This Is AuburnElectronic Theses and Dissertations

Molecular Biology and Epidemiology of Human and Feline Coronaviruses




Barua, Subarna

Type of Degree

PhD Dissertation


General Veterinary Medicine


Coronaviruses (CoVs) are RNA viruses belonging to the Coronaviridae family and known to infect various hosts such as pigs (porcine epidemic diarrhea virus), cats (feline infectious peritonitis virus), birds (avian coronavirus), and even humans (human coronavirus, HCoV) including severe acute respiratory syndrome coronavirus SARS-CoV-2 with different disease severity. Emerged in 2019, the human coronavirus, SARS‐CoV‐2, has caused millions of deaths worldwide which radically changed our lives and influenced our lifestyles and habits. Feline coronavirus (FCoV) causes feline infectious peritonitis (FIP), a disease of felids that has been considered irremediably deadly. A functional surveillance system remains fundamental to understanding the currently circulating virus, the evolution of the virus, risk factors for severe disease, and for taking public health measures. Monitoring mammalian domestic and wildlife populations for coronavirus will not only provide information about the health of threatened or endangered species but also be needed to understand how coronavirus is spread between humans and animals. Designed PCR in combination with fluorescence resonance energy transfer (FRET) could enable efficient diagnosis and active surveillance for mutations and variants. This surveillance effort is essential to provide sustainable platforms for pandemic control, targeted drug discovery, and the development of safe and effective antivirals to combat coronaviruses. Knowing SARS-CoV-2 variants is vital for formulating effective control policies during the pandemic, so we developed a rapid diagnostic technique, a reverse transcription FRET-PCR technique. This technique targeted the two most common mutations in the SARS-CoV-2: A23403G in the genome coding for spike protein and C14408T in the genome coding for RNA-dependent RNA polymerase. The RT FRET-PCRs identified the mutants from the classic variant in ATCC control viruses and feline and human clinical samples based on a high-resolution melting curve analysis. During the COVID-19 pandemic, active surveillance is essential to determine the prevalence of circulating variants of SARS-CoV-2. The Delta RT-FRET-PCR can diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. After confirming the performance of FRET RT-PCR, we also investigated using this technique to see if this target-specific design of RT-FRET-PCR could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. Delta RT-FRET-PCR established in this study could detect as few as ten copies of the DNA target and 100 copies of the RNA target per reaction performed on reference SARS-CoV-2 strains and human nasal swab samples positive for the Delta and non-Delta strains. The melting temperature of the PCR products obtained for SARS-CoV-2 Delta variants (around 56.1 °C) was consistently higher than the same for non-Delta variants (about 52.5°C). To provide more complete data on SARS-CoV-2 infections in dogs and cats in the United States, we conducted a serosurvey on convenience serum samples from dogs (n=1,336) and cats (n=956) collected from 48 states of the USA in 2020. An ELISA targeting the antibody against nucleocapsid identified eleven positive and two doubtful samples in cats and five positive and five doubtful samples in dogs. A surrogate neutralization assay detecting antibodies blocking the attachment of the spike protein to ACE2 was positive, with three of the ELISA positive and doubtful samples and one of 463 randomly selected ELISA negative samples. These four positive samples were confirmed by SARS-CoV-2 virus neutralization testing. The four positives were from cats in New York (n=1), Florida (n=1), and New Jersey (n=2). The serosurvey results, one of the largest yet completed on dogs and cats globally, support the World Organization for Animal Health (OIE) and the Centers for Disease Control (CDC) positions that currently, there is no evidence that pets play a substantial role in the spread of SARS-CoV-2 in humans. While serological and molecular evidence of SARS-CoV-2 infection has been reported in white-tailed deer (Odocoileus virginianus) from the USA, deer sera from the U.K. (n=1,748) were found to be negative by a serosurvey. To further understand the geographical distribution of SARS-CoV-2 infected deer, a serosurvey was performed on archived deer serum samples collected from the Auburn University Captive Facility in Camp Hill, Alabama, between Oct 2019 and Jan 2022. A surrogate SARS-CoV-2 virus neutralization test identified one positive sample, which was later determined to be negative by the virus neutralization testing performed at USDA National Veterinary Services Laboratories. In addition, rectal and nasopharyngeal swabs from deer collected in January and February 2022 were negative by SARS-CoV-2 PCR. Of 72 people who had close contact with the deer over the study period, 29 completed a voluntary questionnaire that showed three had been infected with the SARS-CoV-2 during the study period. Our finding was that the deer we studied appeared not to have been exposed to SARS-CoV-2 despite human infections in the facility. While antiviral drugs such as GS441524 have been used to treat FIP in some countries, such as Australia and the UK, no Food and Drug Administration (FDA)-approved drugs are available to veterinary clinicians for FIP treatment in the USA. In addition, limited data is available for the antiviral efficacy and toxicity of these antiviral drugs against FIPV. We studied six types of antiviral drugs for their cytotoxicity, effect on the cell, and antiviral efficacies in Crandell Reese Feline Kidney (CRFK) cells. The GS441524 molecule showed inhibition of FIPV replication irrespective of initial inocula (2.5 × 103, 2.5 × 102, 2.5 × 101 TCID50) and incubation period (48 and 72 hours). No significant difference was observed in the FIPV inhibition for 24, 48, and 72 hours with 98-99% inhibition by GS441524 (25 µM) as long as the drug was applied at the time of or immediate after the FIPV inoculation. Cytotoxicity assay and viability assays showed that six drugs were safe to be used with essentially no cytotoxicity with the concentration as high as 250 µM for Ruxolitinib, 125 µM for GS441524, 63 µM for Teriflunomide, Molnupiravir, and Nirmatrelvir, and 16 µM for Ritonavir. In the dose-response analysis conducted in CRFK cells, GS441524, Nirmatrelvir, and Molnupiravir were identified as the top three drugs with selectivity for FIPV with SI values of 165.54, 113.67, and 29.27, respectively. In conclusion, we developed and validated specially designed RT-FRET-PCR to detect and differentiate SAR-CoV-2 mutants from classical strains and the Delta variant of SARS-CoV-2. Highly sensitive and specific diagnostic assays established in this work can be used for further active surveillance and longitudinal studies and better understand the ecology of SARS-CoV-2 in domestic and wild animals. In the surveillance conducted on domestic animals, no evidence was found that cats and dogs play a substantial role in the spread of SARS-CoV-2 to humans. In the serological and molecular surveillance conducted on deer, no evidence has been found that deer play a role in spreading SARS-CoV-2 in humans. The FIPV antiviral efficacy study showed that three of six tested drugs (GS441524, Nirmatrelvir, and Molnupiravir) are safe antivirals strongly effective in inhibiting FIPV replication. This data suggest that Nirmatrelvir and Ritonavir may bring new hopes for FIPV treatment besides GS441524 and could be an alternative to treat infection with GS441524-resistant FIPV strains in cats. The in vitro antiviral efficacies of six drugs from this work warrants future studies to explore further their treatment efficacies in vivo and side effects in FIP therapy. The information gained so far on FCoV could be of interest and might give ideas on different pathogenic aspects of SARS‐CoV‐2 that are still unclear and vice versa. This sharing of knowledge may serve as a basis for the rapid development of therapeutics for COVID-19, such as GS441524, as well as for studies on the possible interaction between FCoV and SARS-CoV-2 which may occur due to the close relationship between people and cats as companion animals.