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.

Oral vitamin A supplementation of porcine epidemic diarrhea virus infected gilts enhances IgA and lactogenic immune protection of nursing piglets.

Vitamin A (VA) has pleiotropic effects on the immune system and is critical for mucosal immune function and intestinal lymphocyte trafficking. We hypothesized that oral VA supplementation of porcine epidemic diarrhea virus (PEDV)-infected pregnant gilts would enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. Gilts received daily oral retinyl acetate (30 000 IU) starting at gestation day 76 throughout lactation. At 3-4 weeks pre-partum, VA-supplemented (PEDV + VA) and non-supplemented (PEDV) gilts were PEDV or mock inoculated (mock + VA and mock, respectively). PEDV + VA gilts had decreased mean PEDV RNA shedding titers and diarrhea scores. To determine if lactogenic immunity correlated with protection, all piglets were PEDV-challenged at 3-5 days post-partum. The survival rate of PEDV + VA litters was 74.2% compared with 55.9% in PEDV litters. Mock and mock + VA litter survival rates were 5.7% and 8.3%, respectively. PEDV + VA gilts had increased PEDV IgA antibody secreting cells and PEDV IgA antibodies in serum pre-partum and IgA+ß7+ (gut homing) cells in milk post piglet challenge compared with PEDV gilts. Our findings suggest that oral VA supplementation may act as an adjuvant during pregnancy, enhancing maternal IgA and lactogenic immune protection in nursing piglets.

A highly pathogenic avian-derived influenza virus H5N1 with 2009 pandemic H1N1 internal genes demonstrates increased replication and transmission in pigs.

This study investigated the pathogenicity and transmissibility of a reverse-genetics-derived highly pathogenic avian influenza (HPAI) H5N1 lineage influenza A virus that was isolated from a human, A/Iraq/755/06. We also examined surface gene reassortant viruses composed of the haemagglutinin and neuraminidase from A/Iraq/755/06 and the internal genes of a 2009 pandemic H1N1 virus, A/New York/18/2009 (2Iraq/06 : 6NY/09 H5N1), and haemagglutinin and neuraminidase from A/New York/18/2009 with the internal genes of A/Iraq/755/06 (2NY/09 : 6Iraq/06 H1N1). The parental A/Iraq/755/06 caused little to no lesions in swine, limited virus replication was observed in the upper respiratory and lower respiratory tracts and transmission was detected in 3/5 direct-contact pigs based on seroconversion, detection of viral RNA or virus isolation. In contrast, the 2Iraq/06 : 6NY/09 H5N1 reassortant caused mild lung lesions, demonstrated sustained virus replication in the upper and lower respiratory tracts and transmitted to all contacts (5/5). The 2NY/09 : 6Iraq/06 H1N1 reassortant also caused mild lung lesions, there was evidence of virus replication in the upper respiratory and lower respiratory tracts and transmission was detected in all contacts (5/5). These studies indicate that an HPAI-derived H5N1 reassortant with pandemic internal genes may be more successful in sustaining infection in swine and that HPAI-derived internal genes were marginally compatible with pandemic 2009 H1N1 surface genes. Comprehensive surveillance in swine is critical to identify a possible emerging HPAI reassortant in all regions with HPAI in wild birds and poultry and H1N1pdm09 in pigs or other susceptible hosts.

Identification of several clades of novel single-stranded circular DNA viruses with conserved stem-loop structures in pig feces.

Metagenomic analysis of fecal samples collected from diarrheal swine detected sequences encoding a replication initiator protein (Rep). The genomes of ten novel single-stranded DNA viruses were determined, and they exhibited a similar genome organization. The two putative open reading frames (ORFs) encoding Rep and the capsid protein are bidirectionally transcribed and separated by two intergenic regions. Stem-loop structure(s) typical of genomes that undergo the rolling-circle DNA replication mechanism were observed. Phylogenetically, these ten genomes are in a monophyletic clade with the previously described porcine stool-associated virus (PoSCV) but are divergent enough to be further classified into to six distinct virus clades.

Polymorphisms in the haemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine.

Interactions between the viral surface glycoprotein haemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in HA genes of two pandemic 2009 H1N1 (H1N1pdm09) isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at positions 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published H1N1pdm09 consensus sequence, the P186 genotype was more readily detected in primary infected and contact-naïve pigs when inoculated with a heterogeneous mixed stock of Ca/09. Using reverse genetics, we engineered Ca/09 and Mx/09 genomes by introducing Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either the Ca/09 or Mx/09 backbone resulted in higher and prolonged viral shedding in naïve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occurred within the receptor-binding pocket and the Sb antigenic site, they did not affect serological cross-reactivity. Relative increases of P186 in publicly available sequences from swine H1N1pdm09 viruses supported the experimental data, indicating this amino acid substitution conferred an advantage in swine.

Efficacy in pigs of inactivated and live attenuated influenza virus vaccines against infection and transmission of an emerging H3N2 similar to the 2011-2012 H3N2v.

Vaccines provide a primary means to limit disease but may not be effective at blocking infection and pathogen transmission. The objective of the present study was to evaluate the efficacy of commercial inactivated swine influenza A virus (IAV) vaccines and experimental live attenuated influenza virus (LAIV) vaccines against infection with H3N2 virus and subsequent indirect transmission to naive pigs. The H3N2 virus evaluated was similar to the H3N2v detected in humans during 2011-2012, which was associated with swine contact at agricultural fairs. One commercial vaccine provided partial protection measured by reduced nasal shedding; however, indirect contacts became infected, indicating that the reduction in nasal shedding did not prevent aerosol transmission. One LAIV vaccine provided complete protection, and none of the indirect-contact pigs became infected. Clinical disease was not observed in any group, including nonvaccinated animals, a consistent observation in pigs infected with contemporary reassortant H3N2 swine viruses. Serum hemagglutination inhibition antibody titers against the challenge virus were not predictive of efficacy; titers following vaccination with a LAIV that provided sterilizing immunity were below the level considered protective, yet titers in a commercial vaccine group that was not protected were above that level. While vaccination with currently approved commercial inactivated products did not fully prevent transmission, certain vaccines may provide a benefit by limitating shedding, transmission, and zoonotic spillover of antigenically similar H3N2 viruses at agriculture fairs when administered appropriately and used in conjunction with additional control measures.

Contemporary epidemiology of North American lineage triple reassortant influenza A viruses in pigs.

The 2009 pandemic H1N1 infection in humans has been one of the greatest concerns for public health in recent years. However, influenza in pigs is a zoonotic viral disease well-known to virologists for almost one century with the classical H1N1 subtype the only responsible agent for swine influenza in the United States for many decades. Swine influenza was first recognized clinically in pigs in the Midwestern U.S. in 1918 and since that time it has remained important to the swine industry throughout the world. Since 1988, however, the epidemiology of swine influenza changed dramatically. A number of emerging subtypes and genotypes have become established in the U.S. swine population. The ability of multiple influenza virus lineages to infect pigs is associated with the emergence of reassortant viruses with new genomic arrangements, and the introduction of the 2009 pandemic H1N1 from humans to swine represents a well-known example. The recent epidemiological data regarding the current state of influenza A virus subtypes circulating in the Canadian and American swine population is discussed in this review.

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV, PRRSV or PCV2.

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2-CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vß families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.

Antibody repertoire development in fetal and neonatal piglets. XVI. Influenza stimulates adaptive immunity, class switch and diversification of the IgG repertoire encoded by downstream Cγ genes.

Infection of germ-free isolator piglets with swine influenza (S-FLU) that generates dsRNA during replication causes elevation of immunoglobulins in serum and bronchoalveolar lavage, a very weak response to trinitrophenyl conjugates but an immune response to S-FLU. The increased immunoglobulin levels result mainly from the polyclonal activation of B cells during the infection, but model antigen exposure may contribute. The 10-fold increase in local and serum IgG accompanies a 10-fold decrease in the transcription of IgG3 in the tracheal-bronchial lymph nodes and in the ileal Peyer's patches. Infection results in class switch recombination to downstream Cγ genes, which diversify their repertoire; both features are diagnostic of adaptive immunity. Meanwhile the repertoires of IgM and IgG3 remain undiversified suggesting that they encode innate, natural antibodies. Whereas IgG3 may play an initial protective role, antibodies encoded by downstream Cγ genes with diversified repertoires are predicted to be most important in long-term protection against S-FLU.

Restored PB1-F2 in the 2009 pandemic H1N1 influenza virus has minimal effects in swine.

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts-pigs, humans, or birds-remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1ß in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs.

Live attenuated influenza vaccine provides superior protection from heterologous infection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease.

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.

Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011.

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.

A divergent clade of circular single-stranded DNA viruses from pig feces.

Using metagenomics and molecular cloning methods, we characterized five novel small, circular viral genomes from pig feces that are distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a highly divergent clade of this rapidly growing new viral family. This new clade of viruses, provisionally named porcine stool-associated circular virus 2 and 3 (PoSCV2 and PoSCV3), encodes a stem-loop structure (presumably the origin of DNA replication) in the small intergenic region and a replication initiator protein commonly found in other biological systems that replicate their genomes via the rolling-circle mechanism. Furthermore, these viruses also exhibit three additional overlapping open reading frames in the large intergenic region between the capsid and replication initiator protein genes.

Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils.

The use of immunomodulators is a promising area for biotherapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Cytokines, including granulocyte-colony stimulating factor (G-CSF), have been investigated for potential value as biotherapeutic proteins. G-CSF enhances the production and release of neutrophils from bone marrow and is already licensed for use in humans. A limitation of cytokines as immunomodulators is their short half-life which may limit their usefulness as a one-time injectable in production-animal medicine. Here we report that administration of recombinant G-CSF induced a transient neutrophilia in pigs; however, delivery of porcine G-CSF encoded in a replication-defective adenovirus (Ad5) vector significantly increased the neutrophilia pharmacodynamics effect. Pigs given one injection of the Ad5-G-CSF had a neutrophilia that peaked between days 3-11 post-treatment and neutrophil counts remained elevated for more than 2 weeks. Neutrophils from Ad5-G-CSF treated pigs were fully functional based on their ability to release neutrophil extracellular traps and oxidative metabolism after in vitro stimulation. Since acceptable alternatives to the use of antibiotics in food-animal production need to be explored, we provide evidence for G-CSF as a possible candidate for agents in which neutrophils can provide protection.

Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine.

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

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.

Identification of Pulmonary Infections With Porcine Rotavirus A in Pigs With Respiratory Disease.

While rotavirus (RV) is primarily known to cause gastroenteritis in many animals, several epidemiological studies have shown concurrent respiratory symptoms with fecal and nasal virus shedding. However, respiratory RV infections have rarely been investigated. By screening clinical samples submitted for diagnostic testing, porcine rotavirus A (RVA) was detected by quantitative reverse transcription PCR (qRT-PCR) in 28 out of 91 (30.8%) lungs obtained from conventionally reared pigs with respiratory signs. Among the positive cases, intensive RVA signals were mainly localized in alveolar macrophages (n = 3) and bronchiolar epithelial cells (n = 1) by RNAscope® in situ hybridization (ISH). The signals of RVA in bronchiolar epithelial cells were verified by ISH with different probes, immunohistochemistry, and transmission electron microscopy. Furthermore, additional cases with RVA ISH-positive signals in alveolar macrophages (n = 9) and bronchial epithelial cells (n = 1) were identified by screening 120 archived formalin-fixed and paraffin-embedded lung samples using tissue microarrays. Overall, our study showed a high frequency of RVA detection in lungs from conventional pigs with respiratory disease. Further research is needed to determine if RVA infection in the respiratory epithelium correlates with nasal shedding of rotavirus and its contribution to respiratory disease.

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.

Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008.

Prior to the introduction of the 2009 pandemic H1N1 virus from humans into pigs, four phylogenetic clusters (α-, ß-, γ- and δ) of the haemagglutinin (HA) gene from H1 influenza viruses could be found in US swine. Information regarding the antigenic relatedness of the H1 viruses was lacking due to the dynamic and variable nature of swine lineage H1. We characterized 12 H1 isolates from 2008 by using 454 genome-sequencing technology and phylogenetic analysis of all eight gene segments and by serological cross-reactivity in the haemagglutination inhibition (HI) assay. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. The gene segments from the 2009 pandemic H1N1 formed clusters separate from North American swine lineage viruses, suggesting progenitors of the pandemic virus were not present in US pigs immediately prior to 2009. Serological cross-reactivity paired with antigenic cartography demonstrated that the viruses in the different phylogenetic clusters are also antigenically divergent.