Detection of SARS-CoV-2 and characterization of SARS-CoV-2 S-gene in major companion and food animals

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Project Summary: SARS-CoV-2 is a major human respiratory pathogen that can cause COVID-19, a disease with symptoms similar to those caused by Influenza A (Flu A) and Influenza B (Flu B) viruses. A few major SARS-CoV-2 variants of concern (VOC) have been identified and associated with increased transmissibility in humans, including the UK variant B.1.1.7/Alpha, South Africa variant B.1.351/Beta, Japan/Brazil variant P.1/Gamma, California variants B.1.427/B.1.429/Epsilon, and India variants B.1.6.7.1/B.1.6.7.2/Delta (CDC, 2021a). SARS-CoV-2 has also been found in different animal species, including dogs, cats, tigers, lions, minks, ferrets etc. Transmission among individuals within an animal species has been reported (Halfmann et al, 2020; OIE, 2021), as well as limited reports of animal to human transmission (Hedman et al., 2021), yet more extensive studies may be needed to draw a conclusion. Both Flu A and Flu B are major human and animal respiratory pathogens, and cause symptoms sometimes difficult to differentiate from COVID-19. This proposal has three main goals: 1) Develop and validate a multiplex real-time PCR assay for detection of SARS-CoV-2, Flu A and Flu B viruses; 2) Screening animal respiratory samples submitted to KSVDL from Months 7-30 (2 years) of the grant period; and 3) Sequencing the spike protein- encoding gene (S-gene) of all SARS-CoV-2 positive samples to identify and characterize potential variants and their relationship to human strains. From 2018-2020, Kansas State Veterinary Diagnostic Laboratory (KSVDL) received an annual average of 11,065 animal respiratory samples, including an average of 69 feline, 491 canine, 123 equine, 1,070 swine and 9,296 bovine samples for each year. All feline and canine respiratory samples and selected samples from other species received in Months 7-30 during the grant period will be tested by the multiplex SARS-CoV-2/Flu A/Flu B assay developed in the study. S-gene sequencing of all SARS-CoV-2 positive samples will be attempted with a procedure optimized in this study and results compared to major VOC strains. Full or near-full S-gene sequences will be phylogenetically analyzed to further study the variation and genetic diversity of the SARS-CoV-2 strains identified from animal species. Analysis will also reveal if strains identified within an animal species have a closer genetic relationship than strains between species. The S-gene sequences of animal- and human-derived strains will be compared to determine if animal strains have been observed in humans. Main goals include: 1) Provide comprehensive SARS-CoV-2 positive rates, for a duration of two years, from samples collected from dogs, cats, horses, cattle and pigs; 2) Identify potential SARS-CoV-2 VOC strains from positive samples; 3) Phylogenetically analyze the S-gene from all sequenceable positive samples (Ct ≤ 32) to determine genetic diversity; 4) Identify and phylogenetically analyze similarities and differences of the S-gene of animal- and human-derived SARS-CoV-2 strains.