The application of metagenomic sequencing to detect and characterize emerging porcine viruses

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyRaymond R. R. RowlandEmerging viral diseases threaten the health of the US swineherd and have the potential to impact the industry. Parvoviruses are capable of infecting birds, livestock and humans, however, in swine, parvoviruses cause reproductive failure and contribute to a devastating set of diseases termed porcine circovirus associated disease (PCVAD). Here, a divergent porcine parvovirus, porcine parvovirus 7 (PPV7), distantly related to known parvovirus sequences, was identified in market pigs in the US. The PPV7 non-structural protein displayed 42.4% similarity to Eidolon helvum parvovirus 2 and 37.9% similarity to turkey parvovirus. Conserved parvovirus replicase motifs including three rolling circle replication (RCR), two NTP-binding motifs and a helicase- binding domain, were present in PPV7. Analysis by qPCR of 182 porcine samples found 16 (8.6%) positive, suggesting moderate nucleic acid prevalence in US swine. Paramyxoviruses are capable of infecting various species including cattle, pigs and humans, causing respiratory disease and importantly, can overcome species barriers causing disease. In 2013, a novel paramyxovirus sequence was described in Hong Kong, China in slaughterhouse pigs, and subsequently named porcine parainfluenza virus 1 (PPIV1). The second study identifies two complete PPIV1 genomes in US pigs originating in Oklahoma and Nebraska that display 90.0-95.3% identity to the Chinese strains. Molecular analysis by qPCR resulted in 6.1% prevalence in 279 porcine respiratory samples. Further serological analysis revealed 66.1% of 59 porcine sera samples were positive by PPIV1 F ELISA. Eleven 3-week old nursery pigs from a PPIV1 naturally infected herd were monitored for signs of infection. No clinical signs were seen in the animals, however, six pigs and the lungs of one animal tested qPCR positive by the conclusion of the study. Taken together, PPIV1 is moderately prevalent in US swine-herds. Previously known to infect avian species, canines and swine, recent reports have identified circoviruses in bats, mink, and human feces. In pigs, porcine circovirus 2 (PCV2) is essential to PCVAD, a group of diseases including reproductive failure, respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS) and postweaning multisystemic wasting syndrome (PMWS). Additionally, PCV2 nucleic acid has been detected in mammalian species other than swine such as cattle and mink. The final study focuses on the identification and characterization of a divergent circovirus, porcine circovirus 3, identified in aborted mummies taken from sows displaying clinical and histological signs of PDNS. Putative capsid and replicase open reading frames display 37% and 55% identity to PCV2, respectively. A retrospective study of 48 PDNS cases, PCV2 negative by immunohistochemistry (IHC), identified 45 positive and 60% of a subset, positive for PCV3 by IHC. Molecular and serological prevalence studies revealed 12.5% nucleic acid and 55% antibody prevalence in US swine samples. Collectively, these studies identify emerging porcine viruses with the potential to cause disease using metagenomic sequencing. The results of these studies will help to mitigate the risk attributed to emerging swine viruses causing disease outbreaks

Detection and Investigation of Atypical Porcine Pestivirus within a Breed-to-Finish Farm and Off-Site Nursery and Finisher Locations

Atypical porcine pestivirus (APPV) has been associated with congenital tremors (CT) and splay leg (SL) in piglets of infected dams. The major cost of this virus is the increased pre-weaning mortality due to CT or SL interfering with the piglet’s ability to nurse and move around the farrowing stall. A commercial farrow-to-finish farm with replacement gilts coming from an off-site genetic multiplier farm, and semen delivery from a commercial boar stud began to see an increase of CT and SL in the farrowing room in early 2020. Diagnostics on clinically affected pigs’ samples identified APPV RNA and no other suspected pathogen. At this point, the origin of the virus and means of introduction into the farm was unknown since the farm had no previous clinical cases of CT or SL prior to this investigation. The two hypothesized routes were the introduction of replacement gilts or incoming semen doses. Therefore, the objectives of this investigation were to determine the prevalence of clinical APPV cases at the farrow-to-finish farm, understand the route of introduction of APPV into the farrowto- finish farm, and understand the prevalence of APPV viremia within a population of offspring from a gilt multiplication farm through an off-site nursery and finisher barn. Farrowing records from the farm were analyzed for the presence of CT or SL and parities of females with affected litters. Blood samples were collected at two different times from the new group of replacement gilts and maternal barrows at the isolation nursery barn. Serum and oral fluids were collected from the same pigs at an off-site finisher barn to determine APPV persistence. The APPV sequencing was conducted on a serum sample from a gilt housed at the isolation nursery intended as a replacement gilt for the farrow-to-finish farm, semen dose utilized at the farrow-to-finish farm, and serum of a clinically affected piglet in the farrowing room of the farrow-to-finish farm. Overall, the prevalence of affected litters within batch farrowing groups ranged from 0 to 31%. The prevalence of APPV within samples pooled by pens (5 pigs) ranged from 37.5 to 77.5%, while individual prevalence ranged from 20 to 40%. When followed to the finisher, the same group of pigs had an APPV prevalence in serum ranging from 0 to 26%, while oral fluid prevalence was 100%. Sequencing results indicated that the virus circulating in clinically affected piglets was the most similar to an incoming semen dose. In summary, introduction of APPV into a naïve herd is associated with an increase in clinical CT and SL. While APPV is present in herds previously exposed to APPV, the APPV RNA remains detectable in serum and oral fluids with no clinical disease. To decrease the chance of infection to a naïve herd, quarantines should be implemented for all introductions. Additionally, semen should be screened for APPV presence if there is a recent onset of clinically affected piglets with CT or SL with no other explanation. The APPV RNA was detected in group oral fluids, suggesting the technique may be used to screen incoming animals

Swine Disease Reporting: Report #14

SHIC-funded, veterinary diagnostic laboratories (VDLs) collaborative project, with goal to aggregate swine diagnostic data from participating reporting VDLs, and report in an intuitive format (web dashboards), describing dynamics of disease detection by pathogen or disease syndrome over time, specimen, age group, and geographical space. For this report, data is from the Iowa State University VDL and South Dakota State University ADRDL. University of Minnesota VDL and Kansas State University VDL. Specifically, for PRRSV RFLP data, and syndromic information the results are from Iowa State University VDL. For all “2019 predictive graphs”, the expected value was calculated using a statistical model that considers the results from 3 previous years. The intent of the model is not to compare the recent data (2019) to individual weeks of previous years. The intent is to estimate expected levels of percent positive cases based on patterns observed in the past data, and define if observed percentage positive values are above or below the expected based on historic trends

Comparison of the Effect of Corn-fermented Protein and Traditional Ingredients on the Fecal Microbiota of Dogs

Corn-fermented protein (CFP), a co-product from the ethanol industry, is produced using post-fermentation technology to split the protein and yeast from fiber prior to drying. The objective of this study was to determine the effect of CFP compared to traditional ingredients on the fecal microbiota of dogs. The four experimental diets included a control with no yeast and diets containing either 3.5% brewer’s dried yeast, 2.5% brewer’s dried yeast plus 17.5% distiller’s dried grains with solubles, or 17.5% CFP. The experimental diets were fed to adult dogs (n = 12) in a 4 × 4 replicated Latin square design. Fresh fecal samples (n = 48) were analyzed by 16S metagenomic sequencing. Raw sequences were processed through mothur. Community diversity was evaluated in R. Relative abundance data were analyzed within the 50 most abundant operational taxonomic units using a mixed model of SAS. Alpha and beta diversity were similar for all treatments. Predominant phyla among all samples were Firmicutes (73%), Bacteroidetes (15%), Fusobacteria (8%), and Actinobacteria (4%). There were no quantifiable (p > 0.05) shifts in the predominant phyla among the treatments. However, nine genera resulted in differences in relative abundance among the treatments. These data indicate that compared to traditional ingredients, CFP did not alter the overall diversity of the fecal microbiota of healthy adult dogs over 14 days

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances

Comparison of Pathogenicity and Transmissibility of Influenza B and D Viruses in Pigs

Influenza viruses are important pathogens causing respiratory disease in humans and animals. In contrast to influenza A virus (IAV) that can infect a wide range of animal species, other influenza viruses, including influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV) have a limited host range. Swine can be infected with all four different genera of influenza viruses. IAV infection of pigs causes the well-known swine influenza that poses significant threats to human and animal health. However, influenza virus infection of pigs with IBV, ICV, and IDV are not well-characterized. Herein, we compared pathogenicity of IBV and IDV using intratracheal and intranasal infection of pigs, which are IAV seropositive, and commingled naïve pigs with the infected animals to determine their transmissibility. Both viruses caused fever and some lung lesions, replicated in the lungs of infected pigs, but only IDV transmitted to the contact animals. Although IBV and IDV displayed differing levels of replication in the respiratory tract of infected pigs, no significant differences in pathogenicity of both viruses were observed. These results indicate that both IBV and IDV can replicate, and are pathogenic in pigs