IDENTIFICATION OF NOVEL AND EMERGING VIRUSES ASSOCIATED WITH BOVINE AND PORCINE RESPIRATORY DISEASE AND DEVELOPMENT OF VACCINE COUNTERMEASURES

<b>Project Methods</b><br> Objective 1: Characterize the virome present in nasal swab samples collected from swine and cattle with respiratory disease via metagenomic sequencing The South Dakota State University Animal Disease Research and Diagnostic Laboratory (ADRDL) performs >20,000 PCR tests for PRDC and BRDC pathogens annually. Samples primarily originate from the upper Midwest which includes top states for cattle and swine production. A total of 100 nasal swab pools (five swabs per pool) will be analyzed for both PRDC and BRDC submissions. Nasal swab pools will be assembled from diagnostic cases containing at least five nasal swabs submitted in Mycoplasma/Virus Transport Media from animals with reported clinical respiratory disease. The 100 cases each for PRDC and BRDC swab pools will be selected to ensure maximal seasonal and geographical representation, as well as from different animal age classes and ownership.Pooled nasal swabs will be analyzed by metagenomic sequencing as previously described (Hause et al., 2016). A combination of de novo and reference-based assemblies, along with targeted amplification and resequencing, will be utilized to complete assembly of identified virus genomes when possible, which will subsequently be deposited with Genbank. Genetic and phylogenetic analysis will be performed on the assembled genomes to assess genetic variability. The output from this objective will be a foundational start to understanding the genetic diversity of the virome in PRDC and BRDC.Objective 2: Identify infectious viruses and establish disease etiology using Immunocapture sequencing One challenge with metagenomic sequencing is distinguishing pathogenic from environmental viruses. The nasal swab virome consists of viruses derived from both the host as well as environment, complicating conclusions on metagenomic sequencing results with disease etiology.Following metagenomic sequencing of pooled nasal swabs in Objective 1, pools with novel or emerging viruses will be identified within four weeks of original sample submission. The submitting veterinarian will subsequently be contacted and asked to provide several serum samples from animals in that herd. The serum samples will be pooled and used to immunoprecipitate viruses present in the original nasal swab pool using commercially available protein A/G coupled to magnetic beads. Immunoprecipitated viruses will subsequently be analyzed by metagenomic sequencing to identify viruses which animals in the herd have seroconverted. While not direct proof of causation, the presence of an adaptive immune response to particular viruses will aid in interpretation of the significance of identified viruses.Objective 3: Propagate novel and emerging viruses in cell culture using traditional cell culture based approaches or reverse genetics-launched viruses Virus isolation is a critical prerequisite for virus characterization. With advances in molecular biology, virus isolation is often neglected or overlooked. Isolated virus is needed to fully characterize novel viruses, especially for pathogenesis studies. Additionally, vaccine licensure requires proof of efficacy which nearly always requires vaccination and challenge models in the host animal.Given the critical need for virus which can be propagated in the laboratory, virus isolation will be attempted on a combination of swine and bovine primary cells and immortalized cell lines. Samples positive for atypical viruses will be inoculated on a panel of cells including primary porcine and bovine respiratory epithelial (Sreenivasan et al., 2019) and kidney cells (Takenouchi et al., 2014), as well as alveolar macrophages (Yoon et al., 1992). Swine samples will additionally be inoculated onto immortalized cell lines ST, Vero, and PK15. Bovine samples will be inoculated onto immortalized cell lines MDBK, BT, and HRT18-G. Cells will be monitored for cytopathic effects daily. Additionally, pooled convalescent antisera from the farm where the sample originated will be used to screen for virus isolation by immunofluorescence assay.Reverse genetics will also be employed where suitable systems exist for similar viruses. For example, one likely candidate for further study is bovine rhinitis virus (BRV), which has been associated with BRDC (Ng et al., 2015; Zhang et al., 2019; Hause et al., 2015). BRV is a member of the genus Aphthovirus which notably includes foot and mouth disease virus (FMDV). A FMDV reverse genetic system was initially developed 30 years ago and has been significantly improved (Zibert et al., 1990). Given the high similarity between FMDV and BRV genomes, a reverse genetic approach for BRV seems attainable and represents an alternate path to generate infectious virus for follow up studies should cell culture isolation fail.Objective 4: Characterize virus pathogenesis in the host animal and establish the challenge model Fulfillment of Koch's postulates is a critical step in establishing the significance of novel viruses, as virus detection does not equate to causation. In this objective we will performed controlled host animal infection studies. Naïve, seronegative high health animals will be inoculated with cell culture or reverse genetics-derived virus and virus pathogenesis will be studied by the collection of clinical samples (nasal and fecal swabs, serum, tissue, etc.) at various time points post inoculation. Signs of clinical disease will be recorded. The immune response to the viral inoculum will be monitored over time. Virus isolation and titration will be performed on samples collected inoculated animals.Results from this objective will demonstrate the pathogenesis of emerging viruses and assign them a causative role for respiratory disease. The host animal challenge model developed here will additionally serve as the basis for vaccine development efforts detailed in the following objective.Objective 5: Develop vaccines and demonstrate efficacy in the natural host Our ultimate goal is to improve livestock health and vaccines remain the most effective disease preventative measure. In this objective we will develop vaccines to prevent disease caused by infection with emerging viruses. While the vaccine design will be dictated in part based on the specific biology of the emerging virus, possible vaccines include whole inactivated virus, attenuated live virus and vectored subunit vaccines. Host animal vaccination studies will be carried out to measure the immunogenicity of the vaccine, and protection will be assessed by controlled inoculation of emerging virus followed by monitoring of clinical disease. Prototype vaccines affording significant protection will be considered candidates for further development with an industrial partner. Our long term objective is licensure of these vaccines and mitigation of disease.