Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Experimental porcine astrovirus type 3-associated polioencephalomyelitis in swine.

Porcine astrovirus type 3 (PoAstV3) is an emerging virus in the family Astroviridae that has been recently associated with polioencephalomyelitis/encephalitis. Herein, we describe the experimental oral and intravenous inoculation of an infectious central nervous system (CNS) tissue homogenate containing PoAstV3 to cesarean-derived, colostrum-deprived pigs, and the subsequent development of clinical signs, histologic lesions, specific humoral immune response, and detection of viral particles by electron microscopy (EM) and viral RNA by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) and in situ hybridization (ISH). IgG against a portion of the PoAstV3 ORF2 capsid was first detected at 7 days post-inoculation (DPI) in 2 of 4 inoculated animals and in all inoculated animals by 14 DPI. At 21 and 28 DPI, 2 of 4 inoculated animals developed ataxia, tetraparesis, and/or lateral recumbency. All inoculated animals had histologic lesions in the CNS including perivascular lymphoplasmacytic cuffs, multifocal areas of gliosis with neuronal necrosis, satellitosis, and radiculoneuritis, and PoAstV3 RNA as detected by RT-qPCR within multiple anatomic regions of the CNS. Consistent viral structures were within the soma of a spinal cord neuron in the single pig examined by EM. Of note, PoAstV3 was not only detected by ISH in neurons of the cerebrum and spinal cord but also neurons of the dorsal root ganglion and nerve roots consistent with viral dissemination via axonal transport. This is the first study reproducing CNS disease with a porcine astrovirus strain consistent with natural infection, suggesting that pigs may serve as an animal model to study the pathogenesis of neurotropic astroviruses.

Probability of PRRS virus detection in pooled processing fluid samples.

There has been a tremendous increase in recent years of population-based diagnostic monitoring and surveillance strategies in swine populations. One example is the use of processing fluids (PF) to screen breeding herds for porcine reproductive and respiratory syndrome virus (PRRSV) activity. An important question from practitioners using such methods is on how intensively can the sample be pooled. More specifically, processing fluids of how many litters can be pooled into a single sample for diagnostic testing to preserve a high probability of PRRSV RNA detection at low prevalence situations? The objective of this study was to model the effect of pooling PF samples on the probability of PRRSV RNA detection. For this study, a PRRSV-positive PF field sample with a RT-rtPCR quantification cycle (Cq) value of 28 was selected to represent a litter of 11 pigs with a single viremic piglet. PF samples from a PRRSV-naïve herd were used to perform 6 replications of 8 two-fold serial dilutions of the PRRSV-positive sample, thus modeling the pooling effect (dilution). Each two-fold dilution represented an increase in the number of PRRS-negative pigs in the sample by a factor of 2. Samples were tested for PRRSV RNA by RT-rtPCR and the data was analyzed using linear and probit regression models. There was an average increment of 1.37 points in Ct for each two-fold dilution. The estimated probability of testing positive on RT-rtPCR was 43 %, 80 %, and 95 % when there was a single PRRSv-positive piglet among 784, 492, and 323 PRRSv-negative piglets contributing to the sample respectively. Results from this study support the practice of collecting and aggregating PF samples from multiple litters for PRRSV RNA testing.

PRRSV2 genetic diversity defined by RFLP patterns in the United States from 2007 to 2019.

The genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) increases over time. In 1998, restriction-fragment length polymorphism (RFLP) pattern analysis was introduced to differentiate PRRSV wild-type strains from VR2332, a reference strain from which a commercial vaccine (Ingelvac PRRS MLV) was derived. We have characterized here the PRRSV genetic diversity within selected RFLP families over time and U.S. geographic space, using available ISU-VDL data from 2007 to 2019. The 40,454 ORF5 sequences recovered corresponded to 228 distinct RFLPs. Four RFLPs [2-5-2 (21.2%), 1-7-4 (15.6%), 1-4-4 (11.8%), and 1-8-4 (9.9%)] represented 58.5% of all ORF5 sequences and were used for cluster analysis. Over time, there was increased detection of RFLPs 2-5-2, 1-7-4, 1-3-4, 1-3-2, and 1-12-4; decreased detection of 1-4-2, 1-18-4, 1-18-2, and 1-2-2; and different detection trends for 1-8-4, 1-4-4, 1-26-1, 1-22-2, and 1-2-4. An over-time cluster analysis revealed a single cluster for RFLP 2-5-2, supporting that sequences within RFLP 2-5-2 are still relatively conserved. For 1-7-4, 1-4-4, and 1-8-4, there were multiple clusters. State-wise cluster analysis demonstrated 4 main clusters for RFLP 1-7-4 and 1-8-4, and 6 for RFLP 1-4-4. For the other RFLPs, there was a significant genetic difference within them, particularly between states. RFLP typing is limited in its ability to discriminate among different strains of PRRSV. Understanding the magnitude of genetic divergence within RFLPs helps develop PRRSV regional control programs, placement, herd immunization strategies, and design of appropriate animal movements across borders to minimize the risk of PRRSV transmission.

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.

Ecology of Porcine Astrovirus Type 3 in a Herd with Associated Neurologic Disease.

Astroviruses (AstVs) cause disease in a wide variety of species. Porcine AstVs are highly genetically diverse and conventionally assigned to five genetic lineages (PoAstV1-5). Due to the increasing evidence that porcine astrovirus type 3 (PoAstV3) is a cause of encephalomyelitis in swine and to elucidate important ecologic characteristics, the infection dynamics and environmental distribution of PoAstV3 were investigated in a herd with PoAstV3-associated neurologic disease. Over a 22 week period, the frequency of PoAstV3 fecal shedding varied by pig and age. The peak detection by RT-qPCR of PoAstV3 on fecal swabs (95%; 61 of 64) occurred at 3 weeks of age. The lowest frequency of detection was at 21 weeks of age (4%; 2 of 47); however, the frequency increased to 41% (19 of 46) at the final sampling time point (25 weeks of age). Viremia was rare (0.9%: 4 of 433). Detection in oral fluid was consistent with 75% to 100% of samples positive at each time point. Pens and feeders also had a high rate of detection with a majority of samples positive at a majority of sampling time points. Based on the data presented, PoAstV3 can be consistently detected in the environment with a majority of pigs being infected and a subset intermittently shedding the virus in feces out to 25 weeks of age. These findings suggest the importance of as-yet unidentified risk factors associated with the development of PoAstV3-associated polioencephalomyelitis.

Practical aspects of PRRSV RNA detection in processing fluids collected in commercial swine farms.

Processing fluid samples are easily collected under field conditions and provide the means to test more piglets more frequently in a practical way, thereby improving PRRSV surveillance. However, a deeper understanding of the diagnostic characteristics of this newly described sample type is still required. Therefore, the objective of this field-based study was to determine the relationship between viremic piglets and the detection of PRRSV RNA in processing fluid samples. In two PRRSV-positive breeding herds, processing fluids (n = 77) and individual piglet serum samples (n = 834) were collected from 77 litters in three sampling events and tested for PRRSV RNA. Among the 77 litters in the study, 55 litters (71.4%) contained no viremic piglets and processing fluids tested negative for PRRSV RNA. Among the 22 (28.6%) litters with ≥1 viremic piglets, 10 litters contained a single viremic piglet and 5 of the 10 processing fluids from this group tested positive for PRRSV RNA. Based on a fitted mixed effects logistic regression model, the probability of detecting PRRSV RNA in processing fluids was highly dependent on the number of viremic piglets contributing to the sample. When the within-litter prevalence was ≥39%, the probability of detecting PRRSV RNA in processing fluids was ≥95%. By extension, the results suggest that pooling processing fluids from several litters increases the probability of PRRSV RNA detection because of the greater likelihood of including multiple litters each with ≥1 viremic piglets. In contemporary breeding herds that use processing fluid samples for PRRSV surveillance, the diagnostic costs associated with testing 100% of the processing-age piglet population can be estimated at €0.077 ($0.086 USD) per pig weaned. In contrast, to achieve an equivalent testing coverage with the use of individual piglet serum samples, the diagnostic costs associated would be €4.48 ($5.00 USD) per pig weaned. Processing fluid represents a practical, reliable and efficient method to surveil breeding herds for PRRSV because it allows for continuous surveillance at a low cost.

RNA Extraction from Swine Samples and Detection of Influenza A Virus in Swine by Real-Time RT-PCR.

Real-time reverse-transcription PCR (rRT-PCR) assays are currently the method of choice in many laboratories for the detection and subtyping of influenza A virus (IAV) in swine. Traditionally, nasal swabs and lung tissues (sometimes bronchoalveolar lavage and tracheal tissues) are the primary specimens for IAV testing. However, oral fluids are becoming more common for IAV prognostic profiling. In this chapter, we describe (1) procedures of RNA extraction from the common clinical specimens, (2) two rRT-PCR assays for detection of IAV in swine, and (3) an rRT-PCR assay for subtyping swine IAV. RNA extraction procedures include a magnetic bead method optimized for extraction from nasal swabs and tissue homogenates and a magnetic bead method optimized for extraction from oral fluids. Two rRT-PCR assays for detection of swine IAV include a USDA-validated IAV rRT-PCR targeting the matrix gene and the USDA-licensed VetMAX™-Gold Swine Influenza Virus Detection rRT-PCR kit (Thermo Fisher Scientific) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assay described here is VetMAX™-Gold Swine Influenza Virus Subtyping rRT-PCR kit (Thermo Fisher Scientific) which distinguishes swine IAV H1 from H3 and N1 from N2.

Detection of live attenuated influenza vaccine virus and evidence of reassortment in the U.S. swine population.

Influenza vaccines historically have been multivalent, whole virus inactivated products. The first bivalent, intranasal, live attenuated influenza vaccine (LAIV; Ingelvac Provenza), with H1N1 and H3N2 subtypes, has been approved for use in swine. We investigated the LAIV hemagglutinin (HA) sequences in diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory and potential vaccine virus reassortment with endemic influenza A virus (IAV) in swine. From January 3 to October 11, 2018, IAV HA sequences demonstrating 99.5-99.9% nucleotide homology to the H1 HA or 99.4-100% nucleotide homology to the H3 HA parental strains in the LAIV were detected in 58 of 1,116 (5.2%) porcine respiratory cases (H1 HA A/swine/Minnesota/37866/1999[H1N1; MN99]; H3 HA A/swine/Texas/4199-2/1998[H3N2; TX98]). Nine cases had co-detection of HA genes from LAIV and wild-type IAV in the same specimen. Thirty-five cases had associated epidemiologic information that indicated they were submitted from 11 states representing 31 individual sites and 17 production systems in the United States. Whole genome sequences from 11 cases and another subset of 2 plaque-purified IAV were included in our study. Ten whole genome sequences, including 1 plaque-purified IAV, contained at least one internal gene from endemic IAV detected within the past 3 y. Phylogenetic analysis of whole genome sequences indicated that reassortment occurred between vaccine virus and endemic field strains circulating in U.S. swine. Our data highlight the need and importance of continued IAV surveillance to detect emerging IAV with LAIV genes in the swine population.

Polioencephalomyelitis in Domestic Swine Associated With Porcine Astrovirus Type 3.

In the past decade, different members of the genus Mamastrovirus have been associated with outbreaks of neurologic disease in humans, cattle, sheep, mink, and, most recently, porcine astrovirus 3 (PoAstV3) in swine. We performed a retrospective analysis of 50 cases of porcine neurologic disease of undetermined cause but with microscopic lesions compatible with a viral encephalomyelitis to better understand the role and pathogenesis of PoAstV3 infection. Nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tissue for reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing for PoAstV3. In addition, 3 cases with confirmed PoAstV3-associated disease were assayed by RT-qPCR to investigate PoAstV3 tissue distribution. PoAstV3 was detected in central nervous system (CNS) tissue via RT-qPCR and in situ hybridization in 13 of 50 (26%) FFPE cases assayed. PoAstV3 was rarely detected in any tissues outside the CNS. Positive cases from the retrospective study included pigs in various production categories beginning in 2010, the earliest year samples were available. Based on these results, PoAstV3 appears to be a recurring putative cause of viral encephalomyelitis in swine that is rarely detected outside of the CNS at the time of clinical neurologic disease, unlike other common viral causes of neurologic disease in swine.

Emergence of Salmonella enterica serovar 4,[5],12:i:- as the primary serovar identified from swine clinical samples and development of a multiplex real-time PCR for improved Salmonella serovar-level identification.

Rapid identification of the infecting Salmonella serovar from porcine diagnostic samples is vital to allow implementation of appropriate on-farm treatment and management decisions. Although identification at the serogroup level can be rapidly achieved at most veterinary diagnostic laboratories, final Salmonella serovar identification often takes several weeks because of the limited number of reference laboratories performing the complex task of serotyping. Salmonella serogroup B, currently the dominant serogroup identified from swine clinical samples in the United States, contains serovars that vary from highly pathogenic to minimally pathogenic in swine. We determined the frequency of detection of individual group B serovars at the Iowa State Veterinary Diagnostic Laboratory from 2008 to 2017, and validated a multiplex real-time PCR (rtPCR) to distinguish pathogenic serogroup B serovars from those of lesser pathogenicity. Our results indicate that, since 2014, Salmonella enterica ssp. enterica serovar 4,[5],12:i:- has been the dominant serovar identified from swine clinical samples at the ISU-VDL, with S. Typhimurium now the second most common serovar identified. We developed a rtPCR to allow rapid differentiation of samples containing S. 4,[5],12:i:- and S. Typhimurium from samples containing serovars believed to be of less pathogenicity, such as S. Agona and S. Derby. When combined with enrichment culture, this rtPCR has the ability to significantly improve the time to final serovar identification of the 2 most commonly identified pathogenic Salmonella serovars in swine, and allows rapid implementation of serovar-specific intervention strategies.

Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine.

Porcine parainfluenza virus type 1 (PPIV-1) is a member of the genus Respirovirus in the family Paramyxoviridae. The PPIV-1 was initially detected in 2013 from slaughter pigs in Hong Kong, China although its role in respiratory disease has remained unknown without virus isolates for experimental inoculation in swine. The objective of this study was to determine the relative frequency of PPIV-1 detection in diagnostic samples collected from swine in the United States, describe the cell culture isolation of PPIV-1, and characterize PPIV-1 cell culture isolates in vitro. Among 842 porcine specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 2016-2017, 43.3% were PPIV-1 positive by a real-time, reverse transcriptase PCR suggesting PPIV-1 may be common in swine. Two strains of PPIV-1 were successfully isolated in an LLC-MK2 cell line from a PPIV-1 RT-qPCR positive nasal swab (USA/MN25890NS/2016) and lung (USA/IA84915LG/2017). The PPIV-1 cytopathic effect was demonstrated in tissue culture and enveloped viral particles were observed by electron microscopy. The whole genome, F, and HN gene sequences of both isolates share 98.2%, 98.5%, and 98.2% nucleotide homology, respectively, and phylogenetic analysis indicated they are closely related to other PPIV-1 strains detected in swine from the United States. Whole virus PPIV-1-specific monoclonal antibodies were generated for PPIV-1 detection in infected LLC-MK2 cells by indirect immunofluorescence and immunocytochemistry assays. The virus isolates and monoclonal antibodies obtained in the present study can be used to investigate the pathogenesis of PPIV-1 and develop new diagnostic tests.

Complete Genome Sequences of Two Novel Human-Like H3N2 Influenza A Viruses, A/swine/Oklahoma/65980/2017 (H3N2) and A/Swine/Oklahoma/65260/2017 (H3N2), Detected in Swine in the United States.

Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

Detection and genomic characterization of new avian-like hepatitis E virus in a sparrow in the United States.

Hepatitis E virus (HEV) is a nonenveloped, positive-sense, single-stranded RNA virus that has been detected in a wide variety of animals. In 2017, an avian-like HEV was identified in sparrow feces sampled from around a pig farm in the midwestern United States. Sequence analysis revealed that the sparrow isolate represents a novel HEV that is distantly related to chicken and little egret HEVs.

Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States.

BACKGROUND: Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. METHODS: The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015-2016 were analyzed. RESULTS: A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. CONCLUSION: These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae.

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.

The ontogeny of PRRSV antibody in oral fluids has been described using isotype-specific ELISAs. Mirroring the serum response, IgM appears in oral fluid by 7days post inoculation (DPI), IgA after 7 DPI, and IgG by 9 to 10 DPI. Commercial PRRSV ELISAs target the detection of IgG because the higher concentration of IgG relative to other isotypes provides the best diagnostic discrimination. Oral fluids are increasingly used for PRRSV surveillance in commercial herds, but in younger pigs, a positive ELISA result may be due either to maternal antibody or to antibody produced by the pigs in response to infection. To address this issue, a combined IgM-IgA PRRSV oral fluid ELISA was developed and evaluated for its capacity to detect pig-derived PRRSV antibody in the presence of maternal antibody. Two longitudinal studies were conducted. In Study 1 (modified-live PRRS vaccinated pigs), testing of individual pig oral fluid samples by isotype-specific ELISAs demonstrated that the combined IgM-IgA PRRSV ELISA provided better discrimination than individual IgM or IgA ELISAs. In Study 2 (field data), testing of pen-based oral fluid samples confirmed the findings in Study 1 and established that the IgM-IgA ELISA was able to detect antibody produced by pigs in response to wild-type PRRSV infection, despite the presence of maternal IgG. Overall, the combined PRRSV IgM-IgA oral fluid ELISA described in this study is a potential tool for PRRSV surveillance, particularly in populations of growing pigs originating from PRRSV-positive or vaccinated breeding herds.

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.

Complete Genome Sequence of Porcine respirovirus 1 Strain USA/MN25890NS/2016, Isolated in the United States.

A porcine parainfluenza virus type 1 (species Porcine respirovirus 1) cell culture isolate, USA/MN25890NS/2016, was obtained from porcine nasal swabs, and its complete genome sequence (GenBank accession number MF681710) was determined to help further characterize this virus.

Detection and characterization of an H4N6 avian-lineage influenza A virus in pigs in the Midwestern United States.

H4Nx viruses were reported in swine in Canada and China, but had not been recognized in swine in the USA. In late 2015, an avian-origin H4N6 influenza A virus was isolated from pigs in the United States during a routine diagnostic investigation of clinical respiratory disease in the herd. Serological analysis from additional pigs at the farm and other pigs within the swine production system indicated that the virus did not efficiently transmit from pig-to-pig and the mode of transmission to swine could not be determined. The isolate was characterized at the molecular level and the pathogenesis and transmission was experimentally evaluated in pigs. Although the virus replicated in the lungs of pigs and caused mild pulmonary lesions, there was no evidence of replication in the upper respiratory tract or transmission to indirect contacts, supporting the findings on the farm.