Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples.

The MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research, and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). The ZMAC cell line, derived from porcine alveolar macrophages, has become available, but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI results in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). The PRRSV VI success rate was very low in oral fluids and processing fluids regardless of whether ZMAC cells or MARC-145 cells were used. Success rates of PRRSV VI from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with threshold cycle (CT ) values of <30 had better VI success. PRRSV-2 in genetic lineages 1 and 8 was isolated more successfully in ZMAC cells than in MARC-145 cells, whereas PRRSV-2 in genetic lineage 5 was isolated in the two cell lines with similar success rates. For samples with positive VI in both ZMAC and MARC-145 cells, 14 of 23 PRRSV-2 isolates had similar titers in the two cell lines. A total of 51 of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 cells, and all 46 (100%) MARC-145-obtained isolates grew in ZMAC cells. In summary, ZMAC cells allow better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This report provides important guidelines to improve isolation of PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

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.

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.

Recombination between Vaccine and Field Strains of Porcine Reproductive and Respiratory Syndrome Virus.

We isolated and plaque purified IA76950-WT and IA70388-R, 2 porcine reproductive and respiratory syndrome viruses from pigs in the same herd in Iowa, USA, that exhibited coughing and had interstitial pneumonia. Phylogenetic and molecular evolutionary analysis indicated that IA70388-R is a natural recombinant from Fostera PRRSV vaccine and field strain IA76950-WT.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus.

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

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 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.

Identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the United States.

Two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the USA: US prototype (also called non-S INDEL) and S INDEL PEDVs. In February 2017, a PEDV variant (USA/OK10240-8/2017) was identified in a rectal swab from a sow farm in Oklahoma, USA. Complete genome sequence analyses indicated this PEDV variant was genetically similar to US non-S INDEL strain but had a continuous 600-nt (200-aa) deletion in the N-terminal domain of the spike gene compared to non-S INDEL PEDVs. This is the first report of detecting PEDV bearing large spike gene deletion in clinical swine samples in the USA.

Porcine Astrovirus Type 3 in Central Nervous System of Swine with Polioencephalomyelitis.

Using next-generation sequencing, we identified and genetically characterized a porcine astrovirus type 3 strain found in tissues from the central nervous system of 1 piglet and 3 sows with neurologic signs and nonsuppurative polioencephalomyelitis. Further studies are needed to understand the potential for cross-species transmission and clinical impact.

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality.

We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5' untranslated region (5'-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Role of transportation in spread of porcine epidemic diarrhea virus infection, United States.

After porcine epidemic diarrhea virus (PEDV) was detected in the United States in 2013, we tested environmental samples from trailers in which pigs had been transported. PEDV was found in 5.2% of trailers not contaminated at arrival, , suggesting that the transport process is a source of transmission if adequate hygiene measures are not implemented.

Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States.

Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 10(2) to 2 × 10(5) 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

Comparison of commercial real-time reverse transcription-PCR assays for reliable, early, and rapid detection of heterologous strains of porcine reproductive and respiratory syndrome virus in experimentally infected or noninfected boars by use of different sample types.

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville, MD [TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold, <30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.

Detection of Salmonella enteritidis in pooled poultry environmental samples using a serotype-specific real-time-polymerase chain reaction assay.

While real-time-polymerase chain reaction (RT PCR) has been used as a rapid test for detection of Salmonella Enteritidis in recent years, little research has been done to assess the feasibility of pooling poultry environmental samples with a Salmonella Enteritidis-specific RT PCR assay. Therefore the objective of this study was to compare RT PCR Salmonella Enteritidis detection in individual and pooled (in groups of two, three, and four) poultry environmental drag swab samples to traditional cultural methods. The drag swabs were collected from poultry facilities previously confirmed positive for Salmonella Enteritidis and were cultured according to National Poultry Improvement Plan guidelines. Initial, Salmonella Enteritidis-specific RT PCR assay threshold cycle cutoff values of < or = 36, < or = 30, and < or = 28 were evaluated in comparison to culture. The average limit of detection of the RT PCR assay was 2.4 x 10(3) colony-forming units (CFUs)/ml, which corresponded to an average threshold cycle value of 36.6. Before enrichment, samples inoculated with concentrations from 10(2) to 10(5) CFUs/ml were detected by RT PCR, while after enrichment, samples inoculated from 10(0) to 10(5) CFUs/ml were detected by RT PCR. Threshold cycle cutoff values were used in the subsequent field trial from which Salmonella Enteritidis was cultured in 7 of 208 environmental samples (3.4%). Individual samples were 99.0%, 100%, and 100% in agreement with the RT PCR at threshold cycle (C(t)) cutoff values of < or = 36, < or = 30, and < or = 28 respectively. The agreement for pooled samples also followed the same trend with highest agreement at C(t) < or = 28 (pool of 2 = 100.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), midrange agreement at C(t) < or = 30 (pool of 2 = 99.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), and lowest agreement at C(t) < or = 36 (pool of 2 = 98.1%, pool of 3 = 97.1%, pool of 4 = 98.1%). In conclusion, regardless of the level of pooling after tetrathionate enrichment, sensitivity was very good, and results would be comparable to what would have been found with individual culture or individual RT PCR at C(t) < or = 36.

Genetic characterization of porcine sapoviruses identified from pigs during a diarrhoea outbreak in Iowa, 2019.

Porcine sapovirus (SaV) was first identified by electron microscopy in the United States in 1980 and has since been reported from both asymptomatic and diarrhoeic pigs usually in mixed infection with other enteric pathogens. SaV as the sole aetiological agent of diarrhoea in naturally infected pigs has not previously been reported in the United States. Here, we used four independent lines of evidence including metagenomics analysis, real-time RT-PCR (rRT-PCR), histopathology, and in situ hybridization to confirm porcine SaV genogroup III (GIII) as the sole cause of enteritis and diarrhoea in pigs. A highly sensitive and specific rRT-PCR was established to detect porcine SaV GIII. Examination of 184 faecal samples from an outbreak of diarrhoea on a pig farm showed that pigs with clinical diarrhoea had significantly lower Ct values (15.9 ± 0.59) compared to clinically unaffected pigs (35.8 ± 0.71). Further survey of 336 faecal samples from different states in the United States demonstrated that samples from pigs with clinical diarrhoea had a comparable positive rate (45.3%) with those from asymptomatic pigs (43.1%). However, the SaV-positive pigs with clinical diarrhoea had significantly higher viral loads (Ct  = 26.0 ± 0.5) than the SAV-positive but clinically healthy pigs (Ct  = 33.2 ± 0.9). Phylogenetic analysis of 20 field SaVs revealed that all belonged to SaV GIII and recombination analysis indicated that intragenogroup recombination had occurred within the field isolates of SaV GIII. These results suggest that porcine SaV GIII plays an important aetiologic role in swine enteritis and diarrhoea and rRT-PCR is a reliable method to detect porcine SaV. Our findings provide significant insights to better understand the epidemiology and pathogenicity of porcine SaV infection.

Characterization of a 2016-2017 Human Seasonal H3 Influenza A Virus Spillover Now Endemic to U.S. Swine.

In 2017, the Iowa State University Veterinary Diagnostic Laboratory detected a reverse-zoonotic transmission of a human seasonal H3 influenza A virus into swine (IAV-S) in Oklahoma. Pairwise comparison between the recently characterized human seasonal H3 IAV-S (H3.2010.2) hemagglutinin (HA) sequences detected in swine and the most similar 2016-2017 human seasonal H3 revealed 99.9% nucleotide identity. To elucidate the origin of H3.2010.2 IAV-S, 45 HA and 27 neuraminidase (NA) sequences from 2017 to 2020 as well as 11 whole-genome sequences (WGS) were genetically characterized. Time to most recent common human ancestor was estimated between August and September 2016. The N2 NA was of human origin in all but one strain from diagnostic submissions with NA sequences, and the internal gene segments from WGS consisted of matrix genes originating from the 2009 pandemic H1N1 and another 5 internal genes of triple reassortant swine origin (TTTTPT). Pigs experimentally infected with H3.2010.2 demonstrated efficient nasal shedding and replication in the lungs, mild pneumonia, and minimal microscopic lung lesions and transmitted the virus to indirect contact swine. Antigenically, H3.2010.2 viruses were closer to a human seasonal vaccine strain, A/Hong Kong/4801/2014, than to the H3.2010.1 human seasonal H3 viruses detected in swine in 2012. This was the second sustained transmission of a human seasonal IAV into swine from the 2010 decade after H3.2010.1. Monitoring the spillover and detection of novel IAV from humans to swine may help vaccine antigen selection and could impact pandemic preparedness. IMPORTANCE H3.2010.2 is a new phylogenetic clade of H3N2 circulating in swine that became established after the spillover of a human seasonal H3N2 from the 2016-2017 influenza season. The novel H3.2010.2 transmitted and adapted to the swine host and demonstrated reassortment with internal genes from strains endemic to pigs, but it maintained human-like HA and NA. It is genetically and antigenically distinct from the H3.2010.1 H3N2 introduced earlier in the 2010 decade. Human seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection to improve the ability to prevent respiratory disease in swine as well as the risk of zoonotic transmission.

Considerations in the use of processing fluids for the detection of PRRSV RNA and antibody.

Surveillance is mandatory for tracking the progress of porcine reproductive and respiratory syndrome virus (PRRSV) control and elimination efforts in breeding herds. Processing fluids, the fluid recovered from tissues collected at castration and/or tail docking, are used for breeding herd surveillance by large segments of the industry, but the basic diagnostic characteristics of processing fluids are largely undescribed. We undertook 3 studies to address this information gap. In study 1, we found no differences among the PRRSV RT-rtPCR results obtained with 4 commercial RNA extraction kits. In study 2, we found that PRRSV RNA was highly stable in processing fluid samples at -20°C or 4°C, but detrimental effects were observed at ≥22°C within 24 h. In study 3, using a modified PRRSV ELISA at a sample:positive cutoff of ≥0.5, we found excellent discrimination in the detection of PRRSV antibody (IgM, IgA, IgG) in processing fluids from herds of known PRRSV status. Judicious handling of processing fluid samples from sow herds, and the use of methods available in veterinary diagnostic laboratories, can provide a foundation for reliable PRRSV surveillance.

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.

Pathogenesis of a novel porcine parainfluenza virus type 1 isolate in conventional and colostrum deprived/caesarean derived pigs.

Two experimental challenge studies were conducted to evaluate the pathogenesis of a porcine parainfluenza virus type 1 (PPIV-1) isolate. Four-week-old conventional (CON) pigs were challenged in Study 1 and six-week-old caesarean derived/colostrum deprived (CDCD) pigs were challenged in Study 2. Results indicate that PPIV-1 shedding and replication occur in the upper and lower respiratory tracts of CON and CDCD pigs as detected by RT-qPCR and immunohistochemistry. Mild macroscopic lung lesions were observed in CON pigs but not in CDCD pigs. Microscopic lung lesions were mild and consisted of peribronchiolar lymphocytic cuffing and epithelial proliferation in CON and CDCD pigs. Serum neutralizing antibodies were detected in the CON and CDCD pigs by 14 and 7 days post inoculation, respectively. This study provides evidence that in spite of PPIV-1 infection and replication in challenged swine, significant clinical respiratory disease was not observed.

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.