Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs.

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.

Human Monoclonal Antibody Derived from Transchromosomic Cattle Neutralizes Multiple H1 Clades of Influenza A Virus by Recognizing a Novel Conformational Epitope in the Hemagglutinin Head Domain.

Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans.IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.

Pathogenesis of Senecavirus A infection in finishing pigs.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viraemia was assessed in serum and virus shedding monitored in oral and nasal secretions and faeces by real-time reverse transcriptase quantitative PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 post-inoculation (pi) and persisted for approximately 2-10 days. Vesicular lesions were first observed on day 4 pi on the snout and/or feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viraemia was observed between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and faeces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsils of all SVA-infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (day 5 pi), which coincided with decreased levels of viraemia, virus shedding and viral load in tissues. This study provides significant insights into the pathogenesis and infectious dynamics of SVA in swine.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies.

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.

Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus.

BACKGROUND: A novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection. RESULTS: The nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes. CONCLUSION: These new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Modeling the transboundary risk of feed ingredients contaminated with porcine epidemic diarrhea virus.

BACKGROUND: This study describes a model developed to evaluate the transboundary risk of PEDV-contaminated swine feed ingredients and the effect of two mitigation strategies during a simulated transport event from China to the US. RESULTS: Ingredients imported to the USA from China, including organic & conventional soybeans and meal, lysine hydrochloride, D-L methionine, tryptophan, Vitamins A, D & E, choline, carriers (rice hulls, corn cobs) and feed grade tetracycline, were inoculated with PEDV. Control ingredients, and treatments (ingredients plus a liquid antimicrobial (SalCURB, Kemin Industries (LA) or a 2% custom medium chain fatty acid blend (MCFA)) were tested. The model ran for 37 days, simulating transport of cargo from Beijing, China to Des Moines, IA, US from December 23, 2012 to January 28, 2013. To mimic conditions on land and sea, historical temperature and percent relative humidity (% RH) data were programmed into an environmental chamber which stored all containers. To evaluate PEDV viability over time, ingredients were organized into 1 of 4 batches of samples, each batch representing a specific segment of transport. Batch 1 (segment 1) simulated transport of contaminated ingredients from manufacturing plants in Beijing (day 1 post-contamination (PC)). Batch 2 (segments 1 and 2) simulated manufacturing and delivery to Shanghai, including time in Anquing terminal awaiting shipment (days 1-8 PC). Batch 3 (segments 1, 2 and 3) represented time in China, the crossing of the Pacific and entry to the US at the San Francisco, CA terminal (day 1-27 PC). Batch 4 (segments 1-4) represented the previous events, including transport to Des Moines, IA (days 1-37 PC). Across control (non-treated) ingredients, viable PEDV was detected in soybean meal (organic and conventional), Vitamin D, lysine hydrochloride and choline chloride. In contrast, viable PEDV was not detected in any samples treated with LA or MCFA. CONCLUSIONS: These results demonstrate the ability of PEDV to survive in a subset of feed ingredients using a model simulating shipment from China to the US. This is proof of concept suggesting that contaminated feed ingredients could serve as transboundary risk factors for PEDV, along with the identification of effective mitigation options.

Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus.

BACKGROUND: Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies. RESULTS: A recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. CONCLUSION: Well-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

An evaluation of a liquid antimicrobial (Sal CURB®) for reducing the risk of porcine epidemic diarrhea virus infection of naïve pigs during consumption of contaminated feed.

BACKGROUND: Since its initial detection in May 2013, porcine epidemic diarrhea virus (PEDV) has spread rapidly throughout the US swine industry. Recently, contaminated feed was confirmed as a vehicle for PEDV infection of naïve piglets. This research provides in vivo data supporting the ability of a liquid antimicrobial product to reduce this risk. RESULTS: Sal CURB® (Kemin Industries, Des Moines, IA, USA) is a FDA-approved liquid antimicrobial used to control Salmonella contamination in poultry and swine diets. To test its effect against PEDV, Sal CURB®-treated feed was spiked with a stock isolate of PEDV (Ct = 25.22), which PEDV-naïve piglets were allowed to ingest via natural feeding behavior (ad libitum) for a 14-day period. For the purpose of a positive control, a separate group of piglets was allowed to ingest non-treated (Sal CURB®-free) feed also spiked with stock PEDV (Ct = 25.22). A negative control group received PEDV-free feed. Clinical signs of PEDV infection (vomiting and diarrhea) and viral shedding in feces were observed in the positive control group 2-3 days post-consumption of non-treated feed. In contrast, no evidence of infection was observed in pigs fed Sal CURB®-treated feed or in the negative controls throughout the 14-day study period. In addition, the Sal CURB®-treated feed samples had higher (p < 0.0001) mean PEDV Ct values than samples from the positive control group. CONCLUSIONS: These data provide proof of concept that feed treated with Sal CURB® can serve as a means to reduce the risk of PEDV infection through contaminated feed. Furthermore, the results from the positive control group provide additional proof of concept regarding the ability of contaminated feed to serve as a risk factor for PEDV infection of naïve piglets.

An evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naïve pigs following consumption via natural feeding behavior: proof of concept.

BACKGROUND: Since its initial detection in May 2013, porcine epidemic diarrhea virus (PEDV) has spread rapidly throughout the US swine industry. Initially, contaminated feed was proposed as a risk factor for PEDV; however, data were not available to support this theory. Here we provide proof of concept of this risk by describing a novel means for recovering PEDV-contaminated complete feed material from commercial swine sites and conducting an in vivo experiment to prove its infectivity. RESULTS: For on-farm detection of PEDV RNA in feed, paint rollers were used to collect material from at-risk feed bins from 3 clinically affected breeding herds. This material was tested by PCR and determined to be positive for PEDV-RNA (Ct = 19.50-22.20 range). To test infectivity, this material was pooled (Ct = 20.65) and a Treatment group of 3-week old PEDV-naïve piglets were allowed to consume it via natural feeding behavior. For the purpose of a Positive control, piglets were allowed to ingest feed spiked with stock PEDV (Ct = 18.23) while the negative control group received PEDV-free feed. Clinical signs of PEDV infection (vomiting and diarrhea) and viral shedding were observed in both the Positive control and Treatment group' post-consumption with virus and microscopic lesions detected in intestinal samples No evidence of infection was observed in the Negative controls. CONCLUSIONS: These data provide proof of concept that contaminated complete feed can serve as a vehicle for PEDV infection of naïve pigs using natural feeding behavior.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.

Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

Transmission of porcine reproductive and respiratory syndrome virus in domestic pigs via oral ingestion of feed material.

OBJECTIVE: The purpose of this case study was to describe the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) under field and experimental conditions via the consumption of PRRSV-positive swine feed. ANIMALS: 1 domestic swine breeding herd and 20 laboratory-maintained experimental domestic pigs. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: A 2,500-sow PRRSV-naïve biosecure breeding herd became infected during the autumn months. It experienced a feed outage involving a specific bin on October 23 (day 0), with the bin refilled on October 24 (day 1). From October 28 to 30 (days 5 to 7), signs of anorexia and hyperemia were observed in 30 gestating sows after consuming feed from this bin. On November 1 (day 9), blood samples from 10 affected sows were PRRSV positive by reverse transcriptase PCR. In contrast, sows in the same room that had consumed feed from other bins were clinically normal and PRRSV negative. To investigate whether the feed delivery introduced PRRSV to the herd, on November 2 (day 10) 4 samples of feed material from the interior walls of the index bin were collected and tested by reverse transcriptase PCR. TREATMENT AND OUTCOME: All 4 samples were positive for PRRSV RNA with cycle threshold values ranging from 26 to 29. Nucleic acid sequencing indicated that the open reading frame 5 region of the PRRSV in feed samples was 100% homologous to PRRSV from index cases. To assess viability of the virus, PRRSV-naïve pigs were allowed to consume the index feed bin samples and became infected with PRRSV based on viral RNA in oral fluid samples, clinical signs, and postmortem lesions. CLINICAL RELEVANCE: These results suggest that feed was a likely source of PRRSV introduction to the herd. This is the first report of PRRSV transmission through feed.

Monitoring emerging pathogens using negative nucleic acid test results from endemic pathogens in pig populations: Application to porcine enteric coronaviruses.

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.

Persistent infection of American bison (Bison bison) with bovine viral diarrhea virus and bosavirus.

Bovine viral diarrhea viruses (BVDV) are significant pathogens of cattle, leading to losses associated with reproductive failure, respiratory disease and immune dysregulation. While cattle are the reservoir for BVDV, a wide range of domestic and wild ruminants are susceptible to infection and disease caused by BVDV. Samples from four American bison (Bison bison) from a captive herd were submitted for diagnostic testing due to their general unthriftiness. Metagenomic sequencing on pooled nasal swabs and serum identified co-infection with a BVDV and a bovine bosavirus. The BVDV genome was more similar to the vaccine strain Oregon C24 V than to other BVDV sequences in GenBank, with 92.7 % nucleotide identity in the open reading frame. The conserved 5'-untranslated region was 96.3 % identical to Oregon C24 V. Bosavirus has been previously identified in pooled fetal bovine serum but its clinical significance is unknown. Sequencing results were confirmed by virus isolation and PCR detection of both viruses in serum and nasal swab samples from two of the four bison. One animal was co-infected with both BVDV and bosavirus while separate individuals were positive solely for BVDV or bosavirus. Serum and nasal swabs from these same animals collected 51 days later remained positive for BVDV and bosavirus. These results suggest that both viruses can persistently infect bison. While the etiological significance of bosavirus infection is unknown, the ability of BVDV to persistently infect bison has implications for BVDV control and eradication programs. Possible synergy between BVDV and bosavirus persistent infection warrants further study.

Use of a demonstration project to evaluate viral survival in feed: Proof of concept.

In 2014, the hypothesis that feed ingredients could serve as vehicles for the transport and transmission of viral pathogens was proposed and evaluated by multiple investigators under laboratory conditions. In an attempt to validate these data, we used a demonstration project to test whether three significant viruses of swine could survive in feed ingredients under real-world shipping conditions. Samples of soya bean meal (organic and conventional), lysine, choline and vitamin A were spiked with a mixture of PRRSV 174, PEDV and SVA and transported for 21 days in the trailer of a commercial transport vehicle, encompassing 14 states and 9,741 km. Samples were tested for viral genome and viability at the end of the transit period. Regarding viability, PRRSV, PEDV and SVA were all detected as infectious in bioassays following inoculation with both soy products. In addition, viable PRRSV and SVA were detected by bioassay pigs inoculated with samples of vitamin A, and infectious SVA was detected in pigs inoculated with samples of lysine and choline. These results provide further evidence that select viral pathogens of pigs can survive in certain feed ingredients during commercial transit.

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.

Data standardization implementation and applications within and among diagnostic laboratories: integrating and monitoring enteric coronaviruses.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

Guidelines for Sanger sequencing and molecular assay monitoring.

Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.

Prediction of seasonal patterns of porcine reproductive and respiratory syndrome virus RNA detection in the U.S. swine industry.

We developed a model to predict the cyclic pattern of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by reverse-transcription real-time PCR (RT-rtPCR) from 4 major swine-centric veterinary diagnostic laboratories (VDLs) in the United States and to use historical data to forecast the upcoming year's weekly percentage of positive submissions and issue outbreak signals when the pattern of detection was not as expected. Standardized submission data and test results were used. Historical data (2015-2017) composed of the weekly percentage of PCR-positive submissions were used to fit a cyclic robust regression model. The findings were used to forecast the expected weekly percentage of PCR-positive submissions, with a 95% confidence interval (CI), for 2018. During 2018, the proportion of PRRSV-positive submissions crossed 95% CI boundaries at week 2, 14-25, and 48. The relatively higher detection on week 2 and 48 were mostly from submissions containing samples from wean-to-market pigs, and for week 14-25 originated mostly from samples from adult/sow farms. There was a recurring yearly pattern of detection, wherein an increased proportion of PRRSV RNA detection in submissions originating from wean-to-finish farms was followed by increased detection in samples from adult/sow farms. Results from the model described herein confirm the seasonal cyclic pattern of PRRSV detection using test results consolidated from 4 VDLs. Wave crests occurred consistently during winter, and wave troughs occurred consistently during the summer months. Our model was able to correctly identify statistically significant outbreak signals in PRRSV RNA detection at 3 instances during 2018.