Veterinary and Biomedical Scie
Non Technical Summary
The COVID-19 pandemic has dispersed a new strain of coronavirus (called SARS-CoV-2) throughout the world. During the rapid global spread, the new virus continues to mutate and there are now numerous genetic versions of SARS-CoV-2. The U.S. livestock sector plays an indispensable role in the safe and reliable food supply, employment, and economic development, so it is critical to understand if SARS-CoV-2 viruses can pose a threat to livestock. The goals of this project are to investigate the susceptibility of livestock to SARS-CoV-2 and to determine if the virus may adapt and efficiently spread among livestock. The research will use a combination of experimental infection studies using cell cultures and animals along with computer models to assess the chance for the virus to efficiently infect livestock species. Additionally, the project will develop diagnostic tests and use them to monitor the presence of antibodies to SARS-CoV-2 in livestock animals. Chickens, cattle, and pigs are major animal agriculture species in the U.S., and through this project, a better understanding of how SARS-CoV-2 may affect them will be gained.
Animal Health Component
Research Effort Categories
Goals / Objectives
1). To determine the susceptibility of livestock to SARS-CoV-2 in a BSL3 environment.2). To investigate the potential of transmission of SARS-CoV-2 to livestock and the likely adaptation in animals.
All experiments with live infectious SARS-CoV-2 will be conducted in our animal biosafety level 3 (ABSL-3) facility with appropriate respiratory protection and barrier clothing procedures for personnel.Aim 1: Determine the susceptibility of livestock to SARS-CoV-21a: Investigate the comparative replication of Asian, European and North American SARS-CoV-2 isolates in livestock primary respiratory cells.:We will initially determine the comparative replication ability of three different isolates of SARS-CoV-2 from Asia, Europe, and North America (strains Hong Kong/VM20001061/2020, Italy-INMI1, and USA-WA1/2020). To simulate the real-life settings, we will initially grow the SARS-CoV-2 isolates in human respiratory cells before infecting animal cells. At 6, 12 and 24 hpi, we will fix the cells and immunostain for SARS-CoV-2 viral protein expression. We will also determine the replication kinetics of SARS-CoV-2 in avian, porcine, and bovine cells compared with human cells. Cells will be infected with an MOI of 0.1 or 1.0, and virus production at 12, 24, 48, and 72 hpi will be evaluated by fifty-percent tissue-culture infective dose (TCID50) and RT-PCR. The amount of SARS-CoV-2 protein and virus production will be compared to determine the comparative ability of different strains of SARS-CoV-2 to replicate in livestock cells.1b: Susceptibility of chicken to experimental SARS-CoV-2 infection:We will carry out infection studies in chickens with a SARS-CoV2 isolate that we find to better replicate in chicken cells (Aim 1a). We will investigate susceptibility to experimental SARS-CoV2 infection in two age groups of SPF chicken. We will document the pattern of clinical disease, virus shedding, and seroconversion.Aim 2: To investigate the potential of transmission of SARS-CoV-2 to livestock and the likely adaptation in animals2a: Prevalence of SARS-CoV-2 in poultry, cattle and pigs: As part of this aim, we will raise SARS-CoV-2 specific antisera in chickens, pigs and cattle that will serve as positive control reagents for the indirect ELISA and Luciferase Immunoprecipitation System (LIPS) serological assays. We will monitor the presence of SARS-CoV-2 specific antibody in poultry, cattle and pigs using these two assays using test serum samples submitted for routine diagnosis and surveillance programs (for example Avian Influenza, PA Bull Test sales, etc.) to the Pennsylvania Animal Diagnostic Lab System (PADLS) and networked laboratories.2b. Establishing the mutational landscape in SARS-CoV-2 quasispecies from chicken, pig andbovine cells:We will passage the SARS-CoV-2 in chicken, pig, and cattle primary cells three times, and cell culture supernatants collected from the three passages will be used to analyze the viral quasispecies by Next Generation Sequencing (NGS).2c: Investigate the potential for SARS-CoV-2 adaptation in livestock:? Expanding on our preliminary binding energy analysis, we will use molecular modeling to perform in silico mutation analysis of the SARS-CoV-2 S protein RBD and the resulting effect on Rosetta binding energy. Each residue will be computationally mutated to all possible alternatives to identify the maximum improvement in the binding energy of ACE-2 with SARS-CoV-2 S protein upon a single residue change. Next, the consequences of consecutive mutations (second residue, third residue, etc.) will be explored. Binding energy analysis will also be applied to the mutations observed in quasispecies identified through sequencing in Aim 2b. All calculations will be performed using resources at the Penn State Institute for Cyberscience. Finally, the RBD configurations determined to computationally be best adapted to each species will be experimentally tested. The ideal RBD and spike protein sequence candidates will be expressed as proteins and used to perform binding assays on respiratory and gastrointestinal tissues from chicken, pig, and cattle.