ICTV Virus Taxonomy Profile: Arteriviridae 2021.

The family Arteriviridae comprises enveloped RNA viruses with a linear, positive-sense genome of approximately 12.7 to 15.7 kb. The spherical, pleomorphic virions have a median diameter of 50-74 nm and include eight to eleven viral proteins. Arteriviruses infect non-human mammals in a vector-independent manner. Infections are often persistent and can either be asymptomatic or produce overt disease. Some arteriviruses are important veterinary pathogens while others infect particular species of wild rodents or African non-human primates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arteriviridae, which is available at ictv.global/report/arteriviridae.

Inactivating Three Interferon Antagonists Attenuates Pathogenesis of an Enteric Coronavirus.

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea Virus Suppresses Type I and Type III Interferon Responses.

Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis in vivo We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.IMPORTANCE Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.

Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions.

Viral structural proteins form the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of nonstructural protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom α-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (the PLP2 protease domain [OTU], the hypervariable domain [HV], the putative transmembrane domain [TM], and the C-terminal region [C]) were obtained commercially and validated in PRRSV-infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and Western blot assays using the α-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical virions of uniform size (57.9 nm ± 8.1 nm diameter; n = 50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV protein and reveal that nsp2 exists in or on viral particles as multiple isoforms.

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 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) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 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 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold. CONCLUSIONS: The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Characterization of Senecavirus A Isolates Collected From the Environment of U.S. Sow Slaughter Plants.

Vesicular disease caused by Senecavirus A (SVA) is clinically indistinguishable from foot-and-mouth disease (FMD) and other vesicular diseases of swine. When a vesicle is observed in FMD-free countries, a costly and time-consuming foreign animal disease investigation (FADI) is performed to rule out FMD. Recently, there has been an increase in the number of FADIs and SVA positive samples at slaughter plants in the U.S. The objectives of this investigation were to: (1) describe the environmental burden of SVA in sow slaughter plants; (2) determine whether there was a correlation between PCR diagnostics, virus isolation (VI), and swine bioassay results; and (3) phylogenetically characterize the genetic diversity of contemporary SVA isolates. Environmental swabs were collected from three sow slaughter plants (Plants 1-3) and one market-weight slaughter plant (Plant 4) between June to December 2020. Of the 426 samples taken from Plants 1-3, 304 samples were PCR positive and 107 were VI positive. There was no detection of SVA by PCR or VI at Plant 4. SVA positive samples were most frequently found in the summer (78.3% June-September, vs. 59.4% October-December), with a peak at 85% in August. Eighteen PCR positive environmental samples with a range of Ct values were selected for a swine bioassay: a single sample infected piglets (n = 2). A random subset of the PCR positive samples was sequenced; and phylogenetic analysis demonstrated co-circulation and divergence of two genetically distinct groups of SVA. These data demonstrate that SVA was frequently found in the environment of sow slaughter plants, but environmental persistence and diagnostic detection was not indicative of whether a sampled was infectious to swine. Consequently, a more detailed understanding of the epidemiology of SVA and its environmental persistence in the marketing chain is necessary to reduce the number of FADIs and aide in the development of control measures to reduce the spread of SVA.

Utility of a panviral microarray for detection of swine respiratory viruses in clinical samples.

Several factors have recently converged, elevating the need for highly parallel diagnostic platforms that have the ability to detect many known, novel, and emerging pathogenic agents simultaneously. Panviral DNA microarrays represent the most robust approach for massively parallel viral surveillance and detection. The Virochip is a panviral DNA microarray that is capable of detecting all known viruses, as well as novel viruses related to known viral families, in a single assay and has been used to successfully identify known and novel viral agents in clinical human specimens. However, the usefulness and the sensitivity of the Virochip platform have not been tested on a set of clinical veterinary specimens with the high degree of genetic variance that is frequently observed with swine virus field isolates. In this report, we investigate the utility and sensitivity of the Virochip to positively detect swine viruses in both cell culture-derived samples and clinical swine samples. The Virochip successfully detected porcine reproductive and respiratory syndrome virus (PRRSV) in serum containing 6.10 × 10(2) viral copies per microliter and influenza A virus in lung lavage fluid containing 2.08 × 10(6) viral copies per microliter. The Virochip also successfully detected porcine circovirus type 2 (PCV2) in serum containing 2.50 × 10(8) viral copies per microliter and porcine respiratory coronavirus (PRCV) in turbinate tissue homogenate. Collectively, the data in this report demonstrate that the Virochip can successfully detect pathogenic viruses frequently found in swine in a variety of solid and liquid specimens, such as turbinate tissue homogenate and lung lavage fluid, as well as antemortem samples, such as serum.

Proteolytic products of the porcine reproductive and respiratory syndrome virus nsp2 replicase protein.

The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G(1196)|G(1197) dipeptide in transfected CHO cells. Here the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSVs expressing epitope-tagged nsp2. The data revealed that PRRSV nsp2 exists as different isoforms, termed nsp2a, nsp2b, nsp2c, nsp2d, nsp2e, and nsp2f, during PRRSV infection. Moreover, on the basis of deletion mutagenesis and antibody probing, these nsp2 species appeared to share the same N terminus but to differ in their C termini. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. nsp2b and nsp2c were processed within or near the transmembrane (TM) region, presumably at or near the conserved sites G(981)|G(982) and G(828)|G(829)|G(830), respectively. The C termini for nsp2d, -e, and -f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be definitively predicted. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g., nsp2d, nsp2e, and nsp2f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, named heat shock 70-kDa protein 5 (HSPA5), that was strongly associated with nsp2, which may have important implications for PRRSV replication. Overall, these findings indicate that PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.

Comparison of historical and contemporary isolates of Senecavirus A.

Senecavirus A (SVA) was discovered as a cell culture contaminant in 2002, and multiple attempts to experimentally reproduce disease were unsuccessful. Field reports of porcine idiopathic vesicular disease (PIVD) cases testing PCR positive for SVA in addition to outbreaks of PIVD in Brazil and the United States in 2015 suggested SVA was a causative agent, which has now been consistently demonstrated experimentally. Ease of experimental reproduction of disease with contemporary strains of SVA raised questions concerning the difficulty of reproducing vesicular disease with historical isolates. The following study was conducted to compare the pathogenicity of SVA between historical and contemporary isolates in growing pigs. Six groups of pigs (n = 8) were intranasally inoculated with the following SVA isolates: SVV001/2002, CAN/2011, HI/2012, IA/2015, NC/2015, SD/2015. All isolates induced vesicular disease in at least half of the inoculated pigs from each group. All pigs replicated virus as demonstrated by serum and/or swab samples positive for SVA by quantitative PCR. Pig sera tested by virus neutralization assay demonstrated cross-neutralizing antibodies against all viruses utilized in the study. Cross-neutralizing antibodies from pigs inoculated with historical isolates were lower than those pigs that were inoculated with contemporary isolates. Phylogenetic analysis revealed two clades with SVV001/2002 being in a separate clade compared to the other five isolates. Although differences in the infection kinetics and sequences of these six isolates were found, clinical presentation of vesicular disease was similar between both historical and contemporary isolates.

Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026.

We report the generation of a full-length infectious cDNA clone for porcine deltacoronavirus strain USA/IL/2014/026. Similar to the parental strain, the infectious clone virus (icPDCoV) replicated efficiently in cell culture and caused mild clinical symptoms in piglets. To investigate putative viral interferon (IFN) antagonists, we generated two mutant viruses: a nonstructural protein 15 mutant virus that encodes a catalytically-inactive endoribonuclease (icEnUmut), and an accessory gene NS6-deletion virus in which the NS6 gene was replaced with the mNeonGreen sequence (icDelNS6/nG). By infecting PK1 cells with these recombinant PDCoVs, we found that icDelNS6/nG elicited similar levels of type I IFN responses as icPDCoV, however icEnUmut stimulated robust type I IFN responses, demonstrating that the deltacoronavirus endoribonuclease, but not NS6, functions as an IFN antagonist in PK1 cells. Collectively, the construction of a full-length infectious clone and the identification of an IFN-antagonistic endoribonuclease will aid in the development of live-attenuated deltacoronavirus vaccines.

The United States Swine Pathogen Database: integrating veterinary diagnostic laboratory sequence data to monitor emerging pathogens of swine.

Veterinary diagnostic laboratories derive thousands of nucleotide sequences from clinical samples of swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), Senecavirus A and swine enteric coronaviruses. In addition, next generation sequencing has resulted in the rapid production of full-length genomes. Presently, sequence data are released to diagnostic clients but are not publicly available as data may be associated with sensitive information. However, these data can be used for field-relevant vaccines; determining where and when pathogens are spreading; have relevance to research in molecular and comparative virology; and are a component in pandemic preparedness efforts. We have developed a centralized sequence database that integrates private clinical data using PRRSV data as an exemplar, alongside publicly available genomic information. We implemented the Tripal toolkit, a collection of Drupal modules that are used to manage, visualize and disseminate biological data stored within the Chado database schema. New sequences sourced from diagnostic laboratories contain: genomic information; date of collection; collection location; and a unique identifier. Users can download annotated genomic sequences using a customized search interface that incorporates data mined from published literature; search for similar sequences using BLAST-based tools; and explore annotated reference genomes. Additionally, custom annotation pipelines have determined species, the location of open reading frames and nonstructural proteins and the occurrence of putative frame shifts. Eighteen swine pathogens have been curated. The database provides researchers access to sequences discovered by veterinary diagnosticians, allowing for epidemiological and comparative virology studies. The result will be a better understanding on the emergence of novel swine viruses and how these novel strains are disseminated in the USA and abroad. Database URLhttps://swinepathogendb.org.

The porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities.

The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr(47) to Cys(240)) and aa 47 to 323 (Tyr(47) to Leu(323)), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys(55)- His(124) catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys(111), Cys(142), and Cys(147)). The conserved aspartic acids (e.g., Asp(89)) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G(1196)|G(1197) dipeptide.

Insights into the Porcine Reproductive and Respiratory Syndrome Virus Viral Ovarian Tumor Domain Protease Specificity for Ubiquitin and Interferon Stimulated Gene Product 15.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread economically devastating disease caused by PRRS virus (PRRSV). First recognized in the late 1980s, PRRSV is known to undergo somatic mutations and high frequency viral recombination, which leads to many diverse viral strains. This includes differences within viral virulence factors, such as the viral ovarian tumor domain (vOTU) protease, also referred to as the papain-like protease 2. These proteases down-regulate innate immunity by deubiquitinating proteins targeted by the cell for further processing and potentially also acting against interferon-stimulated genes (ISGs). Recently, vOTUs from vaccine derivative Ingelvac PRRS modified live virus (MLV) and the highly pathogenic PRRSV strain JXwn06 were biochemically characterized, revealing a marked difference in activity toward K63 linked polyubiquitin chains and a limited preference for interferon-stimulated gene product 15 (ISG15) substrates. To extend our research, the vOTUs from NADC31 (low virulence) and SDSU73 (moderately virulent) were biochemically characterized using a myriad of ubiquitin and ISG15 related assays. The K63 polyubiquitin cleavage activity profiles of these vOTUs were found to track with the established pathogenesis of MLV, NADC31, SDSU73, and JXwn06 strains. Fascinatingly, NADC31 demonstrated significantly enhanced activity toward ISG15 substrates compared to its counterparts. Utilizing this information and strain-strain differences within the vOTU encoding region, sites were identified that can modulate K63 polyubiquitin and ISG15 cleavage activities. This information represents the basis for new tools to probe the role of vOTUs in the context of PRRSV pathogenesis.

Progress toward an enhanced vaccine: Eight marked attenuated viruses to porcine reproductive and respiratory disease virus.

Recombinant viruses of strain Ingelvac® PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Δ23 and Δ87) and also the same deletions supplanted with foreign tags (Δ23-V5, Δ23-FLAG, Δ23-S, Δ87-V5, Δ87-FLAG, Δ87-S). The viruses, but one (Δ87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Δ23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Δ87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Δ23-S and Δ87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals.

Porcine reproductive and respiratory disease virus: Evolution and recombination yields distinct ORF5 RFLP 1-7-4 viruses with individual pathogenicity.

Recent cases of porcine reproductive and respiratory syndrome virus (PRRSV) infection in United States swine-herds have been associated with high mortality in piglets and severe morbidity in sows. Analysis of the ORF5 gene from such clinical cases revealed a unique restriction fragment polymorphism (RFLP) of 1-7-4. The genome diversity of seventeen of these viruses (81.4% to 99.8% identical; collected 2013-2015) and the pathogenicity of 4 representative viruses were compared to that of SDSU73, a known moderately virulent strain. Recombination analyses revealed genomic breakpoints in structural and nonstructural regions of the genomes with evidence for recombination events between lineages. Pathogenicity varied between the isolates and the patterns were not consistent. IA/2014/NADC34, IA/2013/ISU-1 and IN/2014/ISU-5 caused more severe disease, and IA/2014/ISU-2 did not cause pyrexia and had little effect on pig growth. ORF5 RFLP genotyping was ineffectual in providing insight into isolate pathogenicity and that other parameters of virulence remain to be identified.

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.

Epidemiologic data from Asian outbreaks of highly-pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) suggest that disease severity was associated with both the virulence of the PRRSV isolates and secondary bacterial infections. Previous reports have indicated that U.S. isolates of PRRSV predispose to secondary bacterial infections as well, but the severity of disease that occurred in Asia in pigs infected with these HP-PRRSV strains has not been reported in the U.S. The objectives of this research were to compare the pathogenesis of Asian and U.S. PRRSV isolates with regard to their ability to cause disease and predispose to secondary bacterial infections in swine. To address these objectives groups of pigs were infected with 1 of 2 Asian HP-PRRSV strains (rJXwn06 or rSRV07) or 1 of 2 U.S. PRRSV strains (SDSU73 or VR-2332) alone or in combination with Streptococcus suis, Haemophilus parasuis, and Actinobacillus suis. Pigs infected with rJXwn06 exhibited the most severe clinical disease while the pigs infected with rSRV07 and SDSU73 exhibited moderate clinical disease, and pigs infected with VR-2332 exhibited minimal clinical signs. The frequency of secondary bacterial pneumonia was associated with the clinical severity induced by the PRRSV strains evaluated. The levels of proinflammatory cytokines in the serum were often lower for pigs coinfected with virus and bacteria compared to pigs infected with PRRSV alone indicating an alteration in the immune response in coinfected pigs. Combined our results demonstrate that severity of disease appears to be dependent on virulence of the PRRSV strain, and development of secondary bacterial infection.

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus.

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].

Neutralizing antibody responses of pigs infected with natural GP5 N-glycan mutants of porcine reproductive and respiratory syndrome virus.

In order to assess the effect of the N-glycans associated with the GP5 neutralization epitope of porcine reproductive and respiratory syndrome virus (PRRSV) on the neutralizing antibody (Ab) response of swine, groups of young pigs were infected with PRRSV strains differing in N-glycosylation pattern. The humoral immune response to strain VR-2332, harboring four potential N-glycan sites, was compared to that of two natural field isolates carrying mutations either abolishing the N-glycosylation site at position 44 (N44) or the two N-glycosylation sites in the hypervariable region upstream of the neutralization epitope (HV-1). The pigs were bled at intervals and their sera were assayed for neutralizing Abs by indirect and competition ELISAs using peptides containing the GP5 neutralization epitope, and selectively for infectivity neutralization of a number of PRRSV strains. In addition, viremia was monitored by quantitative RT-PCR, and anti-N-protein Ab formation was measured by HerdChek ELISA. The neutralizing Ab responses as measured by peptide ELISA varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of peptide binding Abs between 7 and 28 days post infection (p.i.), whereas other pigs had not generated a response by 90 days p.i. However, the HV-1-infected pigs generated Abs to the neutralization epitope more rapidly and to a 5-10 times higher level than VR-2332-infected pigs, and the Abs neutralized the homologous HV-1 virus 10-20 times more efficiently than PRRSV strains VR-2332, N44, MN184, or SDSU73. In contrast, most N44-infected pigs generated neutralizing Abs only after 42 days p.i. and only to low levels. The results suggest that the deletions of the N-glycans or other amino acid substitutions in the GP5 ectodomains of the mutants affect the immunogenicity of the neutralization epitope and the specificity of the Abs raised to it but not the sensitivity of the virions to Ab neutralization.

Status of vaccines for porcine epidemic diarrhea virus in the United States and Canada.

In 2013, porcine epidemic diarrhea virus (PEDV) emerged in the United States as a rapidly spreading epidemic causing dramatic death losses in suckling piglets. Neonatal piglets are most vulnerable to clinical disease and their only protection is passive immunity from their dam. At the end of the third year of the PEDV outbreak, most US sow herds have been infected and many are entering into an endemic disease with much less, but still chronic losses. This endemic state and the occasional naïve herd that breaks with PEDV demonstrate a need to immunize sows to protect piglets. Stimulating PEDV immunity in the sow using safe and efficacious vaccines is the best course of action; however, conducting such studies to develop sow vaccines is very costly and logistically difficult. This manuscript reviews the status of PEDV vaccines available in the United States and Canada, and describes an experiment evaluating the potential use of young pigs as a surrogate model to evaluate potential sow vaccines.

Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for one of the most economically important diseases in swine worldwide. It causes reproductive failure in sows and pneumonia in pigs that predisposes them to secondary bacterial infections. Methods to control PRRSV and/or limit secondary bacterial infections are desired to reduce the impact of this virus on animal health. Neutrophils play a major role in combatting infection; they can act as phagocytes as well as produce and release lytic enzymes that have potent antimicrobial effects leading to the destruction and clearance of bacterial pathogens. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that controls the production, differentiation and function of granulocytes (including neutrophils) from the bone marrow. Recent work from our laboratory has shown that encoding porcine G-CSF in a replication-defective adenovirus (Ad5-G-CSF) and delivering a single dose to pigs induced a neutrophilia lasting more than two weeks. As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with highly pathogenic (HP)-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection. Administration of Ad5-G-CSF induced a significant neutrophilia as expected. However, between 1 and 2days following HP-PRRSV challenge the number of circulating neutrophils decreased dramatically in the HP-PRRSV infected group, but not the non-infected Ad5-G-CSF group. Ad5-G-CSF administration induced monocytosis as well, which was also reduced by HP-PRRSV challenge. There was no difference in the progression of disease between the Ad5-G-CSF and Ad5-empty groups following HP-PRRSV challenge, with pneumonia and systemic bacterial infection occurring in both treatment groups. Given the impact of HP-PRRSV infection on the neutrophilia induced by the Ad5-G-CSF administration, additional studies are warranted to evaluate the timing of Ad5-G-CSF induced neutrophilia and multiple G-CSF inoculations on protection against secondary bacterial infection following PRRSV infection. Nevertheless, this study may provide insight into the pathogenesis of HP-PRRSV.