Performing Department
(N/A)
Non Technical Summary
The secondary spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from humans to other animals has been extensively described in multiple animal species. In particular, natural SARS-CoV-2 infection of White-tailed deer across the USA has been documented. It is well-established that disease-causing microorganisms can pass between deer and livestock species such as cattle. Therefore, continued circulation of SARS-CoV-2 in deer poses a significant risk of infection in cattle and pigs. In this regard, using advanced molecular approaches, this project will address critical questions, Can the SARS-CoV-2 adaptation in deer make it more efficient to infect cattle and pigs? And Have the new SARS-CoV-2 variants spilled over into cattle and pigs in the USA? The outcomes of this project are of significant relevance to US animal agriculture as deer, cattle, and pigs are of substantial economic importance and shape the livelihoods and culture of regions throughout the United States.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to evaluate the risk of SARS-CoV-2 transmission from deer to cattle and pigs with the following independent objectives:Characterize the difference in susceptibility and replication kinetics of recent and emerging SARS-CoV-2 variants in cattle, pig, and WTD respiratory cells.Characterize differential innate immune responses to SARS-CoV-2 variants in cattle, pigs, and WTD respiratory cells.Serosurveillance to monitor SARS-CoV-2 spillover infection of cattle and pigs.
Project Methods
Aim 1: Characterize the difference in susceptibility and replication kinetics of recent and emerging SARS-CoV-2 variants in cattle, pig, and white-tailed deer respiratory cells.Methods:a) Investigate the comparative replication of recent and emerging SARS-CoV-2 variants in deer, cattle, and pig tracheal cells.Using the primary tracheal epithelial cell culture systems for deer, cattle, and pigs established with initial NIFA funding (NIFA: 2020-67015-32175), we will investigate each species' susceptibility to and replication to the delta, omicron, and emerging SARS-CoV-2 variants, including deer SARS-CoV-2 isolates. The susceptibility will be evaluated by measuring the percentage of cells infected and/or replicating, the quantity of viral RNA production, and the amount of live virus production at multiple timepoints after infection to explore viral entry, replication, and egress kinetics.b) Investigate the susceptibility of cattle and pig tracheal cells to deer-adapted SARS-CoV-2. We will simulate the adaptation of SARS-CoV-2 in deer by serially passaging human and deer SARS-CoV-2 isolates in deer tracheal epithelial cells. We will then evaluate the susceptibility of the deer-adapted virus to cattle and pig tracheal epithelial cells as in Aim1a.Aim 2: Characterize the differences in the innate immune response of cattle, pig, and white-tailed deer respiratory cells infected with different SARS-CoV-2 variantsMethods:a). Investigate the innate immune gene expression profiles in deer, cattle, and pig tracheal cells infected with recent and emerging SARS-CoV-2 variants. A set of key innate immune genes will be examined using quantitative real-time PCR following infection of cells with delta, omicron, and emerging variants, including SARS-CoV-2 isolates from deerb) Investigate the innate immune gene expression profiles in cattle and pig tracheal cells infected with deer-adapted SARS-CoV-2. Virus passaged in deer cells will be used to infect cells, and gene expression will be quantified as in Aim 2a.c) Identify viral genetic features corresponding to differential host innate immune response to SARS-CoV-2 in deer, cattle, and pig tracheal cells. Using single-cell RNA sequencing, the host transcriptome and viral subgenomic transcripts produced at key time points after infection will be quantifiedAim 3. Serosurveillance to monitor SARS-CoV-2 spillover infection of cattle and pigsMethods:a) Monitor cattle and pig seropositivity against SARS-CoV-2 using validated indirect ELISA (iELISA). In-house iELISA assays40 developed with prior NIFA funding will be used to screen serum samples from multiple regions of the US. Samples submitted to our existing network of veterinary diagnostic laboratories will be used for testing.b) Characterize the antibody landscapes and antigenic distance to SARS-CoV-2 spike variants in seropositive cattle and pigs. SARS-CoV-2-positive sera will be subjected to a pseudovirus neutralization test to determine the SARS-CoV-2 variant(s) of the animals that were exposed based on the antigenic cartography analysis. We established a lentivirus-basedpseudovirus system and generated pseudoviruses expressing spike proteins of the wildtype and all the major known SARS-CoV-2 variants (B.1, Alpha, Beta, Gamma, Delta, and Omicron)