts were bred with wt boars producing heterozygous, cd +/− fetuses. the hypothesis to be tested was that the presence of the cd ko genotype of the dam would be sufficient to protect fetuses following maternal infection with prrsv. a detailed description of the knockout alleles used in this study is shown in table . each knockout allele possessed a mutation in exon that was predicted to result in a codon frameshift followed by a premature stop codon in the mrna. the matings between wt and cd ko parents are summarized in table . the first group of three dams, which served as positive infection controls, were cd +/+ dams carrying cd +/+ fetuses (++/++ group). a second group (−/+−) were cd −/− dams carrying cd +/− fetuses. in this group, the cd −/− dams are unable to support prrs replication, while the cd +/− fetuses retain susceptibility to prrs infection. and finally, a third group (−/−) consisted of cd −/− dams carrying cd −/− fetuses. for the last group, both dams and fetuses should be resistant to infection. clinical signs in the infected wt dams included lethargy and transient inappetance. the ko dams showed no clinical signs. during the study period, one wt dam, no. , aborted on day of gestation ( dpi). prrsv nucleic acid, measured at dpi, showed a viremia level for dam no. of . log templates per reaction, demonstrating the presence of a productive prrsv infection. between and dpi, all remaining dams were euthanized and uterine horns immediately removed. beginning at the tip of each horn, fetuses and placentas were removed, assessed for the presence of anatomic pathology. a blood sample was obtained from each fetus. if blood was not obtainable, a sample of fluid was collected from the abdominal cavity. the number of fetuses recovered from each dam is listed in table . for the cd wt group (++/++) (including the dam that aborted), the number of fetuses were , and (mean = . ). the cd ko dams carrying the cd +/− fetuses (−/+− group) yielded , and fetuses (mean = . ). for the cd ko dams carrying cd ko fetuses (−−/−−group), the numbers of fetuses were and . the results for fetal viremia and gross pathology are summarized in fig. and table . at the anatomic level, % and % of fetuses derived from the two cd wt (++/++) dams, no. and no. , showed some degree of pathology, including smaller than normal fetuses ( % of all fetuses), fetuses with detached or necrotic placentas ( %), meconium staining ( %), and fetuses table ). below each dam in parentheses is the result for prrs pcr in serum, measured as log templates per reaction. "n" is negative for prrsv nucleic acid (ct > ). fetuses are identified by number and relative position within each uterine horn. asterisks identify fetal pcr samples obtained from abdominal fluid. the number below each fetus is the result for prrs pcr in fetal serum (log templates per reaction). the number within each circle refers to the presence of anatomical pathology: ) normal fetus; ) small fetus; ) placental changes, such as detached placenta and/or necrosis; ) meconium stained fetus; ) fetus is dead and necrotic. lower case letters identify the genotype of the individual fetuses (see table table and fig. . * viremia shown as log virus nucleic templates per pcr. * fetuses showing pathology as described in the legend in fig. . * gilt aborted prior to recovery of fetuses. infection. first, there was a wide variation between fetuses in the concentration of virus detected in serum, the result of fetuses becoming infected at different times. secondly, the level of viremia was not always correlated with pathology. for example, fetus no. from dam no. possessed a high level of viremia ( . log templates per reaction) and yet the fetus appeared unaffected. the reason for the discrepancy between viremia and the pathology is unclear. one possibility is that fetal pathology is the result of tissue damage that occurs on the maternal side and not related to the level of fetal viremia. in the field, these normal, but infected newborn piglets can function as "supershedders", which facilitate the rapid dissemination of prrsv throughout a production system. for the −−/+− group (dams no. , and ), all fetuses appeared normal, with the minor exception of two fetuses that were smaller than the other littermates. the smaller than normal size is likely a consequence of crowding within the uterine horn that decreases the surface area of the placenta, thus restricting the growth of the developing fetus. all dams and fetuses in the −−/+− group were negative for the presence of prrsv nucleic acid. for the last group, −-/−−, there was no visible pathology, and all dams (no. and ) and fetuses were negative for prrsv nucleic acid. the results from this study clearly demonstrate that the absence of cd in the dam is sufficient to protect the prrsv-susceptible fetus. although cd -positive offspring derived from cd ko dams are susceptible to virus immediately after birth, the protection from prrsv in utero provides a means to eliminate a major source of economic loss and animal suffering. cd gene modification. the crispr/cas methods used to generate all of the ko alleles are described in detail in whitworth et al. . the specific edits for alleles a, b, d and e are described in whitworth et al. . the specific edit in allele c ( bp insertion) is described in whitworth et al. . the alleles, described in table were identified based on dna sequencing. the knockout genotype was confirmed by the absence of cd expression, which was measured by staining alveolar macrophages with anti-cd mab, a , as described in wells et al. prrsv infection. the prrsv strain used in this study, nvsl - (nvsl), is a laboratory strain isolated in from a herd in southeast iowa, usa that was experiencing a prrs abortion storm . the virus, maintained as a low passage isolate, was propagated and titered on marc- cells. at to days of gestation, gilts were inoculated with tcid of virus diluted in ml of culture medium. one half of the inoculum was administered by intramuscular injection and the remainder administered intranasally. all gilts were maintained in an environment that allowed for the continuous exposure to virus shed by infected pen mates. blood samples were taken from the gilts prior to infection, days post-inoculation (dpi), and at the time of euthanasia. prrsv nucleic acid was measured by isolation of total rna from serum followed by reverse transcriptase real-time prrsv pcr (tetracore, rockville, md). a standard curve was generated using the quantification standards supplied in the rt-pcr kit. results were reported as log templates per µl reaction, which approximates the number of viral rna templates per ml of blood. ethics statement. experiments involving animals and virus were performed in accordance with the federation of animal science societies guide for the care and use of agricultural animals in research and teaching, the usda animal welfare act and animal welfare regulations, or according to the national institutes of health's guide for the care and use of laboratory animals, and were approved by the kansas state university and university of missouri institutional animal care and use committees and institutional biosafety committees. animals were humanely euthanized by pentobarbital overdose following the american veterinary medical association (avma) guidelines for the euthanasia of animals, and all efforts were made to minimize suffering. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers reproductive failure of unknown etiology lymphotropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero update on abortion storms and sow mortality clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical genetic variation and phylogenetic analyses of the orf gene of acute porcine reproductive and respiratory syndrome virus isolates emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark phylogeny-based evolutionary, demographical, and geographical dissection of north american type porcine reproductive and respiratory syndrome viruses lactate dehydrogenase-elevating virus and related viruses experimental reproduction of swine infertility and respiratory syndrome in pregnant sows the interaction between prrsv and the late gestation pig fetus genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus identification of the haemoglobin scavenger receptor the macrophage scavenger receptor cd cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus substitution of porcine cd srcr domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome (prrs) viruses use of the crispr/cas system to produce genetically engineered pigs from in vitro-derived oocytes and embryos funding for this project was from genus, plc and food for the stcentury at the university of missouri. r.p., k.d.w., k.m.w., m.k., m.s., a.m., l.p., r.r. wrote, critiqued, and edited the manuscript text. r.r. prepared the figure, r.r. and k.m.w. prepared the tables. all authors reviewed the manuscript. competing interests: alan mileham is employed by genus plc, the company that provided funding for the project. randy prather is an inventor on a patent related to the cd knockout pig. the remaining authors, kevin wells, kristin m. whitworth, maureen kerrigan, melissa samuel, luca popescu, raymond rowland declare no potential conflict of interest.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/ . /. key: cord- -bgm smuo authors: li, lan; zheng, qisheng; zhang, yuanpeng; li, pengcheng; fu, yanfeng; hou, jibo; xiao, xilong title: antiviral activity of recombinant porcine surfactant protein a against porcine reproductive and respiratory syndrome virus in vitro date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: bgm smuo porcine reproductive and respiratory syndrome virus (prrsv) has caused significant economic losses in the swine industry worldwide. however, there is not an ideal vaccine to provide complete protection against prrsv. thus, the need for new antiviral strategies to control prrsv still remains. surfactant protein a (sp-a) belongs to the family of c-type lectins, which can exert antiviral activities. in this present study, we assessed the antiviral properties of recombinant porcine sp-a (rpsp-a) on prrsv infection in marc cells and revealed its antiviral mechanism using a plaque assay, real-time qpcr, western blotting analysis and an attachment and penetration assay. our results showed that rpsp-a could inhibit the infectivity of prrsv in marc cells and could reduce the total rna and protein level. the attachment assay indicated that rpsp-a in the presence of ca( +) could largely inhibit marc cell attachment; however, in the penetration assay, it was relatively inactive. furthermore, our study suggested that virus progeny released from infected marc cells were blocked by rpsp-a from infecting other cells. we conclude that rpsp-a has antiviral activity against prrsv, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, rpsp-a holds promise as a novel antiviral agent against prrsv. porcine reproductive and respiratory syndrome virus (prrsv) infection is common worldwide and has become one of the most economically important diseases in the swine industry [ ] . prrsv can cause reproductive failure in sows and is associated with a respiratory disease complex that can affect pigs of all ages [ ] [ ] [ ] . prrsv was discovered almost simultaneously in the late s on the european and north american continents and was divided into type prrsv (european genotype) and type prrsv (north american genotype) [ , ] . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately . kb in size and has at least open reading frames (orfs) [ ] . orf a and orf b encode non-structural proteins, and the other genes encode structural proteins, including gp a, gp b, gp , gp , gp , gp a, matrix protein m and the nucleocapsid protein n [ ] [ ] [ ] [ ] [ ] [ ] . of these, four are glycoproteins: the minor proteins gp a, gp and gp and the major protein gp , which is thought to be important for virus assembly and entry into permissive cells. researchers have made great efforts towards preventing and controlling prrsv infection. currently, vaccination is the main method to control prrsv infection. although many commercially available modified live and inactivated prrsv vaccines have been developed, they fail to provide complete protection against the devastating disease caused by this virus [ , [ ] [ ] [ ] . furthermore, a large amount of literature has reported natural compounds that exhibit activity against prrsv infection, including flavaspidic acid ab [ ] , matrine [ ] , -deoxyphorbol -phenylacetate -acetate (dppa), ouabain, valinomycin and bufalin [ ] , and glycyrrhizin [ ] . additionally, other therapies designed to control prrsv infection have been reported, such as licl [ ] , polyinosinic-polycytidylic acid [ ] , type i and type iii interferons [ ] , double-stranded rna (dsrna) activated caspase oligomerizer (draco) [ ] , and micrornas [ ] . however, none of these aforementioned therapies can effectively treat or prevent prrsv infection, so there is an urgent need to develop new antiviral strategies. surfactant protein a (sp-a) belongs to a family of mammalian c-type lectins, and it is an important innate immune molecule that is involved in a range of immune responses. the primary structure of sp-a is composed of four regions, including an n-terminal region, a collagenlike region, a coiled-coil neck peptide, and a carbohydraterecognition domain (crd) [ ] . the crd can interact with sugar moieties that are present on the surface of many pathogens, including viruses, fungi and bacteria, resulting in inhibition of infectivity in a calcium-dependent manner. previous studies on the antiviral activity of sp-a have been focused on human medicine. in the late s, human sp-a was shown to inhibit respiratory syncytial virus by blocking viral entry and subsequent syncytium formation [ ] . research on porcine sp-a activity against prrsv was limited to one report indicating that recombinant porcine sp-a could exert anti-prrsv activity [ ] . unfortunately, no additional studies on the anti-prrsv potential and mechanism of sp-a were performed. however, studies on the antiviral activity of sp-d, which has a primary structure that resembles that of sp-a, have been reported more frequently. sp-d had an inhibitory effect on hiv infection through inhibition of viral entry by affecting gp -cd interactions [ ] . marine and colleagues demonstrated that recombinant porcine sp-d prevented the attachment of human seasonal h n and h n virus to receptors on epithelial cells of the upper respiratory tract [? ?] . notably, porcine ficolin is also a collagenous lectin that was established to reduce prrsv infection in marc- cells in neutralization assays and to inhibit the replication of infectious viral particles in a glcnac-dependent manner [ ] . in the present study, we report the antiviral properties and mechanism of action of recombinant porcine sp-a in marc- cells. our results indicate that rpsp-a affects virus attachment and blocks the cell-to-cell transmission pathway to suppress prrsv infection in marc- cells, which indicates that rpsp-a may be a useful antiviral agent for the treatment of prrsv infection. marc cells were grown in dulbecco's modified eagle's medium (dmem; gibco, grand island, ny, usa) supplemented with % newborn bovine serum (nbs) at °c and % co . spodoptera frugiperda cells (sf cells), purchased from invitrogen, were grown in grace's insect medium ( ; gibco, grand island, ny, usa) supplemented with % heat-inactivated fetal bovine serum (fbs; sciencell, ca, usa) for recombinant baculovirus production or were grown in sf ii sfm (gibco, grand island, ny, usa) for protein expression at °c. hp prrsv strain nj-a (type prrsv) was isolated from a pig on a farm with an atypical prrs outbreak in nanjing city, jiangsu province, china, in . it was identified and stored by the national research center of engineering and technology for veterinary biologicals. the nj-a strain was propagated and titrated by the plaque method in marc cells to prepare the viral stock used in this study. rpsp-a was obtained using a bac-to-bac baculovirus expression system (invitrogen, ca, usa). an n-terminal in-frame fusion of the psp-a (genbank accession no. nm_ ) gene with an -histidine tag was codon-optimized for baculovirus expression, synthesized, and cloned in the puc vector by genscript (piscataway, nj, usa). the recombinant psp-a gene was subcloned into a pfastbac vector under the control of the ph promoter. once generated in a pfastbac tm construct, the plasmid dna was used to transform max efficiency Ò dh bac tm competent e. coli for transposition into a bacmid. blue/ white selection was used to identify colonies that contained a recombinant bacmid. high-molecular-weight rpsp-a bacmid dna from dh bac tm e. coli was isolated using a bac/pac dna isolation kit (omega, usa). the concentration and purity of the rpsp-a bacmid were estimated using a thermo scientific nanodrop tm spectrophotometer, which was then used to transfect sf insect cells using cellfectin ii reagent. the recombinant baculovirus was harvested days post-transfection and was amplified twice to obtain higher titer virus stocks. recombinant p viral stocks were used to infect sf cells at an moi of . pfu/cell. at days postinfection, cells were harvested and washed twice in phosphate-buffered saline (pbs). cell pellets were immediately stored at - °c. the protein rpsp-a was extracted and purified using a -ml-size ni ? -sepharose histrap hp tm affinity column (ge healthcare, sweden) according to the instructions provided by the manufacturer. imidazole in the eluate, which contained rpsp-a, was removed on a hitrap tm desalting column ( ml) and then exchanged with storage buffer ( mm tris, ph . , mm nacl, . mm pmsf). then, the eluate was filter-sterilized through a . -lm membrane filter (millipore) and analyzed by western blotting using an anti-his antibody (sunshinebio, china). protein concentrations were determined using a bca protein assay reagent kit (pierce, rockford, il, usa). the cytotoxic effect of rpsp-a was examined using an mtt assay. briefly, marc cells in -well plate were exposed to various concentrations of rpsp-a at °c in a % co atmosphere for h. after incubation, the culture medium was replaced with fresh medium containing ll of mtt at a concentration of mg/ml, and the cells were incubated for an additional h. after removal of the supernatant, ll of dmso was added to each well to dissolve the crystals for min. absorbance at nm was recorded using an elisa microplate reader. antiviral activity was detected by plaque assay using marc cells in -well plates. a preliminary study was performed to determine the concentration of infectious virus particles using a - dilution of virus stock ( . pfu ml - ). subconfluent monolayers of marc cells in -well plates were treated with rpsp-a at the indicated concentrations and were simultaneously infected with prrsv. the cells continued to be cultured at °c for h. then, the cells were washed five times with pbs and covered with ml of medium containing a : mixture of dmem and % low-gelling-temperature agarose (sigma, usa). after h, the agarose was removed, and cells were stained with % crystal violet dissolved in % formaldehyde at °c for h. after washing gently with water, plaques were counted. antiviral activity was calculated using the following formula: inhibition rate (%) = À number of plaquestest number of plaquescontrol  % total rna was extracted from samples using a takara minibest universal rna extraction kit (takara, osaka, japan). rna was reverse transcribed into firststrand cdna using primescript tm reverse transcriptase with random primers in a -ll final reaction volume according to the manufacturer's instructions. first, ll of total rna was added to mixture i containing ll of random primer and ll of dntps ( . mm), and then the mixture was heated at °c for min and then kept at °c for min. second, a reaction mixture was prepared by adding the above mixture to mixture ii, containing ll of primescript buffer, ll of rnase inhibitor, ll of primescript reverse transcriptase and ll of rnase-free h o. the reaction was performed under the following conditions: °c for min, °c for min, °c for min and, finally, °c for min. real-time qpcr was performed in a lightcycler ii system (roche, basel, switzerland) using an evagreen x qpcr mastermix-no dye kit (abm, canada). reaction mixtures were prepared according to the manufacturer's instructions and incubated for min at °c, followed by cycles of s at °c and min at °c, followed by s at °c, min at °c, and s at °c. a portion of the orf gene was detected by real-time qpcr, and gapdh was used as a control. the data were analyzed using the -ddct method. customized primers are listed in table . total protein was extracted using cell lysis buffer ( mm tris, ph . , mm nacl, % triton x- , . mm pmsf) and quantified using a bca protein assay reagent kit (pierce, rockford, il, usa). then, lg total protein was subjected to % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to a polyvinyl difluoride (pvdf) membrane, which was and the attachment assay was carried out as described previously [ ] with some modifications. marc cells were seeded into -well plates. cells were washed with cold pbs followed by dmed and then were pre-chilled at °c for min. cells were incubated with prrsv at an moi of . and purified rpsp-a ( lg ml - ) at °c for h. cells were washed with cold pbs to remove unbound virus and rpsp-a, and then, fresh medium containing % fbs was added. the cells were then cultured at °c for h, after which the virus titer was measured. the penetration assay was performed as described previously with some modifications [ , ] . prrsv could be internalized from the surface of marc cells within h to h [ ] . thus, we examined the kinetics of activity against prrsv penetration using time-of-addition assays. marc cells were seeded into -well plates. cells were washed with cold pbs, followed by dmed, and were then pre-chilled at °c for min. cells were incubated with prrsv at °c for h. cells were washed with cold pbs. rpsp-a was added at h, h, and h and removed at h following a temperature shift. the time point when the cell culture was shifted to °c was designated as h. cells were washed with cold pbs to remove unbound virus and rpsp-a, and fresh medium containing % fbs was added. cells were incubated for h at °c, and the virus titer was measured. the data are presented as the mean ± standard deviation from three independent experiments. the data were analyzed using student's t-test with graphpad prism software. the differences between the mean values were considered statistically significant when the p-value was less than . . plaque assay results revealed that rpsp-a had potency against prrsv. as shown in fig. a , the number of visible plaques was significantly reduced in the presence of lg of rpsp-a per ml. furthermore, as shown in fig. b and c, treatment with lg of rpsp-a per ml reduced plaques by % from . pfu ml - to . pfu ml - (p \ . ), whereas treatment with lg of rpsp-a per ml reduced plaques by % from . pfu ml - to . pfu ml - (p \ . ). treatment with lg of rpsp-a per ml in the absence of ca ? was used as a negative control, and no inhibition was observed. to rule out the possibility that nonspecific toxicity caused by rpsp-a was affecting prrsv replication, we investigated its cytotoxicity in marc- cells at concentrations of , , , , , , lg/ml. as shown in fig. , hours after treatment, the cells cultured in medium containing rpsp-a at concentrations below lg ml - retained approximately % viability relative to the control. to elucidate the mechanism of the antiviral effects of rpsp-a, a virus entry assay was utilized. the data showed that rpsp-a could largely inhibit virus attachment; however, it was relatively inactive against virus penetration. in the virus attachment assay, marc cells were incubated with prrsv at an moi of . in the presence of rpsp-a at °c, a condition in which viruses attach to cells but do not penetrate. as shown in fig. , treatment with lg of rpsp-a per ml largely inhibited marc cell attachment. the mean viral titer was . pfu ml - in the absence of rpsp-a, but it was reduced to . pfu ml - in the presence of lg of rpsp-a per ml with the addition of ca ? (p \ . ). no significant difference was observed between and lg ml - of rpsp-a in the absence of ca ? (p [ . ). in the penetration assay, we studied the inhibition kinetics of rpsp-a against prrsv. the mean viral titer in the presence of lg of rpsp-a per ml with the addition of ca ? was . pfu ml - at h (p \ . ), . pfu ml - at h (p \ . ), and . pfu ml - at h (p [ . ) compared to . pfu ml - in the absence of rpsp-a (fig. ) , suggesting that the inhibitory effect of rpsp-a on virus penetration decreased over time, although there was no significant difference at h. additionally, lg of rpsp-a per ml in the absence of ca ? did not affect virus penetration. we next attempted to investigate the kinetics of the rpsp-a-mediated effect on the intracellular viral load. first, marc cells were incubated with prrsv at an moi of . for h, and the medium was replaced with fresh medium containing lg of rpsp-a per ml. then, the total rna in marc cells was extracted for prrsv rna genome detection by real-time rt-pcr after culture for h, h, h or h. as shown in fig. a , the addition of rpsp-a resulted in a significant reduction in the total rna level at h, h and h compared with the non-drug-treated cells (p \ . ), and this reduction reached a peak at h after addition of rpsp-a. however, there was no significant difference at h following rpsp-a addition compared with the non-drug-treated group (p [ . ). moreover, we also detected n protein of prrsv in marc cells by western blotting after treatment with rpsp-a for h. as shown in fig. b , western blotting analysis indicated that rpsp-a reduced the level of n protein in cells in a dose-dependent manner. surfactant protein a, an antimicrobial defense molecule of the innate immune system, has the potential to be exploited as an antiviral drug in the future. for this reason, we sought to develop it as a recombinant product in this study. here, rpsp-a was obtained using a bac-to-bac baculovirus expression system. we found that rpsp-a expressed by sf insect cells could block virus entry. viral entry represents an attractive target for chemotherapeutic intervention [ ] . however, drugs that inhibit virus entry prevent infection of cells by cell-free virus particles and also prevent virus transmission between virus-infected and uninfected cells [ , ] . the capacity to inhibit both modes of infection by a single drug is one of the reasons why sp-a may be superior to other microbicide drugs. thus, sp-a might be a candidate agent for preventing prrsv infection. so far, there have been limited data available regarding the anti-prrsv activity of porcine sp-a. in china, highly pathogenic prrsv appears to be widely prevalent, and therefore, the highly pathogenic prrsv strain nj-a, a type prrsv strain, was used in this study. to explore the anti-prrsv activity of porcine sp-a, further studies need to be performed on type prrsv. our data indicate that rpsp-a significantly suppresses prrsv infection in a dose-dependent manner without causing cytotoxicity in marc cells. we also found that rpsp-a exerts potent antiviral activity in a ca ? -dependent manner, indicating that rpsp-a may interact with prrsv via the carbohydrate-recognition domain, which is consistent with previous studies [ , ] . researchers have confirmed that lectins inhibit virus entry [ , , , ] , and each step of the viral entry pathway is a potential target for antiviral agents. we conducted virus attachment and penetration assays to assess whether rpsp-a also interfered with prrsv entry. the virus entry assay showed that rpsp-a strongly inhibits virus attachment, but its effect on virus penetration was, at most, modest, suggesting that rpsp-a most probably blocks virus attachment, like other lectins. it has been shown that hippeastrum hybrid agglutinin (hha), a mannose-specific lectin, probably interferes with sars coronavirus attachment. when attachment occurred at °c, hha was twice as active (ec = . lg ml - ) as it was in a situation where both attachment and penetration were allowed to occur at °c (ec = . lg ml - ) [ ] . in another study, recombinant porcine sp-d was shown to inhibit iav attachment using human trachea tissue, and it fully prevented binding of human seasonal h n and h n ia at higher dose [ ] . our data indicate that the suppression of prrsv penetration by rpsp-a gradually weakened and even disappeared as the time between infection and addition of rpsp-a increased, suggesting that rpsp-a might act on prrsv entry into marc cells only at the cell surface and that it has no effect once the virus has entered the cell. a better understanding of the underlying mechanisms by which rpsp-a influences viral entry might lead to new antiviral interventions. taking the previously reported data and our own findings together, we hypothesize that rpsp-a inhibits prrsv entry by binding to glycoprotein gp , gp , gp or gp on the surface of prrsv, thereby preventing it from interacting with its receptors on marc cells. the mechanism of the antiviral activity of lectins has been studied by several researchers. it has been shown that human sp-a can bind to the f (fusion) glycoprotein on the surface of respiratory syncytial virus and may neutralize rsv by directly inhibiting the fusion function of this protein, thereby inhibiting viral entry [ ] . in another study, marine et al. reported that the interaction between rpsp-d and the viral ha prevented attachment of the virus to target cells and contributed to the neutralizing effects of rpsp-d [ ] . in addition, other lectins such as sp-d and plant lectins have been shown to bind to glycoprotein gp of hiv- and block access of gp to the receptor protein cd [ , , , ] , thereby blocking viral entry to reduce hiv- infection. the nature of the interactions among prrsv, rpsp-a and cells is not yet known, so this model should be assessed in further studies. interestingly, our data also indicated that rpsp-a had no effect on the total rna level when the virus was allowed to undergo only a single cycle of replication but caused the total rna level to decrease greatly at h, h and h. a possible explanation for this is that virus progeny released from infected marc cells are blocked by rpsp-a and prevented from infecting other cells. since marc- cells are not of porcine origin but of monkey origin, the mechanism of infection of porcine alveolar macrophages (pams) is not identical to that in marc cells [ , ] ; furthermore, a previous study showed that some lectins had a stronger inhibitory effect on virus infection when pams were treated with lectins than when the virus was treated with lectins prior to infection, in contrast to what was observed with marc cells, suggesting potentially complex interactions among pams, lectins and prrsv [ ] . more studies will be needed to characterize the antiviral activity of rpsp-a in pams, which are the main target cells for prrsv in vivo. in conclusion, we show that rpsp-a exerts potent activity against hp-prrsv in marc cells. we clearly demonstrate that rpsp-a interacts at the level of virus attachment and blocks the cell-to-cell transmission pathway, which has never been observed before. rpsp-a most probably interacts with the glycoproteins of prrsv, thereby preventing it from binding to host-cell receptors and subsequently interfering with transmission. a better understanding of the mechanisms by which rpsp-a influences viral infection in porcine alveolar macrophages might lead to new antiviral interventions. porcine reproductive and respiratory syndrome virus vaccine does not fit in classical vaccinology porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis expression and antibody preparation of gp a gene of porcine reproductive and respiratory syndrome virus north american and european porcine reproductive and respiratory syndrome viruses differ in nonstructural protein coding regions inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction pathogenesis of porcine reproductive and respiratory syndrome virus characterization of proteins encoded by orfs to of lelystad virus discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production identification of a novel structural protein of arteriviruses identification and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp encoded by orf is incorporated in virus particles a -kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge inhibition of porcine reproductive and respiratory syndrome virus replication by flavaspidic acid ab antiviral activity and underlying molecular mechanisms of matrine against porcine reproductive and respiratory syndrome virus in vitro natural compounds inhibiting the replication of porcine reproductive and respiratory syndrome virus suppression of porcine reproductive and respiratory syndrome virus proliferation by glycyrrhizin licl inhibits prrsv infection by enhancing wnt/beta-catenin pathway and suppressing inflammatory responses poly(i:c) inhibits porcine reproductive and respiratory syndrome virus replication in marc- cells via activation of ifit antiviral activity of type i and type iii interferons against porcine reproductive and respiratory syndrome virus (prrsv) draco inhibits porcine reproductive and respiratory syndrome virus replication in vitro microrna- inhibits prrsv replication by directly targeting prrsv rna and possibly by upregulating type i interferons surfactant proteins sp-a and sp-d: structure, function and receptors surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity recombinant porcine lung surfactant protein a inhibits porcine reproductive and respiratory syndrome virus infection into host cells in vitro surfactant protein d inhibits hiv- infection of target cells via interference with gp -cd interaction and modulates pro-inflammatory cytokine production porcine plasma ficolin binds and reduces infectivity of porcine reproductive and respiratory syndrome virus (prrsv) in vitro in vitro anti-herpes simplex virus activity of , , , -tetra-o-galloyl-b-dglucose from phyllanthus emblica l. (euphorbiaceae) large-molecular-weight carbohydrate-binding agents as hiv entry inhibitors targeting glycoprotein gp inhibition of hiv entry by carbohydratebinding proteins plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydratebinding agents but not by polyanions effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptormediated endocytosis functional analysis of porcine reproductive and respiratory syndrome virus n-glycans in infection of permissive cells decreased surfactant protein a in patients with bacterial pneumonia ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- -hfau odb authors: wang, rong; zhang, yan-jin title: antagonizing interferon-mediated immune response by porcine reproductive and respiratory syndrome virus date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: hfau odb interferons (ifns) are important components in innate immunity involved in the first line of defense to protect host against viral infection. porcine reproductive and respiratory syndrome virus (prrsv) leads to severe economic losses for swine industry since being first identified in early s. prrsv interplays with host ifn production and ifn-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. prrsv encodes several proteins that act as antagonists for the ifn signaling. in this review, we summarized the various strategies used by prrsv to antagonize ifn production and thwart ifn-activated antiviral signaling, as well as the variable interference with ifn-mediated immune response by different prrsv strains. thorough understanding of the interaction between prrsv and host innate immune response will facilitate elucidation of prrsv pathogenesis and development of a better strategy to control prrs. porcine reproductive and respiratory syndrome (prrs) is an important infectious disease, causing huge economic losses to the swine industry worldwide [ , ] . the prrs clinical signs include respiratory disorders, abortion in pregnant sows, and variable mortality in piglets. prrs was first identified in the usa in and subsequently in europe. the causative agent of the disease is the prrs virus (prrsv), a positive-sense single-stranded rna virus, belonging to the arteriviridae family in the order nidovirales [ ] . according to the genetic differences, prrsv is grouped into two genotypes: european (type ) and north american (type ), represented by lelystad virus (lv) and vr- strains, respectively. the genome of prrsv is about kb in length with open reading frames (orfs) [ ] . orf a and orf b comprise % of the viral genome and encode viral enzymes involved in virus replication. in addition, polypeptides from the two orfs are processed into nonstructural proteins (nsps), including nsp , nsp , nsp , nsp tf, and nsp ∼ [ , ] . orf a, orf b, orf through orf , and orf a code for eight structural proteins: gp , envelop protein (e), gp ∼ , membrane protein (m), nucleocapsid protein (n), and orf a protein [ , ] . swine are the only known host of prrsv. prrsvinfected pigs develop a delayed onset of neutralizing antibodies and a weak cell-mediated immune response [ , ] . the main target cells for prrsv infection in vivo are porcine pulmonary alveolar macrophages (pams), which play a crucial role in host immune response [ ] . in order to successfully invade host, prrsv has evolved various strategies to interfere with host innate immunity. some of the prrsv proteins take part in the modulation of ifn-mediated immune response. host innate immune responses play a key role against early viral infection. interferons are major components of inmate immunity and have diverse biological functions including antiviral activity, antiproliferative activity, stimulation of t cell cytotoxic activity, and modulation of immune response [ ] . there are three types of interferons. in human, type i interferons include ifn-, ifn-, ifn-, ifn-, and ifn- [ , ] . in addition, ifn-, ifn-, and ifn-(or limitin) have been identified as type i ifns in swine, ruminant, and mice, respectively [ ] . almost all cell types are capable of producing ifn-/ ; however, plasmacytoid dendritic cells (pdc) are considered to be the major source of ifn-production during viral infection [ , ] . type ii ifn contains only ifn-, whose production is restricted to activated t cells, natural killer cells, and macrophages [ ] . type iii ifns comprise ifn- , ifn- , and ifn- (also known as interleukin-(il-) , il- a, and il- b, resp.), which are mainly generated by dendritic cells [ ] . considering the major roles in antiviral response by type i ifns, we focus on this type of ifns and discuss the prrsvmediated interference with their production and signaling. this review summarizes the recent advances in the research of prrsv interference with ifn-mediated innate immunity, the viral proteins involved, and their molecular mechanisms, as well as diverse effects by different strains and in different cell types. a few relevant reviews on prrsv interplay with innate immunity were published previously [ ] [ ] [ ] . readers are encouraged to read them if interested as these reviews were written in different angles to address the issue with diverse scopes, though there is some overlap in certain topics. this review is arranged into sections of ifn induction, ifn-activated signaling, ifn-stimulated genes, and perspective. host pattern recognition receptors (prrs) for rna viruses include toll-like receptors (tlrs) and rig-(retinoic acid inducible gene-) i-like receptors (rlrs). tlrs that can detect viral rna are tlr , tlr , and tlr [ ] . the rlr family of prrs comprises rig-i and melanoma differentiationassociated gene (mda- ) [ ] . both rig-i and mda- signal through adaptor ips- (also known as mavs, cardif, and visa) on the outer membrane of the mitochondria [ ] . tlr and rlr can recognize double-stranded rna (dsrna) of viral genome or replication intermediate of rna viruses. activation of tlr and rlr signaling pathways leads to activation of interferon regulatory factor (irf ), irf , and nf-b. these transcription activation factors translocate into the nucleus and result in induction of type i ifns and expression of inflammatory cytokines, which not only lead to an antiviral state of the neighboring uninfected cells, but also serve as key regulators to evoke adaptive immune response. at least functional type i ifn genes have been identified in porcine chromosomes and [ ] . these ifn genes include ifn-subtypes, ifn-, ifn-, ifn-, ifn-, ifn-, and ifn-. prrsv is sensitive to type i ifns and the sensitivity is confirmed in vitro and in vivo. pretreatment of pams with porcine ifn-resulted in significant reduction of prrsv yield [ ] . pretreatment of marc- cells and porcine pulmonary alveolar macrophages (pams) with porcine ifninhibited prrsv replication [ ] . pigs that were inoculated with recombinant adenovirus for ifn-expression and challenged one day later with prrsv had lower febrile responses, reduced lung lesion, and delayed viremia and antibody response compared to controls [ ] . therefore, for invading host immune clearance, prrsv has evolved multiple strategies to antagonize the host ifn induction. cells. prrsv appears to inhibit synthesis of type i ifns in pigs infected with type strains, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifn- [ , ] . ifncould not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least -fold lower than that of prcv [ ] . prrsv infection of pams leads to no ifn-production and when the cells were superinfected with tgev, no ifnwas detected either [ ] . the prrsv suppression of ifn induction correlates with the virus replication. plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifnin vivo. prrsv fails to induce porcine pdcs to produce ifn-, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn- [ , ] . moreover, presence of prrsv markedly reduced the typical ifn-response of pdcs to tgev or toll-like receptor agonist. loving et al. showed that prrsv replicated in monocyte-derived dcs but not lung dcs and the response of both cell types to prrsv was only limited to ifn-transcription [ ] . additionally, for marc- cells prrsv replication also significantly inhibited the dsrna-induced type i ifn expression [ ] [ ] [ ] . these data suggest that prrsv infection directly interferes with type i ifn induction in vivo and in vitro. the prrsv proteins that are found to be antagonists of ifn induction include nsp , nsp , nsp , and n ( figure and table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nsp , a c-like serine protease that is responsible for most of the nonstructural protein processing [ ] , was found to inhibit ifn-promoter activation in a reporter assay [ ] , but no further characterization was reported. further study is needed to elucidate the mechanism. also nsp has been studied in more detail than others. nsp is self-cleaved into nsp and nsp subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-expression by affecting the irf signaling pathway [ ] . ifn-promoter reporter assay was performed in hek t cells in the study. the result showed that nsp and its two cleavage products, nsp and nsp , inhibited the activation of ifn-promoter (p -luc) and an artificial promoter containing three irf binding sites (p -cib-luc) after sv stimulation. prrsv nsp and nsp blocked the induction of ifn-at downstream of irf activation but had no effect on the phosphorylation and translocation of irf . it suggested that nsp and nsp may have a direct effect on the formation of the transcription enhanceosome on the ifnpromoter inside the nucleus. kim et al. showed that nsp inhibited irf association with creb-binding protein (cbp) in the nucleus but had no effect on irf phosphorylation figure : interference of type i ifn production by prrsv proteins. activation of rlr pathway and signaling by viral dsrna is shown. viral dsrna is generated during prrsv replication. "p" besides irf and irf indicates phosphorylation. red-colored blocks indicate prrsv proteins known to inhibit the signaling molecules indicated. prrsv nsp inhibits irf association with cbp, enhances cbp degradation, and interferes with i b degradation. and nsp inhibits irf phosphorylation and nuclear translocation. also nsp inhibits irf phosphorylation and nuclear translocation, interferes with i b polyubiquitination, and prevents its degradation. and nsp inhibits irf phosphorylation and nuclear translocation via degrading ips- mrna. ifn signaling blocking stat /stat nuclear translocation [ ] and nuclear translocation [ ] . immunoprecipitation with anti-irf antibody found that interaction between cbp and irf in prrsv-infected marc- cells or nsp -transfected hela cells was weaker than in cells treated with polyi:c alone [ ] . the expression of nsp also enhanced cbp degradation, which can be rescued by mg treatment, a proteasome inhibitor. no interaction between nsp and cbp was found. the process is independent of the pcp activity of nsp [ ] . beura et al. showed that nsp interfered with irf signaling pathway by inhibiting dsrna-induced irf phosphorylation and nuclear translocation [ ] . the discrepancy is possibly because an nsp that is -residue longer than its authentic form was used in beura's study. another possible reason is that different prrsv strains were used as a couple of other studies showed that prrsv replication significantly blocked dsrna-induced irf activation in marc- cells [ , ] . and nsp was also found to downregulate irf protein level and inhibit its phosphorylation [ ] . in our laboratory, we observed that prrsv infection of marc- cells led to reduction of irf protein level (unpublished data). luo et al. [ ] showed prrsv blocked ifn-production and irf nuclear translocation via significantly inhibiting activation of ips- in rig-i signaling pathway. the structure-function studies of nsp and nsp identified critical motifs of the proteins in inhibition of ifn induction. the zinc-finger (zf) domain in the c-terminus of nsp is critical for this protein to antagonize both ifninduction and nf-b activation, especially the amino acids at c-terminal of the nsp [ ] . shi et al. [ ] screened a series of nsp c-terminal truncated mutants and revealed that the amino acid residue f of nsp is essential for the inhibition of ifn-induction. the residue f played a role in both tlr signaling and rig-i signaling pathways [ ] . double mutations k a/r a (type prrsv) or k a/r a (type prrsv) in a highly conserved motif of nsp , gkylqrrlq, dampened the nsp inhibition of ifn induction [ ] . moreover, recovered recombinant viruses with the nsp mutations by reverse genetics induced higher level gene expression of type i ifns than that of wild type viruses. also nsp inhibits ifn induction by blocking irf phosphorylation and nuclear translocation. the cysteine protease domain (pl ) of nsp was necessary for antagonizing activation of irf pathway [ ] . the cysteine protease domain (pl ) at the n-terminus of nsp , which belongs to the ovarian tumor (otu) protease family, was shown to inhibit type i ifn induction by interfering with the nf-b signaling pathway [ ] . the otu domain possesses deubiquitinase activity, which interferes with the polyubiquitination of i b and subsequently prevents its degradation. recovered recombinant viruses with mutations in the otu domain of nsp by reverse genetics were found to be unable to inhibit nf-b activation as effectively as the wild type virus. nsp is an endonuclease [ ] and ifn antagonist [ ] . nsp suppressed the activation of ifn-promoter and the expression of irf -mediated genes. the endoribonuclease activity of nsp was essential for nsp to inhibit dsrnainduced ifn-induction [ ] . the amino acid residue h of nsp , a presumed catalytic histidine, was involved in the inhibition of irf phosphorylation. it seems that the inhibition of irf activation is due to the nsp endoribonuclease activity, which can cleave mrna of ips- [ ] , the adaptor molecule for rig-i and mda- . the ifn antagonizing activity is not restricted to nonstructural proteins of prrsv. structural proteins, such as the n protein, were found to downregulate ifn-mrna level in polyi:c-treated immortalized pam cells [ ] . n protein interferes with dsrna-induced phosphorylation and nuclear translocation of irf . the multiple components of prrsv are involved in the interference with ifn induction. the nsps are early proteins and n is a late one, which may play roles at different stages of viral replication. induction. the effect of prrsv replication on ifn induction appears to be variable among different strains and different cell types. prrsv field isolates have variable suppressive effect on ifn-induction in pam cultures and the suppression was found at posttranscriptional stage [ ] . this is not unexpected as prrsv strains are divergent in genomic sequences. prrsv infection of monocyte-derived dendritic cells (mo-dc) induced the transcription of ifn-/-but no detectable ifn-in culture supernatant, suggesting a blockage at posttranscriptional stage [ ] . prrsv activated the transcription of ifn-in a pi k-dependent manner in mo-dc cells. prrsv infection of marc- cells inhibits ifn gene expression [ ] by interfering with the ips- activation in the rig-i signaling pathway [ ] . a variety of type and type prrsv strains were found to stimulate ifnsecretion by pdc via tlr pathway and the effect did not require live virus [ ] . the suppressive effect on pdc may be strain dependent. a novel isolate, a mc , induced ifns in both marc- and pam cells and virus replication was needed for ifn induction [ ] . ifn- and elevation of isgs were detected in a mc -infected cells. sequencing analysis indicated that a mc was closely related to vr- and ingelvac prrs mlv with an identity of . % at the nucleotide level [ ] . there were a total of nucleotide (nt) variations when compared to vr- , resulting in amino acid changes scattered from nt to the end of the genome. compared to both vr- and mlv, a mc has unique nucleotides. yet the mechanism of this strain inducing ifns is not known and it is intriguing to note that the first . kb of its genome is identical to vr- . its nsp and nsp proteins should be able to inhibit ifn induction. we hypothesize that the unique nucleotides in a mc genome resulting in special rna structures or unique dsrna formation during early viral replication could evoke ifn production. it is worth to note that the induction of ifns is dose dependent. the virus is able to replicate when the inoculum is at less than . moi, which induces limited ifns that cannot suppress the virus replication [ ] . a mc infection of pigs resulted in earlier onset and higher level neutralizing antibody against homologous and heterologous strain than mlv vaccine strain that is highly homologous in sequence [ ] . type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [ , ] . the activation of ifn signaling leads to the induction of antiviral responses. the signaling of type i ifns is initiated after they bind to their receptors on the cell surface [ ] [ ] [ ] . this receptor binding activates janus kinases (jak), which phosphorylates both the signal transducers and activators of transcription (stat ) and stat . the phosphorylated stat and stat form heterodimers, followed by interaction with interferon regulatory factor (irf ) and subsequently formation of heterotrimers, also known as interferon-stimulated gene factor (isgf ). translocation of isgf into the nucleus followed by binding to consensus dna sequences leads to the expression of ifnstimulated genes (isgs), which then take part in antiviral responses. [ ] [ ] [ ] . prrsv replication in marc- cells suppresses jak/stat signaling stimulated by addition of ifn-to the culture [ ] . transcripts of isg , isg , and protein stat were significantly reduced compared to mockinfected cells. the phosphorylation of both stat and stat was unaffected. immunoprecipitation with stat or stat antibody for marc- cell lysates was performed and the result showed no significant difference for stat /stat heterodimer formation between prrsv-infected and mockinfected cells. further study showed that the nuclear translocation of stat /stat heterodimer was blocked. prrsv infection of pam cells also blocks jak/stat signaling shown by reduction of isg expression after stimulation with external ifn-, while a vaccine strain ingelvac prrs mlv had little effect, possible due to its less efficient replication in the primary cells [ ] . signaling. prrsv proteins that interfere with ifn-activated signaling include nsp , nsp , nsp , gp , and n ( figure and table ) [ ] [ ] [ ] . prrsv nsp was found to block the nuclear translocation of stat and significantly inhibit the expression of isgs [ ] . by observing stat -gfp distribution under fluorescence microscopy, chen et al. [ ] noticed that stat -gfp accumulated in cytoplasm in hek t cells with nsp expression. further studies on nsp revealed that it induced the degradation of karyopherin-alpha (kpna , also called importin-alpha ), which is known to mediate the nuclear import of isgf [ ] . the n-terminal domain of nsp was involved in the ubiquitin-proteasomal degradation of kpna . residue of nsp was found to be essential in inducing kpna degradation and inhibiting ifn-mediated signaling as the residue change from valine to isoleucine diminished the suppressive effect. notably, prrsv infection of marc- cells by vr- and vr- also reduces kpna expression, whereas infection by ingelvac prrs mlv does not. mlv nsp has no effect on kpna expression or ifn signaling but gains the suppressive function when residue is changed to valine [ ] . other prrsv proteins including nsp , nsp , gp , and n were also found to be able to inhibit ifn-activated signaling [ ] . n protein inhibits ifn-activated stat nuclear translocation, albeit less effective than nsp [ ] . signaling. variable effect on ifn signaling among prrsv strains was found [ ] . among six prrsv strains (vr- , ingelvac prrs mlv, vr- , nvsl - , mn , and lelystad) tested, all but mn inhibited ifn signaling in marc- cells, and all but mlv and nvsl - blocked the ifn activation in pams. the result also demonstrated that the interference with ifn signaling by prrsv was variable in different infected-cell types. nsp from the six strains was cloned and all but mlv nsp inhibited ifn signaling when overexpressed [ ] . the ifninducing a mc strain has no effect on jak/stat signaling activated by ifn- [ ] . type i ifns (e.g., ifn-and ifn-) drive the expression of more than genes that encode proteins with antiviral, antiproliferative, proapoptotic, and proinflammatory functions [ ] . among the antiviral isgs, the best studied ones are , -oligoadenylate synthetases (oass), ribonuclease l rnasel, the dsrna-activated protein kinase (pkr), p (isg , interferon-induced protein with tetratricopeptide repeats (ifit )), mx (myxovirus (influenza virus) resistance ), and isg . prrsv nsp was shown to inhibit the antiviral function of ifn-stimulated gene (isg ) by the deubiquitinase activity of the otu domain of nsp ( figure and table ) [ ] . isg is an ubiquitin-like antiviral protein [ , ] . isg conjugation (isgylation) to substrate proteins follows a process similar to that of ubiquitin conjugation. isgylation of many important immune-related molecules leads to the activation of host innate immune response. as mentioned above, the cysteine protease domain (pl ) at the n-terminus of nsp belongs to otu-containing superfamily of proteases (dubs), which possesses deubiquitinating activity [ ] . sun et al. revealed that nsp was an antagonist for the antiviral activity of isg by reducing isg production and conjugation. the n-terminal pl domain of nsp was crucial for the antagonizing function. pkr mediates translational control by phosphorylating the protein translation initiation factor eif , resulting in inhibition of protein synthesis and viral replication [ ] . addition of -aminopurine ( -ap), an inhibitor of pkr, restored prrsv replication in ifn--treated cells [ ] . addition of -ap to recombinant swine ifn--primed marc- cells restored prrsv replication but did not rescue the virus in ifn--primed pam cells [ ] . these results showed the important role of pkr in the ifn-activated antiviral figure : interference with type i ifn-activated jak/stat signaling pathway and antiviral isgs. ifn-/-binds to their receptors ifnar- and ifnar- on cell membrane, which activates jak/stat pathway. "p" besides stat and stat indicates phosphorylation. prrsv nsp inhibits isgf nuclear translocation via inducing degradation of kpna , which is essential for mediating the nuclear import of isgf . n protein also inhibits isgf nuclear translocation. and nsp reduces isg production and conjugation via its deubiquitination activity. signaling. we found that prrsv is able to inhibit the polyi:cinduced activation of pkr, as well as its downstream effector eif (unpublished observation). it is not known if prrsv interferes with other isgs. considering the important roles of the isgs in deterring invading pathogens, one can imagine that prrsv must have evolved strategies to evade them during its replication. further study on the interplay of prrsv and isgs will provide insights into such strategies. prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [ ] , as well as weak cellmediated immune response [ ] . partly the reason is possibly because of prrsv interference with ifn-mediated innate immunity. prrsv infection appears to inhibit synthesis of type i ifns in vivo and in vitro with the exception of some atypical strains that induce ifn production, such as a mc . the mechanism for the interference is at multiple steps from inhibition of irf activation, cbp interaction with irf , and posttranscriptional suppression. prrsv also block ifn-activated signaling, which results in suppression of the expression of antiviral isgs. the mechanism is prrsvmediated blocking of isgf nuclear translocation. the prrsv interference with the innate immunity is at multiple levels, from ifn induction and ifn-activated signaling to activity of isgs. given the divergence of prrsv strains in sequences, variation of these activities in innate immunity is not a surprise, whereas the multifold interplay between the virus and host may determine the consequences. in addition, type i ifns are proinflammatory cytokines. the protective effect of ifns in vivo may be context dependent. the ifns in proper amount at right site and time should be protective. otherwise, its elevation might be a consequence of inflammation during prrsv infection. a typical example is that high-pathogenic prrsv induces high level ifn-but causes high mortality in pigs [ ] . further study is needed to elucidate the contribution of prrsv effect on innate immunity to its pathogenesis and the modulation of adaptive immune responses. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states family arteriviridae arterivirus molecular biology and pathogenesis effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load mystery swine disease in the netherlands: the isolation of lelystad virus the interferons: years after their discovery, there is much more to learn interferons, interferon-like cytokines, and their receptors the receptor of the type i interferon family interferons and viral infections the nature of the principal type interferon-producing cells in human blood ipc: professional type interferon-producing cells and plasmacytoid dendritic cell precursors distribution of interferon-receptor in human tissues interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus interaction between innate immunity and porcine reproductive and respiratory syndrome virus modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus the toll-like receptor (tlr )-specific stimulus loxoribine uncovers a strong relationship within the tlr , and subfamily innate immune recognition of viral infection ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction differential expression and activity of the porcine type i interferon family in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) recombinant swine beta interferon protects swine alveolar macrophages and marc- cells from infection with porcine reproductive and respiratory syndrome virus adenovirus-mediated expression of interferon-delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus interferon-response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc- cells modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells porcine reproductive and respiratory syndrome virus nonstructural protein modulates host innate immune response by antagonizing irf activation identification of two autocleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein antagonizes interferon regulatory factor activation porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferonproduction by inhibiting irf activation in immortalized porcine alveolar macrophages the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-induction structure and cleavage specificity of the chymotrypsin-like serine protease ( clsp/nsp ) of porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonproduction by interfering with the rig-i signaling pathway porcine reproductive and respiratory syndrome virus (prrsv) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in marc- cells nonstructural protein -alpha subunit-based inhibition of nf-b activation and suppression of interferon-production by porcine reproductive and respiratory syndrome virus amino acid at position was essential for porcine reproductive and respiratory syndrome virus (prrsv) non-structural protein (nsp ) as an inhibitor to the induction of ifn targeted mutations in a highly conserved motif of the nsp protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes porcine reproductive and respiratory syndrome virus activates the transcription of interferon alpha/beta (ifn-/ ) in monocyte-derived dendritic cells (mo-dc) impact of genotype and of porcine reproductive and respiratory syndrome viruses on interferon-responses by plasmacytoid dendritic cells induction of type i interferons by a novel porcine reproductive and respiratory syndrome virus isolate enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain interferon signalling network in innate defence immunomodulatory functions of type i interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins transcriptional responses to polypeptide ligands: the jak-stat pathway how cells respond to interferons porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation variable interference with interferon signal transduction by different strains of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nsp inhibits interferon-activated signal transduction by inducing karyopherin- degradation interferons at age : past, current and future impact on biomedicine nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene a human -kda ifn-induced protein induces the secretion of ifn ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses the double-stranded rnadependent protein kinase pkr: structure and function inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with -aminopurine marked differences between marc- cells and swine alveolar macrophages in ifn -induced activation of antiviral state against prrsv experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- - pee i o authors: kang, hyeonjeong; lee, changhee title: sasa quelpaertensis nakai extract suppresses porcine reproductive and respiratory syndrome virus replication and modulates virus-induced cytokine production date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: pee i o although sasa quelpaertensis nakai, a dwarf bamboo, is known to exert a variety of beneficial effects on health, its antiviral effect remains to be elucidated. porcine reproductive and respiratory syndrome virus (prrsv) is one of the most devastating viral pathogens of swine and has a substantial economic impact on the global pork industry. therefore, the present study was conducted to determine whether sasa quelpaertensis nakai extract (sqe) inhibits prrsv infection in cultured porcine alveolar macrophages (pams). our results demonstrated that sqe treatment suppressed the replication of prrsv in a dose-dependent manner. the antiviral activity of sqe on prrsv replication was found to be primarily exerted at early times postinfection. treatment with sqe resulted in marked reduction of viral genomic and subgenomic rna synthesis, viral protein expression, and progeny virus production. notably, pro-inflammatory cytokine production in pam cells infected with prrsv was shown to be modulated in the presence of sqe. taken together, our data indicate that sqe has potential as a therapeutic agent against prrsv. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. porcine reproductive and respiratory syndrome virus (prrsv), a pathogenic macrophage-tropic arterivirus of pigs, is the etiological agent of acute respiratory illness in young piglets and reproductive failure in pregnant sows [ ] . prrsv is a small enveloped single-stranded positivesense rna virus that belongs to the family arteriviridae of the order nidovirales [ , , ] . the prrsv genome contains two large open reading frames (orfs), a and b, which comprise the two-thirds of the viral genome and encode non-structural proteins (nsps). the remaining orfs located in the -terminal region code for structural proteins [ , ] . the initial translation of orf a and orf b yields the a and lab replicase polyproteins, respectively, which are then proteolytically processed into at least functional nsps, including viral rna-dependent rna polymerase (rdrp) [ , , , ] . the rdrpcontaining replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of -coterminal sg mrnas that are individually translated to produce structural proteins [ , ] . prrsv preferentially replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months [ , , , ] . as a result, prrsv infection suppresses normal macrophage functions and immune responses, which may render pigs susceptible to secondary bacterial or viral infections, leading to more-severe disease than caused by either agent alone [ , , , ] . furthermore, a highly pathogenic prrsv infection, which is characterized by high fever and high mortality in pigs of all ages, has recently emerged in china and other asian countries and severely affected local pig husbandries [ ] . although both killed and modified live vaccines against prrsv have been developed to control the disease and are used in the global market, most commercial vaccines have problems related to their efficacy and safety. despite the availability of the vaccine, prrsv has still continued to plague most pork-producing nations, causing enormous financial losses for the swine industry worldwide [ , ] . thus, the lack of effective vaccines increases the need for new compounds against prrsv infection. sasa quelpaertensis nakai, known as jeju-joritdae, is a type of bamboo grass that is widely distributed on mt. halla on jeju island in south korea. its leaf extract possesses a number of health-promoting activities, including anti-ulcerogenic, anti-obesity, anti-inflammatory, and anticancer properties [ - , , ] . to date, there are still no reports on the effectiveness of sasa quelpaertensis nakai extract (sqe) against viral infections in human or veterinary subjects. in the present study, therefore, we tried to investigate the antiviral activity of sqe and its mechanism of action in target cells upon prrsv infection. treatment of target cells with sqe significantly impaired prrsv replication. further experiments revealed that sqe suppresses post-entry steps in the replication cycle of prrsv, including viral genomic and sg rna synthesis, viral protein expression, and virus production. the presence of sqe notably altered expression of cytokine genes in prrsv-infected pam cells, suggesting that sqe activity is involved in the modulation of inflammatory responses during viral infection. collectively, our results suggest that sqe may be an excellent therapeutic option for prrsv infection. cells, viruses, reagents, and antibodies pam-knu cells (immortalized pam cell line; [ ] ) were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; invitrogen), antibiotic-antimycotic solution ( ; invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ; invitrogen) in the presence of lg/ml g (invitrogen). the cells were maintained at °c in a humidified % co incubator. prrsv strain vr- was propagated in pam-knu cells as described previously [ ] . sqe was a kind gift from yong chool boo (kyungpook national university, daegu, south korea). it was prepared and dissolved in distilled water (dw) as described elsewhere [ , , ] . the yield of sqe from dried s. quelpaertensis leaves ( kg) was approximately %. p-coumaric acid was purchased from sigma (st. louis, mo) and dissolved in dimethyl sulfoxide (dmso). a monoclonal antibody (mab; sdow ) against the prrsv n protein was purchased from rural technologies (brookings, sd). anti-bactin antibody and horseradish peroxidase (hrp)-conjugated secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca). the cytotoxic effects of reagents on pam-knu were analyzed by a -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-knu cells were grown to a density of cells/well in a -well tissue culture plate with sqe or p-coumaric acid treatment for and h. after incubation, ll of mtt solution ( . mg/ml) was added to each well, and the samples were incubated for an additional h. the supernatant was then removed from each well, after which ll of dmso was added to dissolve the formazan crystals produced from the mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-knu cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with sqe or pcoumaric acid for h and mock infected or infected with prrsv at a multiplicity of infection (moi) of in the presence of each compound. the virus-infected cells were allowed to grow in the presence of sqe or p-coumaric acid until hpi, fixed with % paraformaldehyde for min at room temperature (rt), and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with an n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on glass microscope slides in mounting buffer ( % glycerol and . % sodium azide in pbs), and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-knu cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv at an moi of . at the indicated times, cells were harvested in ll of lysis buffer ( . % tritonx- , mm b-glycerophosphate, mm q-nitro phenyl phosphate, mm mops, mm, mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, lg/ml e , lg/ml aprotinin, lg/ml leupeptin, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice and clarified by centrifugation at , g (eppendorf centrifuge r, hamburg, germany) for min at °c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with nupage sample buffer (invitrogen) and boiled at °c for min. the proteins were separated on a nupage - % gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immobilon-p membranes (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk (bd biosciences, belford, ma) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at °c for h and reacted at °c overnight with the primary antibody against prrsv n or b-actin. the blot was then incubated with the secondary horseradish peroxidase (hrp)-labeled antibody at a dilution of : , for h at °c. proteins were visualized by addition of enhanced chemiluminescence (ecl) reagent (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify viral protein production, the band densities of prrsv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/ facilities/wcif/imagej/) based on the density value relative to b-actin gene. pam-knu cells were infected with prrsv at an moi of as described above. at - , , , , , , , , , or hpi, sqe was added to reach the indicated final concentration for the remainder of the time course experiment. the virus-infected and sqe-treated cells were further maintained, and the infection was terminated at hpi by fixing the monolayers with % paraformaldehyde for min at rt. the fixed cells were subjected to ifa to assess the presence of prrsv infection as described above. an internalization assay was performed as described previously [ ] . briefly, pam-knu cells grown in -well culture plates were infected with prrsv at an moi of at °c for h in the presence of sqe. unbound viruses were then washed with pbs, and the cells were incubated at °c or °c in the presence of sqe for h, followed by treatment with proteinase k ( . mg/ml) at °c for min to remove the bound but non-internalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added to fresh pam-knu cell monolayers in -well tissue culture plates. at h post-incubation, internalized viruses were titrated by ifa as described above, and the % tissue culture infectious dose (tcid ) was calculated. quantitative real-time rt-pcr pam-knu cells were incubated with sqe for h prior to infection and then inoculated with prrsv at an moi of for h at °c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing sqe for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentration of the extracted rna was measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets as described previously [ ] . the primers used in this study were described elsewhere [ , ] , and primer sequences are available on request. the rna levels of viral and cytokine genes were normalized to that of mrna for the porcine b-actin gene, and the relative quantities (rq) of mrna accumulation were determined using the -ddct method [ ] . to detect alteration of genomic rna and sg mrna levels in the presence of sqe during prrsv infection, the results obtained using sqe-treated samples were compared with those obtained with vehicle-treated samples. to assess the effect of sqe on transcriptional activation of pro-and anti-inflammatory cytokines in virusinfected cells, the relative fold change of each cytokine gene was calculated and compared between virus-infected and mock-infected pam cells and between vehicle-treated and sqe-treated virus-infected cells. pam-knu cells were treated with sqe ( mg/ml) for h prior to infection, followed by prrsv inoculation at an moi of for h at °c. the infected cells were maintained in fresh medium containing sqe or vehicle for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent and treated with dnase i as described above. northern blotting was conducted using a northernmax kit (ambion, austin, tx) according to the manufacturer's instructions. samples of total rna ( lg) were loaded onto a % agarose gel and separated by electrophoresis, using a denaturing buffer system. the separated total rna was then transferred to a brightstar-plus nylon membrane (ambion) for h and cross-linked by exposure to uv light for min. pre-hybridization was performed at °c for min, followed by detection using the genotype-specific probe for the utr ( -aatttcggccgcatggttttcgccaattaaatctt acccccacacggtcgc- ) or porcine s rrna ( -g cggttgcaccatttgggtgtcctg- ). the blot was hybridized to biotin-labeled oligonucleotide probes in ultrahyb reagent at °c overnight and washed twice with low-stringency blocking buffer and wash buffer. target viral rnas were detected using a brightstar biodetect kit (ambion) according to the manufacturer's protocols. the membrane was incubated with alkaline-phosphatase-conjugated streptavidin, followed by reaction with the chemiluminescent substrate cdp-star (ambion). the overlaid films were exposed in a dark cassette box in a dark room, and quantitation was performed by densitometry of the corresponding bands as described above. pam-knu cells were infected with prrsv along with sqe treatment as described above. the culture supernatant was collected at different time points ( , , , , and hpi) and stored at - °c. the titer of prrsv was measured by limiting dilution on pam-knu cells by ifa as described above and then quantified as the tcid per ml. student's t-test was used for all statistical analyses and pvalues of less than . were considered statistically significant. to investigate the antiviral activity of sqe, prrsv was chosen because it is one of the most important viral pathogens that have an economic impact on the global pork industry. based on the mtt assay, the highest concentration of sqe that was noncytotoxic was mg/ml in pam-knu cells. pam-knu cells were pretreated with sqe at concentrations of . to mg/ml or with dw as a vehicle control for h prior to infection, and sqe was present during the entire period of infection. virus production was initially measured by monitoring the cytopathic effect (cpe) after infection and confirmed by immunofluorescence at hpi using an n-protein-specific mab (fig. ) . in vehicle-treated control cells, visible cpe appeared at hpi and became prominent by hpi, and n-specific staining was clearly evident in many cell clusters, indicating infection and the spread of the virus to neighboring cells. in contrast, sqe had an obvious inhibitory effect on virus propagation. as shown in fig. a and b, sqe significantly decreased virus-induced cpe and viral gene expression at concentrations of * mg/ml. n protein staining revealed that the number of cells expressing viral antigen decreased during sqe treatment, resulting in a maximum of * % inhibition at a concentration of mg/ ml ( fig. a and b) . furthermore, the effective dose for inhibiting prrsv infection by % (ed ) was determined to be about . mg/ml. taken together, these results demonstrate that sqe efficiently suppresses the replication of prrsv. to determine the point at which sqe acted during prrsv infection, pam-knu cells were treated independently with sqe at various time points postinfection. at hpi, the level of prrsv replication was measured indirectly using ifa to determine the number of cells expressing the n protein (fig. ) . treatment of cells with mg/ml sqe at - , , and hpi resulted in a more than % decrease in prrsv-positive cells when compared to untreated controls. addition of sqe between and hpi led to a % to % decrease in the number of prrsv-infected cells. in contrast, no significant impairment of prrsv propagation was observed when sqe was added at hpi. these data indicate that the inhibitory effect of sqe was exerted mainly during the initial period of infection, suggesting that its action occurs at early time points after prrsv infection. to further assess the effect of sqe on prrsv replication, virus yield was determined during treatment with sqe. viral supernatants were collected at hpi and viral titers were measured. as shown in fig. a , treatment with sqe inhibited the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be . tcid /ml in the vehicle-treated control for prrsv, but the addition of mg/ml sqe decreased the titer of prrsv to . tcid /ml ([ log reduction compared with the control). a growth kinetics study further demonstrated that the overall process of prrsv replication was significantly delayed when cells were treated with sqe (fig. b) . consequently, these findings revealed that sqe inhibits the optimal release of progeny virus from the natural host cells. to evaluate which steps of the replication cycle of prrsv were targeted by sqe, we started focusing on the earliest step, virus entry. to address this issue, an internalization assay was performed as described previously [ ] . pam-knu cells were inoculated with prrsv at °c for h to allow virus attachment and further maintained either at °c or at °c to permit virus internalization in the presence of sqe. the samples were then treated with proteinase k to remove the remaining viruses from the cell surface. the serially diluted infected cells were subsequently subjected to an infectious center assay on uninfected pam-knu cell monolayers, and virus titers were measured days later by ifa. as shown in fig. c , the titer of prrsv was virtually the same in cells treated with sqe or without sqe at °c and was determined to be . tcid /ml, indicating no differences between those cells. in contrast, only minimal viral production of about tcid /ml was observed in prrsv-infected cells maintained at °c, which was likely due to inefficient removal of the bound virus. these results indicated that sqe has no inhibitory effect on the virus entry process. following the uncoating process, like all positive-sense rna viruses, the genome of prrsv is released into the cytoplasm and immediately serves as a template for viral translation by a cellular cap-dependent mechanism. early viral translation produces replicase polyproteins that are posttranslationally cleaved into nsps by viral or cellular proteinases. subsequently, the replicase proteins mediate de novo synthesis of viral rna, including genomic rna replication and sg mrna transcription. the structural proteins of prrsv are translated later from their respective sg mrna transcripts. thus, to investigate inhibitory effects of sqe on post-entry steps of the prrsv life cycle, we first investigated whether viral protein translation was affected by sqe. to accomplish this, pam-knu cells were treated with sqe for h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. the expression level of the prrsv n protein in the presence or absence of sqe was evaluated at hpi by western blot analysis. the production of the prrsv n protein was drastically diminished during sqe treatment (fig. ) . densitometric analysis of the western blot revealed that the intracellular expression of the viral protein was reduced by sqe treatment, with a maximum of about % inhibition at a concentration of mg/ml (fig. ) . this marked reduction was probably not the result of a nonspecific decrease in translation, since ponceau s staining indirectly indicated that the overall expression levels of cellular proteins remained similar following treatment (data not shown). taken together, these results demonstrated the inhibitory effects of sqe specifically against viral protein expression during prrsv replication. for positive-strand rna viruses, viral nonstructural proteins are required for viral rna replication and transcription. thus, it is conceivable that the impaired viral protein expression was caused by suppression of viral rna synthesis. since prrsv infection produces two rna entities, genomic and subgenomic, we investigated whether sqe specifically affects genome replication and sg mrna transcription. to answer this question, the relative levels of genomic rna and sg mrna were measured by quantitative real-time strand-specific rt-pcr in the presence or absence of sqe upon prrsv infection. as shown in fig. , sqe treatment resulted in a maximal reduction in the synthesis of prrsv genomic rna and sg mrna of % and %, respectively, at a concentration of mg/ ml, when compared with untreated infected cells. the decrease in viral rna levels after the addition of sqe was not due to nonspecific inhibition of transcription, as the mrna level of the internal b-actin control remained unchanged in all samples (fig. c ). the impairment of prrsv transcription in the presence of sqe was confirmed by northern blot analysis (fig. ). in the absence of sqe, prrsv rna synthesis was distinctly observed (lane ). a [ % reduction of rna-synthesizing activity occurred in the presence of mg of sqe per ml (lane ), while viral rna synthesis was almost completely abolished in the presence of mg of sqe per ml (lane ). altogether, these results indicated that sqe specifically suppresses the synthesis of prrsv genomic rna and sg mrna. since sqe has been reported to have anti-inflammatory properties, we examined whether sqe affects the transcriptional activation of immune-response genes upon prrsv infection to determine whether this contributes to b fig. reduction of viral progeny production by sqe treatment. (a) pam-knu cells were pretreated with sqe for h and were mock infected or infected with virus. sqe was present in the medium throughout the infection. at hpi, virus-containing supernatants were collected and virus titers were determined. (b) growth kinetics of prrsv on pam-knu cells treated with sqe, using the same experimental conditions as in panel a. at the indicated time points postinfection, culture supernatants were harvested and virus titers were measured. (c) a virus-internalization assay was conducted by infecting pam-knu cells with prrsv at an moi of at °c for h. after washing with cold pbs, infected cells were maintained in the presence or absence of sqe, either at °c or °c, for an additional hour. bound but non-internalized virus particles were removed by treatment with proteinase k. the infected cells were then serially diluted and plated onto pam-knu cells in -well tissue culture plates. at days post-incubation, internalized viruses were titrated, using ifa for detection, and the % tissue culture infectious dose (tcid ) was calculated. results are expressed as the mean values from triplicate wells, and error bars represent standard deviations. , p \ . fig. inhibition of viral protein translation by sqe treatment. sqetreated pam-knu cells were mock-infected or infected with prrsv for h and were further cultivated in the presence or absence of sqe. at and hpi, cell lysates were collected, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted using a prrsv-specific antibody that recognizes the prrsv n protein. the blot was also reacted with mouse mab against b-actin to verify equal protein loading. the amount of each viral protein was estimated by densitometry in terms of its band density relative to that of b-actin, and sqe-treated sample results were compared to vehicle-control results. values are representative of the mean from three independent experiments, and error bars denote standard deviations. , p \ . inhibition of prrsv by sasa quelpaertensis nakai extract its antiviral activity. we found that numerous immunerelated genes regulated by prrsv infection were significantly altered by treating the cells with sqe when compared with results obtained with vehicle-treated virusinfected cells (fig. ) . among these, upregulation of il- a, il- , il- , il- , tnf-a, and amcf- mrna levels by prrsv infection was dramatically reduced in the presence of sqe. however, chemokine genes that are modulated by prrsv, including mcp- and rantes, were synergistically elevated by sqe treatment. more interestingly, unchanged or slightly increased mrna levels of interferon regulatory factors (irfs), toll-like receptors (tlrs), and antiviral genes including mx and isg- were b fig. inhibition of viral rna transcription by sqe. pam-knu cells pretreated with sqe were mock infected or infected with prrsv for h and were incubated in the presence of sqe. total cellular rna was extracted at hpi (black bars) and hpi (white bars), and viral genomic rna and sg mrna were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna (a) and sg mrna (b) were normalized to mrna for porcine b-actin mrna (c), and relative quantities (rq) of mrna accumulation were evaluated. sqe-treated sample results were compared with untreated results. values are representative of the mean from three independent experiments, and error bars denote standard deviations. *, p = . to . ; , p \ . fig. suppression of viral rna synthesis by sqe treatment. pam-knu cells were treated with sqe, followed by prrsv inoculation, as indicated above the lanes. total rna was extracted from lysates of the infected cells at hpi and subjected to northern blot analysis using genotype-specific biotin-labeled oligonucleotide probes against the utr. porcine s rrna was used as a control to correct for variations in loading during viral rna quantification. the amount of viral sg mrnas was quantified by densitometry and normalized to that in prrsv-infected, untreated control cells ( %). the positions of the genomic rna and sg mrnas are indicated next to the gel significantly increased in prrsv-infected pam cells treated with sqe compared with vehicle-treated infected cells. these results indicated that sqe modulates the cytokine genes for inflammatory responses during prrsv infection. prrsv is not only a devastating viral pathogen in the pig population, but it can also be used as an animal virus model to study nidoviruses that are important in human and veterinary medicine. despite tremendous efforts to control disease caused by this virus, prrsv infection has continuously plagued pig-producing countries and has had a tremendous economic impact on the global swine industry. this is partially due to the absence of efficient vaccines capable of conferring full protection against heterologous viral infection as well as antiviral agents for treating prrsv. although sqe is widely known to possess several health-promoting properties [ , ] , its antiviral activity and mode of action on dna and rna viruses are currently unknown. the present study demonstrated that sqe exerts an adequate antiviral effect against prrsv in vitro, as determined by the ed for viral replication (antiviral activity) of approximately . mg/ml. treatment of cells with sqe resulted in significant inhibition of post-entry steps during the replication of prrsv, as demonstrated by reduced progeny production, diminished viral protein expression, and reduced synthesis of genomic rna and sg mrna. furthermore, a growth kinetics experiment indicated an approximately -log reduction in the titer of prrsv in the presence of sqe at h after infection, and the titer continued to remain at a similar level h after infection. taken together, our data indicate that sqe potently elicits antiviral activity against prrsv in natural target cells. compounds from medicinal plants or natural sources are of interest for their potential to inhibit a variety of viral infectious diseases and cancer. bamboo grasses have been used as traditional medicines throughout asia, and modern studies have reported their beneficial health effects. sasa quelpaertensis nakai is among these and is well known as jeju-joritdae in south korea. sqe, its leaf extract, has a number of biological properties, including anticancer and anti-inflammatory effects. since prrsv infection modulates inflammatory cytokine expression in pam cells, the antiviral activity of sqe may result predominantly from its anti-inflammatory effect. to elucidate potential mechanism(s) responsible for the antiviral effect of sqe on prrsv, we first tested whether sqe affects the induction of cytokine genes by prrsv in immortalized pam cells. the present study provided evidence that various cytokines induced in prrsv-infected cells are transcriptionally modulated in the presence of sqe. our results indicate that treatment with sqe robustly reduced infection-induced expression of pro-inflammatory cytokines, including il- a, il- , il- , il- , and tnf-a. in contrast, sqe resulted in a synergic elevation in gene expression levels of chemokines. notably, the induction of irfs, tlrs, and genes involved in the antiviral immune response was specifically enhanced in sqe-treated virus-infected cells. prrsv is known to possess the ability to escape host defense mechanisms by interfering with innate immune responses to favor its survival and spread in the natural host. thus, sqe appears to disrupt one of the pivotal viral mechanisms of immune evasion by modifying the expression of immune-related genes to facilitate virus infection, resulting in the suppression of prrsv replication. however, we were unable to identify which chemical constituent(s) in sqe is responsible for the regulation of immune-response gene expression that leads to its antiviral activity upon prrsv infection. although p-coumaric acid is known to be a major component of sqe ( . mg/g) and has diverse pharmacological properties, including antimicrobial, anticancer, and antiulcer activities [ , ] , this chemical was incapable of significantly impairing the replication of prrsv, even at the highest noncytotoxic concentration of lm (fig. a ) . it is conceivable that other minor constituents such as tricin, with a content in sqe of . mg/g, may possess the biological characteristics that give sqe its antiviral activity. indeed, recent studies have demonstrated inhibitory effects of synthesized tricin or its derivative on viral infections [ , ] . in addition to prrsv, our experiment was further extended to other nidoviruses to confirm the antiviral activity of sqe. porcine epidemic diarrhea virus (pedv) is one of pathogenic coronaviruses of pigs. like prrsv, it belongs to the order nidovirales, whose members all have a similar genome organization and replication strategy. thus, we attempted to assess whether sqe also affects pedv infection but found that it had no significant inhibitory effect on pedv infection in vitro (fig. a ). this observation might be explained by the tropism of each virus, since prrsv is a porcine macrophage-tropic arterivirus causing acute respiratory illness, whereas pedv is a pathogenic enterocytetropic coronavirus of swine causing acute enteritis and watery diarrhea. although sqe failed to block pedv infection, it still has potential as an antiviral agent against other coronaviruses of human and livestock, for which the modulation of immune-response cytokine gene production in the natural host is related to their replication. in conclusion, our findings described here demonstrate that sqe at subcytotoxic doses effectively prevents the replication of prrsv in natural host cells. additionally, sqe treatment significantly altered prrsv-induced fig. regulation of immune-related genes by sqe during prrsv infection. in the presence of vehicle or sqe ( mg/ml), pam-knu cells were mock infected or infected with prrsv at an moi of . total rna was extracted from lysates of the infected cells at hpi (left bar set) and hpi (right bar set). the mrna level of each cytokine gene was assessed by quantitative real-time rt-pcr and normalized to that of porcine b-actin. the relative fold difference between mock-infected and virus-infected cells (y-axis) was then calculated for each gene and compared between untreated and sqetreated virus-infected cells. data are representative of the mean values from three independent experiments carried out in duplicate, and error bars represent standard deviations. *, p = . to . ; , p \ . cytokine production in pam cells. therefore, we propose that one of the modes of action of sqe to elicit an antiviral effect against prrsv is to regulate the expression of immune-related genes that are associated with the ability of prrsv to escape the host innate immune responses. although further in vivo studies are needed to evaluate the efficacy and safety of sqe, the results presented here indicate that it is a good candidate as an antiviral agent against prrsv. inhibitory effects of tricin derivative from sasa albo-marginata on replication of human cytomegalovirus epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units experimental infection of colostrum deprived piglets with porcine circovirus (pcv ) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv replication ) q-coumaric acid, a constituent of sasa quelpaertensis nakai, inhibits cellular melanogenesis stimulated by alpha-melanocyte stimulating hormone highly pathogenic porcine reproductive and respiratory syndrome virus functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus suppression of coronavirus replication by inhibition of the mek signaling pathway nidovirales: a new order comprising coronaviridae and arteriviridae persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins efficient - frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers anti-inflammatory effect of the hot water extract from sasa quelpaertensis leaves sasa quelpaertensis leaf extracts induce apoptosis in human leukemia hl- cells sasa quelpaertensis nakai extract and its constituent qcoumaric acid inhibit adipogenesis in t -l cells through activation of the ampk pathway anti-obesity properties of a sasa quelpaertensis extract in high-fat diet-induced obese mice combination of sasa quelpaertensis nakai leaf extract and cisplatin suppresses the cancer stemness and invasion of human lung cancer cells straus se (eds) fields virology porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway cytokine production in immortalized porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method inhibition of prrsv by sasa quelpaertensis nakai extract a summary of taxonomic changes recently approved by the ictv assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states histo-chemical studies on the antiulcer effect of bamboo grass in rats porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-b production by inhibiting irf activation in immortalized porcine alveolar macrophages human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus straus se (eds) fields virology family coronaviridae highly pathogenic porcine reproductive and respiratory syndrome effects of sasa health, extract of bamboo grass leaves, on spontaneous mammary tumourigenesis in shn mice proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine acknowledgments we gratefully thank yong chool boo from kyungpook national university for providing sasa quelpaertensis nakai extract. key: cord- -vatfdilt authors: narita, m.; ishii, m. title: encephalomalacic lesions in pigs dually infected with porcine reproductive and respiratory syndrome virus and pseudorabies virus date: - - journal: j comp pathol doi: . /j.jcpa. . . sha: doc_id: cord_uid: vatfdilt four pigs (group ) were infected with an aerosol containing porcine reproductive and respiratory syndrome virus (prrsv) followed days later by pseudorabies virus (prv). three further pigs (group ) received prrsv alone, two (group ) received prv alone, and two (group ) remained as uninfected controls. despite the admittedly small numbers of animals, the experiment appeared to throw light on aspects of synergy. thus, the group pigs showed severe neurological signs characterized by ataxia and muscular tremors. total cell numbers in the bronchoalveolar lavage fluid were increased in all prrsv-infected pigs, and prrsv antigen was detected in the alveolar macrophages. total cell numbers in the cerebrospinal fluid of group pigs were considerably greater than those demonstrated in group , but no prv antigen was found. pigs of groups and showed pulmonary lesions, characterized by interstitial pneumonia and prrsv antigen immunolabelling. non-suppurative encephalitis was found in five of the six pigs of groups and . in particular, one group animal had severe necrotizing encephalitis with intranuclear inclusion bodies and associated immunolabelling of prv antigen. the other three group pigs had prominent malacic lesions, with macrophages. these neuropathological findings strongly suggested that prrsv infection in pigs enhances the severity of brain lesions caused prv. porcine reproductive and respiratory syndrome (prrs) is an arterivirus disease, currently endemic in many swine-producing countries (conzelmann et al., ) . prrs virus (prrsv) primarily infects and destroys alveolar macrophages, which play an important role in pulmonary defence (rossow, ) . prrsv is therefore suspected to have an immunosuppressive effect on pigs. co-infection with prrsv has been reported to increase the severity of disease produced by agents such as porcine respiratory coronavirus, influenza virus, mycobacteria, salmonella choleraesuis and streptococcus suis (groschup et al., ; kawashima et al., ; reeth et al., ; narita et al., ; thacker et al., ; de bruin et al., ; thanawongnuwech et al., ; wills et al., ) . such synergism has been supported by clinical, virological and immunological observations. the immunosuppressive effect of prrsv on the host response to pseudorabies virus (prv) has also been investigated in the respiratory system (shibata et al., ) , but there have been few histopathological studies supporting synergism in dual infections with prrsv and prv, or in dual infection with prrsv and other respiratory viral agents (de bruin et al., ) . the purpose of the present study in admittedly small numbers of specific pathogen-free (spf) pigs was to throw light on the effects (clinical, cytological and pathological) of dual infection with prrsv and prv as compared with single infections. eleven spf pigs aged weeks were used, having been shown to be free from serum antibodies against prv, prrsv and porcine circovirus type . the pigs were divided into four groups (see below), which were housed separately in different blocks to prevent cross-infection. the housing conditions included a filtered air system and regulated temperature. the edrd- strain of prrsv, kindly provided by dr y. murakami of the national institute of animal health, was used after eight passages in swine alveolar macrophages. the ys- strain of prv, also provided by dr y. murakami, was used after three passages in pig kidney cell culture. the infected culture fluids were used as inocula. the pigs were randomly assigned to four groups, as follows: group (pigs - ) were inoculated with prrsv followed by prv; group (pigs - ) received prrsv alone; group (pigs and ) received prv alone; group (pigs and ) were non-infected controls. the inocula were administered as an aerosol produced by a nebulizer, after the animals had been anaesthetized with ketamine hydrochloride ( mg/kg body weight) and xylazine ( mg, intramuscularly). on "day " of the experiment, pigs of groups and received . tcid of prrsv in a dose volume of ml. on day of the experiment, the pigs of groups and received . tcid of prv in a dose volume of ml. the negative control animals of group received ml of non-infected culture medium on day , and ml on day . all animals were killed with an intravenous overdose of pentobarbital sodium on the following days of the experiment: day , pig ; day , pigs , , and ; and day , pigs , , , , and . a csf sample ( ml) was collected by syringe post mortem and placed in a sterile bottle. after removal of the lungs, a cannula ( mm in diameter) was inserted into the right main bronchus, and ml of sterile phosphate-buffered saline (pbs) were introduced into the bronchus and then recovered by suction. counts of nucleated cells per microlitre of csf and balf were made, and . ml of csf and . ml of a in dilution of balf were centrifuged and stained with a diff-quik kit (international reagent corporation, koube, japan). evaluation included a -cell differential count and a morphological description of the cells. the centrifuged csf and balf cells were fixed in cold acetone and stored at c for immunohistochemical examination. specimens from each pig, including parts of the brain (cerebral cortex, frontal lobe, motor area, occipital lobe, corpus striatum, thalamus, colliculus caudalis, cerebellar peduncles, pons, cerebellum and medulla oblongata), spinal cord, trigeminal ganglia, tonsil and lung, were fixed in % neutral buffered formalin. the tissues were embedded in paraffin wax, sectioned ( mm), stained with haematoxylin and eosin (he), and examined by light microscopy. prrsv and prv antigen in the formalin-fixed tissues and cold acetone-fixed csf and balf cells were demonstrated by the streptavidin -biotin (sab) immunoperoxidase (ip) method, with a histofine sab kit (nichirei corp., tokyo, japan). anti-prrsv (chiba - strain) rabbit serum (provided by dr k. kawashima, national institute of animal health, japan) (kawashima et al., ) and anti-prv rabbit serum (provided by dr t. imada, national institute of animal health, japan) (narita et al., (narita et al., , were used as the primary antibodies at dilutions of in and in , respectively. sections were counterstained with methyl green. tissue sections from noninfected control pigs (nos and ) and serum from a non-immunized rabbit were used for control purposes. all four pigs dually infected with prrsv and prv (group ) showed severe neurological signs, characterized by ataxia and muscular tremors, after being infected with prv, and had a severe febrile response (up to c) between days and . the three group pigs, infected with prrsv alone, showed slight respiratory symptoms and had a transient ( -day) febrile response. the two group pigs, infected with prv alone, showed inappetence and pyrexia for a period of days. the two non-infected control pigs (group ) showed no clinical abnormalities during the experimental period. cytology csf cells. the results are shown in table . the total cell number in the csf from pigs infected with prv (groups and ) ranged from to ( £ . )/ ml. the proportions of the various cell types were: neutrophils - %, macrophages - % and lymphocytes - %. the total cell numbers of pigs infected with prrsv alone (group ) and noninfected controls (group ) ranged from to ( £ . )/ml and to ( £ . )/ml, respectively, and no neutrophils or macrophages were found. despite the small numbers of observations, there was an indication that the total number of csf cells may have been considerably higher in group than in group . no prv or prrsv antigen was detected by immunolabelling of the csf cells of any animals. balf cells. the total cell number in the balf from pigs infected with prrsv (groups and ) ranged from to ( £ . )/ml. the proportions of the various cell types were: neutrophils - %, macrophages - %, and lymphocytes - %. the total cell number in pigs infected with prv alone (group ) was to ( £ . )/ml, and in non-infected control pigs (group ) was to ( £ . )/ml. the proportion of neutrophils in pigs infected with prv alone (group ) was - %, that of macrophages was - %, and that of lymphocytes was - %. in non-infected control pigs (group ), the corresponding proportions were as follows: neutrophils - %, macrophages - %, and lymphocytes - %. there was no obvious difference between groups and . prrsv (but not prv) antigen was detected in the balf macrophages of four the seven pigs infected with prrsv (groups and ) (fig. ) . nd, not done. þ, a few immunolabelled cells; , none. see materials and methods for details of inoculation of groups - . the experiment began on "day " and the csf and balf were collected post mortem at the following times: pig (day ); pigs , , and (day ); pigs , , , , and (day ). * £ . for csf, but £ . for balf. macroscopically, all dually infected pigs (group ) showed severe atrophy of the thymus, congestion of the brain, and pneumonia with diffuse tan coloration at the periphery of the lobes. none of the three pigs infected with prrsv alone (group ) or the two pigs infected with prv alone (group ) showed obvious macroscopical lesions, except for slight atrophy of the thymus in the group animals. the two non-infected control pigs (group ) showed no abnormalities. the distribution of microscopical lesions is summarized in table . all dually infected pigs (nos - ) had slight trigeminal ganglioneuritis and slight (pigs - ) to severe (pig ) nonsuppurative encephalitis, consisting of neuronal degeneration and necrosis, neuronophagia, diffuse or focal glial reactions, and perivascular cuffing (fig. ) . the lesions were distributed in the white matter of the lobus frontalis, lobus temporalis and lobus parietalis, but not in the cerebellum or spinal cord. the more severe lesions occurred in the frontalis areas of the cerebral grey matter. some degenerating neuronal and glial cells adjacent to the necrotic areas showed basophilic intranuclear inclusion bodies. three dually infected pigs (nos - ) killed on day of the experiment, had prominent encephalomalacic lesions, localized in table distribution of histopathological lesions in the central nervous system (cns) of pigs infected dually and singly with prrsv and prv encephalitis*/encephalomalacia* in individual pigs of group group group group cns site cerebrum frontalis þþþ/ þ /þþþ þ/þþþ þ/þþþ / / / þ/ / / / temporalis þþ/ þ /þþ þ/þþ þ/þþ / / / / / / / parietalis þ / þ /þ þ/þþ þ/þþ / / / / / / / occipitalis / / / / / / / / / / / hippocampus þ / / / / / / / / / / / thalamus þ / þ / þ / þ/ / / / / / / / cerebellum / / / / / / / / / / / pons þ / þ / þ / þ/ / / / / / / / medulla oblongata þ / þ / þ / þ/ / / / / / / / trigeminal ganglia þ / þ / þ / þ/ / / / / / / / spinal cord / / / / / / / / / / / see materials and methods for details of inoculation of groups - , and for times at which the animals were killed for examination. interstitial pneumonia: moderate (pigs , , , , ), slight (pig ), negative (pigs , , , , ) . tonsillitis: severe (pig ), moderate (pigs , , ), slight (pig ); negative (pigs , , , , , ) . thymic atrophy: severe (pigs - ), slight (pigs , ) , negative (pigs , , , , ). * , negative; þ , slight; þþ, moderate; þþþ, severe. areas near the olfactory bulb and rhinencephalon, including the olfactory tract (fig. ) , olfactory stria, and pyriform cortex. they consisted of cortical cavitation, with macrophages ( fig. ) . pig , infected with prv alone, showed slight perivascular cuffing and glial reaction but no neuronal degeneration or intranuclear inclusion bodies. the four group pigs and two of the three group pigs had slight to moderate interstitial pneumonia (fig. ) , but no necrotizing bronchiolitis. all group pigs had moderate tonsillitis with typical intranuclear inclusion bodies in the degenerating crypt epithelial cells; they also had severe atrophy of the thymus with apoptosis of thymic t lymphocytes (fig. ) . two group pigs also had slight thymic atrophy. such lesions were not observed in the pigs of groups and . the distribution of viral antigens in tissues is summarized in table . in the cerebrum, prv antigen was found in three (nos , and ) of the dually infected pigs. strong immunolabelling was closely associated with the intranuclear inclusion bodies in the neuronal cells of pig , killed on day of the experiment (fig. ) . immunolabelling was observed in a few glial cells of pigs and but not in the macrophages in the encephalomalacic lesions. in the lungs of pigs - , prrsv antigen (but not prv antigen) was detected in macrophages in the alveolar lumina at sites showing interstitial pneumonia (fig. ) . prv antigen was detected in the degenerating epithelial cells of the tonsillar crypts of the four dually infected pigs, and prrsv antigen was detected in the macrophages of two of them (nos and ) and one pig (no. ) that received prrsv alone. neither antigen was found in the thymus of any pig or in any tissue from the two non-infected control pigs. the synergistic effect of dual infection with prrsv and other infectious agents has been investigated previously (groschup et al., ; kawashima et al., ; reeth et al., ; narita et al., narita et al., , thacker et al., ; de bruin et al., ; thanawongnuwech et al., ; wills et al., ) . in this study, pigs infected with prrsv or prv alone did not show severe clinical signs, but there was a febrile response in pigs infected with prv, and there were slight respiratory symptoms in pigs infected with prrsv. the four dually infected pigs (group ), however, showed severe neurological signs characterized by ataxia and muscular tremors. these findings, which resembled those reported by shibata et al. ( ) , suggested that prrsv infection in pigs affects the replication of prv. each of the infectious agents produces characteristic lesions at the various sites of viral replication (narita et al., (narita et al., , (narita et al., , rossow, ) . in the present experiment, seven pigs infected with prrsv (groups and ) had slight interstitial pneumonia but no necrotizing bronchiolitis. these pneumonic lesions were closely associated with the presence of prrsv antigen, and were probably due to prrsv infection since they resembled prrsvassociated lesions described in previous reports (rossow, ; shibata et al., ) . five of six pigs infected with prv (groups and ) had non-suppurative encephalitis and trigeminal ganglioneuritis. one dually infected pig (no. ) had severe necrotizing encephalitis with intranuclear inclusion bodies, while the other three (nos - ) had prominent encephalomalacia, with macrophages. all dually infected pigs had tonsillitis with inclusion bodies and severe atrophy of the thymus. these results strongly suggest that prrsv infection in pigs can increase the severity of brain and tonsillar lesions due to prv replication, and also the severity of lung lesions (shibata et al., ) . moreover, thymic atrophy may be related to dual infection. the total number of csf cells increased -to fold in prv-infected pigs as compared with the numbers in pigs infected with prrsv alone and in the non-infected pigs. moreover, the total number was higher in dually infected pigs that in those infected with prv alone. in prv-infected pigs, neither prv nor prrsv antigen was detected in csf cells. the total cell number in the balf increased by up to -fold in all prrsv-infected pigs, and prrsv (but not prv) antigen was detected in the alveolar macrophages. these results corresponded well with the presence of interstitial pneumonia. thus, the results of csf cell analysis accorded with the severity of the brain lesions in the dually infected pigs, and the results of balf cell analysis with the interstitial pneumonic lesions in prrsv-infected pigs. herpes viruses spread via sensory axons and infect sensory neurons in the ganglia of the peripheral nervous system (cook and stevens, ; narita et al., narita et al., , chowdhury et al., ; yanai et al., ) . in prv infection in pigs, infection spreads from the nasal cavity via the olfactory pathway to produce non-suppurative encephalitis (narita et al., (narita et al., , (narita et al., , (narita et al., , (narita et al., , . in the present experiments, one dually infected pig had severe encephalitis and three had encephalomalacia, characterized by neuronal degeneration with intranuclear inclusion bodies, a diffuse or focal glial reaction, and perivascular cuffing, as well as by cavitation accompanied by large numbers of macrophages. the lesions were mainly confined to the olfactory bulb and rhinencephalon, including the olfactory tract, olfactory stria, and pyriform cortex. the encephalomalacic lesions may therefore have developed from necrotic foci similar to those observed in prv-infected newborn piglets (nunoya et al., ) and in animals with fluctuating temperatures (narita et al., ) . thus, the neuropathological findings strongly suggested that prrsv infection in pigs enhanced the severity of brain lesions caused by prv. this apparent synergy was supported by histopathological findings. neuropathology of bovine herpesvirus type (bhv- ) meningo-encephalitis in a rabbit seizure model molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus serological studies on the potential synergism of porcine reproductive and respiratory syndrome virus and influenza-, corona-and paramyxoviruses in the induction of respiratory symptoms in swine detection of porcine reproductive and respiratory syndrome virus and mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress pseudorabies virus in dexamethasone-treated pigs immunohistological demonstration of spread of aujeszky's disease virus via the olfactory pathway in hpcd pigs immunohistological study of encephalomalacia in pigs infected with aujeszky's disease virus immunohistopathological characterization of pig pneumonia caused by a combined aujeszky's disease virus and actinobacillus pleuropneumoniae infection immunohistochemical characterization of calf pneumonia produced by the combined endobronchial administration of bovine herpesvirus and pasteurella haemolytica immunopathology in aujeszky's disease virusinfected pigs exposed to fluctuating temperatures invasion and spread of equine dual infection of pigs with prrsv and prv herpesvirus in the olfactory pathway of pigs after intranasal inoculation pseudorabies virus infection in piglets acconpanying with the lesion of bilateral encephalomalacia dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study porcine reproductive and respiratory syndrome experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine experimental infection with equine herpesvirus (ehv- ) in cats we thank mr m. kobayashi and miss m. shimada for preparing the histological tissue sections, and dr y. ando and mr t. fujisawa for preparing the photographs. key: cord- -wkisfz m authors: han, mingyuan; yoo, dongwan title: engineering the prrs virus genome: updates and perspectives date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: wkisfz m porcine reproductive and respiratory syndrome virus (prrsv) is endemic in most pig producing countries worldwide and causes enormous economic losses to the pork industry. infectious clones for prrsv have been constructed, and so far at least different infectious clones are available representing both genotypes i and ii. two strategies have been taken for progeny reconstitution: rna transfection and dna transfection. mutations, insertions, deletions, and replacements of the viral genome have been employed to study the structure function relationship, foreign gene expression, functional complementation, and virulence determinants. essential regions and non-essential regions for viral replication have been identified in both the coding regions and non-encoding regions. foreign sequences have successfully been inserted into the nsp and n regions and in the space between orf b and orf a. chimeras between member viruses in the family arteriviridae have also been constructed and utilized to study cell tropism and functional complementation. this review discusses the advances and utilization of prrsv reverse genetics and its potential for future research. porcine reproductive and respiratory syndrome (prrs) was first reported in the united states in and subsequently in europe in and quickly became endemic in most pig producing countries worldwide (benfield et al., ; chand et al., ; murakami et al., ; shimizu et al., ; wensvoort et al., ) . the clinical manifestation of prrs is complicated but is characterized by severe reproductive losses including abortions, mummified fetuses, weak born and stillborn young, post-weaning pneumonia, increased mortality, and growth retardation of young pigs. the etiological agent is prrs virus (prrsv) . prrsv belongs to the family arteriviridae together with lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv). by comparative genome sequence analysis, prrsv isolates are divided into two distinct genotypes: the european type (genotype i) and north american type (genotype ii), represented by lelystad virus (lv) and vr- as the prototype virus for each genotype, respectively (benfield et al., ; wensvoort et al., ) . the sequence similarities between two genotypes are approximately % (allende et al., ; nelsen et al., ; wootton et al., ) . amino acid (aa) sequence alignments indicate that the major differences between two genotypes exist in the open reading frame (orf) a region and the structural protein region (kapur et al., ; murtaugh et al., ; nelsen et al., ) . natural deletions and insertions are observed in some isolates, especially in the orf a region (fang et al., ; gao et al., ; shen et al., ; tian et al., ) . the reverse genetics system has been developed for many rna viruses, and infectious clones have been utilized for the study of biology and the vaccinology of viruses. the availability of such a powerful molecular tool has revolutionized the structure function studies for viral genome and proteins and has facilitated the studies for virulence, pathogenesis, immune responses, and vaccine development. the first full-length genomic cdna clone was constructed for poliovirus more than three decades ago and its infectivity was demonstrated (racaniello and baltimore, ) . infectious clones have since been constructed for picornaviruses, caliciviruses, flaviviruses, togaviruses, influenza viruses, paramyxoviruses, rhabdoviruses, and coronaviruses to name a few (almazan et al., ; boyer and haenni, ; pu et al., ; scobey et al., ; sosnovtsev and green, ; yount et al., ) . for arteriviruses, eav and prrsv are the first for which the reverse genetics system has been developed van dinten et al., ) . this review will summarize our current knowledge on the principles of prrsv infectious clones and the application to the study of arteriviruses. the prrsv genome is a single-strand positive-sense rna of kb in length with a cap and -polyadenylated tail (fig. a ) (meulenberg et al., ; murtaugh et al., ; nelsen et al., ; wootton et al., ) . the prrsv genome is polycistronic and harbors two large open reading frames (orfs), orf a and orf b, followed by orf a, orf b, and orfs through , plus orf a within orf (firth et al., ; johnson et al., ; meulenberg et al., ; murtaugh et al., ; nelsen et al., ; wootton et al., ) . a - ribosomal frame-shifting has recently been identified for expression of nsp tf in the nsp -coding region. the nsp tf coding sequence is conserved in prrsv, ldv, and shfv but absent in eav (fang et al., ) . the coding sequences in the viral genome are flanked by the and un-translated regions (utrs) involved in translation, replication, and transcription (see review in snijder et al., ) . orf a and orf b code for two large polyproteins, pp a and pp ab, with the expression of the latter mediated by the - frame-shifting in the orf a/orf b overlapping region (fig. b; den boon et al., ; snijder and meulenberg, ) . thus, the pp b portion is expressed always as a fusion with pp a. the pp a and pp ab proteins are further processed to generate non-structural proteins (nsps). the polyprotein processing scheme involves the rapid auto-proteolytic release of three nterminal nsps, nsp a, nsp b, and nsp , mediated by papain like proteinase (plp) residing in each of them. the subsequent processing for the remaining portion of polyproteins is mediated by the serine protease in nsp resulting in individual nsps (den boon et al., ; van aken et al., ; ziebuhr et al., ) . the proteolytic cleavages for individual nsps were initially predicted by sequence comparisons in combination with some experimental data from eav, the prototype virus of the family arteriviridae (fang and snijder, ; ziebuhr et al., ) . the exact cleavage sites for prrsv nsp a#nsp b and nsp b#nsp have recently been confirmed to be m #a and g #a mediated by prrsv-plp a and prrsv-plp b, respectively sun et al., ; xue et al., ) . a set of -coterminal nested subgenomic (sg) mrnas, from which structural proteins are translated, is produced during infection (fig. b) . each mrna contains a common -end leader sequence identical to the proximal part of the genome and this sequence is referred to as transcription-regulatory sequence (trs). the fusion of the common sequence (leader trs) to the different -body segments of sg mrnas is mediated by discontinuous transcription which is a common strategy of nidoviruses (sawicki et al., ; snijder et al., ; sola et al., ) . during the negative-strand sg rna synthesis, transcription is attenuated at different body trs regions of the genomic template. the nascent subgenome-length minus-strand rna, having an antibody trs at its end, will then move and base-pair with the leader trs and completes the extension of sg rna. minus-strand sg rnas subsequently serve as a template for plus-strand sg mrna which is subsequently translated for structural protein (music and gagnon, ; sawicki et al., ; snijder et al., ) . the genome of negative-strand rna viruses is noninfectious, and its replication in permissive cells requires the ribonucleoprotein (rnp) complex as the infectious unit. in contrast, the genome from positive-strand rna viruses is fully infectious, and thus the assemly of full-length cdna clones corresponding to the rna genome is the kernel to the construction of infectious clones . the viral genome is polycistronic, harboring orf a and orf b, and structural genes of orf a, orf b, and orfs through , plus orf a within orf . (b) viral gene expression. non-structural proteins (black) are produced from pp a and pp ab after proteolytic processing. the prrsv replicase-processing scheme involves the rapid auto-proteolytic release of nsp a, nsp b, and nsp (yellow boxes), mediated by papain-like proteinase (plp) domains residing in each of them. the remaining polyproteins are processed by nsp , resulting in a set of individual nsps. the cleavage sites by plps and nsp are annotated by curved arrows and blue triangles, respectively. structural proteins (color-coded) are expressed from the subset of sg mrna. the -co-terminal nested set of minus-strand rnas is produced as a template for plus-strand sg mrna synthesis. trs, transcription regulatory sequence. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (boyer and haenni, ; meyers et al., ; moormann et al., ; sosnovtsev and green, ; van dinten et al., ) . non-retroviral rna viruses do not undergo a dna intermediate step in their replication cycle. to obtain a template which can be manipulated by molecular techniques, a full-length cdna clone is first generated using the reverse transcriptase and dna polymerase. once generated, two strategies have been established to generate virus progeny from the full-length copy of viral genome: rna transfection and dna transfection. in the rna transfection strategy, viral rna is synthesized by in vitro transcription using t or sp rna polymerase coupled with the respective promoter located immediately upstream of the viral genome. the synthesized rna genome is then introduced into cells to initiate an infection cycle. in the dna launch strategy, a full-length genomic clone is placed under a eukaryotic promoter such as a cytomegalovirus (cmv) promoter and the entire plasmid is introduced to cells for transcription by exploiting the nuclear function of the cell. the transcribed viral genome in the nucleus is exported to the cytoplasm where viral genome translation and replication occur. this strategy omits the steps of in vitro synthesis of genomic rna and rna transfection, thus the risk of rna degradation during transfection is reduced and transfection efficiency becomes consistent ). an arterivirus infectious clone was first made for eav. the peav full-length clone containing the . kb cdna copy of the eav genome was infectious (van dinten et al., ) , and the first prrsv infectious clone pabv was developed for the genotype i prrsv lelystad virus . subsequently, infectious clones for vr- which is the genotype ii prrsv, and the european-like genotype i prrsv sd - circulating in the us was developed (fang et al., a (fang et al., , b nielsen et al., ) . numerous clones have additionally been developed including the highly-pathogenic prrsv that emerged in china in (guo et al., ; lv et al., ; zhou et al., ) . to date, at least different infectious clones are available for prrsv (table ) . prrsv infectious clones have mostly been developed based on the rna launch strategy. bhk- , ma- , and marc- cells are cells of choice for transfection and progeny production. although bhk- cells are nonpermissive for prrsv infection, they provide a high efficiency of transfection and a good production of progeny . to eliminate the need for in vitro transcription and consistency associated with rna transfection, the cmv promoter has been used for construction of the p infectious clone. the p virus is an isolate recovered from an outbreak of highly virulent atypical prrs in the mid-western usa in yoo et al., ) . the cmv promoter-based infectious clone is convenient and simple to use and provides a consistency of transfection and recovery of progeny virus ). an infectious clone should genetically be identical to the parental virus. however, non-viral nucleotides are occasionally added to the viral genome at the or end to meet the engineering needs without impeding the infectivity of the clones truong et al., ) . to differentiate the reconstituted progeny virus from the parental virus, genetic markers of either restricted enzyme recognition sequences or certain nucleotide mutations have been introduced to infectious clones, and such modifications should be non-lethal and stable. to assure the starting position of the rna polymerase ii-mediated transcription, nucleotides are placed between the tata box and the genome start when constructing pcmv-s-p . the prrsv genome is usually divided to several fragments flanked by restriction sites for subsequent assembly nielsen et al., ) . as a cloning vector, a lowcopy-number plasmid is generally preferred as suggested in some studies sumiyoshi et al., ; nielsen et al., ) but has appeared unnecessary. progeny virus generated from an infectious clone should ideally retain the biological properties of the parental virus, such as growth rate, virulence, and transmissibility meulenberg et al., ; nielsen et al., ; truong et al., ; yuan and wei, ) . like most rna viruses, prrsv genome has evolved to optimal fitness, and most of the genetic information seems to be essential (verheije et al., ) . notably, the proximal portion of the genome is compact and organized to contain eight genes, most of which overlap with neighboring genes (snijder et al., ) . the prrsv genome is complex and the engineering of such a compact viral genome is a challenge. in addition, minor alternations in conserved regions or functional domains in the genome almost inevitably lead to non-viable consequences kroese et al., ; . despite such difficulties, some genetic manipulations for prrsv have been successful. mutation, deletion, insertion, and substitution are major approaches to viral genome manipulation. due to the large genome of prrsv, shuttle plasmids have been used as an intermediate platform to contain the target viral genomic sequence with a pair of unique enzyme sites at each end. mutations are introduced to target sites or sequences in the shuttle plasmid. the biological functions of plp a and plp b in nsp , conserved cysteine residues at c and c in the e protein, n-linked glycosylation sites in gp at n and gp at n , n , and n , cysteines at c , c , and c for homo-dimerization of n protein, and the motif for nuclear localization signal (nls) of n have been mutated to produce prrsv mutants kroese et al., ; pei et al., ; vu et al., ) . alanine scanning and protein surface accessibility predictions were conducted for identification of residues for type i ifns or tnf-a antagonism of nsp , and specific residues have been mutated in the infectious clones li et al., ; subramaniam et al., ) . mutations have also been introduced to knockout genes by changing the translation n/a, not application. a the individual time of prrsv infectious clones construction referred to the date of each publication. b genotype i and ii prrsv represents european and north america strains, respectively. c sequences of full-genome cdna clone rather than complete genome sequences of parental virus are listed. d the genome area in which the restricted enzyme sites are introduced is indicated with brackets. the position where single mutations are introduced is given. e genome-length cdna clones of pabv and pabv encode identical viral protein sequences except for one amino acid at position in orf a, which are a pro in pabv and a leu in pabv . f the abbreviation, na i, in the genotype column represents genotype i prrsv isolated in north america. initiation codon, and this approach destroys the expression of nsp and e protein tijms et al., ) . deletion of genomic sequences has been applied to identifying non-essential regions for prrsv replication or to obtaining attenuated live vaccine candidates (verheije et al., ) . inter-genotypic sequence alignments between genotype and genotype reveal the regions of sequence heterogeneity suggesting the potential to tolerate the deletions, and non-essential regions in the n gene and -utr (table ; sun et al., b; tan et al., ) . the hypervariable regions have been observed in the nsp gene (fang et al., ; gao et al., ; ni et al., ; shen et al., ; tian et al., ) , suggesting the existence of a non-essential region in nsp (chen et al., b; han et al., ; ran et al., ; xu et al., b) . deletion of orf or orf results in the absence of infectivity, suggesting the requirement of gp and gp proteins for prrsv infectivity . insertion of additional nucleotides to the viral genome expands the scope of modifications. an attempt was made to separate overlapping regions of prrsv structural protein genes, and three restriction enzyme sites were inserted between orfs / and orfs / (yu et al., ) , which produced viable viruses. the possibility of expressing foreign genes using prrsv has been explored; the nsp gene was utilized as an insertion site for expressions of green fluorescent protein (gfp) and flag tag (fang et al., b; kim et al., ). an alternate approach was taken to insert foreign genes within a structural gene; for example, a small portion of the influenza virus hemagglutinin (ha) gene into the or end of orf of prrsv (bramel-verheije et al., ) . however, the insertion of ha to n gene resulted in a nonviable virus. a strategy utilizing the mechanism of transcription of prrsv for foreign gene expression is of particular interest. using an infectious clone, two unique enzyme sites have been introduced between orf b and orf , and a copy of the trs sequence was inserted to replace the trs designed to synthesize the mrna for foreign gene expression (fig. ) pei et al., ; yoo et al., ) . the foreign genes including gfp, capsid protein of porcine circovirus type (pcv ), discosoma sp. (sea anemone) red fluorescent protein (dsred), renilla luciferase (rluc), ifn-a , ifn-b, ifn-d , and ifn-v have all been expressed as an independent transcript using this approach (table ; pei et al., ; sang et al., ) . multiple genes, a single gene, or partial sequence of the viral genome have been substituted with corresponding sequences from other arteriviruses for chimeric arterivirus construction. the first chimeric arterivirus was generated using an eav infectious clone as a backbone, and ectodomains of two membrane proteins, gp and m, were substituted with the corresponding sequences from prrsv or ldv (dobbe et al., ) . these chimeric viruses were viable, and additional chimeric arteriviruses have been constructed (table ). the construction of intra-or intergenotypic prrsv chimeras is maneuverable, and the regions of -utr, non-structural genes, and structural genes have been replaced (gao et al., ; lu et al., ; tian et al., tian et al., , vu et al., ; zhou et al., ) . to facilitate the intra-genotypic substitution, a gene-swapping mutagenesis technique has been used to substitute the structural genes (kim and yoon, ) . using this technique, individual replacement of orf a and orf through orf of vr- was successfully carried out with corresponding orfs from other strains of prrsv including ja , sdsu , prrs , and m (kim and yoon, ) . . . . nsp prrsv nsp is a multifunctional protein that undergoes remarkable genetic variations. the nsp protein consists of five regions: hypervariable region i (hv-i), plp cysteine protease core, hypervariable region ii (hv-ii), transmembrane regions, and a c-terminal tail ( fig. a ; han et al., ) . the plp cysteine protease domain possesses cisacting and trans-acting cleavage activities and mediates its rapid release from pp a and pp ab (han et al., ; snijder et al., ) . two sites were initially predicted for nsp /nsp cleavage at g/ g and somewhere at g/ g/ g, and recent studies showed the actual cleavage occurs at g/ g for vr- (allende et al., ; han et al., ; nelsen et al., ) . the corresponding cleavage for europrrsv sd - likely occurs at gg/ a (fang and snijder, ) . plp is as a member of the ovarian tumor domain (otu) family of deubiquitinating enzymes, and has shown to deconjugate ubiquitin (ub) and ifn-stimulated gene (isg) from cellular targets. this is an important viral strategy inhibiting the ub-dependent and isg -dependent host innate immune responses (frias-staheli et al., ; sun et al., a sun et al., , b van kasteren et al., ) besides the proteinase and deubiquitinase functions, nsp contributes to the major genetic differences between genotypes i and ii, sharing only less than % similarity at the amino acid level (allende et al., ; nelsen et al., ) . the nsp gene also contains naturally inserted sequences and deletions ( fig. a) in the hypervariable region (fang et al., ; gao et al., ; ni et al., ; shen et al., ; tian et al., ) . the deletion of amino acids in nsp was first found in a chinese prrsv isolate, hb- (sh)/ , in comparison with other north american isolates (gao et al., ) . sequence analysis of prrsv mn reveals three discontinuous deletions of , , and amino acids at the corresponding positions - , , and - of vr- , respectively (han et al., ) . discontinuous deletions were also identified in the highly pathogenic prrsv (hp-prrsv) associated with the outbreaks of porcine high fever disease in china (tian et al., ) . the amino acids discontinuous deletion consists of aa deletion at position and aa deletions at - , and the deletion region contains bcell epitopes (de lima et al., ) and t-cell epitopes (chen et al., b) . strikingly, cell culture passages of prrsv may generate a deletion in nsp , and a study shows the generation of a large deletion of aa at - in nsp during passages (ni et al., ) . a deletion in nsp is also found in genotype i prrsv. the europrrs sd- - virus in the us shows a aa deletion at positions - of nsp when compared to lelystad virus (fang et al., ) . biological significance of the genetic deletion in nsp remains to be determined. besides deletions, a aa insertion was also observed in the sp strain of prrsv, which is a vaccine strain, located between g and t of the sp nsp (shen et al., ) . given the tolerance of deletions and insertions in the hypervirable region of nsp , this region is considered as a site for foreign gene insertion ( fig. b and c) . the gfp gene was inserted into nsp of the sd - strain and fully infectious virus was rescued (fang et al., b) . the gfp insertion did not affect the growth of the virus, and the infectivity was comparable to that of parental virus. the capacity of deletion in nsp was determined by introducing a series of in-frame deletions (han et al., ) . the plp domain, the plp downstream flanking region, and the transmembrane domain were crucial for virus replication but deletions of - aa from the n-terminal portion of the hypervariable region and - aa from the hypervariable region appeared to be tolerable for viability. in the hypervariable region, the largest deletion that can be achieved was about aa at positions - , although a deletion of up to aa is preferable for infectivity (fig. b) . the insertion of gfp or other genes such as new castle disease virus nucleoprotein (np) gene has been successful as long as insertions reside in the hypervirable regions ( fig. c ) (kim et al., ; xu et al., a) . the deletion of inmmunodominant linear b-cell epitopes (es -es ) were attempted; deletion of es , es , or es allowed the generation of an infectious virus (chen et al., b; oleksiewicz et al., ) . prrsv n is a mutilfunctional protein. the specific domains and residues critical for virus replication have been identified in n (fig. a) . the n protein is comprised of or aa for the north american and european genotypes, respectively (music and gagnon, ) . n consists of the n-terminal rna-binding domain (rbd) at positions - and the c-terminal dimerization domain comprising a four-stranded antiparallel b-sheet floor flanked by a-helices (doan and dokland, ; yoo et al., ) . as the sole component of viral capsid, n interacts with itself via covalent or noncovalent interactions (doan and dokland, ; wootton and yoo, ) . the cysteine at position is responsible for the formation of an intermolecular disulfide bond, and aa - are essential for mediating noncovalent homodimers . a crystallographic study on n shows the imprtance of the c-terminal dimerization domain for n (doan and dokland, ; spilman et al., ) . prrsv n is a serine phosphoprotein which is a common property for n of eav and coronaviruses (music and gagnon, ; wootton et al., ) . one of the phosphorylation sites of n is at position , but its biological significance is still unknown. n contains nls in a stretch of basic amino acids -pgkknkk- which is overlapping with the rna-binding domain and particially with a nucleolar localization signal (nols) at aa - (rowland et al., . the nuclear export signal (nes) is found at positions - and is responsible for the nucleolar-cytoplasmic shuttling of n . the functional structure of n is compact and thus n is sensitive to structural modification. the secondary structure in the c-terminal residues - is an important determinant for conformational epitopes, and the mutations in this region change the monoclonal antibody (mab) reactivity (wootton et al., ) . insertion of a foreign sequence into the n gene was attemped and the influenza virus ha epitope was added at the n-terminus or cterminus. despite the initial rescue of the infectious virus, the ha expression was unsuccessful (bramel-verheije et al., ) . the gfp tag was inserted between orf and orf to moniter the orf mrna synthesis, but no mrna was made, indicating the end of the orf gene is essential for mrna synthesis . the n protein is inter-genotypically conserved but shares only % of its identity between lv and vr- (dea et al., ) . the c-terminus of n is heterogenous, and truncation of up to aa is tolerable (verheije et al., ) . in another study, deletions were made at the inter-genotypic variable region or conserved region of n, and regions at - , - , - , and - , were found to be dispensible for viability ( fig. b ) (tan et al., ) . no foreign gene can be incorporated in these rgions. the prrsv genome is flanked by -and -utr, and the utr sequences play a vital role for genomic replication, mrna transcription, and protein translation (pasternak et al., ; snijder et al., ) . the non-coding regions of the genome have been investigated. by serial deletions, the first nucleotides in -utr appears to be dispensible for viability in type ii prrsv first nucleotides are unique for each genotype, and a stretch of nucleotides is present in vr- but is absent in lv (allende et al., ; verheije et al., ) . a deletion study shows that nucleotides at the end of the -utr is tolerable for genotype i prrsv (verheije et al., ) . the -utr of genotype ii has also been studied, and at least nucleotides immediately following orf is dispensable for virus viability . the genetic information on the structural region of arteriviruses is organized in an extremely efficient manner. the genes for gp , gp , and gp overlap each other, and similarly the genes for gp , m, and n overlap each other for prrsv. this structural complexicity hampers the genetic manipulation of infectious clones. the importance of the overlapping gene arrangement for the life-cycle of virus has been studied (verheije et al., ; yu et al., ) . a series of full-length clones were engineered to separate overlapping genes for eav orfs / or orfs / by inserting small additional sequences containing a termination codon for the upstream gene, a unique restriction site, and a translation initiation codon for the downstream gene. the insertions result in the functional separation of overlapping orfs, and do not impair infectivity (de vries et al., ) . the orfs / separation in genotype i prrsv is also possible and progeny virus is produced (verheije et al., ) . for the north american prrsv, restriction sites were inserted between orfs / , orfs / , orfs / , orfs / , and orfs / -ntr, and progeny viruses are generated from these modifications. this indicates that gene overlap is dispensable for infectivity and that separation of each gene does not interrupt mrna synthesis (yu et al., ) . the development of infectious clones allows the construction of chimeric arteriviruses. an attempt was made to swab the ectodomains of gp and m. in engineered chimeric viruses using the eav clone as a backbone, the ectodomains were replaced by corresponding sequences from other arteriviruses. chimeric viruses containing the gp ectodomain from ldv and prrsv were infectious. these chimeric viruses however retain their cell tropism for bhk- cells, which are susceptible for eav but non-susceptible for ldv and prrsv (dobbe et al., ) . replacement of the m ectodomain of eav with the corresponding sequence from other arteriviruses does not produce infectious virus, but replacement of the m ectodomain of prrsv with the corresponding sequence from ldv, eav, and genotype ii prrsv produced an infectious virus. using the lv infectious clone as a backbone, substitutions with the eav m ectodomain or vr- m ectodomain is impossible, but removal of the gene overlap between the m and gp genes is required before swapping, indicating that the vr- m ectodomain and eav m ectodomain are incompatible with the remaining part of lv m. it is also possible that unintended mutations may have been introduced to gp during the ectodomain swap (verheije et al., ) . substitution of structural genes between arteriviruses has been extremely useful to identify viral factors for viral tropism. the substitution of gp or/and m do not alter their cell tropism (dobbe et al., ; lu et al., ; verheije et al., ) . in contrast, the substitution of minor envelope proteins and e protein using the prrsv infectious clone as a backbone allows the chimeric prrsv to acquire a broad cell tropism but to lose the ability to infect pams. it indicates that the gp /gp /gp minor proteins are determinants for cell entry and tropism (tian et al., ) . intra-genotypic or inter-genotypic gene-swapping have been conducted between eav and prrsv to study the genetic compatibility and viral-specific phenotypes, including neutralization, virulence, and pathogenesis. for neutralization, chimeric eavs were generated in which each construct contained individual orf from different isolates (balasuriya et al., ) . also, the role of individual envelope proteins of gp through m for cross-neutralization was studied using the vr- infectious clone as a backbone (kim and yoon, ) . the prrsv- strain is highly susceptible to serum neutralization and induces atypically rapid and robust neutralizing antibodies in pigs. analysis of structural genes of prrsv- reveals the absence of two n-linked glycosylation sites each in gp and gp . the significance of missing glycans for neutralization has been determined by replacing gp and gp genes from prrsv- (vu et al., ) . the major virulence determinants have also been identified by gene swapping experiments to locate in nsp through nsp and gp . highly pathogenic prrsv contains the aa deletion in nsp sequence (tian et al., ) . by gene swapping studies using nsp from an avirulent prrsv, the deletion in nsp was shown to be irrelevant to virulence and pathogenicity (zhou et al., ) . a recent study identified nsp -and nsp -coding regions together were essential for increased pathogenicity and fatal virulence for hp-prrsv by swapping these regions between the highly and low pathogenic strains (li et al., ) . the intergenotypic -utr swap between genotypes i and ii was investigated and shows that the -utr of genotype ii may be substituted with the corresponding sequence from genotype i, while the substitution of -utr of genotype i with its corresponding sequence from genotype ii is lethal (gao et al., ) . using this approach, the envelope proteins representing gp through gp of genotype i are shown to be fully functional for genotype ii when using genotype ii as a backbone (tian et al., ) . a random sequence shuffling has been employed to generate immunologic variants of prrsv zhou et al., zhou et al., , . gp sequences representing immunologically diverse strains of prrsv are randomly shuffled, and the shuffled gene is incorporated in the infectious clone to generate a new virus that contains a new gp gene, which may improve the cross neutralization . the breeding of gp and m have also been tried, and the rescued virus induces a broad spectrum of cross-neutralizing antibodies . the gp sequence from genetically diverse strains of prrsv and the gp -m sequence from different strains were subjected to breeding, and the shuffled genes were cloned in infectious clones for the generation of new viruses. two representative chimeric viruses, ds by gp shuffling and ds m by gp -m shuffling, were found to be clinically attenuated . this approach allows rapid generation of an attenuated virus and may be useful for vaccine development for antigenetically variable viruses zhou et al., ) . another approach to the rapid generation of attenuated prrsv is referred to as save (synthetic attenuated virus engineering). codon-pair bias is a phenomenon that certain codon pairs appear in a higher frequency in comparison to other synonymous codon pairs for the same amino acid, and the codon-pair bias is host species-dependent related to the efficiency of protein synthesis (coleman et al., ; moura et al., ; mueller et al., ) . by deoptimizing the codon pair of a virulence gene, an expression level of this protein decreases. the computer-aided deoptimization of codon-pairs modifies only naturally optimized pairs of codons and does not change the amino acid sequence (mueller et al., ) . using this approach, the gp gene was codon-pair deoptimized, and a new virus was generated. the modified gp sequence did not affect the viability of prrsv and the engineered virus was clinically attenuated in pigs (ni et al., ) . to fulfill serological discrimination between naturally infected and vaccinated animals, removing an immunodominant epitope has been applied to developing a liveattenuated differentiating infected from vaccinated animals (diva) vaccine against prrsv (de lima et al., ; vu et al., ) . the serologic marker antigen selected for diva vaccine should be highly immunodominant without disrupting protective well-conserved epitopes among prrsv isolates and stability during passages. besides, the removal of a selected epitope should not adversely affect the growth property or virulence of the mutant virus (de lima et al., ; vu et al., ) . two epitopes residing in nsp and m have been identified fulfilling the requirements for prrsv diva vaccine (de lima et al., ; vu et al., ) . two mutants, fldnsp / with a deletion of residues - within nsp , and q r disrupting antigenicity of epitope m in m protein have been designed and constructed accordingly (de lima et al., ; vu et al., ) . the immunogenicity of those two epitopes has been eliminated during infection of prrsv mutants, and both epitopes may be used as an immunologic marker for diva vaccine development (de lima et al., ; vu et al., ) . prrsv may serve as a vaccine vector. prrsv infectious clones have been developed as a gene delivery vector for foreign gene expression. identification of gene insertion sites in the viral genome and viral infectivity is critical for gene delivery. gfp and b-cell epitopes of the newcastle disease virus (ndv) nucleoprotein have been inserted into non-essential regions of nsp of prrsv (fang et al., b (fang et al., , kim et al., ; xu et al., a) . in this approach however, the stability of the inserted gene was of a concern. when prrsv expressing gfp in nsp , prrsv sd - -gfp, was cell-culture passaged, a population of gfp-expression negative-virus appears by the th passage. sequencing shows a deletion of gfp at the n-terminal half ( to ), leading to the loss of gfp expression. insertion of amino acids at position of gfp was also observed in some viral clones (fang et al., b) . the stability of the gfp gene in this recombinant virus was improved by deleting the es epitope located downstream of the gfp gene, and the gfp expression in this virus was stable for passages. however, r c mutation was found in gfp, and this mutation caused the loss of florescence (fang et al., ) . the loss of fluorescence was also observed in two other gfp recombinant viruses during serial passages. in another study, the gfp-coding sequence remained intact but point mutations were identified and these mutations caused amino acid changes to r c and n y (kim et al., ) . the expression of aa b-cell epitope of the ndv nucleoprotein in prrsv nsp remained stable in cell culture up to passages (xu et al., a; zhang et al., ) . the instability of foreign gene insertion in nsp is not fully understood. the length of insertion may be important for stability. an attempt was made to produce an additional mrna for foreign gene expression. the gfp gene was inserted between orf b and orf a for prrsv along with a copy of trs pei et al., ; sang et al., ; yoo et al., ) . compared to insertion in nsp , this site is suitable for foreign gene insertion since the recombinant virus was stable for up to at least passages without the loss of gene or fluorescence (pei et al., ). the genetic stability of genes inserted at this site has been confirmed by expressing other genes including dsred, renilla luciferease, ifna , ifnb, ifnd , and ifnv (sang et al., ) . this approach has the particular advantage of eliminating the need to alter the coding sequence of a viral gene and also of minimizing the effects on expression and post-translational modification of viral proteins (pei et al., ) . infectious clones are important molecular tools to study structure function relationships of proteins and genomic sequences at the infectious virus level in vivo. specific sequence motifs may be mutated or deleted from the virus and their phenotypes may be examined to determine their functions. the removal of n-linked glycosylation at n and n of gp results in a mutant virus with its phenotype of enhanced sensitivity to serum neutralization and high level induction of neutralizing antibodies . elimination of n -linked glycan is not in concert with a high-level neutralizing antibody response to wild type prrsv . meanwhile, introduction of multiple mutations at these n-linked glycosylation sites could significantly reduce virus yields . the e gene knock-out mutation allows for genome replication and transcription but does not produce infectious progeny, indicating that the e protein is essential for virion assembly . prrsv nsp is a multifunctional protein regulating the accumulation of genomic rna and mrnas. it also has the ability to modulate the host innate immunity by suppressing the type i ifn production (nedialkova et al., ; sun et al., a; yoo et al., ) . the motifs for zinc fingers, plps, and nuclease have been identified in nsp (fang and snijder, ; snijder et al., ; xue et al., ) . by deleting from the genome, nsp is shown to be dispensable for genome replication but crucial for mrna transcription. mutation in the catalytic sites of plp impairs both viral genome and mrna synthesis as well as the cleavage between nsp and nsp . mutations in the zinc finger motif abolished the mrna transcription, whereas genome replication was not affected (tijms et al., (tijms et al., , . when the catalytic sites of plp a are mutated using a prrsv infectious clone, the proteinase activity disappears and mrna synthesis is completely blocked. in contrast, mutations at the plp b catalytic sites result in no mrna synthesis and no viral infectivity, indicating that the normal cleavage of nsp and nsp is critical for viral replication (kroese et al., ) . to design effective vaccine candidates that may be useful to overcoming antigenic heterogeneity of prrs, extensive studies have been conducted to eliminate the ifn antagonistic function from the virus (see reviews snijder et al., ; sun et al., a; yoo et al., ) . among viral proteins, nsp a and nsp b have been identified as potent ifn analogists (beura et al., ; chen et al., a; han et al., han et al., , kim et al., ; song et al., ) . subsequent studies have identified specific residues regulating the ifn antagonism, and a mutant virus with a stretch of alanine substitution at positions - of nsp b showed the loss of ifn suppression . in another study, k and r were mutated to release the surface accessibility of nsp b, and mutant prrsv impaired the ifn antagonism (li et al., ) . motifs in the n protein have broadly been studied using mutant viruses. the importance of n protein dimerization has been examined by mutating c s which is responsible for the covalent interaction between n proteins. mutant viruses of c s, c s, and c s were constructed, and with the exception of c s, both c s and c s completely lost their infectivity. in another study however, the replacement of cysteines within n protein, either singly or in combination, did not impair the growth prrsv according . genome replication and mrna transcription were normal for both mutants, suggesting the dimerization of n may be important for particle assembly or maturation . the nuclear localization signal (nls) of n was also mutated to examine the biological consequence of n in the nucleus in prrsv-infected cells. compared to wild-type prrsv, nls-null mutant prrsv was attenuated in pigs and produced a significantly shorter mean duration of viremia and higher titers of neutralizing antibodies pei et al., ) , demonstrating that the n protein nuclear localization is a virulence factor. as an emerged and re-emerging disease in swine, prrs has extensively been studied for molecular biology, immunology, and prevention. the unusual immune responses in pigs and antigenic heterogeneity of the virus are two main obstacles to developing a satisfactory prrs vaccine. the availability of prrsv infectious clones and recent advances in recombinant dna technology have made possible to manipulate the viral genome to introduce specific mutations to targeted sequences and to create genetically modified mutant viruses. extensive efforts have been applied to making mutant viruses with modified phenotypes of immune responses including the evasion of neutralizing antibodies and suppressed innate immune responses. for viral heterogeneity, the rna-dependent rna polymerase (rdrp) is believed to cause frequent mutations in the prrsv genome. genetic swapping or modifications of nsp , the rdrp of prrsv, may be studied to make mutant viruses with reduced mutation rates during replication. the reverse genetics of prrsv is a powerful genetic tool and has the potential to apply to the basic understanding of the biology of prrsv and to the development of genetically modified vaccines and gene delivery. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions engineering the largest rna virus genome as an infectious bacterial artificial chromosome influence of nlinked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cdna clone characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation identification of amino acid residues important for anti-ifn activity of porcine reproductive and respiratory syndrome virus nonstructural protein infectious transcripts and cdna clones of rna viruses expression of a foreign epitope by porcine reproductive and respiratory syndrome virus pathogenesis of porcine reproductive and respiratory syndrome virus identification of two auto-cleavage products of nonstructural protein (nsp ) in porcine reproductive and respiratory syndrome virus infected cells: nsp function as interferon antagonist immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response virus attenuation by genome-scale changes in codon pair bias development of a porcine reproductive and respiratory syndrome virus differentiable (diva) strain through deletion of specific immunodominant epitopes serologic marker candidates identified among b-cell linear epitopes of nsp and structural proteins of a north american strain of porcine reproductive and respiratory syndrome virus genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture development of genetic markers in the non-structural protein region of a us type porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development construction of a full-length cdna infectious clone of a european-like type prrsv isolated in the us heterogeneity in nsp of european-like porcine reproductive and respiratory syndrome viruses isolated in the united states a full-length cdna infectious clone of north american type porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp region the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins efficient- frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses cis-acting structural element in utr is essential for infectivity of porcine reproductive and respiratory syndrome virus replacement of the heterologous untranslated region allows preservation of the fully functional activities of type porcine reproductive and respiratory syndrome virus genomic characterization of two chinese isolates of porcine respiratory and reproductive syndrome virus experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus identification of nonessential regions of the nsp replicase protein of porcine reproductive and respiratory syndrome virus strain vr- for replication in cell culture the porcine reproductive and respiratory syndrome virus nsp cysteine protease domain possesses both trans-and cis-cleavage activities complete genome analysis of rflp isolates of porcine reproductive and respiratory syndrome virus degradation of creb-binding protein and modulation of type i interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp alpha subunit novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states expression and stability of foreign tags inserted into nsp of porcine reproductive and respiratory syndrome virus modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein in marc- and hela cells molecular assessment of the role of envelopeassociated structural proteins in cross neutralization among different prrs viruses the nsp alpha and nsp beta papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis infectious clonederived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties nsp and nsp contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china targeted mutations in a highly conserved motif of the nsp beta protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus chimeric viruses containing the n-terminal ectodomains of gp and m proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome infectious transcripts from cloned genomelength cdna of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cdna constructs infectious rna transcribed from an engineered full-length cdna template of the genome of a pestivirus large scale comparative codon-pair context analysis unveils general rules that fine-tune evolution of mrna primary structure live attenuated influenza virus vaccines by computer-aided rational design isolation and serological characterization of porcine reproductive and respiratory syndrome (prrs) viruses from pigs with reproductive and respiratory disorders in japan comparison of the structural protein-coding sequences of the vr- and lelystad virusstrains of the prrs virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis arterivirus np modulates the accumulation of minus-strand templates to control the relative abundance of viral mrnas porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents establishment of a dna-launched infectious clone for a highly pneumovirulent strain of type porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp region attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of virus envelope genes from genetically divergent strains computer-aided codon-pairs deoptimization of the major envelope gp gene attenuates porcine reproductive and respiratory syndrome virus generation of an infectious clone of vr- , a highly virulent north american type isolate of porcine reproductive and respiratory syndrome virus epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp fragment of the replicase polyprotein contains a cluster of b-cell epitopes nidovirus transcription: how to make sense functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association porcine reproductive and respiratory syndrome virus as a vector: immunogenicity of green fluorescent protein and porcine circovirus type capsid expressed from dedicated subgenomic rnas successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes cloned ppliovirus complementary-dna is infectious in mammalian-cells recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the nsp -encoding region the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus nucleolar-cytoplasmic shuttling of prrsv nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences replication-competent recombinant porcine reproductive and respiratory syndrome (prrs) viruses expressing indicator proteins and antiviral cytokines a contemporary view of coronavirus transcription reverse genetics with a fulllength infectious cdna of the middle east respiratory syndrome coronavirus determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion isolation of porcine reproductive and respiratory syndrome (prrs) virus from heko-heko disease of pigs arterivirus molecular biology and pathogenesis the molecular biology of arteriviruses the arterivirus nsp protease, an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases rna-rna and rna-protein interactions in coronavirus replication and transcription nonstructural protein alpha subunitbased inhibition of nf-kappab activation and suppression of interferon-beta production by porcine reproductive and respiratory syndrome virus rna transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require vpg for infectivity cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid amino acid residues in the non-structural protein of porcine reproductive and respiratory syndrome virus involved in down-regulation of tnf-alpha expression in vitro and attenuation in vivo infectious japanese encephalitis-virus rna can be synthesized from in vitro-ligated cdna templates interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus crystal structure of porcine reproductive and respiratory syndrome virus leader protease nsp -alpha the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein possesses deubiquitinating and interferon antagonism functions nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-simulated gene identification of dispensable nucleotide sequence in ' untranslated region of porcine reproductive and respiratory syndrome virus identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype envelope proteins in the backbone of genotype emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp autoprotease a zinc finger-containing papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp arterivirus and nirovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription viable porcine arteriviruses with deletions proximal to the end of the genome chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting characterization of a serologic marker candidate for development of a live-attenuated diva vaccine against porcine reproductive and respiratory syndrome virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein as well as glycoprotein n-linked glycosylation of gp of porcine reproductive and respiratory syndrome virus is critically important for virus replication in vivo construction and evaluation of genetically engineered replication-defective porcine reproductive and respiratory syndrome virus vaccine candidates mystery swine disease in the netherlands -the isolation of lelystad virus antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages stable expression of foreign gene in nonessential region of nonstructural protein (nsp ) of porcine reproductive and respiratory syndrome virus: applications for marker vaccine design identification of nonessential regions of the nsp protein of an attenuated vaccine strain (hun -f ) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc- cell the crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein nsp -beta reveals a novel metal-dependent nuclease modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin infectious cdna clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus reverse genetic manipulation of the overlapping coding regions for structural proteins of the type ii porcine reproductive and respiratory syndrome virus construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins disulfide linkages mediating nucleocapsid protein dimerization are not required for porcine arterivirus infectivity generation of an infectious clone of hun -f , an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the gp or m genes dna shuffling of the gp genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence virus-encoded proteinases and proteolytic processing in the nidovirales key: cord- -st yw authors: lee, changhee; bachand, aimee; murtaugh, michael p.; yoo, dongwan title: differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus gp and gp glycoproteins date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: st yw the porcine reproductive and respiratory syndrome virus (prrsv) gp and gp proteins are two membrane-associated viral glycoproteins that have been shown to induce neutralizing antibodies. in the present study, the host cell gene expression profiles altered by the gp and gp proteins were investigated by the use of dna microarrays. sublines of marc- and hela cells were established by stable transfection with open reading frame (orf) and orf of prrsv, respectively, and differential gene expressions were studied using microarray chips embedded with human-expressed sequence tags. the genes for protein degradation, protein synthesis and transport, and various other biochemical pathways were identified. no genes involved in the apoptosis pathway appeared to be regulated in gp -expressing cells. the microarray data may provide insights into the specific cellular responses to the gp and gp proteins during prrsv infection. for viruses to replicate, they must enter a host cell and utilize host cell biosynthetic machinery and energy supplies. infected cells activate innate and adaptive immune responses, and host antiviral defense is switched on to eliminate invading viruses. viruses may persist in infected cells when antiviral defenses are insufficient. the series of interactive processes cause the differential expression of cellular genes, and it has been of interest to understand how altered gene expression plays a role during virus infection. rt-pcr, rnase protection assays, and northern and western blot analyses are commonly used techniques to identify altered gene expression. rt-pcr in particular, has been used to study porcine reproductive and respiratory syndrome virus (prrsv)-mediated altered gene expression for ifn-a, ifn-g, il- , il- , and tnf-a (johnsen et al., ; feng et al., ; suradhat and thanawongnuwech, ; thanawong-www.elsevier.com/locate/vetimm veterinary immunology and immunopathology ( ) - nuwech and thacker, ) . such techniques, however, are often time-consuming and labor-intensive and lead to high degrees of experimental variation. for such reasons, little is known concerning the molecular changes in cells upon prrsv infection. the recent development of dna microarray technology allows for the simultaneous assessment of mrna transcription patterns for thousands of genes, and is commonly applied to determine patterns of differential gene expression (schena et al., ) . using this technique, it is now possible to define changes in gene expression that evaluate host cellvirus interaction and to obtain specific insights into the molecular nature of viral pathogenesis (browne et al., ; johnston et al., ) . microarrays are particularly useful in studying whether cellular mrnas, differentially regulated by each viral protein, play a crucial role for virus multiplication in the cell. the prrsv- (north american) genome contains nine open reading frames (orfs). orf a and orf b code for two partly overlapping non-structural polyproteins that are predicted to be post-translationally processed to cleavage products. these nonstructural proteins are believed to participate in viral genome replication and subgenomic mrna transcription. orfs - code for six structural proteins: gp -gp , membrane (m), and nucleocapsid (n) proteins (meulenberg et al., ; snijder and meulenberg, ) . a small internal orf is found within orf , which encodes the e protein (wu et al., ) . the gp protein, consisting of amino acids, is a major glycosylated structural component of the virion. it resembles a type i integral membrane protein with a putative endoplasmic reticulum (er) translocational signal of amino acids at its n-terminus. gp however lacks the typical c-terminal hydrophobic anchor sequence. instead, a triple membrane spanning region is found in the middle of the protein between residues and , leaving a large stretch of a amino acid cytoplasmic tail at the c-terminus (mardassi et al., ; meulenberg et al., ) . gp exists as a heterodimer with the m protein in the virion (mardassi et al., ) , and heterodimerization has been shown to be essential for virus infectivity in ldv (faaberg et al., ) and eav (snijder et al., ) . the gp protein is reported to cause apoptosis (suárez et al., ) and is able to induce neutralizing monoclonal antibodies in mice (weiland et al., ; ostrowski et al., ) . the gp protein is a minor structural protein consisting of amino acids (murtaugh et al., ) , and as with gp , is also able to induce neutralizing antibodies (meulenberg et al., ; weiland et al., ) . the electrophoretic migration of the mature protein incorporated into virions is - kda, suggesting that the gp protein is heavily glycosylated during transport through the er-golgi complex (van nieuwstadt et al., ) . four potential n-glycosylation sites are found on the protein. the amino acid sequence shows that gp contains a putative nterminal signal sequence at positions - , and an additional hydrophobic sequence at positions - at the c-terminus. the hydrophobicity profile of gp suggests that it resembles a class i integral membrane protein. however, gp has a unique feature uncommonly seen in this class of proteins-the lack of a hydrophilic cytoplasmic tail on the carboxy terminus of the hydrophobic transmembrane region. this unique topology of gp is found to mimic a glycosylphosphatidylinositol (gpi) anchored protein (ferguson and williams, ) . indeed, gp has been shown to be a gpi-anchored protein in our laboratory (bachand, ) . the function of gpi anchors is poorly understood, but limited evidence suggests that they are involved in 'lipid rafts' (varma and mayor, ) or in cellular signal transduction (jacobs et al., ) . the present study was designed to examine regulation of specific host cell gene expression by two prrsv structural proteins. we established two independent cell sublines to stably express the gp and gp proteins of the north american type prrsv, and investigated their effect on host cell gene expression using dna microarray technology. marc- cells (a subclone of ma cells (kim et al., ) ) were grown in dmem containing % fetal bovine serum (invitrogen), units/ml of penicillin, and mg/ml of streptomycin. hela-tet-off cells were purchased from clontech. these cells constitutively express the chimeric tetracycline transactivator (gossen and bujard, ) . hela-tet-off cells and marc-gp cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % serum, mm l-glutamine, units/ml of penicillin, mg/ml of streptomycin, and mg/ml of g (geneticin; invitrogen). hela-gp cells were maintained in dmem containing % serum, mm l-glutamine, units/ml of penicillin, mg/ml of streptomycin, mg/ml of g , mg/ml of doxycycline (clontech), and mg/ml of hygromycin b (invitrogen). all cells were maintained at c with % co in a humidified incubator. dna was manipulated according to standard procedures (sambrook and russell, ) . the orf gene of the north american type prrsv strain atcc vr was pcr-amplified from the parental plasmid pgem zf-orf (wootton et al., ) and subcloned into the ecori and hindiii sites of the pci-neo mammalian expression vector (promega) to generate pci-neo-orf . the gp expression plasmid ptre-hyg-orf was constructed by subcloning the vr orf gene from the parental plasmid pgem zf-orf (wootton et al., ) into ptre-hyg (clontech) using the bamhi site such that the orf gene was placed under control of the tetracycline-responsive element along with the hygromycin-resistance gene. marc- cells were grown to approximately % confluence in a mm diameter dish and then transfected for h with . mg pci-neo-orf dna using lipofectin (invitrogen) according to the directions of the manufacturer. after h, the transfection solution was replaced with dmem and incubated for h to allow the cells to divide at least once. cells were then trypsinized and seeded into fresh mm diameter dishes at approximately  cells per dish. the pci-neo-orf plasmid contains a gene conferring neomycin resistance and allows for the selection of cells that have integrated orf into the cellular dna. freshly seeded cells were selected for neomycin resistance using mg/ml of g (invitrogen). selection continued over the course of approximately weeks, with g being replaced at least every days. when the majority of cells died, resistant colonies of cells were picked using cell cloning cylinders and amplified for further characterization. to generate cells expressing gp , the tet-off inducible gene expression system was chosen to prevent cell death that might occur due to the gp protein expression since gp was reported to induce apoptosis. the tet-off inducible cell system was purchased from clontech. hela-tet-off cells were transfected with ptre-hyg-orf for h using lipofectin according to the manufacturer's instruction (invitrogen). the transfection solution was removed and the cells were grown for additional h in dmem. for the transfected hela-tet-off cells, the medium contained mg/ml of doxycycline (clontech). at h of incubation, mg/ml of hygromycin b was added, and cells were further incubated for weeks until the majority of cells died. hygromycin-resistant cell colonies were picked using cell-cloning cylinders and amplified in -well tissue culture plates. cells were grown on microscope slide coverslips placed in mm-diameter culture dishes in the maintaining medium. after h, doxycycline was removed for or h to induce gp expression in gp expressing cells. cells were washed twice in phosphate-buffered saline (pbs) and fixed immediately with cold methanol for min. for immunofluorescence, cells on microscope coverslips were blocked using % bovine serum albumin (bsa) in pbs for min at room temperature. the cells were then incubated with a : dilution of porcine anti-prrsv hyperimmune sera for h. the cells were washed five times in pbs and incubated for h at room temperature with a : dilution of fluorescein isothiocyanate (fitc)-labeled goat anti-swine secondary antibody (kpl). cells were washed five times in pbs and the coverslips were mounted on microscope glass slides in the mounting buffer ( % glycerol and . % sodium azide in pbs). cell staining was visualized by a fluorescent microscope (model ax , olympus). marc-gp and hela-gp cells were seeded in mm-diameter cell culture dishes. hela-gp cells were grown in the presence of mg/ml doxycycline. to induce gp protein expression, doxycycline was removed and incubation was continued. at h post-seeding or h of induction, cells were starved for min in methionine-deficient medium (invitrogen) and labeled for h with mci/ml of easytag express protein labeling mix ([ s]methionine and [ s]cysteine, specific activity, mbq/ml) (perkin-elmer). after labeling, cells were harvested, washed twice with cold pbs, and lysed with lysis buffer ( mm tris-hcl [ph . ], mm nacl, % np- ) containing mm phenylmethyl-sulfonyl fluoride (pmsf). after incubation on ice for min, cell lysates were centrifuged at , rpm for min in a microcentrifuge (model , eppendorf), and supernatants were recovered. for immunoprecipitation, cell lysates equivalent to : of a mm diameter dish were adjusted with ripa buffer ( % triton x- , % sodium deoxycholate, mm nacl, mm tris-hcl [ph . ], mm edta, . % sds) to a final volume of ml and incubated for h at rt with ml of swine anti-prrsv hyperimmune serum. the immune complexes were adsorbed to mg of protein-a sepharose cl- b beads (amersham biosciences) for h at c. the beads were collected by centrifugation at rpm for min, washed twice with ripa buffer and once with wash buffer ( mm tris-hcl [ph . ], mm nacl). the beads were resuspended in ml of sds-page sample buffer ( mm tris-hcl [ph . ], % glycerol, % sds, . % (w/v) bromophenol blue) with % b-mercaptoethanol, boiled for min, and analyzed by sodium dodecyl sulfate (sds)- % polyacrylamide gel electrophoresis (page). gels were dried on filter paper and radiographic images were obtained using phosphor-imager (molecular dynamics phosphorimager si, amersham biosciences). the dna microarray used in this study was comprised of human expression sequence tag (est) clones printed at the microarray centre (toronto, ont., canada). the genes were arrayed in duplicate on one slide. detailed information on the layout of the microarray is found at the microarray centre (http:// www.microarrays.ca/support/glists.html). at h postinduction, total cellular rnas were extracted from each line of established cells, marc-gp and hela-gp , and from their corresponding parental cells, marc- and hela-tet-off, respectively. complementary dnas were synthesized by reverse transcription using units of superscript ii (invitrogen) in a total reaction volume of ml. briefly, mg of total rna was primed with the anct primer (sigma genosys; t( )-v-n ) and reverse transcription was carried out in the presence of . mm each of datp, dctp, and dgtp (invitrogen), . mm dttp, amino-allyl . mm dutp, mm dtt in  firststrand synthesis buffer (invitrogen; mm tris-hcl [ph . ], mm kcl, mm mgcl ). the mixture was heated at c for min followed by c for min. superscript ii was added, and the reverse transcription reaction was further incubated at c for h. the reaction was stopped by heating to c for min, and the rna template was degraded by the addition ml of n naoh followed by incubation at c for min. the mixture was neutralized by the addition ml of m hcl and ml of m tris-hcl (ph . ). the cdna was purified using microcon columns (millipore) and labeled with alexa dyes (molecular probes) at room temperature for h in darkness. for each microarray, control cdna from parental cell lines was labeled with alexa (cy equivalent), whereas cdna from marc-gp or hela-gp cells was labeled with alexa (cy equivalent). the fluorescent labeled cdnas were purified again with the qiaquick pcr purification kit (qiagen) and precipitated by adding one volume of isopropanol and incubating on ice for min. after rinsing with % ethanol, the labeled cdna was resuspended in . ml of dnase-free, rnase-free water (invitrogen). for hybridization, ml of calf thymus dna ( mg/ml) was added to ml of dig easy hybridization buffer (roche) and the solution was heated at c for min. two . ml samples of labeled cdna were combined with ml of the hybridization solution as prepared above. this solution was then incubated at c for min and pipetted onto the chip. the array chip was then covered with a mm  mm coverslip and incubated at c for h. the slides were washed three times in  ssc containing . % sds for min at c, rinsed twice in .  ssc for min each at room temperature, and dried. array chips were scanned on a genpix a scanner (axon instruments inc., union city, ca). the normalization of raw data and the analysis of the data sets were performed using genetraffic microarray data analysis software (iobion informatics, la jolla, ca). the log r/g (where r and g represent cy and cy , respectively) normalized ratio was selected as the value, which was further calculated to a fold change in regulation. to examine the effects of the prrsv gp or gp protein on cell function, cells were first established to express the gp or gp proteins. marc- cells were chosen to establish a cell line stably expressing the gp protein since they are cells susceptible to infection by prrsv. for gp expression, we used the tetracycline-dependent inducible gene expression system (tet-on/tet-off) to avoid a possible cell death that may result from gp expression since the prrsv gp protein was reported to induce cytotoxicity in african green monkey kidney cells (suárez et al., ) . hela-tet-off cells, that were previously transformed with the regulatory plasmid ptet-off and therefore expressing the tetracycline transactivator (tta), were chosen for additional transformation with the orf gene. the presence of tetracycline (tet) or its derivative doxycycline (dox) in the culture medium prevents binding of tta to the promoter, while the removal of tet or dox induces gene transcription placed under control of the promoter. marc- and hela-tet-off cells were transfected with pci-neo-orf and ptre-orf -hyg, respectively, and to confer resistance of transformed cells, neomycin (g ) or hygromycin were added. transfected hela-tet-off cells were maintained in the presence of doxycycline to prevent the gp expression during selection. antibiotic-resistant cell clones were selected and individually confirmed for presence and transcription of the orf or orf gene by pcr and rt-pcr, and designated marc-gp and hela-gp , respectively. marc-gp and hela-gp cells were further examined for their respected protein expression by immunofluorescence ( fig. b and c) . the cytoplasmic fluorescence was evident in these cells when incubated with prrsv-specific hyperimmune sera, whereas no fluorescence was detected in parental marc- and hela-tet-off cells (data not shown), indicating that marc-gp and hela-gp cells expressed the gp and gp proteins, respectively. every cell expressed gp or gp indicating a homogenous population of cells. the gp and gp protein expressions were further confirmed by radioimmunoprecipitation (fig. d) . a specific band of kda protein was identified in marc-gp cells (lane ). this band was absent in the parental marc- cells (lane ) and was considered the gp protein. we were not, however, able to detect the same protein from prrsv-infected cells, and this is probably due to the low abundance of gp in prrsv-infected cells as it is a minor protein. the gp protein was readily produced in hela-gp cells (lane ), and its migration was similar to that of the authentic gp protein seen in prrsv-infected cells (lane ). to investigate the effects of the prrsv gp and gp proteins on cellular gene expressions, a microarray dna chip technology was employed. total cellular rnas were extracted at days post-induction and were reverse transcribed. the cdnas were labeled using fluorescent dyes and used for hybridization of the human . k microarray chip. this chip contained probes designed to detect human transcripts. the slides were scanned and the obtained images were analyzed using genetraffic microarray data analysis software. the log cy /cy normalized ratio . , which corresponds to a two-fold change in regulation, was initially chosen as the cut-off value as used for standard and further calculated to a fold change. when a fold-change calculated by log cy / cy was consistently . or greater in two independent experiments (four hybridizations), the value was considered significant. transcription of a total of cellular mrnas was altered by the gp protein expression in marc-gp cells. of these mrnas, six genes were up-regulated and genes were down-regulated (table ). the classifications of altered genes included those involved in cell adhesion, cell growth, replication, transcription, and protein degradation, and other unknown functional pathways. seven genes associated with synthesis, transport, and biochemical pathway were also identified. microarray analysis of hela-gp cells showed an expression profile of cellular genes altered by the gp protein expression in hela cells, with an increase in transcription of genes and a decrease of eight genes ( table ). the genes that were affected by the gp protein represented those involved in the immune response, cell adhesion and structure, signal transduction pathway, cell cycle and cancer signals, and other functions. five additional genes involved in synthesis, replication, transcription, and protein degradation were found to be altered in gp expressing hela cells. cellular genes involved in apopotis were expected to be regulated in hela-gp , but none of these genes appeared to be regulated by gp expression, suggesting that the gp protein may be irrelevant to prrsv apoptosis (see section ). in this study, we performed a global analysis of the transcriptional response of cells to expression of two prrsv proteins. we established cell lines stably expressing the gp or gp protein, both protein genes under the control of human cytomegalovirus (hcmv) immediate early promoter, and experiments were designed to assess patterns of gene regulation in these cells. in gp -expressing hela cells, actin-related protein (arp ) homologs a and b were identified to be up-regulated. these genes encode a subunit of dynactin that binds to both microtubules and cytoplasmic dynein, and is involved in er-to-golgi transport (lees-miller et al., ) . this may implicate an important function of gp in the transport of the viral components. the bisphosphoglycerate mutase (bpgm) gene was down-regulated by gp by more than six-folds. bpgm is an erythrocyte specific trifunctional enzyme regulating the level of , -bpg in red blood cells. , -bpg is the main allosteric effector of hemoglobin, shifting the equilibrium between the oxy and deoxy conformation of hemoglobins by stabilizing the unliganded form. sickle cell anemia in humans is characterized by polymerization of deoxygenated hemoglobin mutants giving rise to deformed erythrocytes and vasoocclusive complications. , -bpg has been shown to facilitate this polymerization in sickle cell anemia. in humans, deficiency of bpgm has been shown to be associated with anemia (jacobasch and rapoport, ) . rassf was also down-regulated by gp . rassf is a new member of the rassf family and shares the properties of ras effector/tumor suppressors (vos et al., ) . similarly, cyclin d gene expression was found to be suppressed. d-type cyclins are the key regulators along with cyclin e for cell cycle progression from g to s phase. complexes formed between cyclin d or cyclin e and their kinase partners are involved in phosphorylation of retinoblastoma protein, which ultimately leads to activation of e f transcription factor and progression to s phase of the cell cycle. a recent study demonstrates a clear reduction of cyclin d and cell cycle arrest in g /g phase in cells infected with mouse hepatitis coronavirus, a member of nidoviruses (chen and makino, ) . in cell expressing gp , the majority of differentially expressed genes were involved in synthesis, transport, and biochemical pathways. this observation implicates that the gp protein may utilize or change host cell machinery to transport viral or cellular components to the cell surface. zinc finger protein (zfp ) and microsomal triglyceride transfer protein were readily up-regulated, while metallothineins, proliferating cell nuclear protein, and n-acylsphingosine amidohydrolase were down-regulated. zinc finger protein -like is a member of the tristetraprolin family of tandem ccch finger proteins. tristetraprolin can bind to au-rich elements within the -untranslated regions of the mrnas encoding tumor necrosis factor (tnf) and granulocyte-macrophage colony-stimulating factor (gm-csf), leading to accelerated mrna degradation (stumpo et al., ) . tristetraprolin-knockout mice exhibit an inflammatory phenotype that is largely due to increased tnf secretion (taylor et al., ) . microsomal triglyceride transfer protein is a protein complex required for the assembly of lipoprotein particles (gordon et al., ) . it is noteworthy that gp has recently been shown to be a lipid-anchored protein (bachand, ) . metallothionein is a metal binding protein and has been shown to be regulated by a common virus infection (ilback et al., ) . coxsackievirus btype virus infection altered the normal physiological trace element balance in the liver, kidney, spleen, and increased metallothionein in these organs. this may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range. the function of n-acylsphingosine amidohydrolase is unclear. we have observed consistent increases in the expression of cathepsin genes in both marc-gp and hela-gp cells. cathepsin is involved in protein degradation. despite a specific role of cathepsin during virus replication remains to be determined, the up-regulated expression of the gene encoding protease may represent a cellular defense against expression of foreign proteins. interestingly, no pro-apoptotic genes were identified in hela-gp cells. this observation is contradictory to the previous report (suárez et al., ) but is consistent with our recent finding that the gp expressing cells did not show any detectable level of cytotoxicity or cell death (lee et al., ) . zhang et al. ( ) have shown that prrsv infection induced the expression of ifn-inducible gene mx and an ubiquitin-specific protease in porcine alveolar macrophages. these genes were not identifiable in the present study. this difference may be due to the use of different cell types since marc- or hela cells were used in the present study to express the single gp or gp protein rather than using the whole virus to infect porcine macrophages in the previous report. the dna microarray has allowed us to identify the differential effects of prrsv proteins on cellular genes. confirmatory studies are further required as to the significance of the genes that have been identified in the present study. the method of choice to measure and confirm the differential mrna expression mediated by the gp and gp proteins is real-time quantitative rt-pcr. it is also possible that altered mrna profiles may not necessarily reflect altered production of corresponding proteins (gygi et al., ) . in this regard, additional techniques such as western blot analysis or protein arrays may be needed to support our findings. nevertheless, our data obtained from the microarray study will provide future insights into the understanding of host cell virus interactions and eventually of the pathogenic mechanisms of prrsv and the host responses to prrsv infection. characterization of prrsv gp altered cellular mrna levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mrnas murine coronavirus replication induces cell cycle arrest in g /g phase disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus cell surface anchoring of proteins via glycosyl-phosphatidylinositol structures microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles tight control of gene expression in mammalian cells by tetracycline-responsive promoters correlation between protein and mrna abundance in yeast metallothionein is induced and trace element balance changed in target organs of a common viral infection hemolytic anemias due to erythrocyte enzyme deficiencies dengue virus non-structural protein is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction cytokine mrna profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of ifn-g and il- after viral clearance identification of genes involved in the host response to neurovirulent alphavirus infection enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line characterization of the porcine reproductive and respiratory syndrome virus gp in stably expressing cells a vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility molecular analysis of the orfs - of porcine reproductive and respiratory syndrome virus, quebec reference strain intracellular synthesis, processing, and transport of proteins encoded by orfs - of porcine reproductive and respiratory syndrome virus characterization of proteins encoded by orfs - of lelystad virus posttranslational processing and identification of a neutralization domain of the gp protein encoded by orf of lelystad virus comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain molecular cloning: a laboratory manual quantitative monitoring of gene expression patterns with complementary dna microarray the molecular biology of arteriviruses heterodimerization of the two major envelope proteins is essential for arterivirus infectivity chorioallantoic fusion defects and embryonic lethality resulting from disruption of zxp l , a gene encoding ccch tandom zinc finger protein of the tristetraprol family open reading frame of porcine reproductive and respiratory syndrome virus as a cause of virusinduced apoptosis upregulation of interleukin- gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus a pathogenic role for tnf alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin interleukin- , interleukin- , and interferon-g levels in the respiratory tract following mycoplasma hyopneumoniae and prrsv infection in pigs proteins encoded by orfs and of the genome of lelystad virus (arteriviridae) are structural proteins of the virion gpi-anchored proteins are organized in submicron domains at the cell surface rassf is a novel k-ras-specific effector and potential tumor suppressor monoclonal antibodies to the gp of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the gp full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate a kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection the authors are grateful to ryan dowling for his technical assistance for microarray analysis. this study was supported by nserc, ontario pork, the ontario ministry of agriculture and food (animal program). key: cord- -ncvvmkca authors: labarque, g; van reeth, k; van gucht, s; nauwynck, h; pensaert, m title: porcine reproductive–respiratory syndrome virus infection predisposes pigs for respiratory signs upon exposure to bacterial lipopolysaccharide date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ncvvmkca this study examined whether an infection with porcine reproductive and respiratory syndrome virus (prrsv) potentiates respiratory signs upon exposure to bacterial lipopolysaccharides (lps). five-week-old conventional pigs were inoculated intratracheally with the lelystad strain of prrsv and received days later one or two intratracheal lps administrations. the necessary controls were included. after lps administration, pigs were intensively monitored for clinical signs. additionally, some pigs were euthanatized after a second lps administration for broncho-alveolar cell analysis and virological examinations of the lungs. broncho-alveolar lavage (bal) cells were counted and differentiated. lung suspensions and bal fluids were titrated for prrsv. exposure of pigs to prrsv only resulted in a fever for time periods ranging from to days and slight respiratory signs. exposure of pigs to lps only resulted in general signs, characterized by fever and depression, but respiratory signs were slight or absent. prrsv–lps exposed pigs, on the other hand, developed severe respiratory signs upon lps exposure, characterized by tachypnoea, abdominal breathing and dyspnoea. besides respiratory signs, these pigs also showed enhanced general signs, such as fever and depression. lung neutrophil infiltration was similar in non-infected and prrsv-infected pigs upon lps exposure. prrsv quantities were similar in lungs and bal fluids of pigs infected with prrsv only and prrsv–lps exposed pigs. these data show a clear synergism between prrsv and lps in the induction of respiratory signs in conventional pigs. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, causes infections in pigs worldwide. the virus replicates highly in the respiratory tract and shows a distinct tropism for broncho-alveolar macrophages (duan et al., ) . however, a single prrsv infection, particularly under experimental circumstances and with european isolates, fails to induce overt respiratory disease (done and paton, ) . also under field circumstances, most pigs become infected with prrsv at growing age without respiratory disease. still, the frequency and severity of respiratory disease have increased since the enzootic occurrence of prrsv (done and paton, ) . this has stimulated research into the combined effects of prrsv and other infectious agents. consequently, experimental dual infections have been performed with prrsv followed by various bacteria such as haemophilus parasuis, pasteurella multocida, streptococcus suis, bordetella bronchiseptica, and salmonella choleraesuis (cooper et al., ; galina et al., ; brockmeier et al., ; wills et al., ) . we ourselves have performed dual infections with prrsv followed by enzootic viruses, notably porcine respiratory coronavirus (prcv) or swine influenza virus (siv) (van reeth et al., ) . the clinical effects of these combinations were extremely severe in some cases, but almost completely subclinical in other. most important, none of the dual infections mentioned provides a reliable model to study pathogenetic features or to test control measures. we hypothesized therefore that the clinical outcomes of dual inoculations with two infectious agents are influenced by factors that are too difficult to control, such as the stage of replication and the viral or bacterial load. bacterial lipopolysaccharides (lps) or endotoxins, a major constituent of the cell wall of gram-negative bacteria, are released in high concentrations in the lungs upon infection with gram-negative bacteria (lamp et al., ; kadurugamuwa and beveridge, ) and these endotoxins are present in varying concentrations in dust in swine buildings (rylander, ; zejda et al., ) . the release of lps by gram-negative bacteria, such as h. parasuis, p. multocida, b. bronchiseptica, and s. choleraesuis may explain the more severe disease in the experimental dual infections with prrsv and these bacteria (cooper et al., ; brockmeier et al., ; wills et al., ) . van reeth et al. ( ) recently demonstrated that dual inoculations with prcv followed by bacterial lps seriously aggravate respiratory signs in gnotobiotic pigs, while the respective single inoculations were subclinical. therefore, we wanted to examine if exposure of prrsvinfected pigs to lps similarly enhances respiratory signs. prrsv may lend itself excellently as a predisposing agent for synergism with lps, because all pigs become infected at ages varying from weeks to fattening age (albina et al., ; houben et al., ) . also, prrsv persists in the lungs for (labarque et al., ) to (mengeling et al., ) days. we have examined the clinical course of inoculations with prrsv followed by lps, and the effect of the timing and frequency of lps administrations. additionally, some preliminary investigations of cellular and virological aspects in the lungs were performed. a fifth passage on pulmonary alveolar macrophages (pams) of the lelystad strain of prrsv (wensvoort et al., ) was used in this study. the inoculation dose was . tcid /pig. escherichia coli lps (o :b ) was obtained from difco laboratories and used at a dose of mg/kg body weight. this dose was based on data from previous experiments in gnotobiotic pigs, and selected to cause no respiratory signs . forty-six conventional pigs, originating from prrsv-negative sows, were used. pigs were weaned at weeks of age and placed in isolation. they were allowed to acclimatize during days before initiation of the experiments. prrsv inoculations and lps administrations occurred intratracheally as described by van reeth et al. ( ) . briefly, the pigs were held in vertical position with their neck extended. a needle was inserted through the skin cranial to the sternum and the inoculum was injected. the intratracheal administration was chosen to ensure that all the pigs received exactly the same dose in the lungs. three experiments were performed. in the first experiment, pigs were inoculated with prrsv and received one lps administration days later. seven pigs were inoculated with prrsv only. eight pigs, not previously inoculated with prrsv, received one lps administration. clinical monitoring was performed daily during consecutive days after prrsv inoculation and every h during the first h after lps administration. in the second experiment, eight pigs were inoculated with prrsv and, days later they received two lps administrations with a h interval. four pigs, not previously inoculated with prrsv, received two lps administrations with a h interval. clinical monitoring was performed daily during consecutive days after prrsv inoculation and at , , , and h after the second lps administration. in the third experiment, out of the prrsv-lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. these pigs were divided in two subgroups. one subgroup of six pigs was again monitored for clinical signs every h until h after the second lps administration. one subgroup of five pigs was euthanatized between and h after the second lps administration for study of the broncho-alveolar lavage (bal) cell population and for virological and bacteriological examinations of the lungs. from the seven prrsv control pigs, described in the first experiment, four pigs were again monitored for clinical signs every h for h at day after prrsv inoculation. the remaining three pigs were euthanatized at time points corresponding to those of the prrsv-lps exposed pigs and served as controls for the broncho-alveolar cell and virological examinations. all eight lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. four pigs were again monitored for clinical signs every h until h after the second lps administration and four pigs were euthanatized between and h after the second lps administration for broncho-alveolar cell and virological examinations. four non-inoculated pigs were euthanatized for the same purpose. pigs were monitored for general signs, notably fever and depression, and for respiratory signs, notably tachypnoea, abdominal breathing and dyspnoea. scores were given for these five clinical parameters. body temperatures . c were scored as , temperatures between ! . and . c were scored as and temperatures ! . c were scored as . respiration rates were scored as , rates between ! and were scored as and rates ! were scored as . depression, abdominal breathing and dyspnoea were scored as (absent) or (present). scores were added up and a mean of the cumulative general and respiratory scores per group was calculated. at necropsy, the lungs were removed. the right lung was used for broncho-alveolar cell examination after bal using the method described by van reeth et al. ( ) . the bal fluid was centrifuged (  g, min, c) to separate the cells and the cell-free lavage fluid. aliquots of the cell-free lavage fluid were stored at À c until virus titration on pams. bal cells were counted in a türk chamber and cytocentrifuge preparations were stained with diffquik (baxter, düdingen, switzerland) to determine the percentage of mononuclear cells and neutrophils. the left lung was used for virological and bacteriological examinations. twenty percent suspensions of lung lobes were made in a phosphate-buffered saline, clarified by centrifugation and the supernatant was used for prrsv titration. virus titration of lung suspensions or bal fluids was performed on pams, as described by labarque et al. ( ) . for bacteriology, samples of lung tissue were plated on bovine blood agar and cultured aerobically. a nurse colony of coagulase-positive staphylococcus species was streaked diagonally on each plate. plates were inspected for bacterial growth after and h. colonies were then identified by standard techniques. non-parametric tests were used, because of lack of normality in the data. standard twosample mann-whitney tests were used to compare general and respiratory clinical scores between groups. p < : was taken as the level of statistical significance. statistical analyses were performed using spss . . twenty-six of the total of prrsv-infected pigs developed fever for time periods ranging from to days. respiratory signs were absent, except for two pigs, which showed tachypnoea and abdominal breathing. in experiment , six of the seven prrsv control pigs showed fever until the end of the monitoring period. respiratory signs were slight in one pig and absent in the other pigs. the mean respiratory score was . (table ) . in experiment , all four prrsv control pigs showed fever until the end of the monitoring period. respiratory signs, characterized by increased respiration rates, were observed in one of the four pigs. the mean respiratory score was . (table ) . in non-infected pigs, a single lps administration induced transient general signs (fig. ) . respiratory signs were slight or absent and the mean respiratory score was only . ( table ). in prrsv-infected pigs, however, lps induced severe clinical signs with fever table mean general and respiratory scores after the last lps administration in prrsv-lps exposed pigs and their controls in all the pigs and respiratory signs in % of the pigs (fig. ) . respiratory signs were characterized by tachypnoea (peak breaths/min), abdominal breathing and dyspnoea and lasted until the end of the monitoring period. two out of the pigs did not show respiratory signs after lps administration. mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs (table ) . in non-infected pigs, which received two lps administrations with a h interval, both general and respiratory signs were observed (fig. ) . clinical signs were significantly higher in prrsv-infected pigs not only with regard to the number of affected pigs but also with regard to the clinical scores. all pigs reacted severely. the mean clinical scores after the second lps administration are presented in table . non-infected pigs had recovered at the time of the second lps administration, h after the first one. this second lps administration again induced general signs within h, but no respiratory signs (fig. ) . prrsv-infected pigs had not yet recovered h after the first lps administration (fig. ) . the second lps administration however increased the number of pigs with general and respiratory signs and mean clinical scores (fig. ) . here again, mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs. total bal cell numbers and differentials are shown in table . bal cell numbers and differentials in prrsv-lps exposed pigs were essentially similar to those of pigs, exposed to prrsv or lps only. however, there was great individual variation within all three groups. mean prrsv titres in lungs and bal fluids are shown in table . virus titres were similar in lungs and bal fluids of singly prrsv-inoculated pigs and prrsv-lps exposed pigs. the lungs and bal fluids of lps controls and non-inoculated controls were negative for prrsv. bacterial culture of lung tissue yielded negative results for all pigs. it has become generally accepted that prrsv plays an important role in respiratory disease problems in the field, particularly in multi-factorial respiratory disease. however, it has been most difficult to reproduce respiratory signs in experimental infection studies with prrsv and a second infectious agent. the present prrsv-lps combination induces clear respiratory signs in % of the pigs. unlike in our previous studies with prrsv-siv table bal cell study of prrsv-lps exposed pigs and their controls at - h after a second lps administration table virological study of lungs and bal fluids of prrsv-lps exposed pigs and their controls at days after prrsv inoculation exposure number of pigs mean prrsv titres (range) lungs (log tcid /g) bal fluids (log tcid /ml) prrsv-lps . ( . - . ) . ( . - . ) prrsv only . ( . - . ) . ( . - . ) lps only negative negative none negative negative and prrsv-prcv combinations (van reeth et al., ) , mean clinical scores were higher than those of control pigs in every experiment. only two out of the prrsvinfected pigs did not develop respiratory signs upon lps exposure. it is important to mention, however, that individual variation in disease severity is unavoidable with respiratory pathogens. such an individual variation has even been reported in experimental infection studies with primary respiratory pathogens such as actinobacillus pleuropneumoniae (baarsch et al., ) or siv (van reeth et al., ) . we used two lps administrations with the purpose to extend the duration of clinical signs. the clinical effect of a second lps administration was dependent on the time interval between the two lps administrations. in non-infected pigs, a second lps administration at a h interval caused milder clinical signs than the first one. on the other hand, a second lps administration within a h interval seriously aggravated and prolonged general and respiratory signs. these observations suggest that two lps administrations within a short time interval lead to an accumulation of lps in the lungs. indeed, it has been demonstrated that the clinicopathological manifestations of lps are strictly dose-dependent. for example, if sufficient amounts of lps are given to animals and man, cytokine induction, lung inflammation and decreased lung function are observed. slightly smaller lps doses, on the other hand, will cause only a mild lung inflammation (michel et al., ) . in prrsv-infected pigs, the clinical effect of a second lps administration was difficult to assess since pigs had not yet recovered at the moment of the second lps administration, or h after the first one. there is little information on the effect of repeated lps administrations to farm animals in the literature. it appears logical, however, that the mediators or mechanisms responsible for the clinical effects of lps may become exhausted if high lps doses are administered frequently. respiratory signs following prrsv-lps exposure could not be explained by the extent of virus replication or inflammatory changes in the lungs. indeed, virus titres were similar in prrsv-lps or singly prrsv-inoculated pigs. total bal cell numbers and neutrophil infiltration were similar in prrsv-lps or singly lps exposed pigs. also, the two prrsv-lps exposed pigs, which remained healthy, had similar bal cell profiles as their clinically affected group mates. this suggests that inflammatory changes in the lungs have little or no effect on the synergism between prrsvand lps. we hypothesize therefore that functional lung changes, such as bronchial hyper-responsiveness, are more important in the pathogenesis of prrsv-lps induced disease than structural changes. similar findings were made in a previous experimental infection study with prcv followed by lps . in this study, disease development was tightly correlated with lung production of proinflammatory cytokines, among which tumor necrosis factor-alpha (tnfa). interestingly, tnf-a has been shown to cause bronchial hyper-responsiveness in laboratory animal models (kips et al., ; thomas et al., ) . under field circumstances, most pigs become infected with prrsv at growing age and they are continuously exposed to airborne endotoxins. also, during gram-negative infections of the lungs, excessive amounts of endotoxins are released locally. the present prrsv-lps infection model therefore is relevant for the study of prrsv-induced respiratory problems in the field. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units pathophysiologic correlates of acute porcine pleuropneumonia effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs porcine reproductive and respiratory syndrome: neb- prrsv infection did not potentiate bacterial pathogens porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to prrsv interaction between streptococcus suis serotype and porcine reproductive and respiratory syndrome virus in specific pathogenfree piglets pattern of infection with the porcine reproductive and respiratory syndrome virus on swine farms in belgium natural release of virulence factors in membrane vesicles by pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release tumor necrosis factor causes bronchial hyperresponsiveness in rats effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs influence of antibiotic and e monoclonal immunoglobulin m interactions on endotoxin release from escherichia coli and pseudomonas aeruginosa diagnosis of porcine reproductive and respiratory syndrome dose-response relationship to inhaled endotoxin in normal subjects endotoxins tumor necrosis factor-a increases airway responsiveness and sputum neutrophilia in normal human subjects dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study broncho-alveolar interferon-a, tumor necrosis factor-a, interleukin- and inflammation during acute influenza in pigs: a possible model for humans? a potential role for tumour necrosis factor-a in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs mystery swine disease in the netherlands: the isolation of lelystad virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine respiratory health status in swine producers relates to endotoxin exposure in the presence of low dust levels this work was supported by grant a from the belgian ministry of agriculture. the authors would like to thank fernand de backer, krista de winne, lieve sys, chantal vanmaercke and carla de winter for excellent technical assistance. geoffrey labarque was supported by grant o d from the research council of the ghent university. key: cord- - bw sly authors: shi, yuejun; li, youwen; lei, yingying; ye, gang; shen, zhou; sun, limeng; luo, rui; wang, dang; fu, zhen f.; xiao, shaobo; peng, guiqing title: a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: bw sly porcine reproductive and respiratory syndrome virus (prrsv) rna endoribonuclease nsp belongs to the xendou superfamily and plays a crucial role in arterivirus replication. here, we report the first crystal structure of the arterivirus nsp protein from prrsv, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp . however, the structures of the prrsv nsp and coronavirus nsp catalytic domains were perfectly superimposed, especially in the “active site loop” (his to his ) and “supporting loop” (val to thr ) regions. importantly, our biochemical data demonstrated that prrsv nsp exists mainly as a dimer in solution. mutations of the major dimerization site determinants (ser and phe ) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. in the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. these findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. importance porcine reproductive and respiratory syndrome virus (prrsv) is a member of the family arteriviridae, order nidovirales. prrsv is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. the prrsv nsp endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. here, we report the first dimeric structure of the arterivirus nsp from prrsv at . -Å resolution. structural and biochemical experiments demonstrated that nsp exists mainly as a dimer in solution and that nsp may be fully active as a dimer. mutagenesis and structural analysis revealed nendou active site residues, which are conserved throughout the order nidovirales (families arteriviridae and coronaviridae) and the major determinants of dimerization (ser and phe ) in arteriviridae. importantly, these findings may provide a new structural basis for antiviral drug development. n idoviruses are enveloped, positive-sense, single-stranded rna [(ϩ)ssrna] viruses comprising the families arteriviridae, coronaviridae, mesoniviridae, and roniviridae ( ) ( ) ( ) ( ) . nidoviruses (arteriviridae and coronaviridae) are important pathogens that cause significant diseases in animals and humans, typically causing respiratory and enteric disease ( , ) . the genome length of arteriviridae members is approximately . to . kb, among the "small-genome nidoviruses" ( ) . coronaviridae and roniviridae belong to a group of "large-genome nidoviruses," as their genome lengths span . kb to . kb ( ), whereas members of the mesoniviridae have medium-sized ( -to- -kb) genomes, between those of small-and large-genome nidoviruses ( , ) . nevertheless, all nidoviruses are grouped together due to their similar replication/transcription strategies and their relatively close genetic relationship ( , ) . porcine reproductive and respiratory syndrome virus (prrsv) is a member of the family arteriviridae, which also includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv) of mice, and simian hemorrhagic fever virus (shfv) ( ) . prrsv is the causative agent of porcine reproductive and respiratory syndrome (prrs), which has become one of the most important infectious diseases in the swine industry and causes tre-mendous economic losses worldwide ( , ) . the prrsv genome is approximately kb in length, with overlapping open reading frames (orfs), and consists of both nonstructural genes (orf a and orf b) and structural genes (orf to orf ) ( , , ) . orf a and orf b comprise approximately % of the viral genome and encode at least nonstructural proteins (nsps), including nsp ␣, nsp ␤, nsp , nsp tf/nsp n, nsp to nsp , nsp ␣, nsp ␤, and nsp to nsp ( ) ; orf to orf encode the viral structural proteins gp , e, gp , gp , gp , orf a, m, and n ( , ) . the resulting mature nsps direct viral genome replication and subgenomic mrna transcription via a membrane-anchored replicase/transcriptase complex, and these mrnas are then translated to produce structural and accessory proteins ( ) . nsp (rna-dependent rna polymerase) and nsp (helicase) ( ) are the key replicative enzymes in the replicase/transcriptase complex; nsp (endoribonuclease) may also play a key role in arterivirus replication, though the exact function of endoribonucleases in nidovirus replication remains unclear ( , ) . prrsv nsp possesses nidovirus uridylate-specific endoribonuclease (nendou) activity, which is important for arterivirus replication ( ) . xendou, reported to be an endoribonuclease in eukaryotes, is involved in processing the intron-encoded box c/d u small nuclear rna from its pre-mrna ( ) . xendou and nendou, which specifically cleaves = uridine nucleotides of rna substrates to generate a =- =-cyclic phosphate end product, possess common functional characteristics ( ) ( ) ( ) . the endoribonuclease activity of coronavirus (cov) nsp and arterivirus nsp has been confirmed ( ) ( ) ( ) , and recombinant arterivirus nsp displays broad substrate specificity in vitro ( ) . moreover, the nendou activity of coronavirus nsp is stimulated by mn ϩ ( , , ) , whereas mn ϩ was reported to be inhibitory to the activity of arterivirus nsp nendou ( ) . the crystal structures of severe acute respiratory syndrome coronavirus (sars-cov) nsp and murine hepatitis virus (mhv) nsp show that the biological unit of nsp is a hexamer ( , ) and that the n-terminal domain (ntd) is important for oligomerization ( ) . although nendou activity is common to nidoviruses (arteriviridae, coronaviridae, and roniviridae), the nendou domains exhibit considerable variation ( ) . there is no detailed structural information to date for arterivirus nsp . in this study, we performed structural and functional analyses of nsp to elucidate the mechanism underlying the function of prrsv endoribonuclease nsp during arterivirus replication and to identify potential drug targets for controlling prrs disease. we report the crystal structure of prrsv endoribonuclease nsp and demonstrate that the folding of nendou active site residues is widely conserved among members of the order nidovirales (families arteriviridae and coronaviridae). our data also indicate that nsp is fully active as a dimer, and we elaborate on the structural basis underlying this finding. plasmid construction. the sequence encoding the -residue nsp gene corresponds to nucleotides to in the genome of the prrsv wuh strain (genbank accession no. hm ) ( ) . for crystallization, wild-type and mutant (k a) nsp gene sequences were cloned by pcr amplification into pet- b (ϩ) with a c-terminal his tag using the ndei and bamh i restriction sites. mutant plasmid (k a) was used as the template for the generation of expression constructs encoding mutant (s a, f a, and r a) nsp derivatives for dimerization experiments. meanwhile, to obtain high expression in prokaryotic cells, wild-type nsp , flanked by an n-terminal his tag and s tag and a c-terminal his tag, was cloned into pet- a (ϩ) between the ecor i and xhoi restriction sites. in addition, to obtain high expression in eukaryotic cells, wild-type nsp , flanked by an n-terminal hemagglutinin (ha) tag, was cloned into pcaggs vector using the ecori and xhoi restriction sites. point mutations (s a, f a, h a, k a, t a, and y a) were engineered using overlap extension pcr, and the fragments were cloned into pet- a (ϩ) and pcaggs vector according to the same method. all constructs were validated by dna sequencing. protein expression and purification. for analysis of wild-type nsp expression, the recombinant plasmids were transformed into escherichia coli strain trans bl (de ) plyss (beijing transgen biotech co., ltd.). transformed cells were cultured at °c in lb medium containing g/ml kanamycin. induction with . mm iptg (isopropyl ␤-d- -thiogalactopyranoside) was performed when the culture density reached an optical density at nm (od ) of . to . , and cell growth continued for an additional h at °c. for analysis of the expression of the nsp mutant proteins, the recombinant plasmids were transformed according to the same method. when the cells reached an od of . to . , iptg was added to give a final concentration of . mm. then, the cells were grown for an additional h at °c before harvesting. to solve the phase problem, a selenomethionine (se-met)-labeled nsp mutant (k a) was expressed in trans bl (de ) plyss using m salt medium (qingdao rishui biological technology corporation) supplemented with g/ml kanamycin, . % glucose, mm mgso , and . mm cacl at °c until an od of . was reached. then, the amino acid mixture ( mg lysine, phenylalanine, and threonine per liter; mg isoleucine, leucine, and valine per liter; and mg selenomethionine per liter) was added min before induction. iptg was added to give a final concentration of . mm, and the cells were grown for an additional h at °c before harvesting. for protein purification, cells were harvested by centrifugation at , rpm for min in a high-speed refrigerated centrifuge (cr- g; hitachi), resuspended with phosphate-buffered saline (pbs; mm nacl, mm kcl, mm na hpo · h o, and mm kh po , ph . ) and lysed by passage through an ah- homogenizer (ats engineering inc.) at , lb/in . after centrifugation at , rpm for min, the supernatant was filtered with a . -m-pore-size filter and loaded onto a nickel-charged histrap hp column (ge healthcare). the proteins were eluted with elution buffer ( mm tris-hcl, m nacl, and mm imidazole, ph . ). the harvested protein was then concentrated to approximately . ml and filtered using a superdex gel filtration column (ge healthcare) equilibrated with buffer ( mm tris-hcl and m nacl, ph . ). for crystallization, the purified protein was concentrated to approximately mg/ml, flash-frozen with liquid nitrogen, and stored at Ϫ °c. the concentration of the purified prrsv nsp was determined by the absorbance at nm (a ) using a nanodrop c uv-vis spectrophotometer (thermo fisher scientific). crystallization, data collection, and structure determination. crystallization screens for wild-type nsp and the k a mutant protein at a concentration of mg/ml were performed via the hanging-drop vapordiffusion method at °c. crystals of the se-met derivative (k a) mutant were obtained to solve the phase problem. the crystallization conditions were optimized, and the best crystals for both wild-type and se-met-labeled nsp were obtained by vapor diffusion in hanging drops consisting of l of reservoir solution ( . m sodium citrate tribasic dehydrate [ph . ] and % [wt/vol polyethylene glycol ) and l of concentrated protein solution ( mg/ml)- mm tris-hcl- m nacl (ph . ), followed by incubation at °c for days (wild-type nsp ) or days (se-met-labeled nsp ). then, the crystals were flash-cooled in liquid nitrogen in a cryoprotectant solution containing % ethylene glycol and % reservoir solution ( . m sodium citrate tribasic dehydrate [ph . ] and . % [wt/vol] polyethylene glycol ). data collection was performed at the shanghai synchrotron radiation facility (ssrf) with a bl u beam line (wavelength ϭ . Å, temperature ϭ k). reflections were integrated, merged, and scaled using hkl- ( ) , and the resulting statistics are listed in table . the structure of nsp was solved by the use of the single-wavelength anomalous dispersion (sad) method and a se-met derivative k a mutant. all three potential selenium atoms in the nsp monomer were located, and the initial phases were calculated using the autosol program from the phenix software suite ( ) . manual model rebuilding was performed using coot ( ) and then refined in the phenix software suite. structural analysis, sequence alignment, and phylogenetic reconstruction. detailed molecular interactions between the two monomers of nsp were determined using ligplot ( ) , and the other structure figures were generated using pymol (schrödinger). the buried surface areas between the two monomers and the root mean square deviation (rmsd) were analyzed using pdbepisa (http://pdbe.org/pisa/) and pd-befold (http://pdbe.org/fold/), respectively. additionally, cell content analysis was generated using the ccp suite ( ) . the amino acid sequences of arterivirus nsp and coronavirus nsp were aligned using clustalw ( ) and visualized with the espript server (http://espript .ibcp.fr) ( ) . the phylogenetic relationships were analyzed using the maximum likelihood algorithm in the mega package ( ) . the analyzed viruses (abbreviations; ncbi accession numbers) were as follows: hcov-hku (human coronavirus hku ; yp_ ), sars-cov (sars coronavirus wtic-mb; agt ), mhv (murine hepatitis virus strain a ; np_ ), pedv (porcine epidemic diarrhea virus; aim ), hcov- e (human coronavirus e; agt ), tgev (transmissible gastroenteritis virus virulent purdue; abg ), fipv (feline infectious peritonitis virus; agz ), prrsv (porcine respiratory and reproductive syndrome virus strain wuh ; ado ), eav (equine arteritis virus; np_ ), shfv (simian hemorrhagic fever virus; ahh ), and ldv (lactate dehydrogenase-elevating virus; aaa ). in vitro dimerization experiments. as the oligomeric state of the mutant [k a; pet- b (ϩ)] nsp protein is the same as that of the wild type (data not shown), the mutant (k a) protein was purified for size exclusion experiments because the expression of wild-type nsp was low. oligomerization of wild-type ( mg) and mutant (s a, f a, and r a) ( mg) nsp proteins was analyzed using a superdex / gl column (ge healthcare) with a buffer containing mm tris-hcl (ph . ) and mm nacl at a flow rate of . ml/min ( °c). wild-type and mutant (s a, f a, and r a) nsp proteins eluted in different fractions were analyzed by sds-page. equal volumes ( l) of bio-rad size exclusion standards (catalog no. - ; ml/vial), conalbumin ( kda; . mg) (ge healthcare), and mutant (g a, l a, r a, y a, g a, g a, v a, and s a) nsp proteins ( mg) were analyzed under the same buffer conditions. additionally, sedimentation velocity analysis was carried out using an xl-a model centrifuge (proteome lab) at , rpm and °c in -l double-sector cells. the sedimentation boundary was monitored every min using a wavelength of nm for a total of scans. data were interpreted with the modelbased distribution of lamm equation solutions [c(s)] using sedfit software ( ) . the obtained results were analyzed using origin . software. the predicted weight-averaged molar masses were calculated using dnastar (version . ) software. enzyme activity assay. the rna substrate ( =- -carboxyfluorescein [fam]-da-ru-da-da- -carboxytetramethylrhodamine [tamra]- =) was chemically synthesized by the genscript corporation. the endoribonuclease activity of nsp was examined using fluorescence resonance energy transfer (fret) following a previously published protocol ( ) . the assay was performed with m enzyme and m fluorescent substrate at °c in buffer containing mm hepes (ph . ), mm kcl, and mm dithiothreitol (dtt) diluted with . % diethyl pyrocarbonatetreated water. the activity was assayed with an excitation wavelength of nm and an emission wavelength of nm using a fluorescence time scan on a fluoroskan ascent instrument (thermo labsystems, helsinki, finland) and was recorded every min for min. the activity assays for the wild-type and mutant (s a, s a, h a, k a, t a, and y a) nsp proteins were performed under the same conditions. it should be mentioned that purification of the mutant (h a, r a, d a, and d a) proteins was unsuccessful because these mutations rendered the protein insoluble. experiments were performed in triplicate, and the values (Ϯ standard deviations [sd]) of the results of triplicate experiments are shown. in addition, the wild-type and mutant (s a, s a, h a, k a, t a, and y a) nsp proteins were analyzed by sds-page. luc reporter gene assays. hek t cells were seeded into -well plates and incubated until the cells reached approximately % confluence. then, the cells were cotransfected with . g of the reporter plasmid (beta interferon-luciferase [ifn-␤-luc] or irf -luc), . g of plasmid prl-tk (promega) encoding renilla luciferase, . g of wildtype plasmid, or . g of mutant (s a, f a, h a, k a, t a, and y a) plasmids using lipofectamine . total transfected dnas were equalized to . g by the addition of empty pcaggs vector. at h after the initial transfection, the cells were infected with sendai virus (sev). at h posttransfection, the cells were harvested and the luciferase activity was measured using a dual-luciferase reporter assay system (promega) on a glomax / luminometer reader (promega). firefly luciferase activity was normalized to renilla luciferase. experiments were performed in triplicate, and statistical significance was determined using an unpaired two-tailed student's t test. values of Ͻ . were considered statistically significant. western blot analysis. briefly, to analyze the expression levels of the wild-type and mutant (s a, f a, h a, k a, t a, and y a) nsp proteins, hek t cells were transfected with various plasmids using the same method. at h posttransfection, cells were harvested by adding lysis buffer (beyotime), and the protein concentration was measured and adjusted. the same amounts of each protein sample were then analyzed by western blotting with anti-ha antibody (ab; sigma). expression of gapdh (glyceraldehyde- -phosphate dehydrogenase) was de- and f c are the observed and calculated structure factors, respectively; r free is equivalent to r work , but % of the measured reflections have been excluded from the refinement and set aside for cross-validation. tected with anti-gapdh monoclonal ab (mab) (sigma) to confirm loading of equal protein amounts. cell viability assay. hek t cells cultured in white -well plates (corning, tewksbury, ma, usa) were transfected with an empty vector or wild-type or mutant (s a, f a, h a, k a, t a, and y a) nsp plasmids ( . g) using lipofectamine . in addition, the different doses of the wild-type plasmid ( to . g) were transfected, and total transfected dnas were equalized to . g by the addition of empty pcaggs vector. at h posttransfection, cell viability was evaluated using celltiter-glo luminescent cell viability assay reagent (promega, madison, wi, usa) following the manufacturer's protocol. briefly, an equal volume ( l) of celltiter-glo reagent was added and the reaction mixture was shaken for min on an orbital shaker and incubated for a further min at room temperature. the luminescence of each well was measured on a microbeta trilux instrument (perkinelmer, waltham, ma, usa). the percentage of cell viability was calculated as follows: percentage of cell viability ϭ ϫ (luminescence of the experimental group/luminescence of the control group). experiments were performed in triplicate, and statistical significance was determined using an unpaired two-tailed student's t test. values of Ͻ . were considered statistically significant. protein structure accession number and statistical analysis. coordinates and structure factors for prrsv nsp were deposited in the rcsb protein data bank under accession number da . crystal structure of prrsv endoribonuclease nsp . previous studies demonstrated that full-length wild-type endoribonucleases (sars-cov nsp , mhv nsp , eav nsp , and prrsv nsp ) are expressed only weakly; accordingly, these endoribonucleases may be toxic to e. coli and cause slow cell growth and low protein yields ( , , ) . thus, to obtain wild-type nsp , we assessed different expression vectors and used different e. coli strains as hosts; nevertheless, the yield of wild-type nsp remained extremely low. indeed, our experimental results showed that wild-type nsp causes cell cytotoxicity and death after an extended expression time (approximately h). the duration of wild-type nsp expression was examined at different times ( min, min, min, min, min, and min) at °c, with min found to be the best expression time (data not shown). the yields of wild-type nsp from the expression vectors [pet- b (ϩ) and pet- a (ϩ)] were estimated to be approximately . mg and . mg of protein, respectively, per liter of bacterial cell culture. in contrast, the yield of mutant protein reached approximately to mg/liter ( °c, h). previous studies indicated that functional endoribonucleases can cleave the = terminus of the pyrimidines of ssrna and dsrna substrates ( , ) , which might act on both their own and cellular mrna and cause nendou expression to be potentially "suicidal" ( ) . the crystal structure of prrsv nsp (residues gly to glu ) was determined using the sad method and was refined to . -Å resolution, which was of sufficient quality to trace the entire chain (excluding the c-terminal his tags). the matthews coefficient and solvent content are . and %, respectively, as determined by cell content analysis. the solvent content value is high, which may explain why the diffraction of the crystals was poor. the crystal belongs to the space group p and consists of two subunits in an asymmetric unit (fig. b) . interestingly, subunit a is visibly different from subunit b (fig. c , d, and e). in subunit b, residues in the regions asp -gly , ser -lys , and leu -glu could not be traced due to a lack of interpretable electron density (fig. c) . the crystal structure can be divided into two major parts: the n-terminal domain (ntd; gly to phe ) and the c-terminal catalytic domain (arg to glu ). the ntd is formed by six ␤-strands (␤ to ␤ ) and two ␣-helices (␣ to ␣ ) and is connected to the catalytic domain through a linker domain (lkd; val to thr ). the catalytic domain has a typical fold consisting of a compact groove region containing sequentially connected left and right parts (fig. a) : the left part of the catalytic domain consists of two ␣-helices (␣ and ␣ ) and six ␤-strands (␤ to ␤ ), and the right part is formed by three antiparallel ␤-strands (␤ to ␤ ) and one ␣-helix (␣ ). details of the data collection and structure refinement are summarized in table . mutational studies in the dimerization interface. in this study, gel filtration chromatography revealed the dimeric architecture of nsp . our data indicated that nsp eluted primarily in one peak; the calculated molecular mass is approximately . kda, which corresponds to a dimer (fig. c, d, and e) . this finding is consistent with the dimeric crystal structure of nsp (fig. b) . the dimerization interface is shown in fig. a and b. residues gly , leu , arg , tyr , ser , phe , gly , gly , arg , val , and ser were chosen as candidate targets to abolish the dimerization. the mutant (g a, l a, r a, y a, g a, g a, v a, and s a) proteins eluted as a dimer; these mutations could not prevent nsp dimerization (data not shown). however, elution of the mutant (s a and f a) proteins by gel filtration yielded two -nm absorption peaks ( fig. d and e). our results indicated that these two mutations significantly disrupt the dimerization in solution. moreover, the r a mutant existed mainly as an intermediate form (the calculated molecular mass is approximately . kda) compared with the wild type ( fig. d and e) . meanwhile, the oligomerization of wildtype and mutant (s a, f a, and r a) nsp proteins was further analyzed via sedimentation analytical ultracentrifugation (auc), and the results were shown in fig. f and g. the molecular weights of monomers and dimers from the wild-type nsp protein are approximately . (approximately . % of the total population) and . (approximately . %) and are essentially consistent with those of gel filtration chromatography. the sedimentation coefficient (s ,w ) of the mutant (s a, f a, and r a) proteins decreased significantly compared with the wild type, though the relative populations of monomers and dimers of those mutant proteins were not successfully determined. this indicated that the oligomerization of the mutant proteins had markedly changed. therefore, our biochemical data consistently showed that nsp exists mainly as a dimer in solution and that the mutations in the dimerization interface, s a, f a, and r a, disrupt dimerization. in our crystal structure, a total binding surface of , Å is buried at the interface (fig. a) , which is smaller than the subunit a-subunit b binding surface of sars-cov nsp , which is , . Å (fig. c) . regardless, this smaller binding surface may be sufficient to stabilize monomer-monomer interactions because the molecular weight of nsp (approximately . ) is lower than that of sars-cov nsp (approximately . ). in addition, the catalytic domain of subunit a and the ntd of subunit b are associated with a largely hydrophobic and hydrogen-bonding network ( fig. a and b) . a total of residues in the ntd of subunit b interact with residues in the catalytic domain of subunit a. residue phe interacts with residues tyr , leu , pro , gly , val , and ser via the hydrophobic forces ( fig. a and b) ; thus, phe is a key residue within the dimer interface. moreover, residue ser interacts with residues pro , val , and gly and is thus also a key residue within the dimer interface ( fig. a and b ). in addition, interactional residues leu , val , and cys were also observed with residue arg ( fig. a and b) . therefore, these mutations (s a, f a, and r a) may disturb both hydrophilic and hydrophobic interactions between monomers and prevent the formation of stable dimers. to clarify the relationship between dimerization and catalytic activity, we performed fret assays using fluorescence-labeled rna as the substrate. as predicted, the activity levels of the mutants (s a and f a) were significantly decreased (being at least -fold less than wild-type levels) but not completely abolished (see fig. c ) because the mutant proteins were not purely monomeric. in addition, gel filtration chromatography revealed that the -nm absorption peak of the mutant s a protein was obviously lower than that of the mutant f a protein with the same amount of total protein ( fig. d and e) , which indicated that the mutant s a protein is very unstable. this may be the reason why the nendou activity of the s a mutant is lower than that of the f a mutant. in conclusion, the s a and f a mutations severely diminished the catalytic activity, indicating that the dimer is the biologically functional unit. the structural basis for nsp functioning as a dimer rather than a hexamer. our crystal structure indicates that nsp assembles into dimers, which is different from coronavirus nsp ( , ) . the monomer structure of sars-cov nsp includes three domains, the n-terminal domain (ntd), the middle domain, and the catalytic domain ( ) (fig. c) ; the ntd is critical for hexamsites are shown. (b) detailed molecular interactions of the supporting loop (subunit a, magenta) with the active site loop (subunit a, red) and the n-terminal domain (subunit b, red) were determined using ligplot. hydrogen bond interactions and hydrophobic interactions are shown as described for fig. a. (c) surface representation of hexameric sars-cov nsp (pdb code rhb). the individual subunits are colored and marked a to f. the active site loop (residues his to his ) and the supporting loop (residues lys to iie ) are highlighted as described for panel a. the description of sars-cov nsp domains is based on a previous report ( ) . the monomer-monomer buried surface areas of prrsv nsp and sars-cov nsp were analyzed using pdbepisa. the structural comparison of the n-terminal region of prrsv nsp and the middle region of coronavirus nsp . (a and b) the structure of prrsv nsp (subunit a, yellow) superimposed onto the structures of sars-cov nsp (pdb code h , magenta) and mhv nsp (pdb code gth, magenta). the structure of the n-terminal region (gly to gly ) from prrsv nsp superimposed with sars-cov nsp (asn -ser ) and mhv nsp (ser -leu ) is enlarged in panels a and b (the cartoon transparency was set at %). the dimerization site determinants ser and phe (corresponding to val /leu and leu /val in sars-cov nsp and mhv nsp , respectively) are labeled with a ball-and-stick (yellow, prrsv nsp ; magenta, sars-cov nsp and mhv nsp ) representation. the sars-cov nsp domains are colored and marked as described for fig. c . erization and interactions with the middle domain and the catalytic domain of an adjacent monomer ( , ) . however, the ntd structure (approximately n-terminal residues) of coronavirus nsp is missing in nsp , and the ntd of nsp superimposes onto the middle domain of coronavirus nsp (fig. ) . moreover, the major determinants of dimerization (ser and phe ) are significantly different from the key residues involved in the oligomerization of sars-cov nsp ( fig. ; fig. ), which indicates why the active form of nsp is a dimer rather than a hexamer. in addition, subunits a and b in our crystal structure are highly similar, with a root mean square deviation (rmsd) of . Å between the c␣ atoms, though residues asp to gly and ser to lys are missing in subunit b (fig. c ). in analogy with the monomer structure of sars-cov nsp , the loops consisting of residues his to his and val to thr are identified as the "active site loop" and "supporting loop," respectively ( ) . in the dimeric architecture of nsp , the active site loop and supporting loop from subunit a are packed against one another, and their structures are stabilized by monomer-monomer interactions with subunit b (fig. a) . however, the electron density of these loops is missing in subunit b, indicating that they are flexibly disordered. furthermore, interactions among residues nsp oligomerization are shown in a yellow frame. secondary structure elements of prrsv nsp are marked on the top of the alignment (helices with squiggles, ␤-strands with arrows, and turns with tt letters). the sequences were aligned using clustalw , and the alignment was drawn with espript . . the phylogenetic relationships were analyzed using the maximum likelihood algorithm in the mega package. the different subgenotypes are indicated. yellow) superimposed onto the structures of sars-cov nsp (pdb code h , magenta) and mhv nsp (pdb code gth, magenta). the structure of the catalytic domain (ile -glu ) of prrsv nsp superimposed with sars-cov nsp (asp -leu ) and mhv nsp (ser -phe ) is enlarged in panels a and b. the potential catalytic active sites are labeled with a ball-and-stick (yellow, prrsv nsp ; magenta, sars-cov nsp and mhv nsp ) representation. the "supporting loop" and "active site loop" are highlighted with a ribbon representation (the cartoon transparency was set at %) according to structural data for sars-cov nsp ( ) . the sars-cov nsp domains are colored and marked as described for fig. c . val , thr to val , cys , and his in the active site loop and residues val , ser , lys , ala , lys , and cys to thr in the supporting loop are observed in the structure (fig. b) . these extensive interactions between the supporting loop and the adjacent monomer were analyzed. residues val to ser , pro , gly , and lys to ala from the supporting loop interact with residues gly to ser , leu , tyr , val to ser , phe , val , and val of the adjacent monomer (fig. b) , indicating that the supporting loop is involved in dimerization. therefore, the disappearance of these two loops from subunit b may be attributed to the absence of the adjacent monomer. this finding may explain why nsp is fully active as a dimer. interestingly, the potential link between dimerization and catalytic activity is similar to the mechanism of the functional hexamer of sars-cov nsp ( ) . in addition, further research is needed to explore whether the dimer or other oligomers of nsp exist in a functional state during arterivirus replication. the structure of nsp reveals nidovirus-wide conservation of the catalytic domain. multiple-sequence alignment indicated that the amino acid sequence identity between arterivirus nsp and coronavirus nsp is only approximately . % to . %, as demonstrated by their distance on the evolutionary tree (fig. ) . moreover, there are distinct differences between the ntd of nsp and the middle domain of coronavirus nsp (the rsmds with sars and mhv are . and . , respectively) (fig. ) . however, the structures of the catalytic domains can be nearly perfectly superimposed (the rsmds with sars and mhv are . and . , respectively), especially in the active site loop and supporting loop regions (fig. ) . additionally, the structural comparison demonstrated that residues his , his , lys , thr , asp , asp , and tyr from nsp superimpose well onto the corresponding residues of coronavirus nsp (fig. and ), indicating the relative conservation of key active site residues and similar endoribonuclease cleavage mechanisms shared among nidoviruses (families arteriviridae and coronaviridae). in this study, endoribonuclease activity of the wild-type and mutant nsp protein was measured, and the results are shown in fig. c . enzyme activity assays for the wild-type and h a, k a, t a, and y a mutant proteins were performed under identical conditions, and the activity levels of the mutants were significantly reduced compared with the wild-type level (fig. c) , indicating that these residues are located in important nendou active sites. previous results suggested that the catalytic mechanism of the nidovirus endoribonuclease could be consistent with an rnase a-like reaction mechanism that cleaves the rna substrate to form a =, =-cyclic phosphodiester and a =-phosphomonoester by transphosphorylation and hydrolysis ( ) . the catalytic his and his residues of nsp (corresponding to his and his of sars-cov nsp ) are thought to accept and donate protons during production of the =- =-cyclic phosphate ( ) , which may explain why the catalytic activity of the h a mutant is much lower than that of the k a mutant. previous studies also demonstrated that residues his , his , and lys (corresponding to his /his , his /his , and lys /lys in sars-cov nsp and mhv nsp , respectively) are essential for endoribonuclease activity in coronaviruses and arteriviruses ( ) ( ) ( ) ( ) ) . in our crystal structure, these three putative catalytic residues (his , his , and lys ) surround a positively charged cavity in the catalytic domain, with thr located in the middle of the groove (fig. a ). thr (corresponding to ser and thr in sars-cov nsp and mhv nsp , respectively) could also be important for substrate recognition and binding ( , , ) . moreover, tyr (corresponding to tyr in sars-cov nsp ) has been implicated in the orientation and binding of the substrate ( , ) . as predicted, the catalytic activity levels of the t a and y a mutants were significantly decreased but not completely abolished. , together with the wild-type and mutant nsp plasmids ( . g). at h after the initial transfection, the cells were infected with sendai virus (sev). at h posttransfection, the cells were harvested and the luciferase activity was measured. the firefly luciferase activity was normalized to renilla reniformis luciferase, and the untreated empty vector control value was set to . *, p Ͻ . (considered significant compared with the luciferase activity of the cells expressing the wild-type protein); **, p Ͻ . (considered highly significant); ***, p Ͻ . (considered extremely significant). (c) relative luciferase activity of prl-tk. relative luciferase activity ϭ ϫ (the luciferase value for the wild-type and mutant strains/the luciferase value for the control group). (d) western blot analysis of the expression levels from wild-type and mutant nsp . (e and f) cell viability analysis of wild-type and mutant nsp in hek t cells. cell viability of wild-type and mutant nsp was evaluated via the use of a celltiter-glo luminescent cell viability assay. percent cell viability ϭ ϫ (luminescence of the experimental group/luminescence of the control group). in addition, previous studies reported that the endoribonuclease activity of prrsv nsp is essential to inhibit ifn-␤ induction ( ) . we found that the overexpression of wild-type nsp markedly inhibited the activity of the ifn-␤ luciferase reporter induced by sendai virus (sev), while the mutants (s a, s a, h a, k a, t a, and y a) lost the capacity to block the activation of ifn-␤ promoter (fig. a and b) . because the recombinant arterivirus nsp protein displays broad substrate specificity in vitro and is extremely toxic to prokaryotic and eukaryotic cells ( ) , it is possible that the suppression of ifn-␤ induction by wild-type nsp is due to its cytotoxicity. hek t cells expressing wild-type nsp appeared to be in good shape and showed no obvious cytotoxicity in detection experiments performed with the celltiter-glo luminescent cell viability assay (fig. e and f) . however, when we analyzed the ability of wild-type and mutant nsp to inhibit ifn-␤ induction by the use of a dual-luciferase reporter assay system (promega), we found that the value for prl-tk, an internal control reporter, was significantly lower in cells expressing wild-type nsp than in cells expressing nsp mutants or in cells that had received mock treatment (fig. c) , indicating that wild-type nsp inhibits host gene expression. coincidentally, none of the tested nsp mutants without cytotoxicity significantly inhibited ifn-␤ induction (fig. a , b, and c). therefore, we could not exclude the possibility that the potential cytotoxicity of wild-type nsp inhibits ifn-␤ induction. it should be noted that this study involved the individual expression of nsp , outside the context of infection. whether the endoribonuclease function of nsp specifically contributes to the decline of innate immune functions in prrsv infection requires further investigation. conclusions. in summary, we provide the first structural information for arterivirus endoribonuclease nsp , which has a novel dimeric structure that dramatically distinguishes it from coronavirus nsp . our biochemical data showed that mutation of key residues (ser and phe ) in the dimerization interface disrupts dimerization in solution and markedly impairs the endoribonuclease activity in vitro. furthermore, structural analyses showed that the absence of adjacent monomer interactions might damage the structural stability of the catalytic domain, which indicates why the biologically active unit of nsp is a dimer. furthermore, structural conservation of the catalytic domain in members of the order nidovirales (families arteriviridae and coronaviridae) was also observed in this study. these results provide a model that will contribute to an understanding of the structure-function relationship of endoribonucleases in the order nidovirales, and our findings will serve as a structural basis for the development of new nsp -specific antiviral drugs and other inhibitors. nidovirales: evolving the largest rna virus genome site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes the footprint of genome architecture in the largest genome expansion in rna viruses recent progress in studies of arterivirus-and coronavirus-host interactions mechanisms of coronavirus cell entry mediated by the viral spike protein arterivirus molecular biology and pathogenesis discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark the ever-expanding diversity of porcine reproductive and respiratory syndrome virus the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins prrsv structure, replication and recombination: origin of phenotype and genotype diversity efficient Ϫ frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production nidovirus ribonucleases: structures and functions in viral replication purification, cloning, and characterization of xendou, a novel endoribonuclease involved in processing of intron-encoded small nucleolar rnas in xenopus laevis unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp suggests a role for hexamerization as an allosteric switch immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus gp protein encoded by a synthetic orf gene hkl- : the integration of data reduction and structure solution-from diffraction images to an initial model in minutes phenix: building new software for automated crystallographic structure determination coot: model-building tools for molecular graphics ligplotϩ: multiple ligand-protein interaction diagrams for drug discovery the ccp suite: programs for protein crystallography clustal w and clustal x version . deciphering key features in protein structures with the new endscript server mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling rna recognition and cleavage by the sars coronavirus endoribonuclease major genetic marker of nidoviruses encodes a replicative endoribonuclease structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-beta induction this work was supported by the national key basic research plan (grant no. cb ), the national natural science foundation of china (grant no. and ), and the huazhong agricultural university scientific and technological self-innovation foundation (program no. rc , py , and jq ).we thank fang li and ke shi for discussions and comments and the staff at the ssrf bl u beam line for assistance with x-ray data collection. moreover, we also thank research associates at the center for protein research (cpr), huazhong agricultural university, for technical support. this work, including the efforts of guiqing peng, was funded by national key basic research plan ( cb ). this work, including the efforts of guiqing peng, was funded by huazhong agricultural university scientific and technological self-innovation foundation ( rc , py , and jq ). this work, including the efforts of shaobo xiao, was funded by national natural science foundation of china (nsfc) ( ). this work, including the efforts of guiqing peng, was funded by national natural science foundation of china (nsfc) ( ). the dimerization mechanism of prrsv nsp . (a) surface representation of dimeric prrsv nsp . by analogy with the monomeric structure of key: cord- -fgn rps authors: miller, laura c; fleming, damarius; arbogast, andrew; bayles, darrell o; guo, baoqing; lager, kelly m; henningson, jamie n; schlink, sarah n; yang, han-chun; faaberg, kay s; kehrli, marcus e title: analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus date: - - journal: bmc vet res doi: . / - - - sha: doc_id: cord_uid: fgn rps background: porcine reproductive and respiratory syndrome virus (prrsv) is a major pathogen of swine worldwide. emergence in of a novel highly pathogenic prrsv (hp-prrsv) isolate in china necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (tbln) days post-infection with hp-prrsv rjxwn , prrsv strain vr- or sham inocula. rna from each was prepared for next-generation sequencing. amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an rnaseq analysis pipeline to determine differential gene expression. transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library. results: major changes in transcript abundance occurred in response to infection with either prrsv strain, each with over differentially expressed transcripts. the largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid a acute-phase isoforms. however, the degree of up or down-regulation of transcripts following infection with hp-prrsv rjxwn was greater than transcript changes observed with us prrsv vr- . also, of significantly altered transcripts within the hp-prrsv rjxwn library were up-regulated and were down-regulated more than -fold, whilst in the us prrsv vr- library only transcripts were up-regulated and were down-regulated more than -fold. conclusions: the magnitude of differentially expressed gene profiles detected in hp-prrsv rjxwn infected pigs as compared to vr- infected pigs was consistent with the increased pathogenicity of the hp-prrsv in vivo. porcine reproductive and respiratory syndrome virus (prrsv), the causative agent of prrs in swine, is a member of the arteriviridae family in the order nidovirales. prrsv causes highly significant economic losses to the swine industry worldwide [ ] as a result of both reproductive failure (late-term abortions and stillbirths) in pregnant sows and respiratory disease (pneumonia) in nursery and grower/finishing pigs [ ] . infection with prrsv also predisposes pigs to infection by bacterial pathogens as well as other viral pathogens [ ] [ ] [ ] [ ] [ ] , as such, prrsv is a key etiological agent of the porcine respiratory disease complex (prdc). clinical disease caused by prrsv is highly variable, ranging from mild, subclinical infection to acute death of adult animals [ ] . differences in virulence have been attributed to numerous factors including host genetics, management practices, and virus strain heterogeneity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . relatively little is known about the interactions of prrsv and host cells. the lymph node is an anatomic site where the innate immune response and adaptive immune system interface. tracheobronchial lymph nodes (tbln) in swine drain the lung field and provide the focal structure that can reproducibly be identified. although the tbln contains a number of cell types, sampling this tissue allows study of direct and indirect effects of an infectious agent on the lung and cells within the lymph node. in a unique syndrome with high morbidity and mortality was recognized in growing pigs in china that was originally known as porcine high fever disease (phfd) due to its uncertain etiology [ ] . experimental infection of pigs in china with these novel viral isolates reproduced the clinical disease providing strong evidence for the role of prrsv as the causal agent of phfd. however, there was still a question as to whether there was some unknown agent in the prrsv preparations that increased the severity of the clinical disease over what was expected for a "routine" prrsv infection. this question was resolved when phfd was reproduced in china with virus derived from an infectious clone of the jx prrsv isolate [ ] demonstrating that prrsv isolates with a common genetic motif had a causal role in phfd leading to this lineage of virus being called highly pathogenic prrsv (hp-prrsv). we imported a plasmid containing a full-length clone of the jxwn hp-prrsv isolate [ ] from which infectious virus (rjxwn ) was rescued. an animal study was conducted comparing the pathogenicity of hp-prrsv isolate rjxwn with the north american prototype strain vr- prrsv [ ] . the objective of this report was to investigate gene expression profiles in porcine tracheobronchial lymph node (tbln) during viral infection with hp-prrsv rjxwn strain alongside of us prrsv strain vr- at a snapshot of days post-infection using bioinformatics. mapping short rna-seq reads and estimating transcript expression levels genomic short-read nucleotide alignment program (gsnap) was used for alignment and genome construction, and cufflinks to determine if differential expression and changes in transcript abundance were statistically significant [ , ] . the rnaseq yielded , , reads for the control, , , reads for the hp-prrsv, and , , for vr- libraries after quality trimming and excluding any reads less than bp. cufflinks was used to measure transcript abundances in fragments per kilobase of exon per million fragments mapped (fpkm). the cuffdiff output contained normalized fpkm for comparison between libraries (additional file ). these values were used to calculate the fold change (log transformed) in expression between the experimental unit and the control. examination of the rnaseq data indicated that there were major changes in transcript abundance occurring in the prrsv-infected tbln-based unique transcripts [cuffdiff output (additional file )]. of these total transcripts, were found to be significant hits in the hp-prrsv rjxwn library and were significant in the us prrsv vr- library (table ) . of those significant hits within the hp-prrsv rjxwn library hits were up-regulated and were down-regulated more than -fold whilst in the us prrsv vr- library hits were up-regulated and were downregulated more than -fold. this derived catalog of expressed genes represents the first comparative analysis of the hp-prrsv rjxwn and vr- -infected tbln transcript abundance profiles and provides a database that informs us of genes involved in normal tbln physiology, as well as genes whose abundance is altered by prrsv infection. gene annotation of all significant hits (additional files and ) was then carried out using a mysql database matching the ensmbl (sscrofa . ) chromosome location of aligned transcripts to gene names. gene ids and log fold-change expression values for significant hits, that had fpkm values in both the control and the infected differential expression testing for transcripts (cuffdiff output files), were then analyzed using the ingenuity pathway analysis software. when comparing the tbln transcriptome from sham-inoculated controls vs. the hp-prrsv rjxwn -infected pigs, of the gene ids mapped to the ingenuity knowledge base and were up-regulated while were down-regulated. in the tbln of control vs. vr- -infected pigs, of the gene ids mapped to the ingenuity knowledge base and only were up-regulated while were down-regulated. table lists the top ten genes (named by the hugo gene nomenclature committee (hgnc) [ ] ) we detected that had a significant value in both hp-prrsv rjxwn and vr- -infected tbln rnaseq cuffdiff output and a fold-change increase or decrease of greater than . transcripts up-regulated in both hp-prrsv rjxwn and vr- -infected tbln by > -fold and > -fold vs. control tbln, respectively, were three serum amyloid a (saa ) acute-phase isoforms, as well as gene enssscg f s c _pig serum amyloid protein (no hgnc annotation), that are expressed in response to inflammatory stimuli ( table ). other annotated genes ( table ) that were up-regulated in hp-prrsv rjxwn tbln vs. control were resistin (retn) which is secreted by immune and epithelial cells and participates in the immune response by increasing transcriptional events that increase expression of several proinflammatory cytokines [ ] ; three members of the s family (s a , s a , s a ) of calcium-binding proteins localized in the cytoplasm and/or nucleus of a wide range of cells, involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation, and mediators of inflammatory and protective anti-infection responses [ ] ; xanthine dehydrogenase (xdh) a generator of reactive oxygen species and possible cause of hypoxia-mediated lung injury [ ] ; and peptidylarginine deiminase, type iv, (padi ) which may play a role in granulocyte and macrophage development leading to inflammation and immune responses [ ] . also in the top ten up-regulated transcripts were two genes without a hgnc symbol, trem _pig (enssscg ) trigger receptor, which is expressed on myeliod cells, and the interleukin- receptor, type ii gene (il r ) which is associated with host responses to subdue inflammation as a consequence of disease. down-regulated in hp-prrsv tbln vs. control tbln were diacylglycerol oacyltransferase (dgat ) which catalyzes triglyceride synthesis which is critical for formation of adipose tissue [ ] ; perilipin- (plin ) an important regulator of lipid storage; a member of the cytochrome p monooxygenases (cyp b ) of unknown specific function; soluble galactosebinding lectin (lgals ), cell death-inducing dffalike effector c (cidec), tumor suppressor candidate (tusc ), protein phosphatase , regulatory (inhibitor) subunit a (ppp r a), c-type lectin domain family , member g, that encodes a glycan-binding receptor and a member of the c-type lectin family which plays a role in t-cell immune responses (clec g). also in the top ten down-regulated transcripts were the following genes without projected hgnc symbols: ces liver carboxylesterase (enssscg ) and f sty _pig thyroid hormone-responsive protein (enssscg ). in vr- -infected pig tbln vs. control tbln, transcript abundance was down-regulated to a lesser extent and featured genes linked to metabolism in adipose tissue and regulation in neuronal activity functions including dermatopontin (dpt) extracellular matrix protein with possible functions in cell-matrix interactions and matrix assembly which enhances transforming growth factor beta (tgfb ) activity; beta- adrenergic receptor (adrb ); solute carrier family , facilitated glucose transporter member (slc a ); uncharacterized mlx interacting protein-like protein (mlxipl); basic helix-loop-helix transcription factor (tcf ); forkhead box transcription factor protein c (foxc ); protein phosphatase regulatory subunit b also known as dopamine-and camp-regulated neuronal phosphoprotein (ppp r b); potassium voltage-gated channel, kqt-like subfamily, member (kcnq ) that is thought to play a critical role in the regulation of neuronal excitability; plexin domain containing (plxdc ); and adenosine a receptor (adora ). analysis of the genomic data in the context of gene ontology, by ingenuity pathway analysis (ipa), allowed us to ascribe biological functional networks to the differentiated transcript abundance dataset. the top functions identified with the ingenuity canonical pathway list, filtered to apoptosis, cellular immune response, cytokine signalling, humoral immune responses and pathogeninfluenced signalling, based on differentially expressed genes were: granzyme a signalling, crosstalk between dendritic cells and natural killer cells, il- signalling, role of pattern recognition receptors in recognition of bacteria and viruses, il- signalling and production in macrophages, complement system, interferon signalling, communication between innate and adaptive immune cells, il- a signalling in fibroblasts, granzyme b signalling, production of nitric oxide and reactive oxygen species in macrophages, differential regulation of cytokine production in macrophages and t helper cells by il- a lgals and il- f that were above the threshold of p value < . , as calculated by fischer's test representing the ratio of number of genes from the dataset that map to the pathway and the number of all known genes ascribed to the pathway. the genes up-regulated in the hp-prrsv rjxwn infected pigs' tbln were associated in networks: from biological networks with functions associated with cell death, antimicrobial responses and cancer, with the highest network score of , i.e. the likelihood of genes in this network would have approximately a - chance of occurring randomly, and focus molecules, i.e. the starting points for generating biological networks; to networks with functions associated with nervous system development and function, organ morphology and reproductive system disease with a score of and focus molecule. many of the upregulated networks related to cell death and inflammatory response functions fit with the results previously reported [ , ] where hp-prrsv strain rjxwn caused severe disease, resulted in up to x higher abundance of virus and produced an exacerbated release of cytokines, including pro-inflammatory cytokines, when compared to type prototype strain vr- . wide spread tissue damage [ ] and cell death were observed as predicted by up-regulation of celldeath associated genes (circled in orange in figure ) in the network representation of the mostly highly rated network for hp-prrsv rjxwn by ipa. the downregulated network functions in the hp-prrsv rjxwn infected tbln included activities associated with cellular function and maintenance, tissue morphology, metabolic disease, organismal development, carbohydrate metabolism, lipid metabolism, small molecule biochemistry, post-translational modification, protein folding, developmental disorder, which may be associated with cell death and reflects a severe disease state. similarly, the down-regulated network functions in the vr- infected tbln were associated with cellular function, maintenance, development and organization. this study produced transcriptional profiles of tblns from non-infected, hp-prrsv rjxwn and us prrsv vr- -infected pigs that provides insight into immune figure ingenuity pathways analysis summary. to investigate possible interactions of differently regulated genes, datasets representing genes with altered expression profile obtained from the rnaseq data for hp-prrsv rjxwn were imported into the ingenuity pathway analysis tool and the following data is illustrated: the network representation of the most highly rated network (gene expression, cell death, lipid metabolism). the genes that are shaded were determined to be significant from the statistical analysis. the genes shaded red are up-regulated and those that are green are down-regulated. the intensity of the shading shows to what degree each gene was up or down-regulated. a solid line represents a direct interaction between the two gene products and a dotted line means there is an indirect interaction. genes associated with cell death are circled with orange color. dysregulation elicited by the virus on host transcript abundance levels necessary for a effective immune response. this rna-seq compendium extends the analyses of previous gene expression atlases performed using affymetrix genechip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing [ , , ] . it is well established that many pathogens cause changes in expression of specific genes that act to protect the host and clear the infection. prrsv strains differ in their dysregulation of the immune response to infection and delay in development of a protective immune response in vaccinated pigs [ , ] . a higher number of significantly differentially expressed gene instances were detected in hp-prrsv rjxwn than vr- when normalized to control samples at a snapshot of days post inoculation (dpi). as anticipated, some of the genes (e.g., resistin) and pathways identified would be expected to be involved in the host response to a severe disease. in the case of resistin, it would be expected that adipose tissue stores are being mobilized as part of the host response to infection, which includes a high fever typical of infection with hp-prrsv. there are specific cellular proteins that regulate a protective immune response, for example the pro-inflammatory genes that were upregulated to a greater extent in hp-prrsv rjxwn than vr- when normalized to control samples as observed when comparing the pathogenicity of hp-prrsv isolate rjxwn with the north american prototype strain vr- prrsv. at dpi hp-prrsv rjxwn inoculated pigs had an interstitial pneumonia that was significantly more severe than thevr- inoculated group which appeared to be convalescing [ ] . future studies of these differentially expressed genes, their transcript abundance, protein level, and protein function will enhance our understanding of the interaction of prrsv with the host. identification of new virulence mechanisms of prrsv may improve the prospects for rational design of more effective vaccines to limit viral replication and shedding. marc- cells were cultured in minimum essential medium (emem, safc c) with % fetal bovine serum at °c, % co . wild-type (wt) type prrsv strain vr- (genbank u ), passage on marc- cells, was titrated and used for the swine study. virus (rescued jxwn ; rjxwn ) was rescued from a cloned cdna of chinese highly pathogenic type prrsv strain jxwn [pwsk-jxwn; genbank ef , [ ] ] and passaged times on marc- cells for use in the swine study. the animal use protocol was reviewed and approved by the institutional animal care and use committee (iacuc) of the national animal disease center-usda-agricultural research service. thirty-two -week-old cross-bred pigs were obtained from a u.s. high-health herd and were found to be free of prrsv and influenza virus antibodies using commercially available enzymelinked immunosorbent assay (elisa) kits (herdchek prrs xr; idexx laboratories, westbrook, maine) and np elisa (multis elisa, idexx, westbrook, maine), respectively. pigs were also confirmed negative for porcine circovirus type by quantitative real-time pcr [ ] . one day prior to starting the experiment, pigs were bled, weighed and randomly assigned to one of four groups. group (n = ) consisted of negative control pigs, which received an intranasal ml sham inoculum of minimum essential media (mem) on dpi. group pigs (n = ) were challenged intranasally with ml of × % tissue culture infective dose (tcid )/ml of chinese prrsv strain rjxwn in animal biosafety level- -agriculture (absl- -ag) housing, where they remained for the duration of the experiment. group consisted of naïve pigs (n = ) that were placed in contact with group swine on dpi. group pigs (n = ) were challenged intranasally with ml of × tcid /ml of type prototype strain vr- . groups and were housed in separate isolation rooms in an absl facility. animal care and euthanasia were conducted in accordance with the report of the avma panel on euthansia and under the supervision of iacuc of nadc. serum and bronchoalveolar lung lavage fluid (balf) were tested for infectious virus as described previously [ ] . lungs were scored for gross lesions [ ] and sections fixed for histopathology. swabs were collected from balf, and various sites for bacterial isolation [ ] . following humane euthanasia, tracheobronchial lymph nodes (tbln) from in vivo hp-prrsv rjxwn (n = ), us prrsv vr- (n = ), or sham-infected pigs (n = ) were harvested at days post-infection and total cellular rna was prepared as follows. one gram of tbln from each pig was collected immediately upon necropsy, minced and stored in rnalater (life technologies, grand island, ny) at − °c until homogenized for extraction of total rna with magmax ™ - for microarrays total rna isolation kit (applied biosystems, carlsbad, ca) using the manufacturer's protocol. the integrity of the rna was confirmed with a bioanalyzer and rna nano-chip (agilent, santa clara, ca). the samples used had an average rna integrity number (rin) value of . and s: s rrna ratio of . . cdna library construction cdna libraries were constructed from pooled total cellular rna from the tbln in each treatment group using truseq sample prep kits (illumina inc., san diego, ca) and sequenced by × paired-end sequencing on an illumina hiseq instrument. in order to analyze the illumina reads, a series of bioinformatics methods were used to investigate gene expression profiles in tbln during prrsv infection with hp-prrsv rjxwn and us prrsv vr- at a snapshot of dpi. this was carried out with the construction of a rnaseq analysis pipeline ( figure ) comprised of gsnap for alignment and genome construction, and cufflinks to determine if differential expression and changes in transcript abundance were statistically significant. three files of transcriptome data from the sham, hp-prrsv rjxwn and us prrsv vr- inoculated groups were aligned to the ucsc pig genome build using the gsnap alignment program in preparation for differential expression analysis. the next step in the pipeline was to put the gsnap output into the cufflinks program and run it through three separate utilities or tools within the software package; cufflinks, cuffmerge, and cuffdiff. first the three files were run through cufflinks in order to assemble the aligned rna sequence reads into transcripts and estimate the abundances in fpkm of the paired-end reads. the cufflinks q-value was the false discovery rate (fdr)-adjusted p-value of the uncorrected test statistic. the q-value used in this study was . . the significance status was "yes" when p was greater than q after benjamini-hochberg correction for multiple-testing (additional file ). cuffmerge was then used to create a single transcript dataset from the multiple reconstructions. two runs were then conducted using the hp-prrsv rjxwn vs. control and the us prrsv vr- vs. control datasets using the cuffdiff program to test for differential expression and regulation amongst the two disease states. gene annotation of all significant hits was then carried out using a mysql database matching to the ensembl sscrofa . reference genome currently supported by the integrative genomics viewer (broad institute). datasets representing genes with altered expression profile derived from rnaseq analyses were imported into the ingenuity pathway analysis tool (ipa tool; figure computational pipeline. rnaseq analysis pipeline comprised of gsnap for alignment and cufflinks to determine if differential expression, and changes in transcript abundance were statistically significant (adapted from [ ] ). ingenuity w systems, redwood city, ca, usa; http:// www.ingenuity.com). in ipa, differentially expressed genes were mapped to genetic networks available in the ingenuity database and then ranked by score. the basis of the ipa program consists of the ingenuity pathway knowledge base (ipkb) that is derived from known functions and interactions of genes published in the literature. thus, the ipa tool allows the identification of biological networks, global functions within the host and functional pathways of a particular dataset. the program also gives the significance value of the differentially expressed genes, the other genes with which it interacts, and how the products of the genes directly or indirectly act on each other, including those not involved in the microarray analysis. the networks created are ranked depending on the number of significantly expressed genes they contain and also list diseases that were most significant ( figure ). assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states general overview of prrsv: a perspective from the united states porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine laboratory investigation of prrs virus infection in three swine herds a brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in illinois associations between genetics, farm characteristics and clinical disease in field outbreaks of porcine reproductive and respiratory syndrome virus comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrumdeprived pig model reproductive failure of unknown etiology genetic perspectives on host responses to porcine reproductive and respiratory syndrome (prrs) heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development lelystad virus and the porcine epidemic abortion and respiratory syndrome emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus de novo assembly and analysis of rnaseq data transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation genenames.org: the hgnc resources in cloning and characterization of porcine resistin gene porcine s a and s a : molecular characterizations and crucial functions in response to haemophilus parasuis infection effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothelial cells citrullination by peptidylarginine deiminase in rheumatoid arthritis identification of a gene encoding an acyl coa: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis aberrant host immune response induced by highly virulent prrsv identified by digital gene expression tag profiling hp-prrsv challenge of and -week-old pigs indepth global analysis of transcript abundance levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs effect of vaccination with selective bacterins on conventional pigs infected with type porcine circovirus in vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp deletion mutants coinfection of pigs with porcine respiratory coronavirus and bordetella bronchiseptica uncovering the complexity of transcriptomes with rna-seq submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • 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 we thank the following members of the virus and prion research unit at the national animal disease center: j. huegel, j. crabtree, a. burow, d. adolphson, s. anderson, m. kappes and a. vorwald for technical assistance. we also gratefully acknowledge d. alt of the genomics unit at the national animal disease center, and a. severin of iowa state university ngs bioinformatics, for assistance in data analysis. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. additional file : cuffdiff output of all significant hits for comparison, using the hp-prrsv rjxwn vs. control and the us prrsv vr- vs. control datasets, to test for differential expression and regulation amongst the two disease states. gene annotation (symbol) was carried out using a mysql database matching to the ensembl sscrofa . reference genome currently supported by the integrative genomics viewer (broad institute). location (row.names); gene annotation (symbol); entrez gene name; treatment (sample_ , sample_ ); status; abundance in fpkm (value_ , value_ ); differential expression (log _fold_change); test statistic (test_stat); p-value (p_value); false discovery rate (fdr)-adjusted p-value of the uncorrected test statistic (q_value); significance status after benjamini-hochberg correction for multiple-testing (significant).additional file : transcript sequences of all significant hits inthe hp-prrsv rjxwn vs. control and the us prrsv vr- vs. control datasets. the authors declare that they have no competing interests.authors' contributions lcm: study conception, data collection and analysis, research design, manuscript writing. df: research design, data analysis. aa: data analysis. dob: rnaseq interpretation discussions and data analysis. bg: infectious clone and production of virus stocks. kml: study conception, animal study execution, virus stocks, data collection, and manuscript preparation and writing. jnh: data collection. sns: data collection. h-cy: infectious clone of hp-prrsv strain jxwn . ksf: study conception, rescue of hp-prrsv infectious clone, and production of virus stocks. mek: study conception and manuscript preparation and writing. all authors read and approved the final manuscript. key: cord- -cndq aqb authors: xue, chunyi; wang, wei; liu, qiliang; miao, zhongwei; liu, kang; shen, huifang; lv, lishan; li, xiaoming; chen, xiaochun; cao, yongchang title: chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus gp protein and the influenza virus ha and m proteins date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: cndq aqb both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. these diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. in this study, we have developed a chimeric virus-like particle (vlp) vaccine candidate for porcine reproductive and respiratory syndrome virus (prrsv) and h n influenza virus and investigated its immunogenicity in mice. the ha and m proteins from the h n influenza virus and the prrsv gp protein fused to the cytoplasmic and transmembrane domains of the na protein were both incorporated into the chimeric vlps. analysis of the immune responses showed that the chimeric vlps elicited serum antibodies specific for both prrsv gp and the h n ha protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. taken together, the results suggested that the chimeric vlp vaccine represents a potential strategy for the development of a safe and effective vaccine to control prrsv and h n influenza virus. influenza a virus is a segmented, negative-stranded rna virus belonging to the family orthomyxoviridae. among the large variety of species that influenza a viruses infect naturally, swine influenza virus (siv) causes an acute, highly contagious respiratory disease in swine. epithelial cells in the swine respiratory tract have receptors for both avian and mammalian influenza viruses [ ] . therefore, pigs might serve as ''mixing vessels'' for the generation of new reassortant strains with pandemic capacity. three predominant subtypes are prevalent in different countries: h n , h n , and h n . porcine reproductive and respiratory syndrome virus (prrsv), the causative agent of porcine reproductive and respiratory syndrome (prrs), is a member of the family arteriviridae in the order nidovirales [ ] . this virus causes one of the most economically important infectious diseases for the swine industry worldwide [ ] . prrs is predominantly characterized by reproductive failure in breeding swine, pre-weaning mortality, and respiratory disorders in pigs of all ages [ , , ] . vaccination has been an effective way to reduce the incidence of diseases resulting from influenza virus and prrsv infections. compared to conventional vaccines such as killed vaccine and attenuated vaccine, virus-like particles (vlps) have been demonstrated to be a promising alternative candidate [ , , ] ,. as a new form of vaccine candidate, the non-infectious nature of vlps and their lack of viral genomic material are attractive safety features that may be suitable for a variety of viruses [ , , , , , ] . both b cell-mediated antibody and specific t-cellmediated cellular responses were elicited by vlps. vlps not only mimic the overall structure of the virion, but they also present conformational epitopes of surface proteins, which can be readily recognized and processed by antigenpresenting cells [ , , , , ] . the protective effects of vlps have been demonstrated in clinical and preclinical trials [ , , , , ] . an important advance would be the development of new vlps with an enhanced breadth of immunity, which could potentially be used to prevent infection by prrsv and siv. in a previous study, we expressed the gp proteins of prrsv on the surface of chimeric vlps, which elicited a humoral and cellular immune response and a neutralization antibody response to prrsv [ , ] . based on this platform, we hope to generate chimeric vlps for protection against both prrs and influenza. in this study, we chose the h n influenza virus as the basis for vlp production. as expected, the fusion protein na/gp in combination with ha and m effectively formed chimeric vlps. next, we demonstrated that the chimeric vlps induced a potent immune response to both prrsv and h n siv in a balb/c mice model. hence, the results suggested that the chimeric vlp vaccine is a new vaccine candidate for protection against both prrs and h n influenza. spodoptera frugiperda sf cells were maintained in serumfree sf ii medium (gibco) at °c in spinner flasks at a speed of rpm. the prrsv strain gdkp/ / [ ] was propagated on marc- cells that were maintained in dulbecco's modified eagle's medium (dmem, gibco) supplemented with penicillin-streptomycin and % fetal calf serum at °c and % co . the h n strain of the influenza virus (a/swine/guangdong/ / (h n )) was propagated in mdck cells under the same conditions. t cells were maintained in dmem supplemented with penicillin-streptomycin and % fetal calf serum at °c and % co . the ha, na and m genes of the h n influenza virus (accession numbers fj . , fj . and fj . ), the gp gene of prrsv (accession number gq ), and the na/gp fusion gene were inserted into the pfast-bac-dual vector as described previously [ , ] . the na/gp and m genes were cloned into the same vector (pfast-bac-dual) under the control of different promoters. all of the plasmids were confirmed by dna sequencing to ensure that no additional changes were introduced during the pcr. the recombinant baculoviruses (rbvs) were derived from the transfer plasmids pfast-bac-dual-ha, pfast-bac-dual-gp and pfast-bac-dual-na/gp -m using the bac-to-bac baculovirus expression system. the viruses harvested from the supernatant were subjected to three rounds of plaque purification. sf cells were co-infected with rbvs expressing na/gp -m and ha at different ratios ( . , , , , , and ) and then incubated for h at °c. the sf cells showed a high degree of cytopathology. the culture supernatants were collected and centrifuged at g for min at °c and analyzed by western blot. the expressed influenza virus proteins ha and m were detected with mouse polyclonal sera against the h n influenza virus. the fusion protein na/gp was detected with mouse polyclonal sera against prrsv. horseradish peroxidase (hrp)conjugated goat anti-mouse igg polyclonal antibodies were used as the secondary antibody (ptglab, usa). to produce vlps containing ha, m and na/gp , sf cells were co-infected with rbvs expressing the ha and na/gp -m proteins at ratios of and . after incubation for h at °c, the vlps were harvested and purified using %- %- % (w/v) discontinuous sucrose step density gradient ultracentrifugation at , g for min at °c. the fractions were collected and analyzed for the presence of the ha, m , and na/gp proteins by western blot. the na/gp content of chimeric vlps was quantified by grayscale scanning of an sds-page gel. the hemagglutination activity of the vlps was determined using chicken red blood cells. for electron microscopy, the sucrose-gradient-purified vlps were applied to a carboncoated formvar grid for min. then, the grid was immediately stained with % phosphotungstic acid (ph . ) for s. the stained vlps were observed by transmission electron microscopy (jem- cx-ii, jeolltd, japan). four groups of six-week-old, female, inbred spf balb/c mice (n = ) were housed in microisolator units and allowed free access to food and water. the mice were immunized intramuscularly (i.m.) with lg of the chimeric vlps containing lg na/gp proteins with complete freund's adjuvant (cfa) for the primary immunization (weeks ) or incomplete freund's adjuvant (ifa) for subsequent boosts (weeks and ). the positive control groups were immunized i.m. with % formalininactivated prrsv in which the amount of gp was equal to that of the chimeric vlps or % formalin-inactivated h n influenza virus in which the amount of ha was equal to that of the chimeric vlps. pbs was injected as the negative control. blood samples were collected before immunization on the th, th, and nd day after primary immunization. the experiments were carried out in accordance with the ethical guidelines for animal protection in china and approved by the sun yat-sen university animal ethics committee. all procedures were performed under anesthesia, and all efforts were made to minimize suffering. briefly, -well plates were coated with ll of antigens at a concentration of lg/ml in coating buffer ( . m sodium carbonate, ph . ) at °c overnight. the antigens used as targets were extracts prepared from t cells transfected with pfast-bac-dual-ha (to express ha protein) or pfast-bac-dual-gp (gp protein) [ , ] . the plates were then blocked with pbs containing . % tween- and % bsa at °c for h and incubated with serial dilutions of each sample at °c for h. following thorough washing in pbs containing . % tween- , all of the samples were incubated with horseradish peroxidase (hrp)-labeled goat anti-mouse igg with a stock concentration of mg/ml, : , diluted in pbs containing . % tween- and . % bsa at °c for h. the unbound antibodies were removed, and the wells were thoroughly washed. the substrate , , , -tetramethylbenzidine (tmb, sangon, china) in citrate-phosphate buffer (ph . ) containing . % h o was used for color development. the reaction was terminated with . m h s , and the absorbance was determined at nm using a spectrophotometer (bio-tek elx uv, usa). lymphocytes were isolated from the spleens for cytokine elispot assays. briefly, spleens were carefully rinsed with sterile pbs and depleted of erythrocytes by treatment with ammonium chloride ( . m, ph . ). following thorough washing with pbs, cells were isolated from spleens using mouse lymphocyte separation medium (dakewe, china). the collected cells were centrifuged at g for min at room temperature and then resuspended in lympho-spot serum-free medium rodent (dakewe). cell viability was determined by staining with . % trypan blue (sigma). pre-coated anti-mifn-c or anti-mil- ( lg/ml in coating buffer, bd/pharmingen) -well plates (millipore) were incubated with ml lympho-spot serum-free medium rodent at °c for min and then were incubated with splenocytes isolated from vaccinated mice at /well. splenocytes were stimulated with gp protein or ha protein at a concentration of lg/ml. additional wells of cells were stimulated with pma ( ng/ml), and ionomycin ( ng/ml) or were mock stimulated. the plates were incubated for h at °c with % co . plates were thoroughly washed with pbs containing . % tween- and incubated with biotinylated anti-mifn-c or anti-mil- at °c for . h. then, the plates were washed and incubated with streptavidin conjugated to alkaline phosphatase at °c for . h. following extensive washing, antibody-cytokine-antibody complexes were incubated with stable bcip/nbt chromagen at °c for min. the plates were rinsed with ddh o and air dried at °c for h. the spots were counted using an immunospot elispot reader (bio-reader , bio-sys, germany). the sera were complement-inactivated at °c for min before testing, serially diluted twofold in dmem without serum, and then mixed with prrsv ( tcid ). the mixtures were added to prewashed marc- cells growing in -well tissue culture dishes and incubated at °c with % co for h. the cells were examined to observe the appearance of the cytopathic effects (cpe). the neutralization titers were expressed as the reciprocal of the highest serum dilution that neutralized tcid of prrsv in % of the wells. the hemagglutination inhibition (hi) assay was used to assess the ability of functional ha-specific antibodies to inhibit agglutination of chicken erythrocytes. briefly, sera were treated with receptor-destroying enzyme and serially diluted twofold in v-bottom -well microtiter plates. an equal volume ( ll) of four adjusted hemagglutination units of the inactivated h n strain of influenza virus was added to each well. the plates were covered and incubated at room temperature for min followed by the addition of ll of % red blood cells (rbcs) in pbs. the hi titer was determined as the reciprocal dilution of the last row that contained non-agglutinated rbcs. negative serum controls were included for each plate. the geometric mean hi titers and standard error were calculated within each group. all parameters were recorded for individual mice within all groups. statistical comparisons of the data between groups were carried out using an analysis of variance test (anova). statistical analyses were performed using student's two-tailed test with equal variance. p-values less than . (p \ . ) were considered statistically significant. cells were coinfected with recombinant baculoviruses expressing na/gp -m and ha proteins at different ratios. the culture supernatant was harvested and analyzed by western blot (fig. a and b ). different ratios led to different levels of protein expression. the results suggested that infection with rbvs expressing ha and na/gp -m proteins at a ratio of : was optimal for chimeric vlp production. chimeric vlps were produced and released into the culture supernatant of insect cells coinfected with rbvs expressing ha, m , and na/gp as described. the vlps were purified by sucrose gradient ultracentrifugation and characterized by western blot. the fractions containing vlps could be observed at a sucrose density between - %, and the incorporation of ha, m , and na/gp protein into the vlps was confirmed ( fig. a and b) . electron microscopic examination of negatively stained samples revealed the presence of vlps with a diameter of approximately nm (fig. c) . the ha titer was . the immunogenicity of the chimeric vlps was evaluated in a mouse immunization trial in the presence of freund's adjuvants. ha protein was used as an antigen for h n influenza virus antibody detection. mice vaccinated with the vlps and inactivated virus vaccine formulations elicited serum anti-ha protein antibody responses following the first vaccination that were boosted by the second vaccination (fig. b) . the group in which vlps were infected intramuscularly and the positive control group showed considerable igg titers that were significantly higher than those of the negative control group (p \ . , fig. b ). the differences in igg titers between the vlp group and the positive control was not statistically significant (p [ . ). importantly, the chimeric vlps induced significant igg titers against the prrsv gp protein compared with the negative control group (p \ . , fig. a) , and the difference in igg titers between the vlp group and the positive control group was not statistically significant (p [ . ) which was the same as the immune response to the ha protein. these data show that the fusion of the ectodomain of na did not hinder the function of gp as an antigen. meanwhile, the production of an immune response to the ha protein indicated that these chimeric vlps administered via the intramuscular route could effectively induce a response to two different antigens that was comparable to that induced by the positive control vaccine. the numbers of ifn-c and il- cytokine-secreting cells were determined using specific and sensitive elispot assays. spleens were harvested from vaccinated mice, and lymphocytes were isolated after the last immunization. the cells were stimulated with either gp protein or ha protein. our data showed that significant numbers of ifn-cand il- -secreting cells were induced in the chimeric vlp group or the positive control group, but not in the negative control mice (p \ . ), whether gp protein (fig. a and b) or ha protein (fig. c and d) was used as the stimulus. it is notable that the mice immunized with the chimeric vlps elicited both th -and th -type cellular immune responses. this might be helpful to suppress viral replication and reduce viral infection for both the prrsv and h n influenza virus. because neutralizing activities play an important role in the first line of defense against prrsv infection and the clearance of prrsv, most likely conferring protective immunity, we performed neutralization assays. as shown in fig. , prrsv-specific neutralizing antibodies were detected on the th day after primary immunization, they were elevated on the th day, and they increased to the highest level in the nd day. furthermore, no neutralizing antibodies against prrsv were detectable in the pbs group. the hi results demonstrated that a single intramuscular immunization with the vlp vaccine in the presence of adjuvant elicited a relatively modest hi titer against the inactivated h n influenza virus strain, and the two booster immunizations induced a high hi titer, indicating that the vlps were strongly immunogenic (fig. ) . prrsv and siv coinfect pigs under certain conditions. these two agents associated with mycoplasm hyopneumoniae (mhyo), pasturella multocida, and porcine circovirus type (pcv ) have been reported to constitute the porcine respiratory disease complex (prdc) [ ] . a previous study indicated that siv and prrsv are the primary etiological agents associated with respiratory disease in pigs [ ] . the presence of prrsv between vaccinations reduced the efficacy of the vaccine, but it did not negatively impact either the systemic or local antibody response to either siv vaccination or challenge [ ] . previously, we prepared chimeric vlps composed of the m protein from h n influenza virus and a fusion protein, denoted as na/ gp , containing the cytoplasmic and transmembrane domains of the h n virus na protein and the prrsv gp protein. we demonstrated that immunization of balb/c mice with these chimeric vlps could stimulate a robust immune response [ , ] . based on this result, an important advance would be the development of new vaccines, which could be used to prevent coinfection by prrsv and siv. fig. . serum samples were collected on the th, th, and nd day after primary immunization to determine the neutralization antibody titers. the data represent the mean s.d. for eight mice. the differences in the neutralization antibody titers between the experimental groups were statistically significant (**p \ . ) fig. hemagglutination-inhibition (hi) titers. serum samples were collected on the th, th, and nd day after primary immunization of mice ( per group) vaccinated with chimeric vlps, inactivated h n virus, or pbs. the hi responses were assessed compared with the h n viruses. the differences in hi titers between the experimental groups were statistically significant (**p \ . ) the influenza virus virion is surrounded by a lipid membrane containing two major glycoproteins, ha and na. the ha protein is the most abundant glycoprotein and is responsible for the attachment of the virus to terminal sialic acid residues on host cell receptors and mediating fusion between viral and cellular membranes [ ] . influenza vlps could be obtained by the expression of four viral proteins (ha, na, m , m ) [ ] , three viral proteins (ha, m , na) [ , , , , ] , two viral proteins (ha, m ) [ , , ] , or even ha alone [ ] . recently, it has been demonstrated that influenza vlps can be made from m fusion proteins [ ] . therefore, influenza vlps were chosen as a platform on which prrsv proteins (gp ) can be expressed. a previous report showed that, based on the assembly of newcastle disease (nd) vlps [ ] , it was possible to develop chimeric vlps containing respiratory syncytial virus (rsv) g proteins using nd vlps as a platform [ ] , as well as nd vlps containing rsv g and f proteins [ ] . in our study, we showed that the ha protein could assemble with na/gp and m to form chimeric vlps. compared to our previous results, ha had no influence on the formation of chimeric vlps, but the size and morphology of the chimeric vlps were more similar to those of the natural influenza virus. the ha titer of the chimeric vlps indicated that ha was structurally and functionally incorporated into the particles. the results suggested that it might be feasible to generate chimeric influenza vlps in this way. the immunogenicity of chimeric vlps in mice demonstrated that they were effective for inducing immunity. our data showed that both the inactivated virus and the chimeric vlp vaccine generated comparable amounts of prrsvspecific or h n -influenza-virus-specific antibodies, as measured by elisa. splenocytes proliferated vigorously and produced both ifn-cand il- in response to stimulation with gp or ha antigen. following three immunizations, the vlp vaccine induced hi antibodies against the homologous influenza strain in mice. our findings provide evidence that chimeric vlps can induce protective immunity against h n influenza virus similarly to the killed virus. meanwhile, when we measured the neutralizing antibody titers, significantly elevated neutralizing antibodies to prrsv were achieved in the vlp group as well as with inactivated prrsv (p \ . ). this result demonstrated that the prrsv gp protein co-incorporated with influenza h n na and formed chimeric vlps with ha and m , which could be identified and transferred efficiently as a prrsv antigen. it was reported recently that vlps of the hepatitis b virus core protein containing five mimotopes of infectious bursal disease virus (ibdv) protected chickens against ibdv [ , ] . the use of both genetically engineered norovirus vlps incorporating relevant epitopes from multiple strains and multivalent vaccine formulations increases the breadth of the immune response to diverse variants within a genotype [ ] . in summary, we found that the chimeric vlps induced antibodies against two diseases at the same time, in contrast to the chimeric vlps in our previous study. the data presented in this study showed that intramuscular immunization of mice with these chimeric vlps induced systemic immune responses, including both humoral and cellular immune components. similar to our research, chimeric sars-cov s glycoprotein and influenza m efficiently form vlps that protect mice against challenge with sars-cov [ ] . experimental triple-ha vlps containing ha proteins derived from the h n , h n , and h n viruses were immunogenic and protected ferrets from challenge with all three potentially pandemic viruses. additionally, vlps containing ha subtypes derived from the seasonal h n , h n , and type b influenza viruses protected ferrets from three seasonal influenza viruses [ ] . therefore, it is clear that the chimeric vlps were as effective as the killed viruses. the results showed that the immune responses to these chimeric vlps were similar to those to the vlps composed of na/ gp and m proteins. the less an animal needs to be handled to deliver vaccines, the better. the presence of multiple virus targets in a single vaccine will be a new advantage of the chimeric vlp vaccine. such vaccines could also reduce the burden of vaccine production and administration in comparison with regular vaccination. after this basic step, studies of the protective efficacy of chimeric vlps in a swine model need to be carried out. in summary, in this study, we provide a proof of concept for a chimeric vlp vaccine generating an anti-prrsv and anti-h n -influenza-virus immune response. these chimeric vlps could serve as a promising vaccination strategy to control prrs and h n influenza in swine. although the response to immunization with the chimeric vlps was not more effective than the response to inactivated viruses, the fact that chimeric vlps are not infectious and lack viral genomic material means that they could provide a safer method to prevent viral infection. furthermore, the production of the chimeric vlps is faster and presents a lower risk relative to the traditional method. thus, this approach has greater potential for vaccine development. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design chimeric influenza-virus-like particles containing prrsv gp cross-clade protective immune responses to influenza viruses with h n ha and na elicited by an influenza virus-like particle influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin nidovirales: a new order comprising coronaviridae and arteriviridae using plant cells as influenza vaccine substrates retrospective analysis of etiologic agents associated with respiratory diseases in pigs elicitation of protective immune responses using a bivalent h n vlp vaccine chimeric calicivirus-like particles elicit specific immune responses in pigs the receptor-binding and membrane-fusion properties of influenza virus variants selected using anti-haemagglutinin monoclonal antibodies expression of codon optimized major capsid protein (l ) of human papillomavirus type and in pichia pastoris; purification and characterization of the virus-like particles influenza-pseudotyped gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge molecular basis for the generation in pigs of influenza a viruses with pandemic potential influenza virus-like particles as pandemic vaccines influenza vaccines based on virus-like particles production and characterization of virus-like particles and the p domain protein of gii. norovirus formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins genomic analysis of two chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence enterovirus type neutralizing antibodies in the serum of macaque monkeys immunized with ev virus-like particles chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov assembly and immunological properties of newcastle disease virus-like particles containing the respiratory syncytial virus f and g proteins assembly and biological and immunological properties of newcastle disease virus-like particles newcastle disease virus-like particles containing respiratory syncytial virus g protein induced protection in balb/c mice, with no evidence of immunopathology assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states virus-like particles as immunogens immunogenicity and specificity of norovirus consensus gii. virus-like particles in monovalent and bivalent vaccine formulations clinical signs and economic losses caused by porcine reproductive and respiratory syndrome virus in a large breeding farm intranasal vaccination with influenza virus-like particles protects mice and ferrets from lethal and h n influenza virus challenge chinese-like strain of porcine epidemic diarrhea virus recombinant h n virus-like particle vaccine elicits protective immunity in ferrets against the pandemic h n influenza virus influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes influenza virus-like particles comprised of the ha, na, and m proteins of h n influenza virus induce protective immune responses in balb/c mice virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus a bivalent influenza vlp vaccine confers complete inhibition of virus replication in lungs a trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets porcine reproductive and respiratory syndrome immunology of the porcine respiratory disease complex production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus gp protein virus-like particles of hepatitis b virus core protein containing five mimotopes of infectious bursal disease virus (ibdv) protect chickens against ibdv fabrication of influenza virus-like particles using m fusion proteins for imaging single viruses and designing vaccines vaccination with coxsackievirus b virus-like particles elicits humoral immune response and protects mice against myocarditis key: cord- -js l fh authors: zhou, ping; zhai, shanli; zhou, xiang; lin, ping; jiang, tengfei; hu, xueying; jiang, yunbo; wu, bin; zhang, qingde; xu, xuewen; li, jin-ping; liu, bang title: molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo date: - - journal: int j biol sci doi: nan sha: doc_id: cord_uid: js l fh porcine reproductive and respiratory syndrome virus (prrsv) infects mainly the porcine alveolar macrophages (pams) and causes porcine reproductive and respiratory syndrome (prrs). previous studies have analyzed the global gene expression profiles of lung tissue in vivo and pams in vitro following infection with prrsv, however, transcriptome-wide understanding of the interaction between highly pathogenic prrsv (hp-prrsv) and pams in vivo has not yet been established. in this study, we employed affymetrix microarrays to investigate the gene expression patterns of pams isolated from tongcheng piglets (a chinese indigenous breed) after infection with hp-prrsv. during the infection, tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at and dpi. microarray analysis revealed that hp-prrsv infection has affected pams in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. several potential antiviral strategies might be employed in pams, including upregulating ifn-induced genes and increasing intracellular zinc ion concentration. and inhibition of the complement system likely attenuated the lung damage during hp-prrsv infection. transcriptomic analysis of pams in vivo could lead to a better understanding of the hp-prrsv-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to hp-prrs. porcine reproductive and respiratory syndrome (prrs), caused by prrs virus (prrsv) which belongs to the genus arterivirus of the family arteriviridae, is the most economically significant disease effecting commercially bred pigs world-wide [ ] . this disease is characterized by anorexia, increased late-term ivyspring international publisher abortions, increased number of stillborn pigs, mummified fetuses, weak live-born piglets, increased pre-weaning mortality, and delayed return to estrus [ ] . in vivo, prrsv productive infection occurs predominately in alveolar macrophages of the lung [ ] , followed by viremia and subsequent interstitial pneumonia within days [ ] . it was hypothesized that respiratory pathology, especially lung damage during prrsv infection, results from an overproduction of pro-inflammatory cytokines in the lungs [ ] . genome-wide transcriptional responses of lungs of landrace×yorkshire crossbred piglets to a classical north american type prrsv strain infection was analyzed by solexa/illumina's digital gene expression (dge) system, which is a tag-based high-throughput transcriptome sequencing method [ ] . this systematic analysis of the pulmonary gene expression profiles suggested that upregulation expression of pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during prrsv infection processes [ ] . another high-throughput deep sequencing was performed focusing on the pulmonary gene expression profiles after a highly pathogenic-prrsv (hp-prrsv) strain infection [ ] . the system analysis of the pulmonary gene expression provides a comprehensive basis for better understanding the pathogenesis of hp-prrsv [ ] . because prrsv infection occurs predominately in porcine alveolar macrophages (pams) [ ] , the interaction between prrsv and pams have been studied systematically by high-throughput research methods in vitro. pams, lavaged from six piglets, were challenged with the lelystad prrsv strain in vitro, and the gene expression of the pams was investigated using affymetrix microarrays [ ] . the result suggested that the expression of beta interferon (ifn-β), but not of ifn-α, was strongly upregulated in the early stage of prrsv infection [ ] . besides microarray, serial analysis of gene expression (sage) was also employed to examine the global expression of genes in prrsv-infected pams in vitro [ ] . these studies have provided global gene expression profiles of lung tissue in vivo and pams in vitro following infection with prrsv; however, transcriptome-wide understanding of the interaction between prrsv and pams in vivo has not yet been established. in , an unparalleled large-scale outbreak of highly pathogenic prrs (hp-prrs) occured in many areas of china. this outbreak affected more than millions pigs and produced approximately . million fatal cases [ ] . in this study, laboratory infection was performed in tongcheng piglets (a chinese indigenous breed living in tongcheng county of hubei province) using prrsv stain wuh [ ] , a highly pathogenic prrsv isolated in china during the pandemic period of hp-prrs in . we also employed affymetrix microarrays to investigate the gene expression patterns of pams isolated from the piglets after infection. the current study aims at better understanding the interaction between hp-prrsv and the host pams, which may lead to the identification of key host factors for tolerance/susceptibility to the virus and the finding of novel targets for antiviral therapies. all animal procedures were performed according to protocols approved by the biological studies animal care and use committee of hubei province, china. piglets used in this study were free from prrsv, pseudorabies virus (prv) and porcine circovirus type (pcv ) determined by elisa test for serum antibodies. twelve -week-old tongcheng boars (a chinese indigenous breed) were obtained from three litters (four piglets per litter), and raised in pathogen-free facilities. to perform a paired experiment, individuals within a full-sib litter were separated into two groups: one infected group and one control group with piglets in each group. the infected groups were challenged with prrsv-wuh ( ml/ kg, - tcid /ml) by intramuscular inoculation. slaughters were carried out at day post-infection (dpi) for uninfected (control) groups, and at or dpi for infected groups. rectal temperature and clinical signs were recorded daily during the experiment. the serum samples for viremia detection were collected daily from all animals (one ml blood per sampling point). the pams for microarray analysis were collected by bronchoalveolar lavage from three uninfected pigs and three infected pigs at dpi. post-mortem examinations were performed on all pigs. macroscopic lung lesions were given a subjective score to estimate the percentage of the lung affected by pneumonia, following a scoring system described previously [ , ] . for histopathology analysis, samples of the apical segment of the lower lung lobes were collected and fixed in % paraformalclehyde for h. fixed samples were dehydrated, embedded in paraffin, sectioned into μm and stained with hematoxylin and eosin. sections were examined by light microscopy. for viremia detection, serum samples were collected daily from all pigs. total viral rna was extracted from μl serum using trizol reagent (invitrogen, carlsbad, ca). cdna was synthesized using oligo(dt) primer, m-mlv reverse transcriptase (promega, madison, wi) in μl reaction mixture according to the manufacturer's instructions. absolute quantitative-pcr (q-pcr) was performed using primers specific to the orf of prrsv (sense: '-tca gct gtg cca aat gct gg- '; antisense: '-aaa tgg ggc ttc tcc ggg ttt t- '). for absolute quantification, the pet- m plasmid of the known copy number containing the orf fragment generated standard curve. viral copies per ml of the unknown samples were determined by linear extrapolation of the ct value plotted against the standard curve [ ] . trizol (invitrogen) was used for rna extractions following the manufacturer's instructions. rna integrity and concentration were evaluated by denaturing formaldehyde gel electrophoresis and agilent bioanalyzer. the rna samples were sent to genetech biotechnology limited company (shanghai, china) for hybridization to the porcine affymetrix genechip (affymetrix, santa clara, ca). a total of microarray analyses were conducted using the procedure described previously [ ] . the raw data (affymetrix genechip scanner ) was converted to gene signal files by mas . (microarray suite version . , affymetrix). the data points were normalized between slides using the quantile normalization method used by bolstad et al. [ ] . the differentially expressed genes were selected using the sam (significance analysis of microarrays) package (http://www-stat.stanford.edu/~tibs/ sam/), and the false discovery rate (fdr) values were generated using permutations of the repeated measurements to estimate the percentage of genes identified by chance. in the experiment, sam settings were adjusted for a two class paired analysis, using one hundred permutations to calculate the differentially expressed gene list. the fold-change of . and a false discovery rate of approximately % were set as a threshold. all data are miame compliant and have been deposited in ncbi's gene expression omnibus and are accessible through geo series accession number gse (http://www.ncbi.nlm.nih.gov /geo/query/acc.cgi?acc=gse ). differential gene expressions were performed for hierarchical cluster (ver. . ) and treeview (ver. . ) analyses [ ] . the functional annotation of differentially expressed genes was performed by the david (the database for annotation, visualization and integrated discovery) gene annotation tool (http://david.abcc.ncifcrf.gov/) [ ] , as well as by referring to a previous work [ ] . the rna samples prepared for microarray analysis were also used for q-pcr verification. reverse transcriptions were performed using m-mlv reverse transcriptase (promega) according to the manufacturer's instructions. the primers were designed with the primer premier . program. the rpl gene was used as the internal control [ ] . the primer sequences, melting temperatures and product sizes are shown in table . q-pcr was performed on the lightcycler Ⅱ (roche, basel, sweden) using sybr green realtime pcr master mix (toyobo co., ltd, japan) as the readout. data was analyzed by the -ΔΔct method [ ] . the data analysis procedure was performed as described previously [ ] . after infection with prrsv-wuh , the piglets presented typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion. the average rectal temperature rose to above . ºc at dpi and seemed to peak at dpi. the two piglets surviving at dpi showed a slight decrease of rectal temperature in the following two days ( figure a ). to assess the replication and spread of hp-prrsv, the viral copy number/ml in serum was determined by absolute real-time quantitative-pcr ( figure b ). the level of viremia increased rapidly during the first two days post-infection, then increased slowly from to dpi, and approached the plateau phase at or dpi. pathologic examination was carried out on the animals. macroscopic examination detected a mild lung lesion at the apical segment of the lower lobes at and dpi ( figure c ). for estimating the severity of the pneumonia, gross lung lesion scores were made based on the method described previously [ , ] . the low scores indicated a mild regional lung damage at and days after hp-prrsv infection ( figure d ). as compared with the uninfected group ( figure e) , microscopic examination detected a certain extent of congestion as well as interstitial infiltration of leukocytes in the lungs of infected piglets ( figure f ). pams samples collected from three infected piglets at dpi and three uninfected piglets were analyzed. a total of , transcripts ( % of all probesets) were expressed in infected and non-infected pams (supplementary table ). after quantile normalization, genes were identified as differentially expressed (de) genes, with being upregulated and being downregulated, under the threshold of fold change (fc) of . or greater and a false discovery rate (fdr) of approximately % (figure a and supplementary table ). based on the database for annotation, visualization and integrated discovery (david), of the de genes were classified into categories, many of which shared the same genes, according to their functional correlation (figure b and supplementary table ). the majority of the genes related to the virus-host cell interaction could be assigned into the categories including cell death and apoptosis related, response to wounding, response to unfolded protein, response to oxidative stress, response to virus, innate immune response, response to cytokine stimulus, and endoplasmic reticulum (er) overload response. other de genes that were not classified by david were taken into account for further analysis below. in macrophages, prrsv entry into the host cell is mediated by heparan sulphate proteoglycans and the receptor sialoadhesin. upon a ph drop, prrsv is uncoated and its genome is released from the endosomes into the cytoplasm, which allows virus replication [ ] . after hp-prrsv infection the atp v b gene, which encodes a component of vacuolar atpase (v-atpase) that mediates acidification of endosomal organelles [ ] , was upregulated ( figure ). sarm , a negative regulator of trif-dependent toll-like receptor (tlr) signaling [ ] and mapk phosphorylation [ ] , was significantly downregulated (figure ). sbno , a potent inhibitor of nf-κb [ ] , and socs which limits nf-κb signaling by decreasing p stability within the cell nucleus [ ] , were upregulated ( figure ). upon hp-prrsv infection, irf was found to be upregulated in pams at dpi ( figure ) , however, no type-i ifn or ifn-γ induction was observed. a number of ifn-induced genes (ifi , ifi , ifih , ifit , ifit , ifitm , gbp , gbp , mx , gzmb, gzmh, isg , usp , rsad , nmi) were upregulated (table ) . jak-stat pathway seemed to be positively (stat and nmi) as well as negatively (socs ) regulated during hp-prrsv infection (figure ). nine genes (s a , marcks, cacybp, cct a, arhe, cct , ptpn , cct , and twf ) related to actin and tubulin cytoskeleton organization were upregulated and three (rassf , elmo , and kif ) were downregulated (table ). in addition, several exocytosis related genes (rsad , gsk b, lman l, exoc , sels, copz , sec l , and sec l ) were differentially expressed in pams after hp-prrsv infection (table ) . rsad , encoding an ifn-induced protein which inhibits influenza a virus release from the plasma membrane of infected cells by affecting the formation of lipid rafts [ ] , was upregulated significantly. vesicle trafficking between the golgi apparatus and er seemed to be restricted, because copz , a member of the copi coat which helps vesicles transport proteins from the cis end of the golgi complex back to the rough er [ ] , and sec l , a component of the copii vesicle coat that mediates vesicular traffic from the rough er to the golgi apparatus [ ] , were both downregulated. during hp-prrsv infection, homeostasis of isgylation, an ubiquitin-like modification, seemed to be re-established in pams by enhancing the expression of isg , an ubiquitin-like protein, and usp , which is an isg deconjugating protease (table ) . three e ubiquitin ligase genes (cacybp, herc , cul ) were upregulated, and two (herc , g e ) were downregulated ( table ) . as expected, a large set of chaperone genes were upregulated, including heat shock kda protein (hsp ) (dnaja , dnaja , dnajb , dnajb , and dnajb ), hsp (hspd ) , hsp (hspa b, hspa , hspa , and hspa ) , hsp / (hsph ), and subunits of chaperon in containing t-complex polypeptide (cct , cct , and cct a) , as well as npm , a molecular chaperone in the cell nucleus (table ) . upon hp-prrsv infection, several de genes were involved in the intracellular calcium homeostasis in pams (table ) . after hp-prrsv infection, zinc ion concentration in pams seemed to be increased, through upregulating the expression of slc a which encodes a zinc influx transporter [ ] (table ) . several zinc finger protein encoding genes (zdhhc , zfand a, zcchc , zcwpw , zfp , znf ) were also identified as de genes, and all of them were upregulated, except znf (table ) . during hp-prrsv infection, a set of de genes involved in the dynamic regulation of the extracellular matrix and vascular permeability was identified ( table ). infiltration of leukocytes into pulmonary alveoli, as a sign of inflammation, was modulated by upregulating a small number of genes (ccl , ccl l, ccr and csf ) ( table ). three genes, mpp , pf , and ppbp involved in neutrophil infiltration or activation [ ] [ ] [ ] , were all downregulated (table ) . during hp-prrsv infection, complement activation seemed to be inhibited, as expression of c and pfc, a positive regulator of complement activation, were downregulated, and clu, encoding for a complement inhibitor, was upregulated ( table ). seven genes (ccl , slc a , atp v b , c , ddit , glrx and tnf) were selected for q-pcr assay to validate the changes in gene expression observed by microarray analysis. ccl was the main upregulated chemokine gene in this study (table ) . two upregulated genes, slc a and atp v b , were involved in intracellular zinc homeostasis and endosome acidification, respectively. the downregulated c gene is the core member of the complement system which seemed to be inhibited, according to our study ( table ). the q-pcr gene list also contained two de genes (ddit , glrx ) which were not referred to in the discussion, and tnf, an important cytokine gene, which was not differentially expressed in tongcheng pams in response to hp-prrsv infection. the changes of these genes, detected by microarray analysis, was in agreement with the q-pcr validation (figure ) . the results of this study showed that tongcheng piglets exhibited typical clinical signs following infection with hp-prrsv wuh strain. the lung damage caused by the infection was regional and mild at and dpi ( figure c and d), but further observation for a longer period of time was not performed in this study. the slow reproduction rate of the virus (viremia) at to dpi ( figure b ) suggested a near balance between the viral replication and the defense mechanisms in the pams. transcriptomic analysis of the pams at dpi identified de genes under the filter of . -fold change, and the number of upregulated genes ( ) was greater than that of downregulated genes ( ). in comparison, an in vitro transcriptomic analysis of pams revealed that only small numbers (no more than ) of de genes (threshold of . -fold change) were identified at to hours post prrsv infection, and the overall effect of prrsv on the host transcription machinery was downegulation [ ] . it is not sure whether there is a conversion of the overall effect of prrsv on host transcription machinery from downregulation to upregulaton as time goes on, or the change is only the effect of the difference between in vitro and in vivo assays. as compared with the number of de genes in this study, some thousands of de genes (threshold of almost . -fold change) were identified in lung tissues at and dpi following both prrsv and hp-prrsv infection, by high-throughput deep sequencing assays [ , ] . this great number of de genes might result from the huge amount of data obtained by the deep-sequencing method and from the many cell types in lung tissues. prrsv is considered to inhibit the synthesis of type-i ifns and its signaling by blocking stat /stat nuclear translocation [ ] . however, it is also reported that prrsv can phosphorylate ifn-regulatory factor (irf- ) and weakly activate the ifn-β promoter in marc- cells in early infection, but the activations of irf- and ifn-β promoter are rapidly inhibited in the following infection [ ] . the induction of ifn-β mrna, but not ifn-α mrna, is observed in monocyte-derived dendritic cells and primary alveolar macrophages infected by prrsv at dpi [ , ] . in some cases, even the expression of ifn-α can be detected in the lung [ ] or serum [ ] of pigs infected with prrsv during the early days. interestingly, in this study, no induction of type-i ifn was detected in pams at dpi (figure ) , whereas a series of ifn induced genes that are critical for the cell to defend itself against viral infection, were upregulated (table ) . similar results were shown in lung tissues at and dpi following both prrsv and hp-prrsv infection [ , ] , and it was speculated that the ifn induced genes were predominantly expressed by the uninfected cells [ ] . here, another possibility is suggested that a certain amount of type-i ifn might be induced at the early stage of the infection before dpi. during hp-prrsv infection, several aspects of the pams' function were under regulation, such as actin and tubulin cytoskeleton organization, exocytosis, protein degradation, protein folding, intracellular calcium and zinc homeostasis ( table ) . increasing of intracellular zinc concentration impairs the replication of a variety of rna viruses, including poliovirus, influenza virus, coronavirus, arterivirus, rhinovirus, and respiratory syncytial virus [ ] [ ] [ ] . recently, zinc ion has been reported to efficiently inhibit the rna-synthesizing activity of the multiprotein replication and transcription complex of both sars-coronavirus and equine arteritis virus [ ] . upregulation of slc a (also known as zip ) (table ) , a member of the slc (zip) family which transports zinc from the extracellular space or organellar lumen into the cytoplasm [ ] , might be a defense mechanism in pams during hp-prrsv infection. nevertheless, none of the slc family genes was identified as a de gene in a microarray assay of pams infected with prrsv in vitro [ ] . furthermore, the expression of slc a , another member of the slc family, was downregulated in the lungs of landrace×yorkshire crossbred piglets at dpi following hp-prrsv infection [ ] . it has been shown in this study, that modulated inflammatory reaction, with a few proinflammatory cytokines upregulated (ccl , ccl l and its receptor ccr , and csf ) ( table ) , might contribute to the mild regional lung lesion observed at and dpi ( figure c and d) . besides, the complement system is one of the key players in the defense against infections. however, excessive activation of the complement can also exaggerate the disease induced by viral or bacterial infection. in , a new h n influenza a virus caused severe disease in naive middle-aged human individuals with preexisting immunity against seasonal strains, and this disease is reported to be induced through high titers of low-avidity nonprotective antibody and immune complex-mediated complement activation in the respiratory tract [ ] . excessive complement activation can contribute to organ damage in combination with the cytokine storm in the later stages of sepsis caused by bacterial infection [ ] . it is reported that blocking complement activation can ameliorate hepatic inflammation mediated by the hepatitis c virus core protein [ ] . likewise, inhibition of complement with a potent c inhibitor (compstatin) in a baboon model of late-stage sepsis markedly improves organ preservation and other clinical parameters [ ] . as it has been shown here, inhibition of the complement system might also be a contributor to the mild regional lung damage during hp-prrsv infection. interestingly, infection of hp-prrsv in six-week-old crossbred weaned pigs (landrace × yorkshire) induces complement activation accompanied by severe lung damage [ ] . in summary, the data presented in this study suggested that during infection with hp-prrsv tongcheng piglets exhibited typical clinical signs, but displayed mild regional lung damage at and dpi. microarray analysis revealed that hp-prrsv infection has affected pams in vivo in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. several potential antiviral strategies might be employed in pams, including upregulating ifn-induced genes and increasing intracellular zinc ion concentration. furthermore, inhibition of the complement system likely attenuated the lung damage during hp-prrsv infection. this system analysis could lead to a better understanding of the hp-prrsv-host interaction, and to the identification of novel antiviral therapies and identifying genetic components for swine tolerance/susceptibility to hp-prrs. genetic parameters for performance traits in commercial sows estimated before and after an outbreak of porcine reproductive and respiratory syndrome immunohistochemical detection of porcine reproductive and respiratory syndrome virus antigen in neurovascular lesions immunological responses of swine to porcine reproductive and respiratory syndrome virus infection distribution of a korean strain of porcine reproductive and respiratory syndrome virus in experimentally infected pigs, as demonstrated immunohistochemically and by in-situ hybridization the combination of prrs virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing aberrant host immune response induced by highly virulent prrsv identified by digital gene expression tag profiling genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (sage) emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus gp protein encoded by a synthetic orf gene comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge understanding haemophilus parasuis infection in porcine spleen through a transcriptomics approach a comparison of normalization methods for high density oligonucleotide array data based on variance and bias cluster analysis and display of genome-wide expression patterns database for annotation, visualization, and integrated discovery annotation of the affymetrix porcine genome microarray transcriptomic analysis of the dialogue between pseudorabies virus and porcine epithelial cells during infection analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method involvement of proteases in porcine reproductive and respiratory syndrome virus uncoating upon internalization in primary macrophages v-atpase functions in normal and disease processes the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling sarm inhibits both trif-and myd -mediated ap- activation cutting edge: a transcriptional repressor and corepressor induced by the stat -regulated anti-inflammatory signaling pathway suppressor of cytokine signaling (socs ) limits nf{kappa}b signaling by decreasing p stability within the cell nucleus the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts identification and characterization of novel isoforms of cop i subunits human sec b: a family of new mammalian orthologues of yeast sec p that associate with the copii coat structure-function analysis of a novel member of the liv- subfamily of zinc transporters erythrocyte scaffolding protein p /mpp functions as an essential regulator of neutrophil polarity role of the platelet chemokine platelet factor (pf ) in hemostasis and thrombosis canine cxcl and its functional expression in dendritic cells undergoing maturation porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat /stat nuclear translocation porcine reproductive and respiratory syndrome virus (prrsv) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in marc- cells differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus expression of interferon-alpha and mx protein in pigs acutely infected with porcine reproductive and respiratory syndrome virus (prrsv) interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus zn inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture zinc ions inhibit replication of rhinoviruses effect of zinc salts on respiratory syncytial virus replication role of zn + ions in host-virus interactions severe pandemic h n influenza disease due to pathogenic immune complexes complement: a key system for immune surveillance and homeostasis hepatic inflammation mediated by hepatitis c virus core protein is ameliorated by blocking complement activation complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of escherichia coli sepsis the porcine reproductive and respiratory syndrome virus requires trafficking through cd -positive early endosomes, but not late endosomes, for productive infection we would like to thank tinghua huang and lijie su in our lab for their help with microarray data analysis. we are grateful to dr. xiao zhang (uppsala university, sweden) for valuable discussions and improving the manuscript. we thank our lab members for sample collections. the authors have declared that no conflict of interest exists. supplementary key: cord- -ux shvji authors: saade, georges; deblanc, céline; bougon, juliette; marois-créhan, corinne; fablet, christelle; auray, gaël; belloc, catherine; leblanc-maridor, mily; gagnon, carl a.; zhu, jianzhong; gottschalk, marcelo; summerfield, artur; simon, gaëlle; bertho, nicolas; meurens, françois title: coinfections and their molecular consequences in the porcine respiratory tract date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ux shvji understudied, coinfections are more frequent in pig farms than single infections. in pigs, the term “porcine respiratory disease complex” (prdc) is often used to describe coinfections involving viruses such as swine influenza a virus (swiav), porcine reproductive and respiratory syndrome virus (prrsv), and porcine circovirus type (pcv ) as well as bacteria like actinobacillus pleuropneumoniae, mycoplasma hyopneumoniae and bordetella bronchiseptica. the clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. in this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. we discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health. bacterial and viral respiratory diseases are a major health issue in species reared under confined conditions in large groups. most often multiple infectious agents are involved in the development of these clinical conditions making unsuited the common reductionist approach of host-pathogen interactions by the study of single infection [ ] . infection by more than one type of pathogen (viruses, bacteria and parasites amongst others) is described as a mixed infection. however, the term coinfection is frequently used to describe concomitant infection of a cell or a host by separate pathogens [ ] . since in the literature the definitions of coinfection and mixed infection have been both used to describe the same events, we will use the term "coinfection" in the current review. additionally, in virology, the term superinfection is used if one virus infects the cell or the host before infection by the second superinfecting virus. we will also use the term "superinfection" in the review. finally, an opportunistic pathogen is usually considered as a pathogen that would not have infected animals in absence of the primary infection, or alternatively, "pathogen" that would have been asymptomatic in the absence of the primary infection. in some studies, however, the use of the terms "coinfection" is not suitable and "superinfection" should be used instead, as we will see later. this semantic point is responsible for a lot of confusion and makes comparisons between studies sometimes tricky. the outcome of any coinfection or superinfection can be affected by the interactions taking place between the infectious agents, the nature of the cell/host, adverse environmental and management conditions, intestinal and respiratory microbiomes, and the triggered immune response-innate and adaptive-developed afterwards [ , ] . when occurring at the same time or with a delay, infections can impact the virulence of causative pathogens with subsequent consequences on the host immune response and its ability to clear the infections [ ] . the first contact with a pathogen can change the cell/host response against any other second pathogen, possibly causing a more virulent infection, reducing its severity or suppressing it completely [ ] . thus, different scenarios concerning the pathogen interactions can be observed, the first infectious agent can promote the second one, attenuate its effects or simply prevent its establishment. conversely, the second pathogen may also influence the first one directly or indirectly. coinfections have been described in both humans and animals [ , ] . moreover, bacterial and viral infections might be followed by secondary bacterial or viral infections, which in some cases are responsible for the pathology development and the observed clinical signs. in this review, the current knowledge regarding frequent coinfections that occur in the porcine respiratory tract and particularly in the lungs are reviewed. when possible, we focused on the interactions between the mentioned pathogens and the various mechanisms justifying these interactions and their consequences on the host's response. we especially discussed coinfections involving main bacteria and viruses associated with the so-called porcine respiratory diseases, excluding coinfections involving parasites and fungi (including their metabolites, such as mycotoxins). moreover, we do not discuss the impact of adverse environmental and management conditions which have been shown to be of major importance in the modulation of respiratory infections' severity [ ]. respiratory diseases have been formally described in pigs as early as the ′s [ ] and several studies have been carried out to identify associated agents. the role of the infectious pathogens has been assessed by using two main approaches: direct research of the pathogens (by culture or polymerase chain reaction-pcr for instance) from tissue samples of diseased (acute or chronic stage) and non-diseased pigs or indirect detection by serological tests to look for antibodies produced after exposure to specific pathogens. these studies indicated that frequently under field conditions, several infectious pathogens are simultaneously detected from lung lesions (see [ ] [ ] [ ] causative respiratory infectious agents can be divided into primary and secondary or opportunistic pathogens. primary pathogen being defined here as pathogen that can infect the animal as first unique pathogen and then facilitate secondary or opportunistic coinfection. these primary pathogens include common bacteria such as highly virulent actinobacillus (a.) pleuropneumoniae, m. hyopneumoniae, bordetella (b.) bronchiseptica in young piglets and common viruses such as swiav [ ] . prrsv and pcv are not strictly respiratory pathogens as swiav, however, since they also frequently affect the respiratory system and since they can act as facilitators of secondary respiratory infections, they must be considered too. other primary pathogens such as aujeszky's disease virus (adv) and prcov are reported but they are far less frequently encountered today or they have less impact on porcine health [ ]. then, some viruses like the porcine cytomegalovirus can also inhibit host immune functions-particularly the action of t lymphocytes-and promote respiratory diseases such as the porcine reproductive and respiratory syndrome [ ] . among the secondary pathogens common bacteria such as lower virulence strains of a. pleuropneumoniae, a. suis, glaesserella parasuis, pasteurella multocida, and streptococcus (s.) suis are reported. together primary and secondary pathogens are involved in the "porcine respiratory disease complex" (prdc) [ ] . several studies have assessed the nature of the infectious agents directly or indirectly associated with respiratory diseases in pigs [ , , , ] . in one of these studies involving breeding sows in five french farrow-to-finish herds [ ] , results indicated that s. suis, a secondary pathogen, was quite widespread among sows- . % of the animals being positive using a pcr assay-and pcv and swiav infections were highly prevalent ( % of the sows with antibodies against pcv and between . % and % of the sows with antibodies against swiav). other infectious agents such as a. pleuropneumoniae, g. parasuis and p. multocida were detected in %, %, and % of the sows, respectively [ ] . in another study evaluating infectious agents associated with respiratory diseases in farrow-to-finish pig herds in france, it has been shown that m. hyopneumoniae, prrsv, and swiav subtype h n were the major pathogens involved in pneumonia-like gross lesions [ ] . for extensive pleuritis, prrsv was frequently associated with a. pleuropneumoniae [ , ] . regarding bacteria associated with lung lesions in french slaughter pigs [ ] , a report mentioned lesions of pneumonia and pleuritis as the most frequent lesions. in these lesions, bacteria such as m. hyopneumoniae, p. multocida, a. pleuropneumoniae, s. suis, and g. parasuis were detected in . %, . %, . %, . %, and . % of the lungs, respectively [ ] . in a retrospective analysis of the etiologic agents associated with respiratory diseases in pigs in usa, two or more infectious agents were identified in . % of the analyzed cases [ ] . prrsv ( . % of the samples), p. multocida ( . %), m. hyopneumoniae ( %), swiav ( . %), g. parasuis ( . %) and pcv ( . %) were the infectious agents most frequently encountered [ ] . in korean pigs, prrsv and pcv were frequently identified associated or not to various bacteria such as s. suis ( . %), m. hyopneumoniae ( . %), p. multocida ( . %), and a. pleuropneumoniae ( %) [ ] . below we review the main primary pathogens as defined above, common viruses such as prrsv, pcv , swiav, prcov and adv as well as bacteria like a. pleuropneumoniae, m. hyopneumoniae and b. bronchiseptica. conversely, other pathogens involved in the prdc are not presented in the following sections while considered in additional file presenting the different coinfections' situations. prrsv is an enveloped single stranded positive rna virus belonging to the arteriviridae family. two different species, prrsv- (also known as betaarterivirus suid ), from european origin, and prrsv- from american origin, are now distinguished [ ] . this enveloped virus replicates mainly or exclusively in macrophages such as alveolar macrophages (ams), but also macrophages from the nasal mucosa and pulmonary intravascular macrophages (pims) [ , ] . in vitro, prrsv can also replicate in cultured monocytes and monocyte-derived cells including macrophages [ ] and in vitro-derived dendritic cells (dcs) generated either from bone marrow hematopoietic cells (bmdcs) or blood monocytes (modcs), depending on the in vitro culture conditions [ , ] . however, such in vitro generated dcs are not representative of in vivo primary dcs which do not seem to be permissive to viral replication [ ] . in fact, modc and bmdc (at least when generated using granulocyte macrophage colony-stimulating factor, gm-csf) although possessing functional overlaps with the dc family, do not represent bona fide dcs, which represent an own lineage of hematopoietic cells distinct from the monocytic lineage [ ] . different cell surface molecules are involved in prrsv entry and infection of cells: heparan sulfate, porcine sialoadhesin-also known as sialic acid-binding immunoglobulin-type lectin (siglec- ), siglec- , cd and cd [ , ] . heparan sulfate is a glycosaminoglycan (gag) that seems to play a modest or secondary role in prrsv infection since the blocking of this receptor on ams induced only a mild decrease in prrsv infectivity. moreover, this effect was not observed with all the prrsv isolates tested, suggesting that the involvement of heparan sulfate depends on the antigenic diversity of prrsv [ ] . siglec- /cd is a member of the sialic acid-binding lectins (siglecs) family and is expressed on macrophages [ ] and siglec- has been identified as an alternative receptor to siglec- [ ] . binding of prrsv to siglecs induces its internalisation by clathrin-mediated endocytosis. expression of recombinant porcine sialoadhesin is sufficient to induce the internalisation of prrsv by non-permissive cells, but not replication [ ] . cd is a scavenger receptor involved in prrsv infection [ ] . its expression on nonpermissive cells makes them susceptible to infection with prrsv and allows productive replication of the virus [ ] . moreover, cd -ko animals are still susceptible to prrsv- infection [ ] , whereas cd -ko animals are resistant to prrsv- and prrsv- [ , ] . finally, myh has been recently identified as an indispensable partner of cd for prrsv cell entry for both prrsv- and prrsv- [ ] . prrs clinical signs can be nearly absent to severe depending on the considered prrsv species and strains. when observed, there are, amongst the most frequent, lethargy, dyspnea, tachypnea, as well as a reproductive disease [ ] . prrsv can persist in infected pigs for several months after the initial infection particularly in lymphoid tissues and has the ability to alter the host's immune system to escape it (for review see [ ] ). prrsv interferes with the porcine innate immune response through downregulation of type i interferons (ifns-ifnα and ifnβ mostly), which are cytokines known for their antiviral properties [ ] . prrsv-infected macrophages also had a reduced capacity to produce the pro-inflammatory cytokines tnfα and il β [ ] and the production of the anti-inflammatory cytokine il was found enhanced during infection [ ] . nevertheless, the role of cytokine modulation during prrs is unclear considering that the effects appeared to depend on the prrsv species, as well as on the prrsv isolates, since opposite results can be found with different prrsv strains [ , ] . in fact, some prrsv- isolates were shown to enhance ifnα production while other prrsv- isolates suppressed it. results seemed also very variable for the immunoregulatory il along different isolates of prrsv- [ , ] , making general conclusions about how prrsv alters innate immune responses difficult. prrsv impact on adaptive cellular immunity seems also to be highly variable according to the species and the strain [ ] . conversely, whereas non-protective antibody response against the viral nucleocapsid is found within a week post-infection, neutralizing antibodies appearance is highly delayed for all prrsv species and strains, appearing only after or weeks of infection and peaking even later [ ] . pcv is a naked circular single stranded dna virus belonging to the circoviridae family and responsible for porcine circovirus disease (pcvd). the attachment of pcv to target cells occurs through chondroitin sulfate b and probably other receptors [ ] . internalisation is not fully known but it does not seem to involve a specific receptor and the gags could mediate internalisation and binding to the target cells [ ] . most of the time the infection is subclinical but in some circumstances such as coinfections with other respiratory pathogens it can cause the post-weaning multisystemic wasting syndrome (pmws), clinically characterized by wasting respiratory disease, and enteritis [ ] . infection with pcv can occur in utero, resulting in stillborn piglets and mummified fetuses, or death at different ages after birth [ ] . in young and older animals, pcv was found in cells expressing monocytes (cd + ), and t and b cells (cd + , cd + , igm + ) markers [ ] . further results showed that active replication of the virus was supported by t and b cells, with enhanced replication in proliferative cells [ ] . in vitro, pcv can also infect many other cell types including endothelial cells, gut epithelial cells, fibrocytes, and dcs [ ] . in dcs the virus seems to persist and remain infective for a prolonged period without replication indicating that these cells might serve as a vehicle for virus spread in the host [ ] . pmws is characterized by the depletion of lymphoid cells affecting t cells, b cells, and nk cells [ ] . this lymphopenia was also associated with impaired responses of peripheral blood mononuclear cells (pbmcs) to mitogen stimulation with lower levels of il , ifnγ, and il production compared to pbmcs from non-infected pigs [ ] . another feature of pmws is an elevated level of il found in lymphoid organs, especially in the t cells rich areas [ ] . il -mediated immunosuppression could play an important role in the pcv infection and the development of pmws. pcv has also the ability to alter the innate immune response [ ] . even though the virus does not productively infect dcs, evidence shows that it can interfere with the normal plasmacytoid dcs (pdcs) response. upon stimulation with cpg-odn, pdcs' ability to produce ifnα and tnfα was impaired in cells previously infected with pcv [ ] . pcv dna isolated from infected cells induced the suppression of pdc ifnα production [ ] . influenza a viruses are enveloped single stranded negative rna viruses belonging to the orthomyxoviridae family. these enveloped viruses can infect a broad range of hosts, with pigs being one of their natural hosts (for a review see [ ] ). the three main iav subtypes encountered in pigs are h n , h n , and h n [ ] , but many genetic lineages and antigenic variants within these subtypes are co-circulating in the pig population worldwide. subclinical infections with swiavs are common in pigs, but they can also induce a disease similar to what is observed in humans, with upper respiratory tract distress associated with fever, cough, rhinitis, high morbidity, and low mortality [ ] . the main targets of swiavs are epithelial cells of the respiratory tract but iavs can also non-productively infect alveolar macrophages [ ] . two major glycoproteins are present at the surface of the virus: hemagglutinin (ha) and neuraminidase (na). binding of ha with the sialic acid molecules at the surface of the host cells will induce the endocytosis of the viral particle [ ] . the na molecule plays the main role in the budding of the virus by removing the sialic acid, allowing the release of neoformed virus particles from the infected cell [ ] . the innate response against the virus includes production of high levels of pro-inflammatory cytokines such as ifnα, tnfα, and il . dcs, in particular pdcs play an important role in this response [ ] . an important observation was that the production of these cytokines correlated to the viral loads and the severity of the disease. infection with swiav induces cellular and humoral specific immune responses in pigs recovering from the disease and the serum igg and the mucosal iga can protect the animal from re-infection [ ] . ns and pa-x are the main viral proteins that alter the innate immune response, mainly by blocking the type i ifn response [ ] as well as the nlrp inflammasome activation [ ] in infected-epithelial cells and alveolar macrophages. finally, the main mechanisms through which the swiav escapes the adaptive host immune system are the antigenic drift and the antigenic shift concerning mainly ha and na which are also the two major antigenic proteins expressed on the surface of the virus and against which the neutralizing humoral response is directed [ ] . prcov is an enveloped single stranded positive rna virus belonging to the coronaviridae family. in pigs, four alphacoronavirus, one betacoronavirus and one deltacoronavirus have been described [ , ] . thus, most of the porcine coronaviruses are from the genus alphacoronavirus. the only respiratory porcine coronavirus, prcov, is a variant of transmissible gastroenteritis virus (tgev) where a large ′ region deletion (nucleotides - ) in the spike gene of the virus altered the tropism and the virulence. even if pigs have been shown to be susceptible to the first sars-cov (serological evidence and isolation of the virus in a pig farm in the xiqing county of tianjin, china) [ ] they have not been successfully experimentally infected, at this stage, by sars-cov- [ ] . prcov uses aminopeptidase-n (cd ) domain iv to enter cells [ ] and replicates to high titers in the lungs ( × - tissue culture infectious dose -tcid ) specifically in type and pneumocytes. moreover, it can infect epithelial cells of the nares, trachea, bronchi, bronchioles, alveoli, and, occasionally, alveolar macrophages [ ] . infections with the prcov are usually subclinical, but there is variation between strains and some can induce a more severe disease. prcov can infect pigs of all ages by direct contact transmission or aerosol [ ] . the clinical signs are associated to the respiratory system and are mild to severebronchointerstitial pneumonia-depending the strain and the context (environmental and management factors as well as the presence of other pathogens). suid herpesvirus , usually known as prv or adv is the responsible agent of aujeszky's disease in pigs. it is a double stranded enveloped dna virus from the herpesviridae family and alphaherpesvirinae subfamily targeting respiratory and/or genital mucosae for its replication [ ] . adv has a very broad host range varying from domestic animals like pigs, cattle, goats, sheep, cats and dogs to wild animals such as ferrets, foxes, hares, raccoons, and wild deer, and where it induces different diseases [ ] . infected animals usually show fever, sneezing, coughing and vomiting accompanied occasionally with typical nervous manifestations like convulsions, aggressiveness and lack of coordination. mortality rate can reach % in suckling piglets while in mature pigs the infection is inapparent or mild [ ] . adv possesses eleven types of envelope glycoproteins playing major roles in the interaction with host cells and the induction of immune response [ ] . viral binding and fusion with the plasma membrane of the target cell-epithelial cells, neurons and alveolar macrophages-are controlled by a cascade of events orchestred by glycoproteins c (gc), gb, gd, gh and gl. the binding process starts with an interaction of gc with heparin sulfate proteoglycans [ , ] . stabilization of this interaction is then assured by the binding of gd to specific cellular receptors known as herpesvirus entry mediators such as hvea (tnfrsf ), hveb (prr , nectin ), hvec (prr , nectin ), hved (pvr, cd ), and -o-sulfated heparin sulfate [ , ] . at this stage, tyrosine-based or dileucine-based endocytosis in parallel with clathrin-mediated endocytosis occur by the mediation of gb, gh and gl, leading to the penetration of the capsid and the tegument into the cellular cytoplasm. finally, the interaction of the capsid with dynein leads to the release of viral dna into the cellular nucleus after a transport along microtubules from the periphery to the nuclear pores [ ] . porcine humoral immune response is induced by adv and neutralizing antibodies are mainly directed against gc [ ] . specific cell mediated immune responses are also triggered and mhc class i restricted, gc-specific, cytotoxic cells are induced. adv also alters the ifn signaling pathway by suppressing stat tyrosine phosphorylation leading to an inhibition of ifn-stimulated genes (isgs) expression [ , ] . adv may be involved in the prdc and can be isolated alone or with other pathogens. accordingly, a study conducted in taiwan reported the association of adv with pcv in . % of the evaluated pigs using a multiplex pcr [ ] . animals affected with this gram negative bacterium develop a pleuropneumonia characterized by fibrinohemorrhagic necrotizing bronchopneumonia and fibrinous pleuritis which can reach a high mortality rate [ , ] . although the disease is best known in its acute/peracute forms, subacute and/or chronic presentations with low or no mortality are highly prevalent, especially in the presence of antibiotic treatments. many herds are subclinically infected without previous or present episodes of clinical disease and in the absence of suggestive lesions at the slaughter house. animals are, nevertheless, carriers of the pathogen. this happens in several conventional herds which may be simultaneously infected not only with several low/intermediate virulent strains, but also, in some cases, with strains highly likely to cause disease. in the latter case, outbreaks may suddenly appear in the presence of concomitant diseases or as a consequence of changes in management and/or environment [ , ] . eighteen serotypes of the bacterium have been described, which can all induce disease, although clear differences in virulence have been described [ , ] . these bacteria can be found mainly in tonsils of carrier animals; virulent strains have a tropism for the lower respiratory tract where they preferentially bind to ciliated cells of the terminal bronchioli and pneumocytes [ , ] . different virulence factors expressed by a. pleuropneumoniae are involved in the colonization and the development of the disease. adhesion to cells could be mediated by type iv fimbriae that are expressed upon contact with respiratory epithelial cells in vitro and during lung infection [ , ] . adhesion of a. pleuropneumoniae to respiratory epithelial cells also involves the binding of bacterial lipopolysaccharides to glycosphingolipids on the surface of the cells [ , ] . the formation of biofilm by the bacteria is likely to play an important role in the colonization of the host [ ] . after attachment to the target cells, the bacteria can produce four different pore-forming exotoxins (apx i, ii, iii and iv) inducing the lysis of alveolar epithelial cells, thus allowing the acquisition of nutrients by the bacteria, but also participating in the development of the lesions [ , ] . some of the virulence factors expressed by a. pleuropneumoniae interfere with the host's immune response. the toxins apx i, ii and iii induce the lysis of not only respiratory epithelial cells, but also of cells involved in the innate immune response such as macrophages and neutrophils [ , ] . at lower concentrations, these toxins lose their lytic properties but can still impair macrophages chemotactic activity and their phagocytic abilities [ ] . the capsular polysaccharides of a. pleuropneumoniae interfere with macrophage phagocytosis and enable resistance to complement-mediated killing [ ] . a. pleuropneumoniae may also interfere with the antibody response by producing proteases that can degrade porcine iga and igg [ , ] . this cell wall-free bacterium is considered to play a primary role in prdc and is the causative agent of porcine enzootic pneumonia (ep), a disease with high morbidity but low mortality rates [ ] . the main pathological mechanisms involved in m. hyopneumoniae infections are: (i) adhesion to the ciliated cells of the tracheal epithelium inducing ciliostasis, loss of cilia and exfoliation, dysregulation of cellular homeostasis (with increased intracellular calcium concentration) and secretion of cytotoxic factors, (ii) alteration of the mucociliary tract, (iii) inflammatory reactions sometimes exacerbated and prolonged, and (iv) manipulation of the innate and adaptive immune responses [ , ] . among the adhesins described in m. hyopneumoniae, p is reported to be a major determinant of cell adhesion [ ] [ ] [ ] . several other adhesins were reported: p linked to p , lpps, lppt, mgpa, p , p , p , p , p , and p [ , ] . most adhesins are transcribed and translated during m. hyopneumoniae infection and then undergo posttranslational cleavage to result in diverse products on the membrane surface [ , , ] . the diversity of surface proteins can also derive from the variation in the number of repeats in genes encoding adhesins [ ] . these mechanisms of antigenic variation enable the bacterium to escape from immune system recognition and to invade the host [ ] . adhesins can also recruit extracellular matrix components (plasminogen, fibronectin and actin amongst others), and therefore can promote invasion and inflammatory response [ , ] . the immune response induced against m. hyopneumoniae, may have a double action: over-activation of the local immune response resulting in a pathologic inflammatory reaction or local immunosuppression explaining the chronic nature of the associated pathologies [ , ] . acute m. hyopneumoniae infection leads to the recruitment and activation of various innate immune cells, essentially through the involvement of a large range of cytokines: il , il and tnfα in lungs; cxcl , il , il , il , il , tnfα and il in bronchus-associated lymphoid tissue (balt) or tracheobronchial lavage fluid (tblf) [ , , ] . some of these inflammatory cytokines (tnfα, cxcl , il β, il ) are produced chronically in the lungs and play powerful roles in apoptosis (tnfα), differentiation and chemotaxis of neutrophils (respectively, il and il ), and macrophage activation (tnfα, il β). chronic infections are typically associated with intense lymphoid hyperplasia [ ] and are characterized by an accumulation of igg-and iga-expressing plasma cells, cd + t cells, macrophages and dcs in the balt of inflamed lung tissue [ ] . involvement of t cell activation in chronic inflammation is also supported by the presence of t-cell cytokines such as il- and il- in bronchoalveolar exudates [ ] . in vitro studies conducted with macrophages cocultured with m. hyopneumoniae highlighted a strong activation of inflammatory pathways inducing the production of cytokines and chemokines, and expression of receptors or pathways inducing cell apoptosis [ , , , ] . moreover, m. hyopneumoniae is described as an inhibitor of macrophages phagocytic activity, which may explain the chronicity of m. hyopneumoniae infections and the greater host susceptibility to other pathogens [ , , ] . mycoplasma hyopneumoniae was found to activate costimulatory molecule expression on bona fide dcs with poor tnfα production, contrasting with monocytes. interestingly, a strong mitogenic activity for b cells was observed [ ] . altogether, these data indicate that m. hyopneumoniae is well sensed by the innate immune system, but the presence of immune evasion mechanisms targeting antigen presenting cells remains a possibility that needs further investigations. antibody responses after infection develop slowly and do not appear to correlate with protection [ , ] . the literature on m. hyopneumoniae infections coupled with information from mouse models indicates that adaptive immune responses represent a fragile balance between pathogenic and protective th-cell responses, probably belonging to the th or th types [ , ] . this aerobic gram-negative bacterium can be found in the respiratory tract of several animal species and it presents a worldwide distribution in the porcine rearing [ ] . b. bronchiseptica has a strong tropism for ciliated cells from the respiratory tissue and is mostly detected in the apical portion of the ciliated cells of turbinates, trachea and lungs [ , ] . it can also be found in the cytoplasm of neutrophils and macrophages and rarely in the alveolar lumen associated with small tufts of cilia [ , ] . hence, infected pigs show cilia loss in the bronchial and bronchiolar epithelium associated with multifocal erosion, fibrosis, and hyperplasia. neutrophil infiltrates are noted in the peri-conchal meatus and the submucosa of the bronchioles and alveoli, while lymphocyte and plasma cell infiltrations occur at the level of the lamina propria [ , ] . cell adhesion of b. bronchiseptica is a multifactorial process involving two main virulence factors; filamentous hemagglutinin (fha) and pertactin (prn) [ , ] . the expression of both adhesins is controlled by the bordetella virulence genes (bvg)as signal transduction system. fha is an adhesin with several binding domains including a carbohydrate-recognition domain responsible of the adhesion to macrophages and ciliated epithelial cells, a heparin-binding domain that mediates the binding to sulfated polysaccharides, and an arg-gly-asp domain (rgd) regulating the intercellular adhesion molecule (icam ) by epithelial cells after interaction with the nf-κb signalling pathways [ ] . this rgd domain is also present in the structure of prn and contributes to the binding process [ , ] . on the other hand, non-opsonic adhesion mechanisms play a role in binding to the host cells such as carbohydrate-specific mechanisms and those involving sialic acid-containing compounds [ ] . virulence of the bacteria depends on the strains; therefore, clinical signs can be different going from sneezing and transient nasal discharge for moderate and non-toxic strains to bronchopneumonia and atrophy of the nasal turbinate bones for virulent strains, especially if they are associated to other bacteria such as p. multocida [ ] . thus, b. bronchiseptica is usually described as primary lung pathogen in young pigs where it causes necrohemorrhagic bronchopneumonia whereas in older pigs this bacterium is mostly known as an opportunistic pathogen contributing to the prdc [ ] . the immune response against b. bronchiseptica is mainly triggered by the different toxins expressed such as adenylate cyclase, tracheal cytotoxin and dermonecrotic toxin (dnt). in the following section and additional file , we have used the published studies evaluating multiple infections including viral-viral, bacterial-viral, viral-bacterial and bacterial-bacterial respiratory coinfections and superinfections in swine. both in vivo and in vitro studies comparing single to multiple infections were included. studies evaluating vaccinations and the development of diagnostic techniques such as elisa or qpcr were excluded as well as trials testing antiviral or antibacterial molecules when there was no clear comparison between single and multiple infections. an attempt to present a synthetic view of coinfections is depicted in the heat maps (see figures , , ) . however, we recommend readers to refer to additional file for each coinfection couple to get a more detailed view. in these heat maps, we were interested in the effect of the first pathogen on the multiplication of the second one (named "assessed pathogen" in the figures) and on the host immune response and/or clinical signs. these effects were evaluated and a grade from − to + was given to every pathogen depending on the intensity of its impact on the multiplication of the second agent and on the immune response or on the clinical signs. negative grades were given to pathogens decreasing the multiplication of other pathogens, while positive grades were given to those inducing an increase. similarly, negative grades were attributed to pathogens with a tendency to decrease clinical signs or immune response related to the other pathogen. positive grades were given in case of an increase. the sum of the grades was calculated if the same pathogen combination was evaluated in several studies except in the case of discordant results. this grading was represented in the following heat maps and the number of the identified studies for the same pathogen combination is shown on the maps. in the heat maps, other pathogens, that are less associated to prdc such as g. parasuis, m. hyorhinis, m. floculare, p. multocida, and s. suis or even not considered as respiratory pathogens like staphylococcus aureus, classical swine fever virus, hepatitis e virus, porcine rubulavirus, ppv, and torque teno sus virus have also been included. indeed, these pathogens can also impact the outcome of respiratory infections and deserve, at least, to be mentioned. viral/viral respiratory coinfections have always had an important role in the porcine respiratory disease complex [ ] . several studies assessed the presence of two or more viral pathogens in pigs showing respiratory clinical signs in farms located in endemic regions [ , , ] . the main viruses contributing to the porcine respiratory disease are swiav, prrsv, pcv , and to a lower extent the prcov and the adv. due to their fast-spreading and their economic consequences, some viruses were more studied than others in the last years, especially pcv , prrsv, and swiav as shown in additional file a and b. we will thus put more emphasis on these three viruses as causes of primary infections. many in vivo studies were carried out to assess the severity of the clinical signs and the development of the microscopic/macroscopic lesions. this approach enabled a comparison between coinfection/superinfection and single-infection conditions. then, viral interference was progressively more frequently measured as a way to better understand the consequences of coinfections. in the last decades, the strong development of molecular biology and various tools enabled the evaluation of the immune response developed following polyviral infections. in additional file , the selected studies that were carried out on viral coinfections are presented from the oldest in vivo experiments to the latest in vitro and ex vivo experiments (additional file a and b). this data synthesis highlights the major impact of prrsv primary infection, which can both increase the titre of the following virus (pcv , hepatitis e virus-hev) in vitro [ ] and in vivo [ ] [ ] [ ] (figure a ), but can also worsen the clinical score associated to the disease ( figure a ). interestingly, even when the prrsv does not increase the viral production of the other virus, as observed in coinfections involving swiav [ ] or prcov [ ] (figure a) , it can also worsen the associated clinical signs. swiav and pcv as primary infectious agents have been less studied. however, it can be observed that swiav can interfere with other virus productions (prrsv and prcov) [ , ] whereas pcv has some detrimental impact on the clinical outcome of secondary viral infections (prrsv, swiav, and ppv) [ ] [ ] [ ] (see additional file and figure a) . then, regarding the inflammation induced in coinfection conditions, various outcomes were observed depending which viruses were considered (figure a) . many in vitro and in vivo experiments, with different bacterium-virus and virus-bacterium combinations, have been performed to identify the underlying mechanisms of the prdc (see figures b, c, b , c, and b). the main studies are presented in additional file c and d. bacterium-viral coinfections can also involve various primary respiratory pathogens. among them, the most frequently studied bacterium is m. hyopneumoniae, a pathogen that induces a chronic respiratory disease and can influence the outcome of a subsequent viral infection. however, mycoplasma infection needs to be already well established in the respiratory tract at the time of the viral infection to potentiate it. indeed, m. hyopneumoniae inoculated to pigs simultaneously or shortly before the virus did not strongly impact the severity of the viral infections (pcv , swiav, prrsv) [ ] [ ] [ ] , while its impact was clearly evidenced when inoculated weeks before viral infections [ ] . it is well-known that viral infections can induce an ideal environment for a bacterial superinfection through different mechanisms such as the destruction of the epithelial barrier, the over-expression of the receptors involved in the bacterial adhesion to the cells, and the alteration of the host immune response [ , , , ] . the swiav infection has been shown, for instance, as a way to facilitate the colonization of epithelial cells by s. suis, but only for the serotypes containing sialic-acid in their capsule [ ] . the swiav infection induces a loss of ciliated cells leading to the impairment of the mucociliary clearance function, but induces also the presence of the viral ha on the surface of infected cells that interacts with the sialic acid of the bacterial capsule, leading to increased adherence of s. suis [ , ] . although these swiav effects on s. suis have been clearly shown in vitro, no clear in vivo impact of swiav infection on s. suis pulmonary load has been described [ ] . it was clearly shown that the presence of both pathogens significantly induces more inflammation than single infections [ , ] . overall, studies carried out in pigs showed that a bacterium-virus or a virus-bacterium coinfection frequently induces an aggravation of pulmonary lesions ( figure b ) and a higher inflammation ( figure b ) and immune response, with increased production of pro-inflammatory cytokines. in many bacterium-virus and virus-bacterium associations, this worsened outcome seems to be the result of additive effects from both pathogens rather than a real synergy [ , ] . however, a potentiation of the viral infection by bacteria can also be observed in other cases, such as in the m. hyopneumoniae-prrsv coinfection [ ] . in that case, higher amounts of prrsv genomes were detected in lymphoid tissue and blood [ ] and a slower viral clearance was observed [ ] (figure b) , suggesting that the recruitment of immune cells in the lung parenchyma upon established m. hyopneumoniae infection may provide a steady supply of susceptible cells for prrsv [ ] . then, in porcine ams and in the "african green monkey" (originally described as porcine origin) st-jude porcine lung (sjpl) cell line, prrsv infection has been shown to be blocked by a pre-infection with a. pleuropneupmoniae, this antiviral activity being due to the a. pleuropneumoniae metabolites [ ] ( figure b) . given the fact that in vivo studies involving prrsv and a. pleuropneumoniae did not always investigate the impact of an a. pleuropneumoniae pre-infection on the subsequent prrsv infection [ , ] , as done in experiments performed in vitro [ ] , it cannot be easily concluded if this interference would be observed in the target species. however, in vivo, prrsv-a. pleuropneumoniae interactions were reported as absent or mild [ , ] (figure b ). in virus-bacterium coinfections, the dogma usually encountered is that viruses play an immunomodulatory role, which favors bacterial superinfections. nevertheless, a pre-disposing effect is also described for m. hyopneumoniae, which promotes viral but also bacterial superinfections [ ] (figure d ). few studies of experimental coinfections or superinfections with m. hyopneumoniae and/or other bacteria involved in prdc were performed compared to coinfections involving viruses. these studies are reported in additional file e. overall, these coinfections or superinfections induce more clinical signs and lung lesions and poorer technical performances when compared to single infections with the same infectious pathogens ( figure c ). the bacterialbacterial coinfections are also responsible for immune response alterations (for reviews see [ , ] ). for example, macrophages from pigs infected by m. hyopneumoniae decrease their phagocytosis capacity against a. pleuropneumoniae [ , ] . m. hyorhinis and m. flocculare, two mycoplasmas commonly co-isolated with m. hyopneumoniae in gross pneumonia-like lesions, may also impact the immune response by inducing the cytotoxicity of immune cells and/or the secretion of cytokines affecting its outcome [ ] . co-stimulation of porcine bmdcs with m. hyopneumoniae and m. hyorhinis induces a strong il production. in this last in vitro model, m. hyopneumoniae associated with m. flocculare reduces tnfα production compared to bmdcs stimulation by m. flocculare alone producing a tnfα concentration greater than that observed after stimulation with m. hyopneumoniae alone and m. hyorhinis alone [ ] . therefore, m. flocculare might play an initial role in pulmonary inflammation by inducing the production of tnfα by resident myeloid cells. supplementary investigations will be needed to elucidate the role of this cytokine in the pathogenesis of the disease [ ] . other examples of bacterium-bacterium in vivo coinfections are presented in additional file e. regarding coinfections and superinfections, most studies assessed the clinical outcome of the process but less is known about the mechanisms of interactions between pathogens and the consequences for the pathogens themselves, the infected cells and more generally for the host. the outcome of dual infection is variable depending on the antagonism, neutrality or synergy between the infectious agents. on the host side, coinfection can make the host response ineffective, and vice versa. if we look now at the possible interactions that can occur between pathogens we have to consider the nature of the infectious agents (summary provided in figure ) . different situations can be observed and coinfections can involve virus with virus, bacterium with virus and vice versa, and bacterium with bacterium. regarding virus-virus interactions, consequences are diversified and many studies looking at virus replication in coinfection situations have been carried out [ ]. the first consequence of coinfection could be the so-called viral interference, a situation whereby one virus interferes with the replication of the other one making the cells resistant to the superinfecting virus [ ] . the most common way for viral interference is indirect and based on the production of type and ifns which induce the expression of isgs after interacting with their cognate receptors [ , ] . these proteins then activate numerous mediators of the cellular antiviral system that may non-specifically block the replication of viruses. they may also interfere, to a certain extent, with bacterial multiplication since ifn can also be induced by intracellular bacterial pathogens (ibps) or some extracellular bacteria [ , ] . nevertheless, in some situations type ifns can also increase the host susceptibility to subsequent bacterial infection [ ] through impaired macrophage recruitment with a reduced cxcl and cxcl transcription [ ] and a reduced il [ ] production. then, there is also the non-interferon-mediated viral interference (or intrinsic interference) which is a cellular state of resistance induced by the virus to new viral infection by closely related or unrelated viruses [ ] . various mechanisms are described to explain this cellular state of direct or indirect resistance (for examples see [ ]). in this type of interference, which can occur between viruses but also between viruses and bacteria [ , ] , there is a competition between pathogens for the metabolites and all the host factors that allow their multiplication. besides the mechanisms involving a competition for common cellular factors, there are also several other mechanisms of interference described. these relies on viral defective interfering (di) particles [ ] , rna interference (rnai) [ ] , non-specific double stranded rna (dsrna) [ ] as well as trans-acting proteins [ ] . interference can occur at specific steps or multiple steps of the viral replication cycle (attachment, penetration, genome replication and/or budding) and can be direct or indirect. inhibition of superinfection (superinfection exclusion and superinfection suppression) is one of several consequences that can be observed in the interference between related and unrelated microorganisms. in superinfection exclusion, an established infection interferes with a subsequent, closely related infection [ , ] . an example of this phenomenon in pigs is the exclusion of highly virulent classical swine fever virus strain margarita in wild boars persistently infected with this virus upon a challenge infection with the same margarita strain [ ] . superinfection suppression is a quite close concept where this time persistently infected cells resist to a challenge with a heterologous virus [ ] . furthermore, when the host immune response-innate or adaptive-is considered in the study of the complex interactions taking place in viral coinfections, additional mechanisms of indirect interference linked to cellular and humoral cross-protection-resulting from a first viral contact with a wild-type or a vaccine strain-can be described. conversely, in some situations, viral coinfections can directly or indirectly result in enhanced replication and virulence for one or both pathogens as observed in several studies involving porcine viruses [ , , [ ] [ ] [ ] . in other cases, coinfection/superinfection has no effects on virus replications and the viruses can coexist in a relation called accommodation [ ] . besides consequences in terms of viral replication, there are also consequences for the genetic of the viruses and their evolution through events of recombination between closely related viral genomes. recombination, the parameters influencing it and its consequences were reviewed in rna and dna viruses [ , ] . then, as a result of all these possible interactions between viruses, the severity of the resulting disease and its epidemiology can be altered as exemplified in additional file . in the pig studies, most often, however, the exact mechanisms controlling interactions between viruses were not elucidated. several mechanisms explaining bacterium-virus and virus-bacterium interactions have been identified (for reviews see [ , , ] ). the interactions can have either a positive or a negative impact on both pathogens depending on the bacterial and viral species involved. usually, when the interactions are direct they promote viral infection without affecting the bacterial species [ , , ] . examples of these direct interactions are (i) direct binding of the virus to a bacterium or (ii) the utilization of a bacterial product by the virus. an example of direct interactions in the respiratory tract is the cleavage of the iav ha into ha and ha by a staphylococcus aureus protease helping the viral particle to become infectious [ ] . on the contrary, when interactions are indirect they often provide an advantage to bacterial infections. four mechanisms dealing with indirect interactions have been described: (i) viral alteration of the epithelial barrier, (ii) reduction or suppression of the host immune response, (iii) viral alteration/displacement of the microbiota, and (iv) virus-induced alteration of bacterial cellular receptor expression [ ] . all these mechanisms can operate together for the benefit of the superinfecting bacteria. a typical example of these indirect interactions is provided by pcv and swiav and porcine pathogenic bacteria such as a. pleuropneumoniae [ ] and s. suis [ , , ] where the bacteria benefit from the prior viral infections. however, bacteria can also directly benefit from a previous viral infection as observed in a study demonstrating that staphylococcus aureus was able to bind viral ha [ ] . the consequence of that binding was an enhanced bacterial internalisation by two mechanisms: (i) binding to ha exposed at the surface of infected cells, and (ii) binding to free extracellular virions. in some other situations, non-pathogenic bacteria can also directly or indirectly protect the host from viral infection as typically observed with probiotic bacteria which can show antiviral activity through the binding/ capture of the viruses and/or the competition for cell adhesion (for a review see [ ] ). this type of interaction has been frequently observed with enteric bacteria [ ] and an example in pigs is the reduced infection of ipec-j cells by vesicular stomatitis virus after pre-incubation of the cells with multiple probiotic bacteria [ ] . an intriguing relationship is occurring between ibp and viruses where metabolic reprogramming in host cells triggered by viruses might support or conversely limit coinfection by an intracellular bacterial partner (for a review see [ ] ). different possibilities can be identified in that type of interaction [ ] : (i) the first pathogen can reprogram cellular metabolism related to cellular immunity and decrease the defense against the other pathogen, (ii) the metabolic changes triggered by the first pathogen can facilitate the adhesion, the penetration, and the replication of the other, and (iii) the coinfection transform the active replicative state of the first pathogen into a stable persistent state. the first possibility associated to a decrease of the cellular defense is a commonly accepted mechanism [ ] while the second and the third possibilities are less experimentally demonstrated [ ] . looking at bacterium-bacterium interactions, they are extremely complex to assess because of the large diversity of the bacterial world and because little is known about the mechanisms underpinning these interactions during infections. moreover, it is now also clear that intestinal and respiratory microbiomes affect the interactions between pathogenic bacteria and the porcine host [ ] . some examples of the complex interactions occurring in bacteria-bacteria coinfections are presented in additional file e, but little is known about the mechanisms controlling these interactions. however, some mechanisms were provided above and interesting reviews dealing with that subject were published recently [ , ] discussing the possible direct interactions between bacteria-mainly chemical and physical. indirect interactions between bacteria were not reviewed in these articles but were discussed to some extent in other review papers focusing on polymicrobial infections [ , ]. the first observation coming from this review must be, even if several studies have been carried out on the subject, a lack of data about some specific coinfections and many discrepancies between studies. for instance, there are only a few in vivo studies about pcv in virus/virus coinfections and about pcv and prrsv in virus/bacterium coinfections (additional file and heat maps in figures , , ) . discrepancies are not surprising because of the definition of coinfection is not always the same between studies in addition to huge variations in the coinfection parameters amongst studies. in this review we focused on experimental (in vivo and in vitro) coinfections, it is worth to underline that these studies are inspired by field veterinarians and epidemiologists observations. however, the definition of epidemiologist coinfection can also vary between studies. indeed, in some cases there is concomitant direct identification of two microorganisms in the same animals, sometimes in the same farms, while in other cases it is just an indirect identification of the microorganisms' presence at some points through indirect serological assays. moreover, as stated before, the term coinfection is sometimes used to describe some situations of superinfections where the delay can be significant. regarding the experimental parameters, the multiplicities of infection (moi), the strains, the potential delays between successive infections, the routes of inoculations, the types of cellular hosts considered (more or less susceptible to one of the microorganism), the genetic background (breeds) and the sanitary status (specific pathogen free or conventional breeding) of the pigs, and the readout to assess coinfection outcome varied a lot between studies. to fully compare studies, a standardization of the assays would be needed. interestingly also, whereas in vitro studies' usefulness is not in question, it is important to underline here that in vitro observed interplay between pathogens cannot be automatically applied in vivo. for instance, whereas m. hyopneumoniae decreases the prrsv titre in vitro [ ] , it increases prrsv shedding in vivo and indeed worsens the clinical signs upon coinfection [ ] . consequently, the use of intermediary settings, such as co-culture of different cell types (see [ ] for examples), precision cut lung slices (pcls) [ ] or organoids [ ] , could help to understand the complexity of coinfections in the respiratory tissue. in vitro approaches usually consider one or a few cell types with some limitations during the evaluation process of the coinfection consequences. some viruses can contribute to the elimination of other viruses just because of their ability to replicate faster on a particular cell type [ ] . thus, results obtained in vitro cannot mimic the field situation when both agents coinfect the same pig, providing inaccurate conclusions about the coinfection dynamics. under such circumstances, pathogens may simply have different host cells and no longer be under direct competition for resources [ ] . besides the different interactions that infecting agents can have between them through a competition to resources, studies showed clearly that the immune system and the immunological responses can highly affect these interactions by inducing the competitive power of a pathogen or abolishing it and making it less competitive on the resource [ , ] . the effects of the immune system (especially humoral parameters) are often not taken into consideration in selected in vitro models [ ] . on the other hand, in vivo coinfection experiments have to deal with numerous constraints (health status of the animals used, cost, husbandry, and ethics amongst others) and therefore are not always easy to perform. hence, although in vivo experiments are required in this very complex field, they surely need to be combined to in silico/in vitro/ex vivo analyses of potential interactions between pathogens. moreover, multiple parameters of the coinfection protocols appeared difficult to set without any a priori such as the choice of the pathogen that will be inoculated first and the delay between infections. one possibility to deal with multiple parameters is to use intra-host infection mathematical modelling [ ] allowing to play, at limited cost, with the different parameters of the coinfections. however, these models need to be fed with data coming from conventional in vitro experiments as well as more complex in vivo studies. the other possibility is to rely on field prevalence studies monitoring the very presence of the pathogens (isolation, pcr) instead of the sero-conversion, in order to have a clear epidemiological picture of when and where coinfections occur. consequently, ex vivo models such as pcls generated from freshly sacrificed pigs [ ] or organoids [ ] are developing. these models are closer to mimic the in vivo situation than usual in vitro approaches, combining different types of cells and providing the pathogens with a wider range of cell hosts. however, the contribution of the immune response to the interaction between different pathogens is rarely considered [ ] . furthermore, the moi cannot be controlled because the number of infected cells in the slice or the organoid cannot be monitored easily either. another limiting factor in coinfection studies is the cell regeneration, which can vary between in vivo and in vitro models. cell regeneration can highly affect the dynamics of a coinfection, giving some pathogens extra target cells guarantying their longer existence while contributing to the clearance of others [ ] . finally, other potential technical limitations could always be discussed such as the lack of precision or sensitivity in the different diagnostic techniques especially in the presence of multiple agents. hence, the detection of coinfecting pathogens could be compromised or reduced as compared to their detection level in the context of single infections. as shown in this review many works have been dedicated to the study of coinfections and superinfections in pigs. usually, when the experiments were carried out in vivo, the researchers were more interested in the clinical outcome than in the interactions occurring between pathogens. indeed, in most of the cases the fine interactions between pathogens and especially the mechanisms behind these interactions and its potential consequences, at the molecular level, on the immune response were not studied for several reasons including technical limitations. also, in the studies assessing the occurrence of coinfections/superinfections in the fields, coinfection identification based on molecular tools such as pcr would be more accurate than sero-prevalence approaches which are less prone to identify currently present pathogens and thus coinfective pathogens. then, a better knowledge of each pathogen involved is crucial. we thus would like to make recommendations for future studies dealing with respiratory coinfections in pigs: (i) authors should clearly summarize their coinfection or superinfection experimental setup-doses of pathogens, delays between infections-in their materials and methods section; (ii) in this summary they would need to clearly present the pathogens they use and they should, as often as possible, select well-characterized strains; (iii) environmental and management conditions would need a strict control and monitoring; (iv) animal genetic and sanitary status would need to be carefully described and monitored during the study; and (v) the multiplications of all the pathogens shall be followed during the experiment using highly sensitive and specific assays. a clear description of all these parameters would help the scientific community to compare studies and progress in the understanding of the complex interactions between microorganisms. in the last years, the concept of innate immune memory or trained immunity has gained a lot of interest. this concept is coming from old observation, in [ ] , recognizing that the bacterial vaccine strain "bacille de calmette et guérin" (bcg) was protecting not only against mycobacterium tuberculosis but also against antigenically different microorganism causing childhood mortality, suggesting an "adaptation" of the cellular innate immune system. since then, many interesting studies about innate immune memory or trained immunity have been published (for a review see [ ] ) and it is recognized that cells such as myeloid cells, nk cells, and even epithelial cells [ ] can have a higher and quicker response upon re-exposure to a pathogen. trained immunity is accompanied by epigenetic changes and most often associated with modifications in cellular metabolism. a close look at potential epigenetic changes and cellular metabolism modifications would be of high interest in respiratory coinfection studies in the porcine species. recently an alternative to the mechanism of trained immunity in resident lung innate immune cells named "epigenetic legacy" has been described [ ] . in that study, the authors demonstrated that following iav clearance and clinical recovery ( -month post-infection), mice were better protected from streptococcus pneumoniae infection by adult bone-marrow-derived ams displaying transient transcriptional and epigenetic distinct profiles. this newly described consequence of a first viral infection also needs additional studies about prdc with an identification of the mechanisms shaping the complex interactions between pathogens. supplementary information accompanies this paper at https ://doi. org/ . /s - - - . additional file . studies about coinfections in the pig respiratory tract and their consequences. microorganisms associated with pneumonia in slaughter weight swine retrospective analysis of etiologic agents associated with respiratory diseases in pigs infectious agents associated with respiratory diseases in farrow-to-finish pig herds: a cross-sectional study transcriptome analysis of porcine thymus following porcine cytomegalovirus infection interaction between mycoplasma hyopneumoniae and swine influenza virus epidemiological characteristics of pulmonary pneumocystosis and concurrent infections in pigs in jeju island longitudinal study of respiratory infection patterns of breeding sows in five farrow-to-finish herds bacterial pathogens associated with lung lesions in slaughter pigs from herds reorganization and expansion of the nidoviral family arteriviridae porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system infection of monocytes with european porcine reproductive and respiratory syndrome virus (prrsv- ) strain lena is significantly enhanced by dexamethasone and il- phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability porcine reproductive and respiratory syndrome virus type . lena triggers conventional dendritic cells activation and t helper immune response without infecting dendritic cells dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny prrs virus receptors and their role for pathogenesis molecular cloning of porcine siglec- , siglec- and siglec- , and identification of siglec- as an alternative receptor for porcine reproductive and respiratory syndrome virus (prrsv) involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus replacement of porcine cd scavenger receptor cysteine-rich domain with a cd -like homolog confers resistance of pigs to genotype but not genotype porcine reproductive and respiratory syndrome virus direct interaction between cd n-terminal domain and myh c-terminal domain contributes to porcine reproductive and respiratory syndrome virus internalization by permissive cells differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates strain-dependent porcine circovirus type (pcv ) entry and replication in t-lymphoblasts cell tropism and entry of porcine circovirus porcine circovirus diseases characteristics of porcine circovirus- replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection porcine circovirus type (pcv ) distribution and replication in tissues and immune cells in early infected pigs porcine circovirus type displays pluripotency in cell targeting dendritic cells harbor infectious porcine circovirus type in the absence of apparent cell modulation or replication of the virus association of lymphopenia with porcine circovirus type induced postweaning multisystemic wasting syndrome (pmws) cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens distribution and characterization of il- -secreting cells in lymphoid tissues of pcv -infected pigs porcine circovirus type (pcv ) viral components immunomodulate recall antigen responses subset-dependent modulation of dendritic cell activity by circovirus type review: influenza virus in pigs influenza a virus interactions with macrophages: lessons from epithelial cells dendritic cells in innate and adaptive immune responses against influenza virus specificity and functional interplay between influenza virus pa-x and ns shutoff activity protein of pandemic influenza a virus inhibits porcine nlrp inflammasome-mediated interleukin- beta production by suppressing asc ubiquitination porcine coronaviruses. emerging transbounding animal viruses fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin sars-associated coronavirus transmitted from human to pig susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus further characterization of aminopeptidase-n as a receptor for coronaviruses diseases of swine molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine three classes of cell surface receptors for alphaherpesvirus entry the porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gc multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses update on actinobacillus pleuropneumoniae-knowledge, gaps and challenges actinobacillosis biofilm formation is prevalent among field isolates of actinobacillus pleuropneumoniae influence of actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells phagocytosis by pig alveolar macrophages of actinobacillus pleuropneumoniae serotype mutant strains defective in haemolysin ii (apxll) and pleurotoxin (apxiii) functional and structural changes of porcine alveolar macrophages induced by sublytic doses of a heat-labile, hemolytic, cytotoxic substance produced by actinobacillus pleuropneumoniae diseases of swine update on mycoplasma hyopneumoniae infections in pigs: knowledge gaps for improved disease control in vivo expression analysis of the p and p paralog families of mycoplasma hyopneumoniae post-translational processing targets functionally diverse proteins in mycoplasma hyopneumoniae genotype distribution of mycoplasma hyopneumoniae in swine herds from different geographical regions mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with mycoplasma hyopneumoniae cytokine expression in porcine lungs experimentally infected with mycoplasma hyopneumoniae a morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with mycoplasma hyopneumoniae transcription analysis of the porcine alveolar macrophage response to mycoplasma hyopneumoniae differential production of proinfammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model differential innate immune responses induced by mycoplasma hyopneumoniae and mycoplasma hyorhinis in various types of antigen presenting cells diseases of swine a porcine model for the evaluation of virulence of bordetella bronchiseptica contribution of bordetella bronchiseptica filamentous hemagglutinin and pertactin to respiratory disease in swine matrine displayed antiviral activity in porcine alveolar macrophages coinfected by porcine reproductive and respiratory syndrome virus and porcine circovirus type effect of an interferon-stimulated response element (isre) mutant of porcine circovirus type (pcv ) on pcv -induced pathological lesions in a porcine reproductive and respiratory syndrome virus (prrsv) coinfection model experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus hepatitis e virus chronic infection of swine co-infected with porcine reproductive and respiratory syndrome virus vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of siv vaccination dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study comparison of mono-and co-infection by swine influenza a viruses and porcine respiratory coronavirus in porcine precision-cut lung slices expression of toll-like receptor signaling-related genes in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type infection of cesarean-derived colostrum-deprived pigs with porcine circovirus type and swine influenza virus effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type porcine circovirus and porcine parvovirus simultaneous porcine circovirus type and mycoplasma hyopneumoniae co-inoculation does not potentiate disease in conventional pigs experimental dual infection of pigs with an h n swine influenza virus (a/sw/hok/ / ) and mycoplasma hyopneumoniae mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia preinfection of pigs with mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of h n subtype virus-bacteria interactions: an emerging topic in human infection the co-pathogenesis of influenza viruses with bacteria in the lung capsular sialic acid of streptococcus suis serotype binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells dynamic virus-bacterium interactions in a porcine precision-cut lung slice coinfection model: swine influenza virus paves the way for streptococcus suis infection in a two-step process investigation of pathogenesis of h n influenza virus and swine streptococcus suis serotype co-infection in pigs by microarray analysis transcriptional approach to study porcine tracheal epithelial cells individually or dually infected with swine influenza virus and streptococcus suis mycoplasma hyopneumoniae does not affect the interferon-related anti-viral response but predisposes the pig to a higher level of inflammation following swine influenza virus infection cytokine and chemokine mrna expression profiles in tracheobronchial lymph nodes from pigs singularly infected or coinfected with porcine circovirus type (pcv ) and mycoplasma hyopneumoniae (mhyo) the impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs actinobacillus pleuropneumoniae possesses an antiviral activity against porcine reproductive and respiratory syndrome virus enriched housing reduces disease susceptibility to co-infection with porcine reproductive and respiratory virus (prrsv) and actinobacillus pleuropneumoniae (a. pleuropneumoniae) in young pigs dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract polybacterial human disease: the ills of social networking how viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication intra-species and inter-species differences in cytokine production by porcine antigen-presenting cells stimulated by mycoplasma hyopneumoniae viral interference and interferon type i interferons in infectious disease type iii interferons in viral infection and antiviral immunity the roles of type i interferon in bacterial infection a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin influenza "trains" the host for enhanced susceptibility to secondary bacterial infection type i interferon induction during influenza virus infection increases susceptibility to secondary streptococcus pneumoniae infection by negative regulation of γδ t cells intrinsic interference: non-interferon mediated viral interference virus-bacteria interactions: implications and potential for the applied and agricultural sciences the antiviral and antitumor effects of defective interfering particles/genomes and their mechanisms rnai, a new therapeutic strategy against viral infection non-specific dsrna-mediated antiviral response in the honey bee roles of nonstructural polyproteins and cleavage products in regulating sindbis virus rna replication and transcription superinfection prevents recombination of the alphaherpesvirus bovine herpesvirus classical swine fever virus vs. classical swine fever virus: the superinfection exclusion phenomenon in experimentally infected wild boar porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type (pcv ) subtypes pcv a and pcv b by prolonging pcv viremia and shedding microbiome associations in pigs with the best and worst clinical outcomes following co-infection with porcine reproductive and respiratory syndrome virus (prrsv) and porcine circovirus type (pcv ) synergistic effects of sequential infection with highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type mechanisms and consequences of positivestrand rna virus recombination recombination in the alphaherpesvirus bovine herpesvirus role of staphylococcus protease in the development of influenza pneumonia porcine circovirus type promotes actinobacillus pleuropneumoniae survival during coinfection of porcine alveolar macrophages by inhibiting ros production sialic acid-dependent interactions between influenza viruses and streptococcus suis affect the infection of porcine tracheal cells viral hemagglutinin is involved in promoting the internalisation of staphylococcus aureus into human pneumocytes during influenza a h n virus infection a novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria intramacrophage survival for extracellular bacterial pathogens: mgtc as a key adaptive factor role of the microbiome in swine respiratory disease co-infection subverts mucosal immunity in the upper respiratory tract innate immune response to a h n subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices organoids in immunological research interspecific recombination between two ruminant alphaherpesviruses, bovine herpesviruses and plos one :e research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year ready to submit your research ? choose bmc and benefit from dominance of hepatitis c virus (hcv) is associated with lower quantitative hepatitis b surface antigen and higher serum interferon-γ-induced protein levels in hbv/hcv-coinfected patients dual infections of cd expressing nptr epithelial cells with influenza a virus and prrsv why, when and how should exposure be considered at the within-host scale? a modelling contribution to prrsv infection results of bcg immunization in new york city trained immunity: a program of innate immune memory in health and disease trained immunity carried by non-immune cells influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the colleagues who shared their experience regarding coinfections in pigs. fm is supported by an establishment grant from the région pays de la loire (rfi food for tomorrow-cap aliment). gs phd is funded by foundation philippe jabre (liban). all the authors were involved in the writing of the review. gs, nb, and fm generated the figures. gs, cd, jb, cf, cm-c, gsi, nb and fm prepared the additional file . all the authors read and approved the final manuscript. key: cord- - gej h d authors: meier, william a.; husmann, robert j.; schnitzlein, william m.; osorio, fernando a.; lunney, joan k.; zuckermann, federico a. title: cytokines and synthetic double-stranded rna augment the t helper immune response of swine to porcine reproductive and respiratory syndrome virus date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: gej h d immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine initially elicits a weak interferon (ifn)-γ response. to improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (il)- or ifn-α was co-administered during vaccination. in the presence of either adjuvant, at least a three-fold increase in the primary virus-specific ifn-γ response was observed. while this enhancement was only transient ( week) when the il- expressing plasmid was used, the effect was not only still apparent at weeks after vaccination in the presence of the ifn-α expressing plasmid but even after challenge with a virulent genetically divergent prrsv. in contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. despite the apparent augmentation of a t helper (th) type response by the inclusion of ifn-α or il- during vaccination, this modulation did not necessarily correlate with a reduction in viremia. since a similar increase in the degree of the ifn-γ response to the prrsv vaccine could be achieved by substituting polyinosinic–polycytidylic acid in lieu of either cytokine, exposure to prrsv in the presence of a variety of th polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. conceivably, such intervention could be applied to improve the formulation of anti-prrsv vaccines. porcine reproductive and respiratory syndrome (prrs) is an economically important disease of swine characterized by abortion, stillbirth and weak-born pigs. in its non-reproductive form, this syndrome affects younger pigs more severely than older animals, causing reduced growth and pneumonia that can be made more severe by co-infection with other pathogens (thacker, ) . the etiologic agent for this disease is an rna virus (prrsv) that belongs to the family arteriviridae, which targets macrophages for infection (wensvoort et al., ; collins et al., ) . prrsv exhibits a significant degree of genetic diversification (murtaugh et al., ; key et al., ; goldberg et al., ) and this characteristic probably contributes to the difficulty in controlling infectious outbreaks caused by it (meng, ) . under experimental conditions, the currently available modified live virus (mlv) vaccine against this pathogen has been shown to provide immunized pigs adequate protection from subsequent challenge with the homologous strain (lager et al., a (lager et al., , b . however, despite the demonstrated ability of this vaccine to afford pigs partial protection against infection by a genetically divergent prrsv strain (osorio et al., ; lager et al., ; mengeling et al., a mengeling et al., , b , the overall protective immunity provided to swine in commercial settings is generally inadequate (halbur, ) . infection of pigs with wild type prrsv (nelson et al., ; loemba et al., ; vezina et al., ; yoon et al., ; albina et al., b; gonin et al., ) or their vaccination with a live attenuated form of this virus (labarque et al., ; ostrowski et al., ) elicits an exuberant production of nonneutralizing antibodies. in contrast, a transient t cell mediated prrsv-specific lymphoproliferative response is detected at weeks post-infection and lasts an additional (bautista and molitor, ) to weeks (lopez-fuertes et al., ) . moreover, during this time interval, limited quantities of ifn-g secreting cells (sc) are also generated (meier et al., ; xiao et al., ) . interestingly, in the absence of additional antigenic stimulation this polarity reverses within the ensuing months, as manifested by a decreasing antibody response and a gradual increase in the intensity of the ifn-g response (meier et al., ) . the initial antibody-dominated immune response is not the result of insufficient antigenic stimulation, since neither the inclusion of a commercial adjuvant during primary vaccination (meier et al., ) nor booster immunizations of previously heavily vaccinated pigs (bassaganya-riera et al., ) enhances virus-specific cell-mediated immunity (cmi). thus, prrsv seems to inherently stimulate an imbalanced immune response characterized by an abundance of humoral immunity and a limited, but potentially protective, th -like ifn-g response . one characteristic of prrsv infection that probably contributes to the retarded development of a specific cell-mediated immune response is the lack of induced ifn-a production (albina et al., a; buddaert et al., ; van reeth et al., ) . usually, virus-infected cells secrete type i ifn and the released cytokine interacts with a subset of naïve t cells to promote their conversion into virus-specific ifn-g sc (cella et al., ; cousens et al., ; kadowaki et al., ; biron, ; levy et al., ) . however, to counteract a virus that is a poor inducer of type i ifn, the development of a th response can be generated by a pathway that utilizes interleukin (il)- (cousens et al., ; biron, ) . indeed, the injection of recombinant at the time of vaccination and three additional times during the following week enhanced the host cellular immune response to prrsv (foss et al., ) . based on the success of this intervention and the noted absence of ifn-a generation during prrsv infection, the present studies were undertaken to determine if the antiviral adaptive immunity induced by vaccination with prrs mlv could be modified simply by the co-administration of ril- protein directly, or of either porcine ifn-a or il- indirectly via plasmids expressing these cytokines. in addition to these two elements that can deliver th polarizing signals (biron, ; kadowaki and liu, ; kapsenberg, ) , the effect of stimulation with a known inducer of ifn-a production, namely double-stranded rna, was examined. although all three adjuvants positively influenced the intensity and rate of development of the virus-specific ifn-g response, the results indicate that a stronger immunomodulator will be needed to overcome the tendency of prrsv not to elicit this type of th response. prior to use for immunization, the ingelvac prrs mlv vaccine (boehringer ingelheim vetmedica inc., st. joseph, mo) was reconstituted from lyophilized vials according to manufacturer's instructions. the highly virulent, ''atypical'' prrsv strain ia- - - ( - , genbank accession number af ) was originally isolated from a severe case of reproductive failure in an iowa swine herd in and was used as the challenge virus in the animal studies. the extent of nucleotide sequence identity in orf between this wild-type strain and the parental strain of the vaccine (vr ) used in this study is approximately %. for elispot assays of prrsv-specific ifn-g sc, prrsv strains vr- (american type culture collection, manassas, va) and ia- - - were used as sources of antigen. when required, prrsv was propagated and titrated in monolayers of marc- cells as previously described (meier et al., ) . porcine peripheral blood mononuclear cells (pbmc) were isolated from venous blood that had been anti-coagulated with mm heparin and the cells were subsequently maintained as described by meier et al. ( ) . for generating a plasmid capable of expressing ifn-a in mammalian hosts, an intact cdna encoding porcine ifn-a was first prepared by rt-pcr using rna isolated from pig lymphocytes previously infected with pseudorabies virus (to stimulate ifna production). forward (tctgcaaggttcccaa-tg) and reverse (gtctgtcactccttcttcctg) primers were designed based on the nucleotide sequence of porcine ifn-a cdna (lefevre and la bonnardiere, ) . products of the anticipated size ( bp) were cloned into the pcr . plasmid (invitrogen corp., carlsbad, ca), and an insert having the predicted restriction enzyme sites was sequenced. a comparison of the coding region within the selected amplicon to the previously reported ifn-a cdna revealed three nucleotide differences. these variations resulted in two amino acid changes of a tyrosine to a cysteine at position and of an arginine to a leucine at position of the intact protein. the ifn-a cdna was then excised from the recombinant pcr . plasmid and placed under the transcriptional regulation of the cytomegalovirus promoter in pcdna (invitrogen) to generate pina. to verify that an active cytokine was encoded by the amplified cdna, chinese hamster ovary (cho) cells were transfected with pina and single cell clones resistant to . mg geneticin per ml growth medium were prepared. supernatant medium from the clones were tested for the ability to inhibit the replication of an interferoninducer negative strain of vesicular stomatitis virus (sekellick and marcus, ) in madin derby bovine kidney (mdbk) cells as previously described (rubinstein et al., ) . clones producing from to greater than , units ( unit inhibits % of vsv replication in mdbk cell monolayers) of ifn-a were detected. for generating a plasmid capable of expressing a composite il- moiety consisting of the p and p subunits joined in tandem by an inert amino acid stretch in mammalian cells, cdna encoding the amino acid long leader region and amino acids of the mature form of the il- p subunit was first derived from plasmid p - race # (provided by dr. m.p. murtaugh, university of minnesota) that contains approximately the half of il- p subunit cdna. this fragment was then inserted into pcdna and ligated to a segment that was excised from the yeast expression plasmid ppic scil- (foss et al., ) and consisted of the remainder of the il- p subunit cdna linked by a nucleotide stretch to cdna encoding the mature form of the il- p subunit. the resultant plasmid, pcpil , utilizes the cytomegalovirus promoter to regulate expression of a complete il- complex consisting of one of each of the two subunits. to verify that a biologically active, secreted cytokine could be produced by pcpil , cho cells were either mock-transfected or transfected with the plasmid and after h the overlaying medium was subjected to a bioassay designed to demonstrate the presence of porcine il- based on its ability to increase the ifn-g response of memory t cells to recall viral antigen (meier et al., ) . by comparison to a yeast-derived porcine ril- standard (provided by dr. m. moody, endogen, woburn, ma), > ng/ml bioactive cytokine was found in the transfected cell medium whereas no activity was found to be associated with the control supernatant. escherichia coli strain dh a (invitrogen) was transfected with either pina or pcpil and grown in l of lb medium supplemented with mg/ml ampicillin (sigma, st. louis, mo) for h at c with constant shaking ($ rpm). plasmid purification was conducted using a qiagen plasmid maxi kit (qiagen inc., valencia, ca) according to manufacturer's instructions. plasmid pina or pcpil was precipitated onto the surface of gold particles (average diameter of mm; bio-rad laboratories, inc., hercules, ca) at a concentration of . mg dna/mg gold. plastic tubing was then coated with . mg of the dna-bound gold particles using a tubing prep station (bio-rad) following the manufacturer's instructions and cut to yield cartridges containing . mg dna. immediately prior to use as a standard in the il- bioassay or as an adjuvant in the animal studies, lyophilized yeast-derived porcine ril- (endogen) was reconstituted in low endotoxin-tested pbs (mediatech, herndon, va) to a concentration of mg/ml. stabilized polyinosinic:polycytidylic acid (poly i:c) was prepared by the method of levy et al. ( ) with minor modifications. briefly, poly i:c (sigma) at mg/ml in pyrogen-free . % nacl was denatured at c for h and allowed to re-anneal while cooling slowly to ambient temperature. the annealed poly i:c solution was then mixed with equal volumes of . mg poly-l-lysine/ml pyrogen-free . % nacl and % carboxymethylcellulose in pyrogen-free . % nacl. the final preparation was stored at c until needed. in the first study, -week-old yorkshire x landrace cross-bred pigs were obtained from a prrsv-free herd and randomly segregated into five groups (n = ) and a sixth group of only two individuals. the latter group was kept in a prrsv-free environment and was not vaccinated or challenged. all other animals were immunized in their adductor muscles (inner thigh) with . ml of ingelvac prrs mlv vaccine. at the same time, some of the pigs were inoculated intramuscularly (i.m.) by needle with either ml of saline (group ), mg pina (group ) or pcpil (group ) per animal or intradermally (i.d.) with mg of pcpil /animal (group ) via biolistic delivery with a gene gun (bio-rad) at locations adjacent to the site of vaccination. twenty micrograms of porcine ril- in a ml volume were co-administered to the members of group , which also received a second i.m. injection of the cytokine h later. at weeks postimmunization, all pigs receiving only the vaccine except one (group ), or the vaccine in conjunction with an i.m. application of either plasmid pina (group ) or pcpil (group ) were transferred to a biocontainment facility together with five additional prrsv-naïve pigs (group ) obtained from the same herd mentioned above. at this time all of the transferred animals were challenged with . tcid / . ml ( . ml/nostril) of prrsv strain ia- - - . fourteen days later all pigs were euthanized. for the second study, twelve -week-old yorkshire  landrace cross-bred pigs were obtained from the same prrsv-free herd described above and were randomly assigned to one of two groups (n = ). while all pigs were immunized i.m. with . ml of ingelvac prrs mlv vaccine, . mg poly i:c/kg of body weight was co-administered to the animals of one group only. this dose of poly i:c was selected based on its demonstrated ability to induce the maximum presence of ifn-a in the sera of -week-old pigs (loewen and derbyshire, ) . pigs were bled at , , and h after poly i:c administration and differential white blood cell numbers were determined with a cell-dyne (abbott laboratories, abbott park, il) and confirmed by microscopy by an experienced technician. eight weeks later, members of both groups were given a secondary ''booster'' immunization identical in composition to that of the primary vaccination. the extent of the host cell-mediated immune response to prrsv was measured by using a single cell elispot assay meier et al., ) to enumerate virus-specific ifn-g-sc in the pbmc population. the presence of prrs virus-neutralizing (vn) antibodies in the sera was determined as previously described by benfield et al. ( ) . briefly, twofold serial dilutions of serum ( ml) in modified eagle's minimal essential medium (mem) were mixed with an equal volume of mem containing tcid of the selected prrsv strain. after incubation at c for h, the mixture was added to monolayers of marc- cells in -well tissue culture plates. the cells were then examined daily during a -day interval, and the end point titer was expressed as the reciprocal of the highest serum dilution that neutralized the development of a prrsv-induced cytopathic effect. quantities of non-neutralizing prrsv-specific antibodies in the sera were measured by using a commercial elisa assay (herd-chek prrs, idexx, westbrook, me). sample to positive (s/p) ratios greater than . were considered positive and were calculated based on the manufacturer's instructions utilizing the formula: s/p = (test sample a( ) against prrsv antigen À test sample a( ) against normal host cell antigen)/(positive control against prrsv antigen À positive control against normal host cell antigen). virus was detected in serum samples collected on days , and post-prrsv challenge and from tonsil biopsies obtained at the termination of the experiment. prior to use, tonsils ( . g) were first homogenized by grinding the tissue with a pestle in a . ml microfuge tube, adding ml of mem supplemented with mg of gentamicin per ml, and vigorously shaking the sample with the pestle in the tube. after removal of the pestle, an additional ml of mem was added, and the homogenate was again mixed vigorously. the suspension was then passed through a . mm pore-size filter and stored at À c. to detect infectious prrsv, quadruplicate sets of marc- cell monolayers in -or -well plates were incubated with either ml of serial -fold dilutions of serum or tonsil homogenate (starting with : dilution) in mem supplemented with mg of gentamicin per ml for h at c in % co . afterwards, the inocula were removed and replaced with ml mem supplemented with % fetal calf serum and mg of gentamicin per ml. the cells were then incubated for days at c and checked daily for signs of cytopathic effect. results are expressed as the mean ae the standard error of the mean (s.e.). statistical significance was determined by analysis of variance. when significant differences at the . % confidence level were present, individual differences between treatment groups were determined with fisher's protected least significant difference (plsd). the comparison between the quantity of virus in sera and tonsils was done by using the fisher's exact test. the analyses were performed with the statview program v . (abacus concepts, berkeley, ca). immunization of pigs with only prrs mlv vaccine induced a relatively mild cmi response during the ensuing weeks as evidenced by the low frequency of prrsv-specific ifn-g sc in the vaccinated pigs' pbmc populations (fig. ) . although a similar response was evident during this time period for vaccinated pigs also i.m. injected with exogenous il- either directly as protein or indirectly via il- cdna, immunized animals that were i.d. inoculated with il- cdna exhibited an overall . -fold transient increase in the frequency of prrsv-specific ifn-g sc at weeks post-vaccination. likewise, a slightly greater enhancement of three-fold was found in those vaccinated animals that had received ifn-a cdna. however, in this case, the ifn-g response remained elevated at about this extent throughout the following weeks. since the pigs immunized with prrsv mlv in the presence of pina exhibited a significantly enhanced and sustained ifn-g response as compared to those in the other vaccinated groups, these animals as well as those receiving the vaccine only or the vaccine plus an i.m. injection of pcpil were transferred to a biocontainment facility and challenged with prrsv strain ia- - - . as controls, an additional group of age matched prrsv-naïve pigs were included and also infected with the virulent virus. during the weeks after challenge, the animals originally immunized in the presence of ifn-a cdna continued to display a statistically significant, greater ifn-g response relative to that measured in the other pigs. all mlv-vaccinated pigs exhibited strongly positive anti-prrsv antibody titers (s/p ratios ranged from . to . ) at weeks following immunization and their levels of humoral immunity had increased when measured weeks later (s/p ratios ranged from . to . ) (fig. ) . however, there were no significant differences between the mean s/p ratios of the cohort receiving the vaccine alone and of any of those groups whose vaccination was supplemented with a cytokine adjuvant. in contrast, vn antibodies were not detected until weeks after immunization and then only at low titers ( : ; v:v) and in at most one or two pigs per group (table ) . differences between the various cohorts in regards to the proportion of pigs that contained vn antibodies were not found to be significant. immediately prior to challenge with the virulent prrsv ia- - - strain at weeks postvaccination, vn antibodies were not detected in serum collected from any member of the three cohorts being challenged (mlv only, mlv + pcpil i.m., and mlv + pina i.m.; fig. ) . although subsequently by weeks post-challenge the majority of the pigs had developed a detectable vn antibody response, the three groups could still not be statistically differentiated based on their average vn titers. despite use of the challenge strain in lieu of prrsv isolate vr- as an antigen source appearing to result in a slightly more sensitive assay for the presence of vn antibody, this visual difference was determined not to be statistically significant. as expected due to the short interval between exposure to virus and performance of the assay, prrsv-neutralizing antibodies were not injections of porcine ril- at a daily interval (group ). an additional group served as unvaccinated controls (group ). at weeks post-immunization, vaccinated animals in groups - as well as five, newly acquired prrsv-naïve pigs (challenge control, group ) were challenged with prrsv strain ia- - - . pbmc were isolated from the pigs at the indicated times post-vaccination and the presence of virus-specific ifn-g sc was determined by using an elispot assay. asterisks indicate significant differences (p < . ) between the frequencies of ifn-g sc in the blood of the animals immunized with the mlvalone as compared to the other immunized groups. each value represents the mean response of five animals ae s.e.m. except for group that was downsized to four pigs immediately prior to challenge and group that consisted of two animals. unvaccinated / / / / nd a serum was drawn from all members of groups - described in the legend for fig. at the indicated times after vaccination and assayed for the presence of virus neutralizing (vn) antibodies against prrsv isolates vr and ia- - - . b the numerator values represent the number of vn positive pigs, and the denominator values represent the total number of pigs in each respective group. the vn titer in positive samples was : against either virus isolate. c not done. fig. . comparison of the presence of prrsv-neutralizing antibodies in the serum of vaccinated pigs after a challenge with wild type prrsv. immediately prior to and weeks after challenge with prrsv strain ia- - - , serum was drawn from four members of group and all pigs in groups , and described in the legend to fig. and assayed for the presence of prrsv-neutralizing antibodies. results are presented as the reciprocal of the lowest two-fold dilution of serum that inhibited the replication of either prrsv strain vr- or ia- - - in marc- cell monolayers. each value represents the mean prrsv neutralizing antibody titer in the blood of five animals ae s.e.m. except for group that was downsized to four pigs immediately prior to challenge. detected in the sera of the challenged, non-vaccinated pigs. at days post-prrsv challenge, one of four pigs immunized with mlv vaccine alone (group ) and three of five pigs immunized in conjunction with an i.m. injection of pcpil (group ) had detectable viremia (level of sensitivity . tcid /ml serum) (fig. ) . in contrast, at this time all of the unvaccinated pigs (group ) and all of those that received pina in combination with the vaccine were viremic (group ). three days later, only three of the five animals injected with pina (group ) as well as all of the nonimmunized pigs (group ) remained viremic (level of sensitivity . tcid /ml serum). by days post-challenge, virus could be found in the serum of only one of the unvaccinated pigs and none of the vaccinated pigs (level of sensitivity . tcid /ml serum). prrsv was also detected in the tonsils of three of the five non-immunized animals, but not in biopsies removed from any of the vaccinated pigs, when the experiment was terminated at days after challenge (level of sensitivity . tcid /g tissue). the lower sensitivity of the virus detection assay at days after challenge was due to the toxicity of the serum at a : dilution. this toxic effect was not present in the serum samples collected or days after challenge. to evaluate the impact of poly i:c on the host response to prrsv, the immune status of pigs receiving the mlv vaccine alone or in conjunction with this synthetic double-stranded rna and then revaccinated with the same formulations weeks later was monitored for a total of weeks. a nearly immediate effect of poly i:c on the pigs was observed as evidenced by an approximately % reduction in the quantity of white blood cells in their peripheral blood as compared to the other animals at h after the fig. . comparison of the virus titers in the sera and tonsils of vaccinated pigs after a challenge with wild type prrsv. serum was drawn from four members of group and all pigs in groups , and described in the legend to fig. at , and days post-challenge with prrsv strain ia- - - , while tonsil biopsies were obtained at days after challenge for all virus-challenged pigs. virus titers (log tcid per ml of serum or per gram of tonsil) are presented for each pig (circle). the limit of detection for each assay is indicated by the dotted line and values at this level have been placed on the line. bars indicate the mean of four or five measurable titers within a group. statistical significance (p < . , p < . ) is based on differences on the percent of viremic pigs between the indicated treatment groups as determined by fisher's exact test. administration of this compound. this leukopenia was transient as it was no longer apparent h later (data not shown). at week post-immunization, the poly i:c-treated pigs also exhibited a significant . -fold increase in the frequency of their prrsv-specific ifn-g sc as compared to the untreated pigs (fig. ). this enhancement tapered off but was still > -fold when measured and weeks later. from then until the time of the second immunization, significant differences between the frequencies of the ifn-g sc in the two groups were not detected. however, week after the booster immunization, once again the frequency of virus-specific ifn-g sc in the poly i:c-treated animals increased in comparison to the untreated animals-in this case a statistically significant . -fold. during the ensuing -week interval until the termination of the experiment, no differences between the two groups in regards to this parameter were observed. although prrsv-specific antibodies were readily detected in all of the pigs' sera when collected at and weeks post-primary immunization, no significant differences were found to exist between the average antibody titers of the poly i:ctreated and untreated groups (data not shown). the data presented here demonstrate the adjuvant effects of ifn-a when provided exogenously in the form of an expressible cdna (pina) or circuitously via induction of ifn-a synthesis (poly i:c) on the vaccine-induced ifn-g response to prrsv. however, despite the positive modulation of th immunity, no significant alteration in the development of the humoral immune response was observed. thus, even with such intervention at the initiation of prrsv immunization, the usual rapid onset of anti-prrsv antibody production and delayed appearance of vn antibodies (labarque et al., ; ostrowski et al., ; meier et al., ) still occurred. although in previous studies a similar increased presence of prrsv-specific ifn-g sc was afforded to pigs receiving multiple injections of il- at the time of vaccination and during the ensuing week (foss et al., ) , the use of a conventional oil-in-water adjuvant was found to be ineffective in this regard (meier et al., ) . moreover, in that study, even though this compound demonstrably enhanced the genesis of vn antibodies recognizing pseudorabies virus in addition to specific ifn-g sc, the intensity of either type of immune response to prrsv was not affected. apparently prrsv possesses inherent structural elements that prevent the timely development of protective innate or adaptive immunity capable of inhibiting its infectious process. thus, the ultimate outcome of the interaction between this virus and its host will be determined by the elicited host response which is highly variable, as evidenced by the inconsistency of the clinical outcomes seen upon challenge of naïve or immune pigs with prrsv (labarque et al., ; mengeling et al., a mengeling et al., , b . this is likely due to the significant variability within the swine population in regards to their innate and adaptive immune responses to prrsv (xiao et al., ; royaee et al., ) . fig. . comparison of the intensities of the virus-specific ifn-g sc response of pigs to immunization with a prrs mlv vaccine in the absence/presence of poly i:c. groups of pigs were vaccinated with prrs mlv alone or in conjunction with poly i:c at an -week interval. pbmc were isolated from the pigs at the indicated times post-vaccination and the presence of virus-specific ifn-g sc was determined by using an elispot assay. significant differences (p < . ) between the frequencies of ifn-g sc in the blood of the two groups are indicated by an asterisk. each value represents the mean response of six animals ae s.e.m. the ability of prrsv to not initially elicit protective immunity in an infected host is relatively novel among viruses that even after being inactivated can usually still promote a th -like response (de wit et al., ) . in this regard it is notable that the ifn-a response to exposure to prrsv is nearly non-existent. for example, ifn-a production in the lungs of pigs acutely infected with prrsv was either almost undetectable, or -fold lower than that induced by another pathogen, porcine respiratory coronavirus (prcv) (buddaert et al., ; van reeth et al., ) . such lack of efficient stimulation of ifn-a production by a pathogen has a significant impact on the nature of the host's adaptive immune response, since ifn-a up-regulates ifn-g gene expression, and thus controls the dominant pathway that promotes the development of adaptive immunity, namely, t cellmediated ifn-g responses and peak antiviral immune defenses (cousens et al., ; levy et al., ) . in this regard, it has become evident that the link between innate and adaptive immunity in viral infections occurs through the interaction of dendritic cells with type i interferon (montoya et al., ; tough, ) and the dendritic-cell controlled polarization of t-cell function (kapsenberg, ) . the production of ifna by plasmacytoid dendritic cells/natural ifn-a/bproducing cells (nipc) has an autocrine effect that promotes their functional and phenotypic activation, which is necessary for their optimal expression of costimulatory molecules and subsequent ability to cause naïve t cells to differentiate into ifn-g-sc (cella et al., ; kadowaki et al., ; fitzgerald-bocarsly, ; montoya et al., ; honda et al., ) . presumably, prrsv is a poor inducer of ifn-a production by nipcs since unlike transmissible gastroenteritis virus (charley and lavenant, ; nowacki et al., ) and type-a cpg oligonucleotides (guzylack-piriou et al., ) it fails to stimulate the secretion of ifn-a from cultured porcine pbmc (albina et al., a; zuckermann et al., unpublished observations) . thus, direct examination of the outcome of the interaction of prrsv with porcine nipcs will likely reveal important information on the immunobiology of this virus, especially since this virus is susceptible to the antiviral effects of ifn-a (albina et al., a) . the application of molecular tools such as real-time pcr assays to measure the expression of key immune mediators that regulate the development of th responses in swine will be particularly useful in this endeavor. it should be noted that in the absence of ifn-a/b production, the cytokine il- whose synthesis is not induced by most viral infections (orange and biron, ) can increase ifn-g production by t cells (cousens et al., ) . thus, two alternative routes (il- -or type i ifn-dependent) can lead to an adaptive th cell-mediated immune response with potent antiviral effects (biron, ) . according to a scenario involving the presence of less than a requisite amount of ifn-a, il- could provide the necessary impetus for the development of an anti-viral ifn-g response. in this regard, il- mrna has been detected in porcine macrophages infected with prrsv (thanawongnuwech et al., ) , and transiently in the lungs of prrsv-infected pigs (chung and chae, ) . however, this pathogen is also apparently a poor stimulator of il- production, since a negligible quantity of il- mrna or protein was produced by porcine pbmc exposed in vitro to prrsv zuckermann et al., unpublished observations) . the observation that the inclusion of either il- and ifn-a during immunization increased the intensity of the ifn-g response to prrsv validates the proposed role of these two innate cytokines in directing the in vivo differentiation of swine th cells, and helps explain the poor virusspecific ifn-g response that normally develops as a result of the exposure of pigs to prrsv (meier et al., ; xiao et al., ) . to compensate for the lack of stimulation of innate cytokine expression by prrsv, novel adjuvants have been used during immunization. the administration of il- in combination with a live or killed prrsv vaccine resulted in an increased lymphoproliferative response to this virus (wee et al., ) . inclusion of either a combination of il- and il- or cholera toxin correlated with an enhanced response to prrsv but only at weeks after the initial exposure (foss et al., ) . moreover, in that study, the frequency of virusspecific ifn-g sc did transiently increase between and weeks post-vaccination of pigs also injected with porcine ril- . although similar results were obtained in the current study, albeit after biolistic injection of expressible il- cdna in lieu of protein, the provision of ifn-a cdna had a more pronounced and sustained effect on the intensity of the cell-mediated immune response. it was notable that the i.d. administration of il- cdna with a gene gun was more successful at enhancing the vaccine-induced ifn-g response than the i.m. injection of the same plasmid. this observation is in agreement with the reported higher efficiency of in vivo dna transfection by biolistic delivery (fynan et al., ; colosimo et al., ) . the greater effectiveness of the plasmid encoding ifn-a rather than il- at enticing an ifn-g response could be attributed to the relatively low amounts of complete il- receptor on swine lymphocytes and the limited up-regulation of expression of the il- receptor ß subunit gene as compared to other species (solano-aguilar et al., ) . in this regard, it should be noted that the injection of bioactive il- into naïve pigs did not stimulate a strong t cell response (solano-aguilar et al., ) . likewise, the introduction of a known inducer of ifn-a production in pigs, poly i:c (derbyshire and lesnick, ) , during vaccination temporarily amplified the quantities of prrsvspecific ifn-g sc, but was not as efficient as the ifn-a encoding plasmid at enhancing the ifn-g response to the vaccine. this difference could be attributed to the presence of immunostimulatory cpg motifs in the eukaryotic expression vector pcdna , which was used in this study to express the cytokine genes, and has been shown to induce ifn-g expression by porcine leukocytes (magnusson et al., ) . it is plausible that the combination of direct stimulation of nipc by the cpg motifs in the ifn-a-encoding plasmid, presumably through their toll-like receptor (shimosato et al., ) , in combination with the plasmid-driven production of ifn-a would provide the necessary stimulatory signals to promote the maturation of dendritic cells and the creation of a microenvironment conducive for a sustained ifn-g response to prrsv. although poly i:c induces ifn-a production, it does not entice porcine nipc to differentiate (guzylack-piriou et al., ) , and thus might not provide enough impetus to promote a sustained t-cell-mediated ifn-g response to this virus. this notion is in agreement with the role attributed to cpg-containing oligonucleotides in promoting the maturation of nipc in humans (krug et al., ) and swine (guzylack-piriou et al., ) . the inability of ifn-a cdna to enhance the vn antibody response to prrsv is in contrast to the positive effect exerted by the co-administration of adenovirus expressing ifn-a in combination with a foot-and-mouth disease virus (fmdv) subunit vaccine into pigs . the notion of an structural inherent property unique to prrsv that deters the elicitation of a strong vn antibody response is in accord with the observation that in the case of prrsv-neutralizing antibodies, the close association of a nearby n-glycan with a recognized epitope in the virus's envelope glycoprotein may impede neutralization of this virus (plagemann et al., ) . in addition, decoy epitopes that delay the development of vn antibodies also exist (ostrowski et al., ) . remarkably, as we have shown here, the titer of vn antibodies increased equally in all vaccinated groups as a result of the pigs being challenged with a genetically divergent, virulent virus. similarly, augmented titers of vn antibodies against prrsv have been observed in pigs previously vaccinated with prrs mlv after they received a booster immunization with an inactivated genetically divergent virus but not when exposed to the same attenuated virus (osorio et al., ; bassaganya-riera et al., ) . as compared to the control (prrsv naïve) pigs, a lower proportion of those vaccinated with prrs mlv exhibited viremia at and days after challenge. however, no further reduction was observed in animals also receiving either il- or ifn-a. rather, injection of pina increased the percentage of viremic pigs, although the mean virus titer in their sera was intermediate between those values determined for the vaccinated and un-vaccinated groups at days after challenge. the significance of this observation with regards to protective immunity is unclear. this is due to the fact that while vaccination can decrease the duration and magnitude of viremia following an experimental challenge (van woensel et al., ; verheije et al., ) , the reduction in viremia is not necessarily associated with commensurate amelioration of the severity of other clinical parameters associated with prrs such as a lessened rate of weight gain, fever, respiratory distress or virus transmission to sentinel pigs (nodelijk et al., ; labarque et al., ; mengeling et al., a) . a similar lack of correlation between viremia and clinical signs was also noted when two different age groups of non-immune pigs were infected with prrsv. whereas a greater frequency of viremia with an accompanying higher virus titer was found in the younger animals ( months of age), the older pigs ( months of age) exhibited more severe clinical signs . the difficulty of deciphering prrsv biology is further revealed by the marked degree of variability and irreproducibility of consecutive trials conducted by the same investigators (labarque et al., ; mengeling et al., a mengeling et al., , b . moreover, although a positive association between protection from disease and the intensity of the ifn-g response in swine has been observed in regard to pseudorabies virus van rooij et al., ) , a similar relationship concerning prrsv was not apparent from the results of this study. thus, the identification of an immunologic mechanism responsible for mediating protective immunity against prrsv poses a significant challenge and will perhaps require monitoring the extent of reproductive failure caused by this virus as a measurement of the degree of protection (lager et al., ) . notably, in recent, related field studies we have noticed a positive correlation between the reduction of abortion/still births in sows and the relative frequency of prrsv-specific ifn-g sc in their blood (lowe et al., ) . in any event, perhaps a more marked enhancement of the vaccine-induced ifn-g response will be needed in order to improve protective immunity against prrsv as we found to be the case when il- was used as an adjuvant for an inactivated pseudorabies virus vaccine, which like the prrs mlv vaccine stimulates a weak ifn-g response . clearly, further studies will be required to clarify how the outcome of a prrsv infection is influenced by the intensity of the ifn-g response by memory t cells. nevertheless, the potential importance of eliciting a strong ifn-g response against this pathogen might not be limited to preventing virus replication (bautista and molitor, ; rowland et al., ) . since ifn-g can restrain b cell differentiation and even polyclonal b cell activation (cowdery and fleming, ) , it is reasonable to propose that a strong ifn-g response could mediate protective immunity by thwarting the polyclonal b cell activation that is associated with prrsv infection and results in immunopathology (lemke et al., ) . the uncontrolled b cell activation induced by prrsv could also contribute to the observed weak cell-mediated immune response, since activated b cells can release il- (burdin et al., ) , a cytokine that inhibits both ifn-g production by porcine t cells (waters et al., ) , and ifn-a synthesis by human pbmc in response to viral stimulation (payvandi et al., ) . in the accompanying manuscript we demonstrate that pbmc obtained from pigs at weeks after immunization with prrs mlv spontaneously secrete il- , il- , and il- and that the production of these cytokines are indeed affected by the administration, at the time of vaccination, of the same ifn-a expressing plasmid utilized in the present study. such repression may be critical since unabated prrsv infection will actually stimulate synthesis of il- (asai et al., ) , a known inhibitor of th cell development (diehl and rincon, ) and promoter of polyclonal immunoglobulin production following b cell activation by the murine arterivirus, lactate dehydrogenase-elevating virus (markine-goriaynoff et al., ) . strategies designed to shift the bias of the initial reaction to prrsv from the strong elicitation of nonneutralizing antibody production towards a greater th -like immune response are worth exploring since they could conceivably lead to the development of an improved vaccine against this pathogen. the need to develop such methodology is made palpable by the unusual kinetics of the immune response to this virus, as evidenced by the lack of a marked cmi response upon vaccination and subsequent challenge with virulent virus. the ifn-g response to prrsv therefore appears to be determined at the time of the first exposure to this pathogen and is only minimally affected by re-exposure. although it is also possible that we did not monitor the immune response long enough post-challenge to detect a significant increase in the number of virus-specific ifn-g sc, this is unlikely since in the accompanying manuscript a similar minimal enhancement was observed when the immune response was measured for up to weeks after a booster immunization . in addition, similar limited changes in the ifn-g response have been observed upon challenge of vaccinated pigs with wild-type virus (foss et al., ) or booster immunization with mlv vaccine (meier et al., ) . remarkably, the t-cell proliferative response to prrsv was also not increased by a booster immunization in pigs that had previously been repeatedly exposed to a mlv vaccine, but rather appeared to be suppressed as compared to that elicited by the same vaccine in naïve pigs (bassaganya-riera et al., ) . the mechanism responsible for this unusual effect is currently unknown, but might be related to the persistence of the virus in lymphoid tissues associated with the site of infection (xiao et al., ) . such sustained presence of virus could adversely affect a subsequent response due to an inherent and yet unknown property of prrsv. in summary, it is apparent that prrsv elicits in swine a polarized immune response characterized by an abundance of non-neutralizing antibodies and a paucity of ifn-g sc. the molecular pathway responsible for generating this type of immunity is unknown at this time, but based on the results presented it likely involves the limited induction of ifn-a and il- production and/or inherent structural elements of the virus that promote such a response. we propose that the strong humoral immunity bias of the host response to prrsv is mostly responsible for the difficulties in the development of a vaccine deemed effective in the field. although our use of porcine cytokines, especially ifn-a, as adjuvants did not radically affect prrsv's inherent ability to promote this type of polarized immunity, a positive impact on the transition of t lymphocytes to ifn-g sc was noticed. the limited up-regulation of cellular ifn-g responses (presented here) or gene expression (described in the accompanying paper, royaee et al., ) indicates that more substantial stimulants of innate immunity are to modify the host response to prrsv. perhaps a more potent immunomodulator, such as type a cpg oligodeoxynucleotides (klinman, ) , will overcome the tendency of prrsv to stimulate a strong non-neutralizing antibody response and simultaneously promote a th (ifn-g dominated) response. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) elevated serum haptoglobin in pigs infected with porcine reproductive and respiratory syndrome virus impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of cd + t cells cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) interferons alpha and beta as immune regulatorsa new look in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) b-cell-derived il- : production and function plasmacytoid dendritic cells activated by influenza virus and cd l drive a potent th polarization characterization of blood mononuclear cells producing ifn alpha following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus) novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease expression of interleukin- and interleukin- in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (prrsv) isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs transfer and expression of foreign genes in mammalian cells interferonalpha/beta inhibition of interleukin and interferon-gamma production in vitro and endogenously during viral infection two roads diverged: interferon alpha/beta-and interleukin -mediated pathways in promoting t cell interferon gamma responses during viral infection in vivo depletion of cd t cells increases b cell sensitivity to polyclonal activation: the role of interferon-gamma identification of key immune mediators regulating t helper responses in swine the effect of interferon induction in newborn piglets on the humoral immune response to oral vaccination with transmissible gastroenteritis virus host-dependent type cytokine responses driven by inactivated viruses may fail to default in the absence of il- or ifn-alpha/ beta the two faces of il- on th /th differentiation natural interferon-a producing cells: the plasmacytoid dendritic cells. cytokines: receptors and cell signaling (supplement) in vitro and in vivo bioactivity of single-chain interleukin- adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the gp major envelope glycoprotein type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-alpha, tumour necrosis factor-alpha and interleukin- factors that influence the severity of clinical disease selective contribution of ifnalpha/beta signaling to the maturation of dendritic cells induced by double-stranded rna or viral infection natural interferon alpha/beta-producing cells link innate and adaptive immunity natural type i interferon-producing cells as a link between innate and adaptive immunity dendritic-cell control of pathogen-driven t-cell polarization genetic variation and phylogenetic analyses of the orf gene of acute porcine reproductive and respiratory syndrome virus isolates immunotherapeutic uses of cpg oligodeoxynucleotides toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd ligand to induce high amounts of il- effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine homologous challenge of porcine reproductive and respiratory syndrome virus immunity in pregnant swine evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenge-exposed with an antigenically distinct prrsv isolate molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs a modified polyriboinosinicpolyribocytidylic acid complex that induces interferon in primates ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity interferon induction in piglets with polyinosinic:polycytidylic acid complexed with poly-l-lysine and carboxymethylcellulose kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection correlation of cellular immunity to porcine reproductive respiratory syndrome virus and clinical disease during outbreaks of prrs in commercial swine herds the plasmid pcdna differentially induces production of interferon-alpha and interleukin- in cultures of porcine leukocytes distinct requirements for il- in polyclonal and specific ig production induced by microorganisms quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen characteristics of the immune response of pigs to prrs virus gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductive and respiratory syndrome strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus type i interferons produced by dendritic cells promote their phenotypic and functional activation immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine genetic variation in the prrs virus immunological responses of swine to porcine reproductive and respiratory syndrome virus infection serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus a quantitative assessment of the effectiveness of prrsv vaccination in pigs under experimental conditions age-related increase of porcine natural interferon alpha producing cell frequency and of interferon yield per cell an absolute and restricted requirement for il- in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections prrsv: comparison of commercial vaccines in their ability to induce protection against current prrsv strains of high virulence passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain exogenous and endogenous il- regulate ifn-alpha production by peripheral blood mononuclear cells in response to viral stimulation the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr- is located in the middle of the gp ectodomain inhibition of porcine reproductive and respiratory syndrome virus by interferongamma and recovery of virus replication with -aminopurine deciphering the involvement of innate immune factors in the development of the host responses to prrsv vaccination convenient assay for interferons persistent infection. ii. interferon-inducing temperature-sensitive mutants as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus swine toll-like receptor recognizes cpg motifs of human cell stimulant limited effect of recombinant porcine interleukin- on porcine lymphocytes due to a low expression of il- b receptor clinical manifestations of prrs virus differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model type i interferon as a link between innate and adaptive immunity through dendritic cell stimulation virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity vaccine-induced t cellmediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type ) infection in pigs effect on viraemia of an american and a european serotype prrsv vaccine after challenge with european wild-type strains of the virus safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested serpulina hyodysenteriae bacterin efficacy of porcine reproductive and respiratory syndrome virus vaccine and porcine interleukin- mystery swine disease in the netherlands: the isolation of lelystad virus the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection use of interleukin to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine interleukin- enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine the research work was supported by illinois agricultural experimental station swine research funds to fz and usda ars cris funds to jl. we gratefully acknowledge drs. tony goldberg and elizabeth greeley for helpful discussions and suggestions in the preparation of this manuscript. key: cord- -n w f rr authors: lee, sang-myeong; schommer, susan k.; kleiboeker, steven b. title: porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: n w f rr type i interferons (ifn-α and -β) play an important role in the innate host defense against viral infection by inducing antiviral responses. in addition to direct antiviral activities, type i ifn serves as an important link between the innate and adaptive immune response through multiple mechanisms. therefore, the outcome of a viral infection can be affected by ifn induction and the ifn sensitivity of a virus. north american porcine reproductive and respiratory syndrome virus (prrsv) field isolates were studied with regard to ifn-α sensitivity and induction in order to understand the role of type i ifn in prrsv pathogenesis. prrsv isolates were differentially sensitive to porcine recombinant ifn-α (rifn-α) and varied in their ability to induce ifn-α in porcine alveolar macrophages (pam) cultures as measured by a porcine ifn-α specific elisa on cell culture supernatants. fifty-two plaques were purified from three prrsv isolates (numbers , , and ) and tested for ifn sensitivity and ifn induction. plaque-derived populations were composed of heterogeneous populations in terms of ifn-inducing capacity and sensitivity to rifn-α. when macrophages infected with isolates , , or were treated with polycytidylic acid (polyi:c), ifn-α production was enhanced. cells infected with isolate and treated with polyi:c showed the most consistent and strongest enhancement of ifn-α production. it was demonstrated that the relatively low concentrations of ifn-α produced by isolate contributed to the enhanced ifn-α synthesis in response to polyi:c. isolates and significantly suppressed the enhanced ifn-α production by isolate in polyi:c treated cells. to determine if suppression was at the level of ifn-α transcription, quantitative rt-pcr was performed for ifn-α mrna and compared to gapdh and cyclophilin mrna quantification. however, the relative number of ifn-α transcript copies did not correlate with ifn-α protein levels, suggesting a post-transcriptional mechanism of suppression. in summary, these results demonstrate that prrsv field isolates differ both in ifn-α sensitivity and induction. furthermore, a prrsv field isolate strongly enhance polyi:c-induced ifn-α production in pam cultures and this priming effect was suppressed by other prrsv isolates. porcine reproductive and respiratory syndrome (prrs) is one of the most economically important diseases of swine. this disease was first detected in the u.s. in (keffaber, ) and in europe in (wensvoort et al., ) . the etiologic agent for this disease, prrs virus (prrsv) is an enveloped, positive-stranded rna virus that is a member of the arteriviridae family, order nidovirales. the fulllength genomic sequence has been determined for prrsv isolates of both european and north american lineage (allende et al., ; meulenberg et al., ; nelsen et al., ; shen et al., ; wootton et al., ) . molecular analysis of the prototype prrsv vr- and lelystad strains (u.s. and european isolates, respectively) has suggested that divergently evolved strains emerged on two continents almost simultaneously, perhaps due to similar changes in swine management practices (murtaugh et al., ; nelsen et al., ) . analysis of partial genomic sequence data for hundreds of prrsv strains reveals extensive diversity but also well-conserved regions (andreyev et al., ; meng et al., ; reviewed by meng, ) . disease caused by prrsv is characterized by severe and sometimes fatal respiratory disease and reproductive failure. infection with prrsv also predisposes pigs to infection by bacterial pathogens as well as other viral pathogens (benfield et al., ) and prrsv is a key etiologic agent of the economically important porcine respiratory disease complex (prdc). the most consistent pathological lesions caused by prrsv during acute infection are interstitial pneumonia and mild lymphocytic encephalitis plagemann, ; rossow et al., rossow et al., , . tissue macrophages and monocytes are the major target cells during both acute and persistent infection (molitor et al., ) , although pneumocytes and epithelial germ cells of the testes have also been shown to be infected (sur et al., (sur et al., , . it is important to note that clinical disease caused by prrsv is highly variable, ranging from mild, subclinical infections to acute deaths of adult animals . the differences in virulence have been attributed to numerous factors including host genetics, management practices, and virus strain heterogeneity (halbur et al., , (halbur et al., , keffaber, ; wensvoort, ) . after the acute phase of prrsv infection, which is typically characterized by viremia and clinical disease, many pigs fully recover yet carry a low-level viral infection for an extended period of time. under experimental conditions, persistent infection with prrsv has been well documented (albina et al., ; allende et al., ; christopher-hennings et al., ; horter et al., ; sur et al., ; yoon et al., ; wills et al., ) . these ''carrier'' pigs are persistently infected with prrsv and shed the virus, either intermittently or continuously, and may infect naïve pigs following direct or indirect contact. most notably, infectious virus has been recovered for up to days postinfection . producers often vaccinate swine against prrsv with modified-live attenuated strains or killed virus vaccines. however, current vaccines do not provide satisfactory protection, possibly due both to strain variation and inadequate stimulation of the immune system. a protective immune response is possible since it has been demonstrated that previous exposure can provide protection when pigs are challenged with a homologous strain of prrsv (lager et al., ) . however, protective immunity has never been consistently demonstrated for challenge with heterologous strains (lager et al., ; mengeling et al., ) . the ability of prrsv to routinely establish a persistent infection in pigs coupled with experimental evidence of suboptimal humoral and cellular immunity to prrsv suggests that the adaptive immune response to prrsv is often ineffective (reviewed by murtaugh et al., ) . the innate immune response, of which type i ifn is a major component, plays a key role in establishment of an effective adaptive immune response. the type i ifn response serves as an important link between the innate and adaptive immune response through multiple mechanisms (reviewed by biron, ) . type i ifn: (i) promotes the development of cd + t cell response (reviewed by boehm et al., ) , (ii) regulates the expression of many proteins responsible for generating antigenic peptides to be displayed in association with mhc class i (reviewed by york and rock, ) , (iii) enhances differentiation of dendritic antigen-presenting cells (luft et al., ) , (iv) stimulates the division of memory t cells by inducing il- (tough et al., ) , (v) contributes to prolonging the lifespan of activated t cells (marrack et al., ) , and (vi) enhances igg production and down-regulates ige secretion in b cells (finkelman et al., (finkelman et al., , . in addition to a role in the establishment of an adaptive immune response, type i ifn also plays an important role in innate host defenses against viral infection. type i ifn has been shown to activate or induce the synthesis of several proteins including , oligoadenylate synthase (oas), double-stranded rna-dependent protein kinase (pkr), mx and ribonuclease l (rnase l) (vilcek and sen, ) , all of which result in induction of an antiviral state (reviewed by samuel, ) . not surprisingly, many viruses have evolved specific mechanisms to counteract the ifn response. for example, the hepatitis b virus orf-c product and terminal protein (whitten et al., ) , the human papillomavirus (hpv) e and e proteins (park et al., ; ronco et al., ) , and the influenza a virus ns protein (talon et al., ) are viral proteins that have been shown to inhibit ifn synthesis. some viral proteins, such as the adenoviral e a (zhang et al., ) , sendai virus c proteins (komatsu et al., ) and simian virus protein v (didcock et al., ) , block ifn signaling via the jak-stat pathways. additionally, herpes virus, sv and emcv are known to block ifn-induction of the , -oas/rnase l system (vilcek and sen, ) . better understanding of the type i ifn response following prrsv infection will provide an important basis for understanding the adaptive immune response to this pathogen. previous work (albina et al., a; buddaert et al., ; van reeth et al., ) has demonstrated that european prrsv strains do not induce a strong ifn-a response, yet they are sensitive to the effects of ifn and can suppress a viral-induced ifn response. in this study, north american field isolates of prrsv were evaluated with respect to induction, sensitivity and suppression of an in vitro type i ifn response. differences in ifn phenotype were observed among north american prrsv field isolates. the pam cultures were obtained by bronchoalveolar lavage of - week-old domestic piglets from a prrsv seronegative herd. the lungs were removed from the piglets immediately after death and rpmi- medium (life technologies, grand island, ny) was introduced through the main stem bronchi. bronchoalveolar lavage fluid was centrifuged at  g for min. when cell pellets contained obvious contamination with red blood cells (rbc), pam cultures were purified by histopaque- (sigma inc., st. louis, mo) according to manufacturer's directions to remove red blood cell contamination. after centrifugation, cell pellets were resuspended in rpmi- medium supplemented with % fetal bovine serum (fbs), mm lglutamine, . mg/ml fungizone, u/ml penicillin, mg/ml streptomycin sulfate and mg/ml gentamicin (biowhittaker, walkersville, md) and plated at a density of -  cells/well in a -well primaria plate (becton dickinson and company, franklin lakes, nj). the pam cultures were confirmed to be prrsv-negative by rt-pcr before use in subsequent experiments. pam cultures were incubated for h at c in a humidified % co incubator and washed once with complete rpmi- media before use. the marc- cell line is a clone of the african green monkey kidney cell line ma- which is highly permissive to prrsv infection (kim et al., ) . cells were cultured and maintained in dulbecco's modified eagle medium (dmem) supplemented with % fbs, . mg/ml fungizone, u/ml penicillin, mg/ml streptomycin sulfate and mg/ml gentamicin (biowhittaker inc., walkersville, md) and then held at c in a humidified % co incubator. swine testicular (st) cells were used to grow and titrate tgev. st cells were grown in dmem supplemented with % heat-inactivated fbs, mm l-glutamine, . mg/ml fungizone, u/ml penicillin and mg/ml streptomycin sulfate (bio-whittaker inc., walkersville, md). all cells were maintained at c in a humidified % co incubator. fifteen prrsv field isolates, numbered from to , were obtained from clinical cases submitted to the university of missouri's veterinary medicine diagnostic laboratory. genomic sequences of open read-ing frames - of prrsv isolates , , and were submitted to genbank and assigned accession numbers ay (prrsv b), ay (prrsv ), and ay (prrsv ), respectively. virus stocks of prrsv isolates were prepared in pam cultures. a low multiplicity of infection (moi < . ) was used to prepare viral stocks, and the third to fifth passages were used for all experiments. transmissible gastroenteritis virus (tgev) (purdue strain) was obtained from the university of missouri's veterinary medicine diagnostic laboratory serology section. to purify plaques from prrsv isolates, confluent marc- cells (kim et al., ) in -well plates were infected with prrsv at a very low moi (< . ). after h, cells were rinsed and overlayed with cell culture medium containing . % agarose and incubated for - days until plaques were visible. plaques were harvested by aspiration into a micropipette and diluted into a final volume of . ml of culture medium and used for experiments without further propagation. a total of plaques were harvested, from isolate , from isolate and from isolate . the titers of original stocks of prrsv plaques were analyzed by quantitative real-time rt-pcr using purified rna from a titered virus stock to establish the standard curve. titers from isolate , , and plaques were . ae . tcid /ml (mean ae s.e.m.), . ae . tcid /ml, and . ae . tcid /ml, respectively. the moi used in experiments with plaque-purified stocks was approximately . for plaques from isolates and , and . for isolate . pam cultures in -well plates were pre-incubated with or u/ml rifn-a (r&d systems inc., minneapolis, mn) in cell culture media for h before virus infection. cells were washed with cell culture media and infected with prrsv field isolates at moi = . . after h post-infection (p.i.), supernatants were harvested, frozen and thawed one time. infectious virus titers in cell culture supernatants were determined by serial dilution in -well plates and calculated by the method of reed and muench ( ) . for ifn-a sensitivity experiments of prrsv isolates and , rifn-a ( , or u/ml) was added h prior to prrsv infection (moi = ) and virus growth curve experiments were performed. cell culture supernatants were harvested at , , , , h p.i. infectious virus titers in cell culture supernatants were determined by serial -fold dilutions of viral stocks with % tissue culture infectious dose (tcid ) titers calculated by the method of reed and muench ( ) . ifn-a was measured with a porcine ifn-a specific elisa by using f monoclonal antibody (mab) and k mab (r&d systems inc., minneapolis, mn) as previously described (diaz de arce et al., ) . mab k was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). flat-bottomed -well plates (fisher scientific, houston, tx) were coated overnight at c with f at a concentration of mg/plate in coating buffer ( mm carbonate buffer, ph . , sigma inc., st. louis, mo). after blocking with % non-fat dried milk, . % tween in phosphate buffered saline (pbs) for h at c, the plates were washed five times with . % tween in pbs. samples ( ml) were added into each well containing ml of % non-fat dried milk, . % tween in pbs and incubated for h at c. following five washes, ml of peroxidase conjugated k was added to each well. after h incubation and five washes, ml of substrate solution, tetramethylbenzidine (sigma inc., st. louis, mo), was added to each well. after min, the reaction was stopped with n hcl and the optical density was measured at nm by an elisa plate reader. quantified recombinant porcine ifn-a (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifn-a concentrations were calculated based upon a standard curve. one unit/ml of rifn-a is equivalent to pg/ml. pam cultures were infected with prrsv isolate , or at a moi = for h. after h p.i. the cell culture media was replaced with media containing polyi:c (sigma inc., st. louis, mo) at mg/ml. at h after the initial prrsv infection, supernatants were collected and the ifn-a concentration was measured by elisa. for suppression experiments, isolate was inoculated at h p.i. into pam cultures that were previously infected with either isolate , or . after h cell culture media was replaced with medium containing polyi:c at mg/ml. supernatants and cells were harvested at h p.i and stored at À c for subsequent analysis. prrsv isolates were uv-inactivated by exposing microcentrifuge tubes containing virus stocks to uv light (wavelength nm) for min. stocks of uvinactivated virus were demonstrated to be completely non-infectious for pam cultures. pam cultures were inoculated with either prrsv or uv-inactivated prrsv at moi = , incubated for h then washed with cell culture media. polyclonal antibody against porcine ifn-a ( neutralizing units/ml, r&d systems inc., minneapolis, mn) was added to cell culture media at the time of infection ( h) and again after infection ( h). at h p.i. cell culture medium was replaced with media containing polyi:c ( mg/ml). after an additional h incubation, cell culture media was collected and analyzed for ifn-a production by using porcine specific elisa. at h p.i. with isolate , or (moi = ), pam cells were harvested and resuspended in pbs at a concentration of cells/ml and assessed for cell viability. propidium iodide (sigma inc., st. louis, mo), which will positively stain dead cells with a disturbed cellular membrane, was added into . ml of cell suspension at mg/ml and incubated at room temperature. after min, cell viability was analyzed by flow cytometry. a second aliquot of infected cells with isolate , or was stained with . % trypan blue (bio-whittaker, walkersville, md), and then quantified microscopically with a hemocytometer to determine the total number of cells and the number of dead cells, i.e., those retaining trypan blue. the celltiter aq ueous one solution cell proliferation assay (promega corp., madison, wi) was performed as described by the manufacturer. briefly, ml of the celltiter solution reagent was added into each well of -well plate and the plate was incubated for h at c in a humidified, % co incubator. absorbance was measured at nm. the od measured at nm represents the amount of tetrazolium dye (mts)-toformazan conversion, which represents the number of viable cells. extraction of rna from samples of ifn suppression experiments was performed using trizol (invitrogen, carlsbad, ca) and the nucleospin rna ii kit (bd biosciences inc., palo alto, ca) with dnase i digestion performed directly on the spin column according to the manufacturer's instructions. extractions of the purified competitor rna were performed using the qiagen rnaeasy kit (qiagen inc., valencia, ca). heterologous competitor rna for quantification of swine ifn-a, cyclophilin or gadph was synthesized using the respective real-time rt-pcr primer sequences in a methodology previous described (kleiboeker, ) . the concentration of purified competitor rna was estimated by measuring the absorbance at nm and the purity was assessed by determining the ratio of absorbance at nm to the absorbance at nm. samples were considered to be relatively pure and suitable for use as quantification standards if the ratio was > . . following purification the rna was serially diluted in rnase-free dh o and stored as aliquots at À c. the number of molecules of competitor rna/ml was estimated based on the rna concentration and the molecular weight of the transcript. amplification of ml rna was performed using the qiagen quantitect probe rt-pcr kit (qiagen inc., valencia, ca) with thermocycling and detection performed in a stratagene mx (stratagene inc., la jolla, ca). samples were analyzed in triplicate. thermocycling conditions were: c ( min), c ( min), followed by cycles of denaturation ( c, s) and annealing/extension ( c, s). genbank accession numbers of sequences used for porcine ifn-a, porcine cyclophilin, and porcine gapdh real-time rt-pcr assays were nm , ay , and af , respectively. primers used for -exonuclease (taqman) amplification of swine ifn-a were (forward, position - ) -tctcatgcaccagagcca- , (reverse, position - ) -cctggaccacagaaggga- , and for amplification of swine cyclophilin were (forward, position - ) -atggcactggtggcaagt- , (reverse, position - ) -gatgccag-gacccgtatg- . primers used for -exonuclease (taqman) amplification of for swine gapdh were (forward, position - ) -tgcccagaacat-catccc- and (reverse, position - ) -ggatgaccttgcccacag- . all oligonucleotide primers were used at a final concentration of . mm. the dual-labeled probe used for detection of ifn-a transcript was - -fam-cttgagccttctggac-ctggttgc-bhq - (position - ), for detection of swine cyclophilin transcript was - -fam-catctatggagagaaatttgatgatgaga-bhq - (position - ), and for detection of swine gapdh transcript was -fam-cttct-accggcgctgccaag-bhq - (position - ). the dual-labeled probe used for detection of heterologous swine ifn-a, cyclophilin, or gapdh competitor rna was: -hex-tgtgctgcaaggc-gattaagttgggt-bhq - . each probe was used at a final concentration of . mm. all oligonucleotide primers and probes were synthesized by integrated dna technologies inc. (coralville, ia). the relative copy numbers of ifn-a mrna compared to cyclophilin or gapdh were determined as previously described (stordeur et al., ) . the student's t-test was used for the statistical analyses. p-values of less than . were considered statistically significant. to determine the sensitivity of north american prrsv field isolates to exogenous rifn-a, viral replication of field isolates was assessed in pam cultures that were pretreated with or u rifn-a/ ml cell culture media (fig. ) . for most isolates, viral titers were either unchanged or slightly decreased by the addition of u/ml of rifn-a. however, replication of one isolate (number ) was reduced by > log tcid /ml at this dose. all isolates demonstrated greater declines in replication following pretreatment with u/ml of rifn-a, with titers for of the isolates declining to the detectable limits of the assay. this experiment was repeated twice independently and results were consistent between experiments. to further characterize differences in sensitivity to rifn-a, two isolates, numbers and , were selected for use in a dose-response growth curve experiment. a representative experiment from independent experiments is shown (fig. ) . a dose-dependent reduction in viral titers was noted for both isolates. when pam cultures were pretreated with rifn-a at , and u/ml there was a significant (p < . ) reduction in viral titer from to h p.i. for isolate whereas for isolate only and u/ml of rifn-a resulted in a significant reduction in viral yield. for pam cultures that were pretreated with u/ml of rifn-a, titers of isolate were -fold lower than isolate at h p.i. compared to the untreated controls for each isolate. at , , and h p.i. the titers of isolate in u/ml rifn-a-treated cells were approximately . - . log % tissue culture infectious dose (tcid )/ml lower than in untreated cells. in contrast, the titers for isolate were reduced by about . - . log tcid /ml. when pam cultures were pretreated with u rifn-a/ml, virus yields of isolate were inhibited throughout the time course while virus yields of isolate increased through the time course and reached titers of the untreated control at h p.i. taken together, these results demonstrate that north american field isolates have different sensitivities to exogenous rifn-a in pam cultures. to investigate whether prrsv isolates are composed of heterogeneous populations with measurable differences in rifn-a sensitivity and ifn-a induction, plaque-derived populations of field isolates were prepared and tested. as shown in fig. , plaquepurified populations of all three isolates demonstrated a range in sensitivity to exogenous rifn-a. for isolates and , the range of reduction in viral titers was approximately . - . log tcid /ml. for isolate , the range of reduction in viral titers was - . log tcid /ml. in preliminary experiments, isolate was the most consistent ifn-a inducer compared to other field isolates tested. more than u/ ml of ifn-a was consistently synthesized in cells infected with isolate . in contrast ifn-a was typically undetectable in cultures infected with other isolates, although very low concentrations were detected in some experiments. plaque-derived populations of field isolates were also characterized with respect to ifn-a induction (fig. ) . while individual plaques from all three isolates induced relatively low concentrations of ifn-a compared to tgev, ifn-a production varied among plaques from each isolate. ifn-a induced by plaque-derived populations from isolates , and were . ae . , . ae . , and . ae . , respectively (mean ae s.d.). plaques from isolate induced a higher concentration of ifn-a than plaques of the other two isolates (p < . ). to investigate the ability of prrsv field isolates to suppress ifn-a induction by an ifn inducer, pam cultures were infected with prrsv then treated with polyi:c ( mg/ml) after h. in this experiment, no measurable amount of ifn-a was induced by prrsv infection alone with any of the three prrsv isolates (fig. a ). in the absence of prrsv, polyi:c failed to induce ifn-a at h, although ifn-a was induced at later time points. however, in the experiments shown samples from later time points were not used in order to avoid the influence of prrsv-induced cytopathic effect on ifn-a production. when prrsv-infected cells were treated with polyi:c, ifn-a production was consistently enhanced to the greatest extent for isolate . therefore it was tested whether isolates or could suppress ifn-a induced by a combination of isolate infection and polyi:c treatment of pam cultures. both isolates and significantly (p < . ) decreased ifn-a production by % and fig. . ifn-inducing capacity of prrsv plaque-derived populations. pam cultures were inoculated with ml/well of each plaque and supernatants were harvested after h and analyzed for ifn-a concentration using a porcine ifn-a-specific elisa. plaques from isolate induced a higher mean concentration of ifn-a than plaques of the other two isolates (p < . ). one unit/ml of rifn-a is equivalent to pg/ml. %, respectively. in contrast, ifn-a production was significantly (p < . ) increased by % in isolate superinfected cells. to demonstrate that suppression of ifn was not due to decreased cell viability, three cell viability assays (propidium iodide (pi) staining, trypan blue exclusion, and cell titer aq ueous cell proliferation and viability assay) were performed. when cell viability was analyzed by flow cytometry after pi staining, cell viability of prrsv-infected cells at h p.i. was . %, . %, and . % for isolates , , and , respectively. uninfected cells demonstrated . % viability. this result was in excellent agreement with the other two independent assays used to assess cell viability. to determine the effect of prrsvon ifn-a mrna synthesis, quantitative real-time rt-pcr was performed with samples from the suppression experiments. normalization of ifn-a mrna copy number was performed both with cyclophilin and gapdh transcript, with equivalent results obtained. as shown in fig. b , prrsv isolates induced variable quantities of ifn-a mrna in pam cultures. the quantity of ifn-a mrna did not correlate with the ifn-a protein level detected by elisa (fig. a) . consistently, ifna mrna synthesis was increased in prrsv-infected pam cultures (p < . ), despite a lack of detectable ifn-a protein. as a control, real-time pcr without the rt step was performed to determine if levels of contaminating genomic dna following dnase i treatment affected real-time rt-pcr quantification. relative amounts of dna in each sample were always less than % of the total signal, demonstrating that the signal analyzed was from rna rather than dna. uv-inactivated prrsv stocks were tested to determine whether virus binding to the cells is responsible for enhanced ifn-a production by polyi:c in cells infected with isolate (fig. ) . uvinactivation of virus reduced ifn-a in these cells to . % of control values obtained using fully infectious virus (p < . ). thus, virus binding to the cells alone was not sufficient to amplify ifn-a synthesis by polyi:c. to determine if ifn-a secreted after initial infection with isolate was responsible for enhanced ifn-a production in respond to polyi:c, neutralizing antibody against porcine ifn-a was used (fig. ) . neutralizing antibody against pig ifn-a reduced ifna production by polyi:c in cells infected with isolate - % of control values (p < . ) when added into cell cultures at a concentration of neutralizing units/ml prior to polyi:c treatment. pretreatment of macrophages with low concentration of rifn-a ( u/ fig. . effect of prrsv on ifn-a induced by polyi:c treated pam cultures. pam cultures infected with each isolate were infected with isolate at h p.i. (expressed as [# ]) and then treated with polyi:c at h p.i. supernatants were collected at h p.i. (a). ifn-a protein was measured by elisa. one unit/ml of rifn-a is equivalent to pg/ml. (b) quantitative real-time rt-pcr was performed by using total rna extracted from pam cultures. results are expressed as relative copy numbers of ifn-a mrna using cyclophilin as an internal control. values are shown as the means ae s.d. from duplicate wells and represent at least two independent experiments. *, significant difference compared with [# ] infected, polyi:c treated cells (p < . ). #, significant difference compared with polyi:c only treated cells (p < . ). no significant differences were detected in the copy numbers of ifn-a mrna among prrsv isolate infected cells and control cells. ml) also enhanced ifn-a production from u/ml to more than u/ml by polyi:c. thus these experiments suggest that even low concentrations of ifn-a induced by isolate are sufficient to strongly enhance the ifn response to polyi:c treatment. the type i ifn response plays an important role in host defenses against viral infection by performing immunoregulatory functions that link innate and adaptive immune responses by multiple mechanisms (reviewed by biron, ) as well as through direct antiviral effects. in this study, north american prrsv field isolates were evaluated with respect to induction, sensitivity and suppression of an in vitro ifn response in pam cultures. our results show that these isolates are sensitive to rifn-a in a dosedependent manner, but are generally poor inducers of ifn in vitro. notably the extent of the sensitivity and induction of ifn not only differed between prrsv isolates, but also showed variation among the plaquederived populations within each isolate. two of the field isolates studied were able to suppress the ifn-a production of cells dually-treated with polyi:c and a third north american prrsv isolate. while considerable variation was noted for the levels of ifn-a measured between replicates, most likely due to the variability of alveolar macrophage function among pigs (du manoir et al., ) , the trends observed were consistent between experiments. taken together, these results demonstrate that north american prrsv isolates differ in the ability to induce or suppress ifna, which may contribute to both virulence differences commonly observed among prrsv infections and the failure of prrsv infection to consistently induce a rapid and robust sterilizing immune response. the role of type i ifn in prrsv pathogenesis has been addressed by previous research (albina et al., a; buddaert et al., ; van reeth et al., ) , however this work was performed with two isolates (lelystad and sdrpi) of the european prrsv lineage, which is quite divergent ($ % sequence identity) compared to north american strains. in the present study, it was demonstrated that north american prrsv isolates were sensitive to rifn-a in a dose-dependent manner and were poor ifn-a inducers in vitro. these results are consistent with previous studies using european prrsv isolates (albina et al., a; buddaert et al., ; van reeth et al., ) . similar to results with type i ifn, north american prrsv replication was also blocked by a type ii ifn (ifn-g) and virus replication was restored by the addition of -aminopurine, an inhibitor of pkr (rowland et al., ) . biological, antigenic, pathogenic, and genetic variation among prrsv field isolates has been well documented (reviewed by meng, ) . the data presented herein demonstrated that north american prrsv field isolates were differentially sensitive to the in vitro antiviral effects of rifn-a and the use of plaque-derived populations from these field isolates demonstrated that closely related variants of prrsv may differ in their ifn responses. similarly, it has been shown that the sensitivity of reovirus, hepatitis c virus and lymphocytic choriomeningitis virus (lcmv) to type i ifn antiviral activity differs among viral strains (enomoto et al., ; moskophidis et al., ; reviewed by samuel, ) . a previous study suggested that significant differences of ifn-inducing capabilities could be a quasispecies marker of fig. . effect of uv-inactivated virus and anti-ifn-a antibodies on ifn-a production. pam cultures were infected with either prrsv or uv-inactivated prrsvat moi = . for h. polyclonal antibody against porcine ifn-a ( neutralizing units/ml) was added into cell cultures at the time of infection. at h p.i. cell culture media was replaced with media containing polyi:c ( mg/ml). after additional h incubation, cell culture media was collected and analyzed for ifn-a production by using porcine specific elisa. one unit/ml of rifn-a is equivalent to pg/ml. *, significant difference compared to polyi:c treated and isolate infected cells (p < . ). vesicular stomatitis virus (vsv) (marcus et al., ) . in contrast to the results presented herein, a study using the european prrsv strains sdrpi and sdrpii found no strain differences in ifn-a sensitivity or induction, despite differences in clinical virulence (albina et al., a) . previous experimental work has demonstrated evidence of a relationship between ifn phenotypes and virulence for other viruses. for example, virulent measles virus induces lower levels of ifn than attenuated strains and ifnresistant strains can establish persistent infections of the central nervous system (carrigan and knox, ). it has also been shown that the capability of noncytopathic bovine viral diarrhea virus to establish persistent infections in the early fetus is related to its ability to suppress type i ifn synthesis (charleston et al., ) . future in vivo studies will be needed to determine if the ifn phenotypes described in the present study are related to prrsv virulence or persistence. in the present study it was shown that polyi:c treatment of prrsv-infected cells resulted in greatly enhanced ifn-a production, especially in pam cultures infected with prrsv isolate . similar phenomenons have been reported by other studies. ifn-inducing listeria monocytogenes (havell, ) and type i ifn pretreatment (rosztoczy and megyeri, ) enhanced ifn production by polyi:c, sendai virus, or endotoxin. a plausible mechanism of this enhancement is by positive feedback regulation of type i ifn synthesis, in which a weak ifn-a/b signaling contributes to the enhancement of ifn-a/b synthesis due to the accumulation of transcription factor irf- (reviewed by taniguchi and takaoka, ) . this positive feedback loop of ifn synthesis could be efficiently induced by a small amount of ifna produced during the first h after isolate infection, resulting in amplification of the ifn-a response to polyi:c. consistent with this possibility was the observation that neutralizing antibody against ifn-a remarkably reduced ifn-a production by polyi:c in cells infected with isolate , and that u/ml of rifn-a added to poly i:c treated cells greatly increased the levels of ifn-a detected. tolllike receptor (tlr ) which recognizes polyi:c (alexopoulou et al., ) could be also involved in the enhancement of ifn production. measles virus strains with ifn-b inducing properties up-regulated the expression of tlr resulting in enhanced ifn-b production in response to polyi:c (tanabe et al., ) . therefore ifn produced by isolate may act through a similar mechanism to up-regulate tlr expression followed by increased ifn-a production after polyi:c treatment. for viruses such as sendai virus and vsv, low ifninducing strains have been shown to suppress ifn induction of high ifn-inducing strains of the same virus (marcus et al., ; mattana and viscomi, ) . in the present study, the levels of prrsv ifn induction were typically too low to use in a similarly designed suppression experiment. therefore the suppressive effect of prrsv isolates and on ifn-a production was evaluated in cells infected with isolate and treated with polyi:c, and significant suppression of ifn-a was demonstrated. european prrsv was also shown to suppress ifn-a production by tgev, both in vitro and in vivo (albina et al., a) . however, this result was not corroborated in vivo by another group using the closely related porcine respiratory coronavirus (prcv) (buddaert et al., ) . microarray experiments have indicated that polyi:c, ifn and viruses induced different subsets of the same cellular genes by activating diverse signaling pathways (geiss et al., ) . since ifn-a production was enhanced by the addition of polyi:c, it is likely that prrsv isolates and suppress an ifn-a synthesis pathway activated by isolate and polyi:c but not polyi:c alone. it has been shown that viruses which are resistant to the effects of ifn or can suppress ifn production have increased opportunities to spread before activation of the adaptive immune response (moskophidis et al., ; naniche et al., ) . mouse models of viral infection have clearly demonstrated that disrupting the ifn response leads to higher levels of viral replication. mice deficient in an ifn receptor exhibited increased susceptibility to lcmv (van den broek et al., ) and extensive spread of lcmv infection correlated with an isolate's relative resistance to ifn-a/b and ifn-g (moskophidis et al., ) . viruses can suppress ifn-a synthesis both at the transcriptional and post-transcriptional level. many viruses prevent mrna synthesis of ifn-a through various mechanisms but vsv inhibits ifn-a synthesis by shutting down the host protein synthesis (ahmed et al., ) . the present study showed that prrsv stimulated ifn-a mrna synthesis but inhibited ifna protein expression. this result suggests that the mechanism by which prrsv isolates suppress ifn-a production is mediated by generally inhibiting host protein expression or by specifically inhibiting ifn-a protein expression. half-life times of ifn mrna and protein could be different and that might be responsible for differences between mrna and protein levels of ifn. however, increases in ifn-a mrna expression for prrsv infected pam cultures were consistently detected at multiple times postinfection, yet for cells infected with isolate or very low to undetectable levels of ifn-a protein were typically observed at all times p.i. thus, although the mechanisms of ifn-a suppression by prrsv remain to be elucidated, data presented herein show that it is likely to be post-transcriptional in nature. while the immune response against prrsv is poorly understood, experimental work has demonstrated that the adaptive immune response of prrsvinfected pigs is generally ineffective (horter et al., ; mengeling et al., ; reviewed by murtaugh et al., ; wills et al., wills et al., , . specific evidence of this includes a slow neutralizing antibody response, which is typically not detected until weeks p.i. (albina et al., b) and does not reach maximum levels until - weeks p.i. (nelson et al., ; yoon et al., ) . wide variation has been shown in individual animals, with some studies demonstrating that many infected animals fail to develop a neutralizing antibody response (loemba et al., ; nelson et al., ) . while the importance of a cell-mediated response for protection against prrsv has not been debated, the effectiveness of this response during the early phases of disease also appears to be suboptimal (reviewed by murtaugh et al., ) . for example, the t-cell response to prrsv is weak and transient and cannot be restimulated for more than weeks post-challenge (meier et al., ; molitor et al., ) . additionally, ifn-g responses of prrsv-infected pigs were relatively weak and increased slowly in comparison to pseudorabies virus infected pigs (meier et al., ) . although the precise mechanisms for the ineffective nature of the adaptive immune response to prrsv is not known, prrsv evasion of the innate immune responses, such as the type i ifn response, may set the stage for subsequent subversion of the adaptive immune response. in conclusion, the data presented in this study demonstrates that north american prrsv field isolates are distinctively different in their in vitro ifn phenotypes, a difference that could contribute to the variable clinical signs and pathology observed in prrsv infections. moreover, the lack of ifn response following infection with prrsv could be involved in the establishment of persistent infections or secondary bacterial and viral infection, both of which are characteristic of prrsv infections in swine. however, in vivo studies are required to fully define the role of type i ifn in prrsv pathogenesis and virulence. the mechanisms involved in enhancement of ifn-a by prrsv infected and polyi:c treated cells remains to be elucidated. additionally, it will be important to determine whether prrsv generally inhibits host protein synthesis or specifically downregulates ifn expression and to identify the viral proteins that contribute to the different ifn response among prrsv isolates. knowledge about the role of ifn in prrsv pathogenesis will provide new hypotheses for improved vaccines or methods to modulate the immune response in order to more effectively control prrsv infection. ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units interferon-a response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) recognition of double-stranded rna and activation of nf-kappab by toll-like receptor north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection genetic variation and phylogenetic relationships of porcine reproductive and respiratory syndrome virus (prrsv) field strains based on sequence analysis of open reading frame characterization of swine infertility and respiratory syndrome (sirs) virus isolate atcc vr- role of early cytokines, including alpha and beta interferons (ifn-alpha/beta), in innate and adaptive immune responses to viral infections cellular responses to interferon-gamma in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) identification of interferonresistant subpopulations in several strains of measles virus: positive selection by growth of the virus in brain tissue establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type i interferon persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars a sensitive immunoassay for porcine interferon-alpha the v protein of simian virus inhibits interferon signaling by targeting stat for proteasome-mediated degradation variability of neutrophil and pulmonary alveolar macrophage function in swine mutations in the nonstructural protein a gene and response to interferon in patients with chronic hepatitis c virus b infection lymphokine control of in vivo immunoglobulin isotype selection regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice a comprehensive view of regulation of gene expression by double-stranded rna-mediated cell signaling comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a week-old cesarian-derived colostrums-deprived pig model differences in susceptibility of duroc, hampshire, and meishan pigs to infection with a high virulence strain (vr ) of porcine reproductive and respiratory syndrome virus (prrsv) augmented induction of interferons during listeria monocytogenes infection characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection reproductive failure of unknown etiology applications of competitor rna in diagnostic rt-pcr enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogenous subpopulation of ma- cells sendai virus blocks alpha interferon signaling to signal transducers and activators of transcription evaluation of protective immunity in gilts inoculated with the nadc- isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenge-exposed with an antigenically distinct prrsv isolate kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus type i ifns enhance the terminal differentiation of dendritic cells interferon induction as a quasispecies marker of vesicular stomatitis virus populations type i interferons keep activated t cells alive variations in the interferoninducing capacity of sendai virus subpopulations characteristics of the immune response of pigs to prrs virus gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination sequence comparison of open reading frames to of low and high virulence united states isolates of porcine reproductive and respiratory syndrome virus heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development diagnosis of porcine reproductive and respiratory syndrome using infected alveolar macrophages collected from live pigs strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav immunity to prrsv: double-edged sword resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon comparison of the structural protein coding sequences of the vr- and lelystad virus strains of the prrs virus immunological responses of swine to porcine reproductive and respiratory syndrome virus infection evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of alpha/beta interferon production serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents inactivation of interferon regulatory factor- tumor suppressor protein by hpv e oncoprotein. implication for the e -mediated immune evasion mechanism in cervical carcinogenesis lactate dehyrogenase-elevating virus and related viruses simple method of determining fifty percent enpoints human papillomavirus e oncoprotein binds to interferon regulatory factor- and inhibits its transcriptional activity pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs fetal microscopic lesions in porcine reproductive and respiratory syndrome virus-induced abortion essentially pure murine interferon-alpha/beta primes poly ri:rc and sendai virus-induced interferon production in mice inhibition of porcine reproductive and respiratory syndrome virus by interferongamma and recovery of virus replication with -aminopurine reoviruses and the interferon system antiviral actions of interferons determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion cytokine mrna quantification by real-time pcr in vivo detection of porcine reproductive and respiratory syndrome virus rna by in situ hybridization at different times postinfection porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis and induces germ cell death by apoptosis influenza a and b viruses expressing altered ns proteins: a vaccine approach mechanism of up-regulation of human toll-like receptor secondary to infection of measles virus-attenuated strains a weak signal for strong responses: interferon-alpha/beta revisited stimulation of naive and memory t cells by cytokines antiviral defense in mice lacking both alpha/beta and gamma interferon receptors differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity interferons and other cytokines mystery swine disease in the netherlands: the isolation of lelystad virus lelystad virus and the porcine epidemic abortion and respiratory syndrome identification of the hepatitis b virus factor that inhibits expression of the beta interferon gene prrs virus: a persistent infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate persistent and contact infection in nursery pigs experimentally infected with prrs virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection antigen processing and presentation by the class i major histocompatibility complex two contact regions between stat and cbp/p in interferon gamma signaling general overview of prrsv: a perspective from the united states key: cord- -y fyke u authors: jiang, yunbo; fang, liurong; luo, rui; xiao, shaobo; chen, huanchun title: n-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro date: - - journal: vet res commun doi: . /s - - - sha: doc_id: cord_uid: y fyke u nitric oxide (no) was proposed to be an important molecule against some microorganisms. in this study, we investigated the inhibitory effect of no on the infection by porcine reproductive and respiratory syndrome virus (prrsv) in vitro and the role of no in the defense against prrsv. our results indicated that exogenous no did not inhibit prrsv infection. unexpectedly, n-acetylpenicillamine (nap), a commonly used compound as negative control for no-producing reagents, inhibited prrsv replication. thus, the inhibition effect of nap on prrsv replication was further explored. we found that the maximal inhibition effect of nap on prrsv replication was achieved upon treatment h after virus infection and the virus yield was reduced by approximately fold in the presence of μm nap. an obvious inhibitory effect on viral rna and protein synthesis was also observed. however, the inhibitory effect was only achieved at early phase of virus infection. the normal virus yield could be restored upon the removal of nap treatment. the inhibitory effect might be caused by sulfhydryl-reducing capacity and metal chelating properties of nap. these studies suggested that (i) no production or no synthase (nos) expression profiling may not be a reliable index for the immune response to prrsv; (ii) nap could inhibit the replication of prrsv. distress in piglets and growing pigs (pejsak and markowska-daniel ) . the prrs virus (prrsv), the causative agent, is a positive-strand rna virus belonging to the family arteriviridae (meulenberg ) . this family also includes murine lactate dehydrogenaseelevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv) (meulenberg ) . currently, prrsv isolates can be divided into two distinct groups, which are represented by lelystad virus in europe and vr- in the united states, respectively (wensvoort et al. ; benfield et al. ) . the viral genome contains nine open reading frames (orfs): orf a, orf b, orf a, orf b and orfs - (wu et al. ) . nitric oxide (no) is a highly reactive molecule generated by nitric oxide synthase (nos), which converts l-arginine to citrulline and no (moncada et al. ) . the no molecule mediates numerous physiological functions as a vasodilator, neurotransmitter, immune regulator, antimicrobial and antiviral agent (bogdan ; cherayil and antos ; reiss and komatsu ) . in addition, no plays an important role in the defense against herpes simplex virus type , vesicular stomatitis virus, japanese encephalitis virus, murine hepatitis virus, vaccinia virus, influenza virus, and severe acute respiratory syndrome coronavirus (Åkerström et al. ; bi and reiss ; harris et al. ; lin et al. ; pope et al. ; rimmelzwaan et al. ) . moreover, no also participates in immune responses and possesses inhibitory properties against various pathogens (harris et al. ; lin et al. ; pope et al. ) . n-acetylpenicillamine (nap) is the acetylated form of d-penicillamine (dpa). in some experiments, due to the absence of no-donating s-nitro group, nap was used as the negative control for no donor snap. dpa is a potent metal chelator and belongs to the amino acids containing a thiol group. dpa can be used as antidotes for toxic metals, such as mercury poisoning. nap, the acetylated dpa, retains these properties. to date, nap is only used as the negative control for snap to study the inhibitory effect of no on virus replication in previous studies, and its antiviral activity was not reported. in the present study, we first investigated the potential inhibitory effect of no on prrsv replication in marc- cells. although exogenous no was released by no donor, snap, in marc- cells, the replication of prrsv remained unaffected. unexpectedly, nap exhibited an inhibitory effect on prrsv replication. therefore, this intriguing inhibitory effect of nap on prrsv replication was further investigated. virus and cells prrsv strain ya used in this study was isolated from lungs of field pigs at the acute stage of prrsv infection in china in and later identified as a highly virulent north american type isolate. the prrsv was propagated and titered in marc- cells. reagents snap (a no-producing compound), nap (a negative control for snap due to the absence of no-donating s-nitroso group) and dpa (an analog of nap without n-acetylation) were purchased from sigma (usa). the solutions were prepared by dissolving mm snap, nap and dpa into dimethyl sulfoxide-h o with a ratio of : in volume and stored at − °c for the future experiments. the amount of no produced in medium was determined by measuring its stable form, no - (lin et al. ) . the sample was added with equal volume of greiss reagent ( % sulfanilamide, . % n- -naphthylethy-lenediamine and % h po ) (sigma, usa) and incubated in -well microtiter plates at room temperature for min. the color development was measured at the wavelength of nm with an automated microplate reader (model el ; bio-tek, inc, usa). mtt assay for cytotoxicity cell cytotoxicity was determined by standard mtt assay with appropriate concentrations of different reagents (lin et al. ) . in order to infect cells with prrsv, marc- cells in -well plates were first incubated with prrsv at a multiplicity of infection (moi) of . at °c for h. then, unbound viruses were removed by washing with pbs (ph . ) for three times, and fresh medium was added to each plate for further incubation at °c. during experiments, the culture medium was supplemented with , and μm snap, nap or dpa. the medium containing the same concentrations of agents were replenished every - h during the culture period. as a negative control, the solvent alone (dimethyl sulfoxide-h o) was added to the culture medium. infectious prrsv in the supernatant was quantitatively evaluated by plaque-forming assay in marc- cells. various virus dilutions were added to the monolayer of marc- cells and incubated at °c for h. after the incubation, the cells were washed with pbs (ph . ) and overlaid with % methyl cellulose containing dmem f- medium plus % fbs. after an incubation of days, the cells were stained with . % crystal violet. total rna was extracted from marc- cells with trizol® reagent (omega, usa) according to the manufacturer's instruction. rt-pcr was performed with one-step rna pcr kit (amv) (takara, japan). the primer pair for prrsv orf was as follows: p - , '-cccctagtgagcggcaatt- '; and p - , '-agtcccagcgccttgattaa- '. the housekeeping gene, β-actin gene, was used as a constitutively-expressed control. its primers were as follows: pβ-actinf, '-actgtgcccatctacgag- '; and pβ-actinr, '-gttgcgttacaccctttc- '. the -bp orf fragments were visualized and photographed under uv illumination. the cells were washed twice with pbs (ph . ) and lysed in lysis buffer ( mm tris-hcl [ph . ], mm nacl, % nonidet p- , . % sodium deoxycholate, . % sodium dodecyl sulfate [sds] ). solubilized proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). proteins in the gel were transferred to a nitrocellulose membrane and probed with rabbit hyperimmune sera against n protein followed by horseradish peroxidase-conjugated goat anti-rabbit igg (southern biotechnology). the housekeeping gene, β-actin gene, was used as a constitutively-expressed control in western blot. blots were developed by the enhanced chemiluminescence western blot detection system (amersham) and directly exposed to image in molecular image station mm (kodak). after the treatment with/without chemicals and prrsv infection, the infected cells were detected with rabbit anti-prrsv n protein polyclonal antibodies as described previously (tian et al., ) . the labeled cells were examined by fluorescence microscope (olympus ix , japan). in order to investigate no release in marc- cells after snap treatment, the cells were treated with different concentrations of snap and nap or culture medium over a period of h. cell culture supernatants were collected and no production were measured. after snap treatments, the release of no in medium was observed in a concentration-dependent manner, but neither nap nor culture medium released a detectable amount of no. all drug treatments did not exhibit any negative impact on cell growth by mtt assay. compared with untreated cells, snap could not inhibit prrsv replication in marc- cells based on the no release. unexpectedly, nap, the "commonly used" negative control for no-donor snap had a significant antiviral activity against prrsv replication (fig. a ) at low infectious dose (moi= . ). the inhibitory effect was observed from to h post-infection. at h post-infection, virus titer was decreased from to , and pfu/ ml under , and μm nap treatments, respectively. however, the inhibition effect was lost at higher infectious doses (moi= . and . ) (fig. ) . in order to investigate the inhibitory kinetics of nap on prrsv replication and its inhibitory mechanisms, the optimal inhibition conditions were also investigated. in our experiments, inhibition analysis of nap on prrsv replication was carried out by μm nap at low infectious dose (moi= . ). the inhibitory effect of nap in marc- cells was investigated by determining the kinetics of prrsv production under different nap treatments. approximately -fold decrease of virus yield in prrsv-infected cells was observed in the presence of μm nap, as shown in fig. a . at the early stage of experiments, nap could completely inhibit the detectable propagation of prrsv. the inhibitory effect on viral release was observed in a concentration-dependent manner of nap. however, it lost at higher infectious doses (moi= . and . ) (fig. ) . in order to investigate inhibitory kinetics of nap on prrsv and inhibition mechanisms, the optimal condition was also investigated in following tests. the analysis of nap inhibitor to prrsv was conducted using μm nap at a low infectious dose (moi= . ). the inhibitory kinetics of nap to prrsv was stable and easily observed which provided sufficient conditions for analyzing inhibitory mechanisms. meanwhile, to better understand the inhibition mechanism of nap, the inhibition of dpa, an analog of nap without n-acetylation, was also investigated. when the cells were incubated with nap for h prior to the infection, total virus yield remained unaffected (fig. b) , indicating that nap had no effect on the capability of host cells to support subsequent viral replication. compared with the inhibitory effect of nap, prrsv remained unaffected by dpa (fig. c) . in order to investigate whether nap treatments reduce the amount of viral rna in infected cells, total rna was extracted from infected marc- cells with or without nap treatment. as shown in fig. a , the amount of viral rna was significantly reduced at h post-infection, indicating that nap could decrease viral rna in a dose-dependent manner. since nap could reduce the yield of viral rna, a significant inhibitory effect on viral protein synthesis should be observed. at h post-infection, n protein expression level of prrsv was examined by western blot with a polyclonal antibody against n protein (fig. b) . beta-actin expression was used as the loading control. in addition, as shown by immunofluorescence analysis (ifa) (fig. c-e) , compared with prrsv-infected cells without treatment, nap treatments on prrsv-infected cells resulted in an obvious decline in the number or intensity of stained cells against n protein. effector phase and reversibility of nap inhibition nap inhibition on prrsv replication at the early and late phases of virus infection as well as its reversibility was also studied. at the inhibitory phase, nap was added to infected cells at , and h post-infection. the viral yield was significantly reduced at and h post-infection (fig. a) . when nap was added to infected cells at h post-infection, virus titer was detected until h post-infection. in contrast, at h post-infection for adding nap, the viral yield did not exhibit significant difference between treated and untreated-cells, which indicated that nap inhibitory effect was abolished at this time point, and inhibitory effect of nap could be achieved only at early phase of virus infection. to determine the inhibition reversibility, nap was added to the infected cells at h postinfection and the treatment was removed after h by washing the treated cells. the cell culture was then replenished in fresh medium without nap treatment. the results showed that virus titers from nap treated cells followed by washing treatment were similar to those of the controls (fig. b) . therefore, the inhibitory effect was reversible. in this study, snap treatment was used to induce exogenous no, but its inhibitory effect on prrsv infection was not observed. unexpectedly, nap, originally used as the negative control of snap, exhibited an inhibitory effect on prrsv replication in a concentrationdependent manner. nap treatment could inhibit both prrsv viral rna and protein fig. the inhibitory effect of nap on prrsv growth at the higher infectious doses (moi= . and . ). the inhibition effect of nap at the concentrations of , , and μm on prrsv replication at the higher infectious doses was investigated. a moi= . , b moi= . synthesis and its inhibitory effect was achieved at to h after virus infection. moreover, the normal virus yield could be restored after the removal of nap treatment. no was reported to be a crucial effector molecule against microorganism infection (nathan and xie ) . in addition, no could modulate the gene expression of multiple signal transduction pathways (huang et al. ) . therefore, no production or nos expression profiling was often considered as an important index of immune responses against some pathogens (gamba et al. ). however, previous experimental evidences indicated that no activity varied among different cells or tissues and no formation could not be detected in porcine immune cells. in addition, no level was not changed in lung macrophages of the prrsv-infected pigs (boissel et al. ) . recently, the reports also confirmed that no was not able to significantly inhibit prrsv replication in vivo (jung et al. ) . exogenous or endogenous no and inducible nos expression exhibited no apparent antiviral effect on sendai virus (z strain) and influenza a virus (h n ) in mdck cells (yoshitake et al. ) . similarly, our results demonstrated that sufficient no production could not suppress the propagation of prrsv in vitro. based on these results, it is reasonable to propose that no production or nos expression profiling may not be a key index for the immune response to prrsv. unexpectedly, we found that nap, a structural analog of cysteine and a metal chelator, could inhibit the replication of prrsv in marc- cells. due to nap treatment, a significant reduction of viral yield and a remarkable inhibition of both viral rna and protein synthesis were observed. however, the inhibitory activity was not attributed to the involvement of marc- cells. nap could result in the maximal inhibitory effect on the replication of prrsv at the h post-infection and total abolishment of prrsv replication at h post-infection. this observation strongly suggested that nap only interrupted prrsv replication during the early phase of virus infection. the inhibitory effect was reversible, and the normal virus yield could be recovered after the removal of the drug. in addition, dpa, an analog of nap with the free amino group, could not inhibit prrsv replication (fig. c) . the results indicated that acetylation may play an important role on prrsv replication. previous studies demonstrated that dpa could prevent trans-activation of human immunodeficiency virus type- (hiv- ) ltr (chandra et al., ) . a docking study further revealed a selective binding of dpa to the cysteine-rich region of tat protein in hiv (kanyalkar et al. ) . here we found that the inhibitory activity of nap and the inactivity of dpa on prrsv replication. these results enriched our understanding of nap and dpa, although the exact mechanisms of inhibition by nap still need to be further studied. the inhibitory effect may result from the disruption of disulfide bonds by the free thiol group of nap. because the thiol group of snap is nitrosated, snap exhibits no disulfide reducing activity (fig. ) . disulfide bonds play an important role in assembling and stabilizing viral capsid structure of several viruses with icosahedral nucleocapsids (jeng et al. ; li et al. ; wootton and yoo ) . human cytomegalovirus and vesicular stomatitis virus (vsv) are also demonstrated to be vulnerable to sulfhydryl reagents (baum et al. ; beatrice and wagner ) . alternatively, the inhibitory effect of nap on prrsv replication could be related to its metal chelating properties. it was reported that the replication of human immunodeficiency virus (hiv), hepatitis c virus (hcv) and flavivirus can be inhibited by different chelating agents (chyan-jang et al. ; koch et al. ; van-asbeck et al. ) .the exact inhibitory mechanisms of prrsv replication need to be verified. in summary, our studies provide two crucial evidences. first, no have no effect on prrsv replication in marc- cells. second, nap can inhibit the replication of prrsv. these findings could contribute to the development of effective anti-prrsv agents. nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus inhibition of human cytomegalovirus ul protease by specific intramolecular disulfide bond formation effect of sulfhydryl reagents on the infectivity of vesicular stomatitis virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) inhibition of vesicular stomatitis virus infection by nitric oxide nitric oxide and the immune response neuronal-type no synthase: transcript diversity and expressional regulation d-penicillamine inhibits transactivation of human immunodeficiency virus type- (hiv- ) ltr by transactivtor protein inducible nitric oxide synthase and salmonella infection cholesterol effectively blocks entry of flavivirus early inhibition of nitric oxide production increases hsv- intranasal infection gamma interferon-induced nitric oxide-mediated inhibition of vaccinia virus replication nitric oxide regulates th cell development through the inhibition of il- synthesis by macrophages differential formation of disulfide linkages in the core antigen of extracellular and intracellular hepatitis b virus core particles nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus docking studies reveal a selective binding of d-penicillamine to the transactivator protein of human immunodeficiency virus type -thienyl)- , -dihydroxy- -carboxypyrimidines as inhibitors of the hepatitis c virus ns b polymerase: discovery, sar, modeling, and mutagenesis formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerzation of simian virus vp in the cytoplasm inhibition of japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on rna virus replication prrsv, the virus nitric oxide: physiology, pathophysiology, and pharmacology nitric oxide synthase: roles, tolls, and controls losses due to porcine reproductive and respiratory syndrome in a large swine farm resistance to murine hepatitis virus strain is dependent on production of nitric oxide does nitric oxide play a critical role in viral infections? inhibition of influenza virus replication by nitric oxide an attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) protects piglets against hp-prrs anti-hiv effect of iron chelators: different mechanisms involved mystery swine disease in the netherlands: the isolation of lelystad virus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages a kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf b nitric oxide as an endogenous mutagen for sendai virus without antiviral activity key: cord- -qea b i authors: eck, melanie; durán, margarita garcía; ricklin, meret e.; locher, samira; sarraseca, javier; rodríguez, maría josé; mccullough, kenneth c.; summerfield, artur; zimmer, gert; ruggli, nicolas title: virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: qea b i porcine reproductive and respiratory syndrome virus (prrsv) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. the vaccines currently available on the market elicit only limited protection. recombinant vesicular stomatitis virus (vsv) replicon particles (vrp) have been used successfully to induce protection against influenza a virus (iav) in chickens and bluetongue virus in sheep. in this study, vsv vrp expressing the prrsv envelope proteins gp , m, gp , gp , gp and the nucleocapsid protein n, individually or in combination, were generated and evaluated as a potential vector vaccine against prrsv infection. high level expression of the recombinant prrsv proteins was demonstrated in cell culture. however, none of the prrsv antigens expressed from vrp, with the exception of the n protein, did induce any detectable antibody response in pigs before challenge infection with prrsv. after challenge however, the antibody responses against gp , gp and gp appeared in average weeks earlier than in pigs vaccinated with the empty control vrp. no reduction of viremia was observed in the vaccinated group compared with the control group. when pigs were co-vaccinated with vrp expressing iav antigens and vrp expressing prrsv glycoproteins, only antibody responses to the iav antigens were detectable. these data show that the vsv replicon vector can induce immune responses to heterologous proteins in pigs, but that the prrsv envelope proteins expressed from vsv vrp are poorly immunogenic. nevertheless, they prime the immune system for significantly earlier b-cell responses following prrsv challenge infection. infections with porcine reproductive and respiratory syndrome virus (prrsv) causes reproductive failures in sows [ ] and respiratory disorders particularly in young pigs [ ] , which results in important economic losses worldwide [ , ] . recently, highly pathogenic prrsv strains have emerged in china [ ] and eastern europe [ ] . prrsv is an enveloped positive sense singlestranded rna virus belonging to the family arteriviridae within the order nidovirales [ ] . two prrsv genotypes can be distinguished, type prrsv of european origin and type prrsv originating from north america and china, both spreading worldwide with high genetic and antigenic diversity [ , ] . the prrsv genome consists of at least open reading frames (orf). orf a and b encode the non-structural proteins from two polyproteins pp a and pp ab that are further processed proteolytically, as well as two proteins nsp tf and nsp n resulting from ribosomal frameshifts within the nsp gene (for a detailed review see [ ] ). the remaining orfs encode the structural proteins on subgenomic messenger rnas. orf a, b and - , encode the glycoprotein (gp ) also termed gp a, the non-glycosylated protein b also termed e, the glycoproteins gp , gp , gp , the non-glycosylated membrane protein m (from orf ) and open access *correspondence: nicolas.ruggli@ivi.admin.ch institute of virology and immunology ivi, sensemattstrasse , mittelhäusern, switzerland full list of author information is available at the end of the article the nucleocapsid protein n (from orf ), respectively (reviewed in [ ] ). recently, an alternative orf a protein was identified as a minor component of the equine arteritis virus (eav) [ ] and the prrsv virions [ ] . gp and m form a disulphide-linked heterodimer that is essential for the formation of infectious particles [ , ] . for eav, the glycoproteins gp , gp and gp form a heterotrimeric complex that is stabilised by disulphide bonds, which has not been demonstrated for prrsv yet (reviewed in [ ] ). the prrsv gp -m and gp -gp -gp complexes are linked essentially through non-covalent interactions between gp and gp [ ] . the basic protein n associates with the viral rna genome to form the nucleocapsid. n is the most immunogenic prrsv structural protein. it elicits a strong antibody response a few days post infection (pi). these antibodies do however not neutralize the virus and are therefore not protective [ ] . the major neutralizing epitopes are found on gp [ ] [ ] [ ] [ ] and gp [ ] [ ] [ ] which are also the most diverse structural proteins between isolates [ ] . neutralizing epitopes were also found on m, gp and gp [ ] [ ] [ ] , but their contribution to protection is unclear. gp co-expressed with m elicits a better neutralizing ab response than gp alone [ , ] . however, neutralizing antibodies appear typically several weeks only after the onset of the first antibody response, simultaneously with clearance of the virus from the bloodstream [ , ] . the development of vaccines against prrsv has been only partially successful so far and remains a challenging task (for comprehensive reviews, see [ ] [ ] [ ] ). there are currently two types of prrsv vaccines on the market: modified live-virus vaccines (mlv) and inactivated vaccines [ ] [ ] [ ] . mlv are typically more efficacious than inactivated vaccines [ , ] . numerous alternative prrsv vaccine approaches have been explored with limited success so far (reviewed in [ , ] ). these efforts include for instance dna vaccines, subunit and peptide vaccines, viral vector vaccines and plant-derived vaccines [ , [ ] [ ] [ ] [ ] [ ] [ ] . propagation-incompetent recombinant vesicular stomatitis virus (vsv) represents yet another vector vaccine approach. recombinant vsv replicons lacking the vsv glycoprotein (g) gene and carrying genes of interest instead can be packaged in virus replicon particles (vrp) with high infectious titres using a complementing g-expressing cell line [ ] . such vrp were used successfully in the past to induce protection against sars coronavirus in a mouse model [ ] , influenza a virus (iav) in chickens and mice [ ] [ ] [ ] , and bluetongue virus (btv) in sheep [ ] . vsv vrp are safe due to the lack of glycoprotein g expression, preventing assembly and spread of virus particles. pigs have typically no pre-existing immunity against vsv. thus, vsv replicons represent an attractive novel vaccine platform for pigs. they have however not been evaluated in pigs yet. in the present study, vsv vrp were generated to express different combinations of the major and minor prrsv glycoproteins. the immunogenicity and protective potential of these vrp were assessed in pigs. marc- cells (atcc, lgc standards) were grown in dulbecco's modified eagle medium (dmem; life technologies) supplemented with % foetal bovine serum (fbs; biowest). bhk- cells were obtained from the german cell culture collection (dszm) and grown in earle's minimal essential medium eagle (mem; life technologies) supplemented with % fbs. bhk-g , a transgenic bhk- cell clone expressing the vsv g protein in a regulated manner, were maintained as described previously [ ] . the type prrsv strain olot/ was kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). this virus was a marc- -adapted variant of the original olot/ virus and was therefore propagated and titrated in marc- cells. for the detection of prrsv proteins, monoclonal antibody (mab) e directed against prrsv n, and mab vii d directed against amino acids - of prrsv gp were kindly provided by hans nauwynck (university ghent, belgium). a polyclonal rabbit serum against prrsv gp was obtained from luis enjuanes (centro nacional de biotecnología, madrid, spain). this serum was generated at biogenes (germany) with purified recombinant gp from prrsv olot/ expressed with the baculovirus system. the mab e c directed against prrsv m, and the mab ah against prrsv gp were a gift from ingenasa (madrid, spain). the rabbit anti-myc antiserum c , the mouse anti-flag m antibody and the anti-α-tubulin mab were purchased from sigma-aldrich. the mouse anti-ha antibody ca was from roche. the secondary antibodies goat anti-mouse igg alexa and goat anti-rabbit igg alexa were from molecular probes. the anti-swine igg antibody conjugated with rhodamine was purchased from rockland. the polyclonal rabbit anti-mouse igg coupled with horseradish peroxidase was from dako. the cdna of gp and m and the codon-optimized cdna of gp , gp and gp from the prrsv olot/ strain (genbank accession number kc ) were derived from psl-orf -orf and psl-gp - - , respectively, kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). the codonoptimized cdna of n from the olot/ strain (genbank accession number agw ) was synthesized by genscript (piscataway). for generation of recombinant vsv replicons, gp or n were inserted into the plasmid pvsv* using mlui and bsteii restriction sites upstream and downstream of the fourth transcription unit, replacing the g gene of vsv in analogy to a previous report [ ] . this replicon contained an additional transcription unit at position , expressing the green fluorescent protein (gfp) referred to by an asterisk (*) in the vector nomenclature. the resulting plasmids were designated pvsv*Δg(gp ) and pvsv*Δg(n), respectively. for generation of a dual antigen expression vector, the m cdna was inserted into pvsv*Δg(gp ) using xhoi and nhei restriction sites, thereby replacing the gfp gene in the fifth transcription unit. the resulting plasmid was designated pvsvΔg(gp /m). for generation of a triple antigen expression vector, the cdna of gp , gp and gp was inserted into a vsvΔg vector containing transcription units described recently [ ] using the mlui (in case of gp ), xhoi (in case of gp ) and nhei (in case of gp ) restriction sites. the resulting plasmid was designated pvsvΔg(gp /gp /gp ). for expression of a modified m and gp containing a short peptide epitope at the c terminus, the m and gp gene, respectively, were inserted without stop codon into the pcmv- tag- a plasmid vector (agilent technologies) upstream and in frame with a triple flag epitope (dykddddk)coding region followed by a stop codon. the m-flag and gp -flag open reading frames were amplified by pcr and inserted into the fourth transcription unit of pvsv*Δg, resulting in the plasmids pvsv*Δg(m-flag) and pvsv*Δg(gp -flag), respectively. the antigens gp and gp were modified by fusing a short ha epitope (ypydvpdya) to the c terminus, while gp was modified at the c terminus with a short myc epitope (eqkliseedl). the orfs were inserted into the fourth transcription unit of pvsv*Δg resulting in the plasmids pvsv*Δg(gp -ha), pvsv*Δg(gp -ha), and pvsv*Δg(gp -myc), respectively. for expression of the gp ectodomain (gp ecto) without the c-terminal transmembrane domain, a gene cassette encoding the amino acids - of gp (numbering according to genbank accession number agw ) was inserted in frame with the igκ leader sequence and an optimal signalase cleavage site in the mammalian expression vector psectag- a (invitrogen). in this way gp ecto was fused to a myc peptide epitope and a histidine tag ( xhis) at the c terminus, followed by a stop codon. this igκ-gp ecto-myc- xhis construct was then amplified by pcr and inserted into the fourth transcription unit of pvsv*Δg, resulting in the plasmid pvsv*Δg(gp ecto-myc). for expression of iav proteins, the cdna encoding ha, na, np and m of iav a/swine/belzig/ / (h n ) were kindly provided by jürgen and olga stech (friedrich-loeffler-institut, greifswald-insel riems, germany). the genes were inserted into the plasmid pvsv* using the mlui and bsteii restriction sites, replacing the g gene of vsv as described [ ] . all nucleotide sequences were confirmed by sanger sequencing. the recombinant replicons were propagated in the bhk-g helper cell line providing the vsv g protein in trans, yielding infectious vrp with titres of - infectious units (iu)/ ml as described previously [ ] . for the titrations, gfp expression was used as readout. for the detection of vrp that did not express gfp, infected cells were fixed with pbs containing % paraformaldehyde (pfa) for min, washed with pbs containing . m (w/v) glycine and then permeabilized with . % (v/v) triton x- . the cells were incubated with a rabbit anti-vsv serum and subsequently with a goat anti-rabbit horseradish peroxidase conjugate (dako) and stained with -amino- -ethylcarbazole (aec)/h o as substrate. an overview of all constructed vsv vrp is provided in table . marc- cells grown on -mm-diameter cover slips were inoculated for min at °c with recombinant vrp using a multiplicity of infection (moi) of iu/cell. at h post infection, the cells were fixed with % pfa for min and washed with pbs containing . m (w/v) glycine. the cells were permeabilized with . % (v/v) triton x- for - min and subsequently incubated with primary and secondary antibodies, diluted in pbs containing % bovine serum albumin (bsa). after each incubation period ( min, room temperature), the cells were washed three times with pbs. finally, the cells were washed with distilled water and embedded in mowiol - mounting medium (sigma-aldrich). all pigs were obtained from the specific pathogen free (spf) breeding facility of the institute of virology and immunology ivi. three experiments were performed. for each experiment the pigs were randomly assigned to treatment groups housed in separate stables. the groups were immunized by intramuscular injection of ml of cell culture supernatant containing - iu/ml recombinant vrp. in the first experiment, groups of pigs (n = ) were immunized three times at ½, ½ and ½ weeks of age with vsvΔg(gp /m) or with the control vsv*Δg vrp, respectively. in the second experiment, one group of pigs (n = ) was immunized twice at ½ and ½ weeks of age with a mixture of vsvΔg(gp /m) and vsvΔg(gp /gp /gp ) and the second group (n = ) received the control vsv*Δg vrp. in the third experiment, groups of pigs (n = ) were immunized three times at , and months of age with two different vsv vrp each injected at two different sites, i.e. vsv*Δg(n) and vsv*Δg(ha belz ), vsv*Δg(gp ecto-myc) and vsv*Δg(m belz ), vsv*Δg(gp -ha) and vsv*Δg(na belz ), vsv*Δg(control) and vsv*Δg(np belz ), respectively. in all experiments, the pigs were challenged via the intranasal route with tcid /animal of the prrsv strain olot/ - weeks after the last vaccination. the challenge virus was diluted in ml mem and administered dropwise intranasally. blood was taken before each vaccination and at regular intervals after vaccination and after the challenge infection. serum was stored at − °c. body temperature and clinical score were monitored daily according to a defined scoring system [ ] . the experiments in pigs were performed in compliance with the swiss animal protection law and approved by the animal welfare committee of the canton of berne, switzerland (authorization number be / ). titration by end point dilution was performed in marc- cells grown in -well plates. the cells were inoculated with tenfold serially diluted serum samples. at h pi, the cells were fixed and immunoperoxidase staining with the anti-n mab e was performed according to standard protocols. the % end point titre was expressed as tissue culture infectious dose % (tcid )/ ml, with a limit of detection of . log tcid /ml. total cellular rna was extracted from pig sera using the nucleospin multi virus kit (macherey-nagel) on a freedom evo robot (tecan) and stored at − °c. for detection of viral rna, a reverse transcriptase quantitative pcr (rt-qpcr) based on the amplification of the conserved region of orf was performed in duplicates employing gfp messenger rna as internal control [ ] . the results were expressed as the total number of cycles minus the quantification cycle (cq)-value. the elisa prrs x (idexx laboratories) was used for measuring anti-prrsv igg antibodies. samples to positive (s/p) ratios higher than . were considered positive. antibodies against prrsv gp , gp and gp were detected using in house competitive (gp ) and indirect (gp and gp ) elisas (ingenasa, madrid, spain). these elisas were performed with plates coated with recombinant gp , gp and gp antigens from prrsv olot/ . for the gp elisa, the percentage of competition was calculated using the formula [(odneg − odsample)/(odneg − odpos)] × . positive and negative controls were provided by ingenasa (spain). samples were considered positive if the percentage of competition was > %. for the indirect elisas, appropriate cut-offs were set. for the detection of c-reactive protein (crp; genway), haptoglobin (hp; genway) and ifn-γ (mabtech) in pig sera, commercially available elisas were used according to the manufacturer's protocols. porcine interferon-α (ifn-α) was determined by elisa as described previously [ ] . serum samples were heat inactivated at °c for min prior to performing the serum neutralization assay. µl of two-fold serially diluted sera were mixed with an equal volume of tcid of prrsv olot/ and incubated in -well tissue culture plates for h at °c. after h, marc- cells were added to each well. after days, the culture plate was fixed with % pfa. the presence of virus was detected by cpe and by staining with the anti-n mab and aec/h o . the virus neutralization titre was expressed as the reciprocal of the serum dilution leading to % reduction of infection. data were analysed with the graphpad prism . software. significant differences between groups were assessed by multiple t tests. p < . was considered significant. in previous work, a propagation-incompetent vsv replicon vector was generated by replacing the gene of the glycoprotein g in the fourth transcription unit of the viral genome with influenza virus genes. this replicon contained the gfp gene [referred to by an asterisk (*) in the vector nomenclature] in an additional transcription unit at position [ ] . based on this vector, we generated several vsv vrp expressing the individual structural antigens of prrsv olot/ or combinations thereof for evaluation as prrs vaccine candidates (see "materials and methods"; table ). the empty parental vsv*Δg vector served as control [ ] . the vsv vrp-mediated expression of the recombinant prrsv antigens was studied in marc- cells, taking advantage of the broad tropism of the vsv particles. this allowed direct comparison of the recombi- marc- cells infected with either vsv*Δg(gp -flag) or vsvΔg(gp /gp /gp ) reacted specifically with the anti-gp mab, while cells infected with either vsv*Δg(gp -ha) or vsvΔg(gp /gp /gp ) reacted with a polyclonal anti-gp immune serum (figure ). since a specific antibody against gp was not available, the expression of gp could not be demonstrated. any tag was omitted on purpose in the vsvΔg(gp /gp / gp ) to preserve the natural conformation of the three proteins. nevertheless, myc-tagged gp expression was detected from vsv*Δ(gp -myc) using the anti-myc serum (not shown). with the aim of evaluating whether protein secretion into the supernatant may enhance b-cell responses, the vsv*Δg(gp ecto-myc) replicon was constructed for the expression of the ectodomain of gp fused to a igκ leader sequence and an optimal signalase cleavage site. the myc-tagged ectodomain of gp reacted with the anti-gp mab and anti-myc serum as expected ( figure a ). western-blot analysis with the anti-myc serum demonstrated the presence of a kda protein in the cell lysate (ly) which was however missing in the supernatant (sn) of vsv*Δg(gp ecto-myc) infected marc- cells ( figure b ). this showed that the ectodomain of gp was retained in intracellular compartments despite the igκ signal sequence and the lack of the transmembrane domain. finally, expression of the prrsv n antigen by vsv*Δg(n) was demonstrated by immunofluorescence ( figure a ) and by western blot ( figure b ) using the anti-n mab. a kda protein was detected in the lysate of both, vsv*Δg(n)-and prrsv olot/ -infected cells. the gp and m complex constitutes the major protein component of the prrsv envelope against which neutralizing antibodies are formed [ , , ] . therefore, we first determined the immunogenicity of gp /mrecombinant vsv replicons in pigs. two groups of five pigs were immunized three times with vsvΔg(gp /m) or with the control vsv*Δg respectively, and were challenged weeks later with the homologous prrsv olot/ strain. following fever ( . - . °c) on day pi, whereas the control pigs had no fever despite slightly elevated body temperature ( figures a and b) . all animals developed a low viremia of short duration ( figures c and d) . a maximum mean virus titre of . log tcid /ml was reached in the serum at day post challenge. the virus was detectable up to day and viral rna up to day after challenge. no significant differences in viremia and viral rna in serum were observed between the vaccinated group and the control group at any time. before challenge infection, vaccination with vsvΔg(gp /m) did not induce any detectable antibody response against gp as measured by elisa (not shown). the first gp -specific antibody responses were detected by elisa in the vsvΔg(gp /m)-vaccinated group in out of five pigs on day after the challenge, and all pigs of this group were positive on day pi. all pigs of the vsv*Δg control group remained gp antibody negative at this time (table ). in the vsv*Δg control group, gp specific antibodies were detected for the first time on day pi in out of the pigs. seroconversion against n in response to the prrsv challenge infection was similar in the two groups, with the first n-specific seroconversion observed on day pi (table ) . virus neutralizing antibodies were not detected in any of the animals, neither before nor after the challenge (day and day pi). ifn-α and ifn-γ could not be detected in the serum at any time (data not shown). specific t-cell responses were not detected before challenge (not shown) and were therefore not further investigated. the acute phase proteins crp and hp which are early and sensitive markers of disease and inflammation including prrsv infection [ ] were increased in both groups between days and pi ( figures e and f) . however, no significant differences could be detected between vaccinated and control animals at any time. thus, apart from priming pigs for gp specific antibody responses after challenge infection, the vsvΔg(gp /m) vector could not induce any seroconversion against gp before challenge nor any sign of protection from viremia after infection. in order to determine whether the immune responses could be enhanced and induced before challenge by providing the three minor glycoproteins gp , gp and gp together with gp and m, pigs (n = ) were immunized twice with a mixture of vsvΔg(gp /m) and vsvΔg(gp /gp /gp ) or with vsv*Δg as control (n = ) followed by prrsv olot/ challenge. on day after challenge, two out of the four vaccinated pigs developed mild fever ( °c; figure a ) while all control animals remained free of fever (< °c; figure b ). all animals developed a short viremia following challenge infection with no significant differences in viral rna load and virus titres between the vaccinated and control groups (figures c and d) . as in the previous experiment, a maximum mean virus titre of . log tcid /ml was reached at day post challenge. again, before challenge infection, vaccination with vrp expressing the structural proteins of prrsv did not induce any detectable antibody response against gp , gp and gp as measured by elisa (not shown). following challenge infection, gp -specific antibody responses were detected on day pi in out of pigs, and all pigs vaccinated with vrp expressing the prrsv proteins seroconverted to gp at day pi (table ). all pigs of the vsv*Δg control group remained negative until day pi. seroconversion to gp (table ) vsv*Δg control group remained negative until day pi. a gp -specific immune response could be detected only in out of vaccinated animals on day pi. here also, the seroconversion against n following challenge was similar in the two groups ( table ) . none of the animals developed any neutralizing antibodies before nor after challenge (day and day pi). ifn-α and ifn-γ were not found in any of the sera in this experiment either (data not shown) whereas the serum crp and hp levels increased significantly in both groups between days and after challenge and declined subsequently. on day pi, the crp and hp levels were significantly different between the vaccinated and the control group ( figures e and f) , suggesting that the vaccinated group developed a slightly reduced inflammatory response after co-vaccination with vsvΔg(gp /m) and vsvΔg(gp / gp /gp ). together, these two vaccination trials show that vsv vrp cannot induce any detectable seroconversion against prrsv before challenge nor any protection against virus infection and viremia. nevertheless, these vectors can clearly prime pigs for earlier prrsv-specific antibody responses after challenge infection. the poor immunogenicity of vsv vrp expressing prrsv envelope proteins contrasts with previous reports showing protection against iav and btv infections in chickens and sheep, respectively [ , , ] . thus, the lack of antibody induction after immunization of pigs with vrp expressing the five prrsv envelope proteins raises the questions whether this vector vaccine is suitable for induction of antibody responses in pigs or whether the prrsv proteins are poorly immunogenic per se. in order to test this, the immunogenicity of prrsv and iav proteins expressed from vsv vrp was assessed in the same animal by co-vaccination with two different vsv vrp constructs expressing a prrsv protein (n, gp ecto, gp or empty vector) and a iav protein (ha, m , na, np), respectively, injected at two different sites (see "materials and methods"). after vaccinations, the pigs were challenged with the homologous prrsv olot/ strain. while, antibody responses were detected against all influenza virus proteins on day after vaccination (including neutralizing antibodies, not shown), no seroconversion against any of the prrsv proteins except n could be detected before challenge (table ). pigs vaccinated with vsv*∆g(n) seroconverted on day after vaccination ( days after the third vaccination). following challenge infection with prrsv olot/ , the control group seroconverted on day pi. there was no evidence of protection from virus infection after prrsv challenge as indicated by the kinetics of viremia (data not shown). nevertheless, a gp -specific immune response was induced earlier after challenge (day pi) in the vsv*Δg(gp ecto-myc) vaccinated pigs compared with the vsv*Δg-vaccinated pigs (no response on day pi). a gp -specific immune response was also induced on day pi whereas the vsv*Δg-vaccinated pigs stayed seronegative against gp . in line with the two previous immunization trials, these data show again a priming effect. more importantly however, simultaneous vaccination of the same pigs with two different vrp show clearly that the prrsv antigens are far less immunogenic than any of the iav antigens tested. the current prrsv vaccines on the market are of limited efficacy and come with several drawbacks [ ] . avoid the biological risks of virus spread and to circumvent potential immune modulating properties of modified live prrsv vaccines [ ] . therefore, we explored the immunogenic and protective potential of vsv replicon particles as a vectored vaccine approach against prrsv. such vrp were successfully used before as efficacious experimental vaccines against sars, iav and btv [ ] [ ] [ ] [ ] [ ] . in the present study, vsv replicons were engineered to express prrsv structural proteins, individually or in combination (table ) . emphasis was on gp , m, gp and gp , since these proteins are important targets for neutralizing and protective antibodies [ , , , ] , with the major neutralizing epitopes residing on gp [ ] . since several studies have shown that co-expression of prrsv antigens resulted in better humoral and cellular immune responses than expression of the individual proteins [ , ] , gp , m, gp , gp and gp proteins were co-expressed to partly mimic the formation of the gp /m and gp / / oligomers. vaccination of pigs with vrp expressing two or five prrsv envelope proteins in total did not result in any significant reduction of viremia compared with the control group. a tendency for reduced prrsv-related inflammatory responses was observed only when all five proteins were expressed. interestingly, vaccination with gp /m induced fever up to . °c for day following challenge infection with prrsv olot/ whereas mockvaccinated pigs remained asymptomatic. fever was less pronounced when all envelope proteins were included in the vaccine. enhanced disease following vaccination with recombinant gp and m was observed previously [ , ] . this was attributed to antibody-dependent enhancement of disease during natural infection, which is probably mediated by non-neutralizing antibodies [ , ] . such antibodies were shown to increase virus infection in porcine alveolar macrophage cultures and in vivo [ ] involving different fcγr isoforms [ , ] . neutralizing antibodies were not found at any time following vaccination and challenge infection. nevertheless, the vaccinated pigs seroconverted - days earlier than the mock-vaccinated animals after challenge virus infection, indicating that immunization with the recombinant vrp primed the immune system. in naïve pigs, the delayed seroconversion against the envelope proteins as opposed to the n protein after prrsv infection or after immunization with vectored vaccines is well table documented [ , , , ] . of note, with the marc- -adapted olot/ virus used in this study, seroconversion against n became detectable - days after challenge only (tables , , ) as opposed to the reported occurrence of anti-n antibodies as early as to days after prrsv infection [ , ] . this is probably related to the low level and short duration of replication of the marc- -adapted virus in pigs ( figures c, d, c, d) . despite the poor replication of this virus in vivo, vaccination of pigs with vrp did not result in any significant reduction of viremia. a better immunogenicity of prrsv proteins was reported when the antigens were modified, coupled to immunostimulatory molecules or administered as purified proteins along with adjuvants [ , ] . this raises the question whether the immunogenicity of the prrsv envelope proteins expressed from vrp is affected by glycosylation, subcellular localization, intracellular retention or low stability. in order to elaborate on this, gp was modified with the aim of obtaining secreted gp by replacing the leader sequence with a canonical igκ signal sequence followed by an optimal signalase cleavage site and by deleting the hydrophobic transmembrane anchor. however, the modified gp was retained in the cell, and no seroconversion against gp ecto was obtained in absence of challenge virus infection. the lack of gp secretion may be related to the fact that gp is naturally retained in intracellular compartments despite the n-terminal signal sequence [ ] . in order to address the question whether prrsv structural proteins are immunogenic at all when expressed by vsv vrp in pigs, a vector expressing prrsv n protein was included in one vaccination trial. n was chosen because it is the most immunogenic structural protein after prrsv infection [ , ] . indeed, vsv*Δg(n) was the only vrp capable of inducing a detectable antibody response in the absence of a viral challenge ( table ). the co-vaccination experiments with vrp expressing iav and prrsv envelope proteins corroborated these results. the iav proteins were strongly immunogenic while the prrsv envelope proteins were not, clearly demonstrating that the vsv vector per se is functional in pigs. although several vector vaccines failed to induce a protective immune response, immunizations essentially primed the immune system to a challenge infection [ , ] . these reports and the present study altogether suggest that the prrsv envelope proteins are expressed in a way to hide partially from the immune system. whether this immune evasion strategy is due solely to glycan shielding [ ] or to a particular intracellular topology remains to be investigated. future efforts in vectored vaccine development should consider these aspects to enhance the immunogenicity of the prrsv antigens. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states origin of highly pathogenic porcine reproductive and respiratory syndrome virus pathogenesis and antigenic characterization of a new east european subtype porcine reproductive and respiratory syndrome virus isolate nidovirales: evolving the largest rna virus genome the ever-expanding diversity of porcine reproductive and respiratory syndrome virus molecular evolution of prrsv in europe: current state of play arterivirus molecular biology and pathogenesis membrane proteins of arterivirus particles: structure, topology, processing and function discovery of a small arterivirus gene that overlaps the gp coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf present in all arteriviruses intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus the minor envelope glycoproteins gp a and gp of porcine reproductive and respiratory syndrome virus interact with the receptor cd immunological responses of swine to porcine reproductive and respiratory syndrome virus infection the amino acid residues at and in gp of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody role of neutralizing antibodies in prrsv protective immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain the primary gp neutralization epitope of north american isolates of porcine reproductive and respiratory syndrome virus gp -specific neutralizing antibodies might be a driving force in prrsv evolution posttranslational processing and identification of a neutralization domain of the gp protein encoded by orf of lelystad virus a variable region in gp of european-type porcine reproductive and respiratory syndrome virus induces neutralizing antibodies against homologous but not heterologous virus strains heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp and gp proteins and susceptibility to neutralization characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus co-expressing gp and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) pathogenesis of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) taming prrsv: revisiting the control strategies and vaccine design porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv vectored vaccines to protect against prrsv antigenic structures stably expressed by recombinant tgev-derived vectors immunization with dna vaccines containing porcine reproductive and respiratory syndrome virus open reading frames , , and may be related to the exacerbation of clinical disease after an experimental challenge vaccine production in plant systems: an aid to the control of viral diseases in domestic animals: a review baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain olot/ . involvement of orf and orf proteins in protection immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp /gp of porcine reproductive and respiratory syndrome virus and swine il- ctla mediated targeting enhances immunogenicity against prrsv in a dna prime/killed virus boost strategy use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector a recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (h n ) vaccination with recombinant rna replicon particles protects chickens from h n highly pathogenic avian influenza virus vesicular stomatitis virus vectors expressing avian influenza h ha induce cross-neutralizing antibodies and long-term protection vesicular stomatitis virus replicon expressing the vp outer capsid protein of bluetongue we thank luis enjuanes, centro nacional de biotecnología, madrid, spain, for supplying the marc -adapted prrsv olot/ strain, the cdna for orf to and the rabbit anti-gp serum; we thank also hans nauwynck, university ghent, belgium for providing the mabs e and vii d, and ingenasa, madrid, spain for the mabs e c and ah . we thank markus gerber and nuria de la roja for excellent technical assistance, and daniel brechbühl and veronika ayala for animal care. the authors declare that they have no competing interests. authors' contributions nr, kcm, gz and as conceived the study. me, gz, nr, as, mgd and mjr designed the experiments. me performed most of the experiments. mgd, js and mjr performed the glycoprotein-specific elisas. sl and mer contributed with iav vrp. me, nr, gz and mgd wrote the manuscript. all authors read and approved the final manuscript. key: cord- -u ggw authors: gao, peng; chai, yue; song, jiangwei; liu, teng; chen, peng; zhou, lei; ge, xinna; guo, xin; han, jun; yang, hanchun title: reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u ggw the unfolded protein response (upr) in the endoplasmic reticulum (er) constitutes a critical component of host innate immunity against microbial infections. in this report, we show that porcine reproductive and respiratory syndrome virus (prrsv) utilizes the upr machinery for its own benefit. we provide evidence that the virus targets the upr central regulator grp for proteasomal degradation via a mechanism that requires viral glycoprotein gp a, while both ire -xbp s and perk-eif α-atf signaling branches of the upr are turned on at early stage of infection. the activated effector xbp s was found to enter the nucleus, but atf was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp / to promote viral rna synthesis. rnai knockdown of either atf or xbp s dramatically attenuated virus titers, while overexpression caused increases. these observations reveal attractive host targets (e.g., atf and xbp s) for antiviral drugs and have implications in vaccine development. the unfolded protein response (upr) in the endoplasmic reticulum (er) represents an ancient but critical cellular mechanism in response to various deleterious stresses within the er lumen. it maintains er homeostasis by increasing protein-folding capacity, reducing global protein synthesis, and clearing misfolded proteins [ ] [ ] [ ] . protein loads and quality within the er lumen are monitored by three transmembrane proteins: pkr-like er kinase (perk), activating transcriptional factor (atf ) and inositol requiring enzyme (ire ) [ , ] . these sensors are normally kept in an inactive state by grp (also known as bip or hspa ), which is an er-resident chaperone and the master regulator of upr [ , ] . under er stress conditions, grp detaches from the sensors to allow their activation as it proceeds to binds to exposed hydrophobic regions of nascent polypeptides [ , ] . the three sensors regulated by grp control distinct pathways. activated perk phosphorylates eif α. although this leads to global translational attenuation, it does allow the preferential translation of a subset of stress-related mrnas, including the activating transcription factor (atf ) [ ] [ ] [ ] , which is a pro-survival protein that regulates the expression of stressrelated genes involved in anti-oxidation, amino acid biosynthesis and transport, apoptosis, etc [ ] [ ] [ ] [ ] . sensor atf can detect folding perturbations such as disruptions in disulfide bonding and protein under-glycosylation, and its activation leads to its translocation and cleavage within golgi by host proteases to release a transcriptionally-active fragment that induces expression of genes involved in protein folding, such as grp , pdi and grp , etc [ , [ ] [ ] [ ] [ ] . lastly, the ire -xbp branch is evolutionarily conserved from yeast to humans and responds to all types of er stress [ , ] . activated ire has the endoribonuclease activity and splices many mrna targets destined for translation at the er, which causes ire -dependent mrna decay [ , ] . although most of the target mrnas are degraded, the unconventional splicing of xbp mrna produces a functional transcript that encodes the transcription factor xbp s, which controls the expression of many target genes involved in lipid synthesis, erassociated degradation (erad), and chaperones [ ] [ ] [ ] . the cellular upr has been increasingly recognized as an intrinsic line among innate defenses that can result in either degradation of microbial components, translational inhibition, or apoptosis [ ] [ ] [ ] . moreover, the upr can regulate autophagy, inflammation, and type i interferon responses [ ] [ ] [ ] . consequently, investigations of how pathogens interface with this cellular response is required for a thorough understanding of how they replicate and cause disease [ ] . the experiments described in this report address the interplay of upr signaling with porcine reproductive and respiratory syndrome virus (prrsv), a positive-strand rna virus within the arteriviridae family in the order nidovirales [ , ] . prrsv mainly causes reproductive failure of sows and respiratory diseases of young pigs, and it has remained a major threat to global pork production since the first outbreak in late s [ ] . currently, there are no effective vaccines or anti-viral drugs available for this virus [ ] . persistent infections, dysregulation of host immunity, and rapid genetic mutation/recombination all contribute to the difficulty of disease control and the emergence of many highly virulent strains, including the deadly chinese highly pathogenic prrsv in [ ] [ ] [ ] [ ] . clearly, a better understanding of the interactions between this pathogen and its host is needed for developing effective prophylactic and therapeutic measures. prrsv has recently been shown to induce the activation of the perk and ire branches of the upr in porcine alveolar macrophage cell line zmac [ ] , but the significance of these events for virus replication is unknown. here, we show that the virus encodes a protein, gp a, which targets grp for degradation. moreover, we show that the upr does not inhibit but instead stimulates efficient replication of prrsv. in particular, the virus appears to hijack atf to viral replication and transcription complexes (rtcs) to facilitate viral rna synthesis. thus, our results reveal that prrsv exploits upr signaling for its own advantage. to investigate the mechanism of upr induction by prrsv, we initially focused on grp because it is the master regulator that binds to and negatively controls sensors perk, atf , and ire [ , ] . when levels of misfolded proteins rise, grp is released, resulting in activation of the upr [ ] . for many dna and rna viruses, their infections cause increased levels of grp , which is also a chaperone, so that the protein folding capacity of the cell can be enhanced [ ] [ ] [ ] . in the case of prrsv, we observed an elevation of grp at the mrna level in both prrsv-infected marc- cells (used for in vitro propagation of the virus; fig a) and primary pams (porcine alveolar macrophages, the major in vivo cell target; fig c) . but in spite of these increases, we found that grp protein levels actually declined as the infection proceeded (fig b and d ). in contrast, treatment with the potent er-stress inducer thapsigargin (tg) resulted in a dramatic increase of grp (fig b) , indicating that uninfected cells have the capacity to produce large amounts of this protein. the mechanism of prrsv-induced grp decay was probed by treating mock or infected marc- cells with either mg- (a proteasome inhibitor), bafilomycin a (a lysosomal inhibitor), z-vad-fmk (an apoptosis inhibitor), or dmso before collecting samples for analysis. additions of dmso, bafilomycin a , or z-vad-fmk did not have a significant effect, but treatment with mg- restored grp to a level similar to that of uninfected cells ( fig e) . thus, the proteasomal degradation pathway contributes to the reduced levels of grp seen in prrsv-infected cells. to identify the viral protein(s) needed for grp decay, we used transfection assays in marc- cells and hek- ft cells. expression of nonstructural proteins (s fig) and the major envelope membrane proteins (s fig) did not decrease grp expression but seemed to activate the upr, as indicated by the apparently increased levels of this protein. in contrast, the minor envelope protein gp a did not increase grp expression (s a fig) but instead caused a decrease of this protein in about % of the cells (s b fig). this activity was confirmed by western blot analyses in a dose-response experiment, which showed that increasing levels of gp a caused decreasing levels of grp (fig f) , and this effect could be reversed by treatment of the cells with mg- ( fig g) . because subgenomic mrna contains the open reading frames for both gp a and the e protein, we made constructs that eliminate the start codon for e (construct gp aΔe-ha) or express only the e protein (e-ha). the results show that gp a but not e protein decreased levels of grp (fig h) . to verify that marc- cells will exhibit the expected responses upon induction of the upr, we transfected them with an sirna that knockdowns expression of grp . in contrast to the mock-transfected control, rnai silencing of grp (fig a) resulted in an increased amount of xbp s (fig b) , elevated phosphorylation of eif α (fig c) , and increased expression of (fig c and d ). as expected, the scrambled-sequence control (sinc) did not induce these effects. while it seemed likely that the reduced levels of grp in prrsv infections (fig b and d ) would result in elevated expression of downstream components of the upr, it was possible that these would be targeted for degradation, too. to investigate this, the expression levels of the various upr components were examined. in marc- cells, we found that both the perk-eif α-atf and ire -xbp s branches of the upr were activated (fig a, c and e ). in particular, there was a gradual increase in the phosphorylation of perk and eif α, a concurrent increased in expression of atf , and an elevation of the spliced form (xbp s) of xbp at both the protein and mrna levels. in contrast, atf was not activated, and the cleaved fragment of atf was not detectable during infection. similar results were observed in infected, primary pams (fig b, d and f); however, the antibodies to atf did not work on pams extracts, and pdi was used as a surrogate since its expression is subject to regulation by atf [ ] . the degradation of grp and strong induction of downstream components raised the possibility that prrsv might utilize some of the upr machinery for its own benefit. to begin exploring this, we measured the temporal relationship between upr induction and infectious virion production. upregulation of atf and xbp s was detectable around hpi in marc- cells ( fig e) and hpi in pams ( fig f) . this coincided with detectable viral protein expression (here assayed for n; fig a and b ), but it occurred earlier than peak virus replication, which was at hpi in marc- cells ( fig g) and hpi in pams (fig h) . the observed correlation of the timing of upr induction and virus production in both cell types is consistent with the possibility of upr machinery being needed for prrsv replication. the importance of both atf and xbp s for prrsv replication was subsequently investigated with an rnai knockdown approach. all sirnas could efficiently silence the expression of the respective protein at h post transfection ( fig a) without affecting cell viability ( fig b) . infection of these cells with prrsv revealed a reduction in virus titers when either atf or xbp s expression was reduced, but depletion of atf did not have this effect ( fig c) . also, overexpression of atf or xbp s in marc- cells by means of lentivirus transduction significantly increased the virus yields ( fig d) . thus, both atf and xbp s, two downstream effectors of the upr, are critical for prrsv replication. to explore the roles of atf and xbp s in prrsv replication, we began by using a cytoplasmic-nuclear fractionation assay to measure their cellular distribution. marc- cells were either infected with prrsv or mock infected, and duplicates of these cultures were then treated with either thapsigargin (tg) or dmso at hpi for . h. the cytoplasmic and nuclear fractions were isolated and probed for the presence of atf and xbp s (fig a) . the quality of the fractionation was checked by assaying for cytoplasmic gapdh and nuclear histone subunit h . as expected, tg-treatment and prrsv infection upregulated the expression of both atf and xbp s (fig a, compare lanes and with lane ). in the case of xbp s, the protein was enriched in the nuclear fraction whether the cells were treated with tg (compare lanes and ) or infected (compare lanes and ). to our surprise, atf behaved differently. in tg-treated cells, this protein was almost exclusively found in the nuclear fraction (compare lanes and ), but in infected cells, it was found almost exclusively in the cytoplasmic fraction (compare lanes and ). in addition, prrsv infection blocked the tg-induced atf (fig a, compare lanes and ) . thus, prrsv appears to have a mechanism for retaining atf in the cytoplasm. to verify these observations in situ, an immunofluorescence assay (ifa) was used to detect the subcellular location of atf . (unfortunately, the antibodies against xbp s did not work in this assay). we found that atf was not within nuclei but instead was present at perinuclear regions in prrsv-infected cells (fig b, right panels) , and the tg-induced atf nuclear translocation was blocked by prrsv infection (s a fig). moreover, atf was found to colocalize very well with prrsv nsp and nsp ( fig b, right panels) , which are key components of viral replication and transcription complexes (rtcs) [ ] , suggesting that atf is hijacked to viral rtcs during infection. similar results were obtained in prrsv-infected primary pams (s fig) . consistent with cytoplasmic retention, the canonical atf -target genes gadd and asns [ ] were not upregulated during infection as compared with the mock or tg-treated cells (s b and s c fig) . also, we found that this phenomenon is independent of prrsv strain used (s fig to gain insight to how atf is diverted to viral rtcs, individual nonstructural proteins (nsps) were screened for their ability to cause cytoplasmic retention of atf in marc- cells that were treated with tg for . h at h post-transfection (s a fig) . we found that prrsv nsp was able to partially retain atf in the cytoplasm ( fig c and s b fig) , but coexpression with nsp , which is known to form heterodimer with nsp , dramatically increased the retention efficiency, even though nsp by itself-like nsp -did not have any activity ( fig c) . the interaction between nsp and endogenous atf was also confirmed with a co-ip assay using infected cell lysates ( fig d) . these results indicate that nsp / contributes to the recruitment of atf , which is also consistent with the proposed role of nsp / as membrane scaffolding proteins for viral rtcs assembly [ , ] . to learn when atf is redirected to viral rtcs, we used immunofluorescence staining at different times after infection. the results showed that atf became detectable around hpi in marc- cells, concurrent with the expression of the nsp replicase protein, and both colocalized well at the perinuclear region ( fig a) . similar finding was observed in primary pams ( fig b) . the detection of atf was earlier than observed with western blots (fig ) , and this is likely because a low moi was used ( . ). because most cells are not infected, the ifa would be expected to have greater sensitivity because it enables the detection of protein expression at the single cell level. in any case, it is clear that recruitment of atf to viral rtcs is an early event in prrsv infection. infection, the cells were collected and subject to western blot analysis with antibodies against the indicated proteins. nucleocapsid protein n and β-actin served as the indicator for infection and the loading control, respectively. (c and d) detection of xbp mrna splicing. prrsv-infected cells were harvested at the indicated times post infection. the xbp mrna sequence was amplified by rt-pcr followed by digestion with pst i and the products were analyzed by electrophoresis on a . % agarose gel. (e and f) quantification of the proteins or mrnas during prrsv infections. the abundance of pperk, peif a, atf , xbp s, atf , and pdi were expressed as fold changes compared to mock-infected control after being normalized against β-actin. having found that atf is critical for high virus titers and co-localizes with viral rtcs in prrsv infections, we hypothesized that this component of the upr facilitates viral rna synthesis. consistent with this, rnai knockdown of atf greatly reduced the level of total viral rnas, as measured by rt-qpcr analysis (fig a) , and this correlated well with the impaired virus production (fig c) . to investigate which step of viral rna synthesis was affected, we measured the relative abundance of negative-and positive-stranded rnas in marc- cells that had been transfected with either sirna-atf or the scrambled sinc h prior to infection. rna levels were analyzed during a single viral replication cycle (i.e., within hours) by rt-qpcr amplifying orf rna and calculating the amount present relative to the rna from gapdh, a house-keeping gene. the relative abundance of each viral rna was then calculated as the ratio of the sirna-atf -treated group compared to the sinc-treated control. as expected, no negative-stranded viral rnas could be detected in either the control or siatf -treated cells at hpi (fig b) . at - hpi, this rna became detectable, but its accumulation was greatly reduced when atf was depleted. positive-stranded rna could be detected at hpi due to the input of virions used to start the infection, but a gradual, obvious decrease was observed over time ( fig c) . we also measured the abundance of all subgenomic mrnas with primers that specifically amplify the leader-body junctions. the results show that reduced atf expression had an effect on all rna species regardless of the strands (s a and s b fig) , suggesting that this protein is a host factor required for viral rna synthesis. in support of our hypothesis, antibodies to atf , but not the isotype control (igg), could immunoprecipitate viral rna from infected marc- cells, as detected by rt-qpcr of the prrsv 'utr region ( fig d) . also, a purified gst-atf chimera, but not gst alone, could pull down viral rna from purified infected-cell rna (fig f) , which suggests that an atf -rna interaction can occur in the absence of other viral factors. as negative controls, neither the atf antibodies nor the gst-atf chimera pulled down gapdh rna (fig e and g ). lastly, confocal microscopy of infected cells showed that atf exhibited remarkable co-localization with negative-stranded viral rna (detected by rnascope in situ hybridization), as well as the viral polymerase nsp in infected cells (fig h) . together, the above results provide strong evidence for a critical role of atf in prrsv rna synthesis via an interaction with viral rna. the endoplasmic reticulum has complex roles in autophagy, apoptosis, and innate immunity, and thus, is an important organelle in viral pathogenesis [ ] . this is also the location where the upr originates, and previous studies have revealed an association of this response with prrsv-induced apoptosis and dysregulation of tnf-α production in cell culture [ , ] . in this study, two unexpected findings were made that together reveal a novel way in which prrsv reprograms the cellular upr to its own advantage. in particular, the virus targets grp for degradation to induce or potentiate the upr, and it then hijacks atf to viral strain jxwn at an moi of . . at the indicated times, the total titer of infectious virus present in each culture was measured by the endpoint dilution assay. (d) marc- cells were transduced with lentivirus expressing atf -myc, xbp s-myc, or the empty vector. at h post transduction, the cells were infected with prrsv at an moi of . . at hpi, the total viral titer in each culture was measured by the end-point dilution assay (right panel), and the expression of the transduced proteins was analyzed by western blot (left). data information: statistical analysis was performed by two-tailed student's t-test, and asterisks ( � ) indicate the statistical significance: ns, no significance; � , p < . ; �� , p < . ; ��� , p < . . data were presented as means ± standard deviations (sd) of three independent experiments. https://doi.org/ . /journal.ppat. .g repurposing cellular upr by prrsv the first unexpected finding from this study was the downregulation of grp in a manner that is proteasome dependent and requires a viral membrane protein gp a. it is likely that the erad pathway is utilized to retrotranslocate gpr into the cytosol, where proteasomes reside [ ] , but the details of how gp a might cause this remain to be elucidated. in any case, downregulation of grp was surprising because many viruses, especially enveloped viruses and er-tropic viruses (e.g., the flaviviruses and coronaviruses), quickly produce large amounts of membrane proteins and thereby place a large burden on the folding machinery in the er [ ] [ ] [ ] . in response to this stress, most viruses tend to upregulate chaperon grp and other factors to meet the increased demand for folding and post-translational modifications [ ] . this is not the case for prrsv. because grp is the master regulator, its reduced accumulation likely prevents any modulation of the upr, keeping the er stress pathways induced for the abundance of viral rna was assessed by rt-pcr of orf , normalized against the house-keeping gene gapdh, and then compared the benefit of the virus. in addition to providing downstream factors such as atf that this virus utilizes for replication, the reduced levels of grp will likely result in increased levels of misfolded cellular proteins, potentially aiding in immune evasion [ , , ] . what remains unclear is how prrsv promotes the folding of its own proteins in this situation. the second unexpected, and perhaps most exciting, finding from this study is the dependence of prrsv on two downstream upr effectors, atf and xbp s, which were induced in the reduction of gpr . with regard to the perk-eif α-atf signaling branch of the upr, previous studies of how viruses utilize atf have only revealed effects that depend upon its activity as a dna transcription factor. for example, in the cases of west nile virus (wnv) and infectious bronchitis virus (ibv) [ , ] , atf upregulates transcription factor chop to induce apoptosis and virus release. in these cases, activation takes place during the late stages of infection, after high titers of viruses have been made. other viruses (e.g., hsv- and dengue virus) use atf to upregulate gadd to antagonize eif α phosphorylation [ , ] . in the case of retroviruses (e.g., hiv), atf has been found to be diverted and bound to viral promoters to mediate transcriptional activation, and thus virus replication [ ] . in our studies, atf was found to be sequestered in the cytoplasm during prrsv infections, and consistent with this observation, the canonical atf target genes such as gadd and asns [ ] were not upregulated and eif α remained phosphorylated. this property of prrsv appears to be similar to that of mouse hepatitis virus (mhv) [ ] , where high levels of phosphorylated eif α and activation of atf were found without subsequent expression of gadd ; however, in that work, the authors did not examine possible roles for, or the intracellular location of, atf . mechanistically, we found that atf is recruited by nsp / to viral rtcs to promote viral replication. when this protein was depleted with sirnas, the accumulation of viral rna was severely attenuated for both the positive and negative strands. indeed, the effect seems to be equal on the accumulation of all viral rna species at the transcriptional level, suggesting that atf serves as a general facilitator for viral rna biogenesis. this is unlike host rna helicase ddx , which is relocated to the cytoplasm in coronavirus infections [ ] and is needed for the synthesis of much longer viral rna species [ ] . further evidence supporting the association of atf with the replication machinery of prrsv includes its interaction with viral rna in pulldown assays and co-localization with negative-strand rnas during infection. whatever its exact role, depletion of this component of the upr led to a dramatic attenuation of prrsv titers, whereas overexpression increased the viral yield. future studies are needed to dissect how exactly atf promotes rna biogenesis and to identify the atf -interacting domain or motif within prrsv so that a mutant virus can be tested for vaccine development. to sinc control at to hpi. (b and c) the same as (a) except the abundance of positive-and negative stranded rnas were analyzed by rt-qpcr at , , and hpi. (d) control, rabbit igg or antibodies specific for atf were used for immunoprecipitations from lysates of infected marc- cells (moi = . , hpi). the presence of viral rna was assayed by rt-qpcr targeting '-utr sequence or (e) gapdh as a negative control, and the fold enrichment was calculated against the normal rabbit igg group. (f) gst-atf or gst were purified from e. coli (left panel) and added to rna extracted from prrsv-infected marc- cells in an in vitro binding assay. rt-qpcr with primers specific for a 'utr sequence was used to detect viral rna (middle panel) and the fold of enrichment of viral rna was expressed against the gst control. (g) in parallel, the pull down of cellular gapdh mrna was used as a negative control. (h) to look for atf within viral replication compartments in infected marc- cells (moi = . , hpi), the negative-strand rna was detected by the rnascope in situ hybridization method, and atf and nsp were detected by immunostaining. data information: statistical analysis was performed by two-tailed student's t-test, and error bars indicate standard deviations (sd) of means. asterisks ( � ) indicate the statistical significance: ��� , p < . ; ���� , p< . ; ns, no significance (n = experiments in each condition). confocal images were acquired with nikon a confocal microscope. oil objective: x; zoom in x. https://doi.org/ . /journal.ppat. .g interestingly, atf has also been implicated in cancer progression [ ] . in tumors, atf expression is detected in hypoxic-and nutrient-deprived regions where it promotes metabolic homeostasis and cancer cell survival by transcriptionally regulating amino acid uptake and biosynthesis, autophagy, redox balance and angiogenesis [ , [ ] [ ] [ ] . as atf is normally expressed at low levels and is not essential for normal cells, this molecule serves as a potential target for drug development in both human and veterinary medicine. currently, there are no approved drugs for this target, and a search for one is warranted. we also found that prrsv is dependent on xbp s. the ire branch of the upr is linked to many functions, such as apoptosis through the ire -mediated ridd and jnk pathways, inflammation through ire -ask pathway, autophagy, erad, and lipid synthesis through activation of xbp s [ , , , , ] . our study showed that ire is activated in infected cells, consistent with two previous reports [ , ] . while it is clear that the downstream effector xbp s is important for virus replication the mechanism was not further explored here. there are several ways that xbp s might be important. one is for fulfilling the increased demand for lipid synthesis, as the replication of some viruses, including prrsv, often involves extensive the activated effector xbp s enters nucleus, but atf is diverted to viral replication complexes by nonstructural proteins nsp / to promote viral rna synthesis. to maintain a favorable environment, prrsv targets grp for partial proteasomal degradation by viral glycoprotein gp a, which creates a positive feedback loop to sensitize and potentiate the upr signaling. https://doi.org/ . /journal.ppat. .g modulation, rearrangement and capture of intracellular membranes into virions [ , , ] . second, xbp s may be important for regulation of autophagy [ ] . indeed, xbp s has been shown to be an important regulator of genes involved in autophagy biogenesis, such as lc , ulk , atg , beclin , and bcl [ , , ] . third, xbp s may facilitate the degradation of misfolded proteins via the erad pathway and thereby increase the folding capability via protein chaperones [ , ] . prrsv appears to be different from closely-related coronaviruses in its requirement for xbp s signaling. for example, mhv induces splicing of xbp but suppresses the activation of its downstream target genes [ ] . also, transmissible gastroenteritis virus (tgev) triggers the activation of ire -xbp s signaling, but this pathway is not required for viral replication [ ] . in any case, since xbp s is a stress-inducing gene product and normally expressed at low level, it represents a potential antiviral target for prrsv. in summary, the findings reported here provide further insight into the mechanisms by which prrsv interacts with cellular pathways and reveal a novel paradigm for how pathogens can repurpose components upr for their own use. our results also identify potentially druggable targets (atf and xbp s) and provide new information for making attenuated vaccines to control prrsv. the sampling of primary porcine pulmonary alveolar macrophages (pams) derived from onemonth-old spf pigs was performed according to the chinese regulations of laboratory animals-the guidelines for the care of laboratory animals (ministry of science and technology of people's republic of china) and laboratory animal-requirements of environment and housing facilities (gb ± , national laboratory animal standardization technical committee). the license number associated with this research protocol was cau , which was approved by the laboratory animal ethical committee of china agricultural university. the chinese highly pathogenic prrsv strain jxwn (genbank accession no: ef ), low pathogenic prrsv strain hb / . (eu ), the nadc -like prrsv chsx (kp ) [ , ] , porcine epidemic diarrhea virus (pedv) bj c strain [ ] , and encephalomyocarditis virus (emcv) strain (bjc ) [ ] have been documented previously. antibodies used in this study were obtained from a variety of sources. mouse anti-ha monoclonal antibody (mab) (#h ), rabbit anti-myc polyclonal antibody (pab) (#c ) and mouse anti-β-actin mab (#a ) were all purchased from sigma-aldrich (mo, usa). rabbit anti-grp mab (#cs- ), rabbit anti-pdi pab (# ), rabbit anti-perk mab (# ), rabbit anti-eif α pab (#cs- ), and rabbit anti-phospho-eif α (ser ) mab (#cs- ) were obtained from cell signaling technology (beverly, ma). rabbit anti-atf pab (#nbp - ) and rabbit anti-xbp pab (#nbp - ) were obtained from novus biologicals (littleton, usa). rabbit anti-atf pab (# - -ap), anti-gadd pab (# - -ap), anti-gapdh pab (# - -ap) and histone-h pab (# - -ap) were purchased from proteintech (chicago, il). normal mouse igg used as negative control was purchased from beyotime (#a ). mouse anti-n (prrsv), anti-nsp β (prrsv), anti-nsp (prrsv), anti-nsp (prrsv), mouse anti-n (pedv) and anti-vp (emcv) mab were produced by our lab at china agricultural university and used as previously described [ , ] . the plasmids expressing nsp α, nsp β, nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , gp a, gp , gp , gp , m and n were constructed by cloning the corresponding coding region from prrsv strain jxwn genome into the vector pcmv-ha (clontech, # ). gp aΔe-ha was created by introducing the corresponding mutations into the plasmid pgp a-ha with the quikchange site-directed mutagenesis kit (agilent technologies, # ). the atf gene from marc- cells was cloned into the vector pgex- p- (ge healthcare # - - ) for expression of the fusion protein gst-atf in e. coli bl cells (takara, # ). transfection of plasmid was performed with lipofectamine™ ltx reagent (thermo fisher, #a ) according to the manufacturer's instructions. marc- cells were infected with prrsv strain jxwn at an moi of . . at hpi, the cells were washed twice with cold phosphate-buffered saline (pbs) ( mm nacl, . mm kcl, mm na hpo , . mm kh po ), harvested with trypsin-edta digestion, and then centrifuged at , rpm for minutes. the cell pellets were resuspended with pbs, and the cytoplasmic and nuclear fractions were prepared by using ne-per nuclear and cytoplasmic extraction reagents (thermo fisher, # ), following the manufacturer's instruction. gapdh and histone-h were used as the respective cytoplasmic and nuclear makers for monitoring fraction quality by western blot. total rnas from cultured cells were extracted with trizol reagent (thermo fisher, # ) according to the manufacturer's instruction. the concentration of the extracted rna was measured using a nanodrop spectrophotometer (thermo fisher). reverse transcription was performed using a fastking rt kit (with gdnase) (tiangen, #kr - ) following the user guide. the xbp gene was amplified by pcr with the forward primer (f ) '-aaacagagtagcagcgcagactgc- ' and the reverse primer (r ) '-ggatctctaa gactagaggcttggtg- '. to resolve the spliced forms (xbp s) and unspliced (xbp u) of xbp , the pcr products were digested with the restriction enzyme pst i-hf (neb, #r l) and separated on the . % agarose gel. the cdna was synthesized from the total cellular rna by reverse transcription using the indicated primers. the relative quantitative pcr (qpcr) was performed with the applied biosys-tems™ sybr™ select master mix (thermo fisher, # ) according to the manufacturer's recommendations. the pcr was assembled in a μl reaction containing . μm gene specific primer, μl sybr™ select master mix, and μl cdna template. the pcr parameter was set up as follows: ˚c for min, ˚c for min; cycles of ˚c for s, ˚c for s, and ˚c for s. to measure viral total rna, a pair of internal primers in the orf gene were used to amplify all sgmrnas and grna. to measure grna and each viral sgmrna, a pair primers in the corresponding orf were used for the qpcr. the cellular gapdh was quantified as the internal control to normalize the cdna amounts. the -ΔΔct method was used to calculate the relative abundance of target genes compared to gapdh. the primers used for qpcr are listed in s and s tables. the lentiviral packaging system containing plasmids pwpxl (# ), pmd .g (# ) and pspax (# ) was purchased from addgene. the genes coding for xbp s and atf were cloned from marc- cells into the plasmid pwpxl and expressed as c-myc epitope fusion protein (xbp s-myc and atf -myc). the recombinant viruses were rescued by co-transfecting the three plasmids into hek- ft cells. at h post-transfection, the supernatants containing lentiviruses were harvested, filtered and concentrated. titers of the lentiviruses were determined using a quicktiter™ lentivirus titer kit according to the manufacturer's instruction (cell biolabs, #vpk- ). marc- cells in -well plates were transduced with × lentiviral particle (lp). at h post-transduction, the cells were infected with prrsv strain jxwn at an moi of . . the expression level of xbp s and atf was assessed by western blotting with rabbit anti-myc polyclonal antibodies to measure the effect of overexpression of atf and xbp s on viral replication, and the total virus titer was assessed by end point dilution assay. for rna interference, small interfering rnas (sirnas) was designed to target two different coding regions for each given gene. the sequences of sirnas are listed in s table. sirnas were transfected with lipofectamine rnaimax (thermo fisher, # ) according to the manufacturer's instruction. cell viability was assessed at h post transfection with celltiter aqueous one solution reagent (promega, #g ), and the knockdown effect was examined by western blot analyses of the whole cell lysate with antibodies to atf , atf , grp or xbp s, and β-actin was used as a loading control. for transfection/infection assay, marc- cells were infected with prrsv strain jxwn at h post transfection of sirnas, and total viruses were collected at the indicated time points for titration, and total rnas were extracted for analyzing the viral rna abundance by relative qpcr. the amount of total cellular proteins was quantified by using pierce™ bca protein assay kit (thermo fisher, # ), and - μg of the whole lysate per sample was subject to western blot analysis. briefly, the protein samples were separated by sds-page, transferred onto pvdf membranes, blocked with pbst (pbs with . % tween- ) containing % milk for . h, and then probed with appropriate primary antibodies at room temperature. for detection of phosphorylated proteins, bovine serum albumin (bsa) was used instead of milk. the membranes were then washed with pbst and incubated with appropriate hrp-conjugated secondary antibodies with dilution ratio : , . the membranes were again washed and then developed with the pierce ecl western blot substrate (thermo fisher, # ). marc- cells in six-well plates were infected with prrsv strain jxwn at an moi of . . at hpi, the cells were harvested and lysed in np- lysis buffer ( . % np- , mm nacl, mm tris-hcl ph . ) containing protease inhibitor (sigma, #p ). after clarifying by centrifugation at , rpm for min, the supernatants were precleared with protein a/g sepharose beads (santa cruz, #sc- ) and then incubated with μl mouse anti-nsp mab (mouse isotype antibody as a negative controls) and μl protein a&g sepharose beads overnight at ˚c with gentle rotation. the beads were washed five times with the np- lysis buffer, and the proteins bounded to the beads were separated by sds-page, followed by western blot analysis. marc- cells grown to - % confluence on coverslips in six-well plates were infected with strain jxwn at an moi of . . at indicated time points post infection, the cells were fixed with . % paraformaldehyde for min at room temperature, permeabilized with pbs containing . % triton x- and % bovine serum albumin (bsa) for min, and then blocked with % bsa-pbs ( % bovine serum albumin in pbs) for min. the cells were then incubated with primary antibodies as indicated in a humid chamber for h at room temperature or overnight at ˚c. after being washed times with pbs for min each, the cells were incubated with appropriate second antibody (alexa , or -conjugated) for additional h. nuclear dna was stained with ', -diamidino- -phenylindole (dapi) (thermo fisher, # ) for min and then washed with pbs three times for min each. the cells were imaged using a nikon a confocal microscope. to detect negative-stranded viral rnas, in situ hybridization was employed by using the rnascope multiplex fluorescent detection reagents v kit (acd, # ). a total of double-z branched pairs targeting the regions of orf , orf and 'utr of the prrsv genome (nt position , - , ) as rna probe (acd, # ) to detect negative-strand prrsv rnas. the hybridization procedure was performed according to the manufacturer's instruction. briefly, marc- cells on lab-tek ii chamber slides (thermo fisher, # ) were infected with prrsv at an moi of . . at the indicated time points post infection, the cells were fixed with % neutral buffered formalin (nbf), followed by dehydration and rehydration steps with suggested concentrations of ethanol and subsequent treatment by hydrogen peroxide (acd) and protease iii (acd). the cells were then incubated with rna probes for h at ˚c in hybez™ oven (acd), followed by a cascade of signal amplification and a series of washing procedures. hybridization signals were detected by tsa plus cyanine (perkinelmer, #nel e kt). for triple staining, the cells were fixed again with . % paraformaldehyde and then processed following the normal immunofluorescence assay procedure as described above. rna immunoprecipitation was carried out using the ez-magna rip™ rna-binding protein immunoprecipitation kit by following the manufacture's protocol (merck millipore). briefly, marc- cells in mm petri dish were infected with prrsv strain jxwn at an moi of . for h and then washed twice with ice-cold pbs. the cells were then scraped off the plates, collected by centrifugation, and lysed by complete rip lysis buffer with protease inhibitor cocktail (sigma, #p ) and rnase inhibitor (thermo fisher, #am ), followed by a step of centrifugation at , rpm for min at ˚c to remove the cell debris. to immunoprecipitate atf -rna complexes, magnetic beads were mixed with rabbit anti-atf polyclonal antibodies or normal rabbit igg (isotype control), and then added to the clarified cell supernatants for incubation overnight at ˚c. after washing with cold rip wash buffer six times, the beads-protein-rna complexes were digested with proteinase k at ˚c for min. afterwards, the supernatants were collected on a magnetic separator and transferred to a new tube for rna exaction with trizol reagent (thermo fisher, # ). the abundance of viral rnas was analyzed by relative rt-qpcr targeting prrsv 'utr or gapdh (as a control). e. coli bl cells containing the plasmid pgst-atf or pgex- p- vector were grown in ml x yt (yeast extract and tryptone) media at ˚c. protein expression was induced for h with isopropyl β-d- -thiogalactopyranoside (iptg) at a concentration of . mm when the optical density at nm reached . . the cultures were pelleted at , rpm for min, resuspended in pbs supplemented with bacterial protease inhibitors, sonicated, and lysed for min on ice with % triton x- . the lysates were cleared twice at , rpm for min at ˚c, and the supernatants were incubated with glutathione-sepharose b beads at room temperature for h, washed times with pbs, and resuspended in μl pbs. to test the interaction with viral rna in vitro, total rna was extracted from prrsvinfected marc- cells using trizol reagent, and μg were used for incubation with gst-atf or gst beads in μl np buffer ( . % np- , mm nacl, mm tris-hcl, ph . ) with protease inhibitor cocktail (sigma, #p ) and rnase inhibitors (thermo fisher, #am ) overnight at ˚c. after washing six times with np- buffer, the protein-rna complexes were eluted from beads with μl elution buffer ( mm tris-hcl, ph . , mm edta, ph . , . % sodium dodecyl sulfate), and the proteins were digested at ˚c for . h using proteinase k at a concentration of . mg/ml. afterwards, the supernatants were collected by centrifugation at , rpm for min and transferred to a new tube where the abundance of viral rna was analyzed by relative rt-qpcr targeting prrsv 'utr or gapdh as a control. following treatment with sirnas or lentivirus transduction, marc- cells were infected with prrsv strain jxwn . after incubation at ˚c for h, unbound viruses were washed off with serum-free dmem three times, and the cell cultures were supplemented with maintenance medium containing % fbs. at indicated time points post infection, the whole cell culture was collected, and the virus was titrated by the end point dilution assay. all the graphs and relevant statistical tests used in the work were created by graphpad prism version . (la jolla, ca, usa). statistical significance between two groups was analyzed by two-tailed unpaired student's t-test or one-way anova, and asterisks indicate the statistical significance: ns, no significance; � , p < . ; �� , p < . ; ��� , p < . . error bars indicate means ± standard deviations (sd). the unfolded protein response: from stress pathway to homeostatic regulation the unfolded protein response intracellular signaling by the unfolded protein response signal integration in the endoplasmic reticulum unfolded protein response the unfolded protein response and cell fate control dynamic interaction of bip and er stress transducers in the unfolded-protein response master regulator of the unfolded protein response and crucial factor in flavivirus biology dissociation of kar p/bip from an er sensory molecule, ire p, triggers the unfolded protein response in yeast coping with stress: eif kinases and translational control reinitiation involving upstream orfs regulates atf mrna translation in mammalian cells regulated translation initiation controls stress-induced gene expression in mammalian cells an integrated stress response regulates amino acid metabolism and resistance to oxidative stress transcription factor atf directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver trb , a novel er stress-inducible gene, is induced via atf -chop pathway and is involved in cell death activating transcription factor in vitro reconstitution of er-stress induced atf transport in copii vesicles the luminal domain of atf senses endoplasmic reticulum (er) stress and causes translocation of atf from the er to the golgi underglycosylation of atf as a novel sensing mechanism for activation of the unfolded protein response atf is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum er stress signaling by regulated splicing: ire / hac /xbp intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals decay of endoplasmic reticulum-localized mrnas during the unfolded protein response rna surveillance is required for endoplasmic reticulum homeostasis xbp : a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum the multiple roles of xbp in regulation of glucose and lipid metabolism a review of the mammalian unfolded protein response innate sensing of influenza a virus hemagglutinin glycoproteins by the host endoplasmic reticulum (er) stress pathway triggers a potent antiviral response via er-associated protein degradation antiviral activity of an isatin derivative via induction of perk-nrf -mediated suppression of cap-independent translation roles of endoplasmic reticulum stress in immune responses cross talk between er stress, oxidative stress, and inflammation in health and disease crosstalk of er stress-mediated autophagy and er-phagy: involvement of upr and the core autophagy machinery fxr inhibits endoplasmic reticulum stress-induced nlrp inflammasome in hepatocytes and ameliorates liver injury the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis nidovirales: a new order comprising coronaviridae and arteriviridae reorganization and expansion of the nidoviral family arteriviridae mystery swine disease in the netherlands: the isolation of lelystad virus pathogenesis and control of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome virus: a persistent infection genotype strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response the er chaperone and signaling regulator grp /bip as a monitor of endoplasmic reticulum stress japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response endoplasmic reticulum stress is induced and modulated by enterovirus induction of the unfolded protein response (upr) during marek's disease virus (mdv) infection. virology protein disulfide isomerase-associated is an atf -inducible er stress response protein that protects cardiac myocytes from ischemia/reperfusionmediated cell death the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins atf -dependent transcription mediates signaling of amino acid limitation orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex involvement of unfolded protein response, p and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation new insights into the physiological role of endoplasmic reticulum-associated degradation er stress, autophagy, and rna viruses how does the stressed out er find relief during virus infection? coronavirus infection, er stress, apoptosis and innate immunity viruses, endoplasmic reticulum stress, and interferon responses the molecular chaperone grp contributes to toll-like receptor -mediated innate immune response to hepatitis c virus in hepatocytes grp plays an integral role in tumor cell inflammationrelated migration induced by m macrophages west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis upregulation of chop/gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway herpes simplex virus infection activates the endoplasmic reticulum resident kinase perk and mediates eif- alpha dephosphorylation by the gamma( ) . protein the role of the unfolded protein response in dengue virus pathogenesis hiv exploits antiviral host innate gcn -atf signaling for establishing viral replication early in infection coronavirus infection modulates the unfolded protein response and mediates sustained translational repression the cellular rna helicase ddx interacts with coronavirus nonstructural protein and enhances viral replication nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription targeting the atf pathway in cancer therapy psat is regulated by atf and enhances cell proliferation via the gsk beta/beta-catenin/cyclin d signaling pathway in er-negative breast cancer role of atf in regulation of autophagy and resistance to drugs and hypoxia the endoplasmic reticulum stress sensor ire alpha protects cells from apoptosis induced by the coronavirus infectious bronchitis virus xbp mrna splicing triggers an autophagic response in endothelial cells through beclin- transcriptional activation mouse hepatitis virus infection activates the ire /xbp pathway of the unfolded protein response japanese encephalitis virus activates autophagy through xbp and atf er stress sensors in neuronal cells a regulatory link between er-associated protein degradation and the unfolded-protein response the perk arm of the unfolded protein response negatively regulates transmissible gastroenteritis virus replication by suppressing protein translation and promoting type i interferon production nadc -like strain of porcine reproductive and respiratory syndrome virus the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence genomic analysis of two porcine encephalomyocarditis virus strains isolated in china an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription mapping the nonstructural protein interaction network of porcine reproductive and respiratory syndrome virus targeting swine leukocyte antigen class i molecules for proteasomal degradation by the nsp alpha replicase protein of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus strain jxwn we sincerely thank dr. eric j. snijder (leiden medical center, netherlands) for providing eav infectious cdna clone and dr. qin wang for providing csfv and the antibodies to e protein. we also sincerely thank dr. john w. wills (pennsylvania state university college of medicine) for language editing of the manuscript.