UC Davis

Coronaviruses are enveloped RNA viruses capable of causing a spectrum of diseases including respiratory, enteric, and/or systemic diseases in a wide variety of hosts. The major structural proteins of coronaviruses include the nucleocapsid (N), spike (S), membrane (M), and envelope (E) proteins. The viral Spike protein largely determines host range and tissue/cellular tropism and is responsible for promoting fusion of the viral and host cell membranes, leading to cell entry and infection. A literature review of coronaviruses in companion animals is included in this thesis (Chapter 1), which highlights the virology, epidemiology, clinical and pathologic features of coronaviruses of the cat, ferret, dog, horse, and alpaca.Feline infectious peritonitis (FIP) is one of the most complex and interesting viral diseases of domestic cats, caused be the FIP virus (FIPV), a biotype of feline coronavirus (FCoV). FIP has an immune-mediated pathogenesis involving multiple host and virologic factors. FIP presents as a spectrum of clinical signs including cavitary effusions, anorexia, persistent fever, lymphopenia, and lesions of pyogranulomatous vasculitis and peri-vasculitis that may or may not include central nervous system or ocular involvement. Untreated, FIP is generally considered to be fatal once clinical signs appear. FCoV includes two biotypes, feline enteric coronavirus (FECV) and FIPV. FIPV spreads systemically and is thought to arise from a discrete set of mutations in the more benign FECV, which is confined to the alimentary tract. These coronaviruses are further classified into serotypes I and II based on Spike-associated antigenic differences. Serotype II FCoV initially arose as the result of a series of recombination events between feline and canine coronavirus (CCoV) and is the less prevalent serotype naturally circulating in feline populations worldwide. As a result of recombination, the serotype II FIPV Spike protein has its origin in CCoV, which serves as the viral ligand for cell entry. Although FIPV serotype I is the more prevalent viral serotype in cats with naturally occurring FIP, serotype II has been more extensively studied in vitro due to the relative ease in propagating this virus in tissue culture systems. Consequently, more is known about the biology of serotype II FIPV than the more biologically relevant serotype I. Serotype II FIPV utilizes the cell surface protein feline aminopeptidase N, the ligand of the viral Spike protein, while the receptor for serotype I remains unknown. An understanding of viral receptor biology is a key facet in decoding viral pathogenesis, informing mechanisms of disease, viral dissemination, and potential vaccine strategies. The recent development of a feline cell line that effectively propagates serotype I FIPV, FCWF-4 CU, offers an opportunity to expand our understanding of serotype I FIPV biology. FCWF-4 CU is a culture adapted feline cell line derived from FCWF-4 cells available through the ATCC. Importantly, these two FCWF cell lines are variably permissive to the propagation of serotype I and II FIPV. In Chapter 2, we determined the targeted gene expression patterns in four feline cell lines, utilized normal feline tissues to determine the immunohistochemical expression patterns of two known coronavirus receptors, ACE2 and DC-SIGN, and compared the global transcriptomes of the two closely related FCWF-4 cell lines. We identified six differentially expressed transcripts with potential to explain the differential FIPV replication kinetics. The discovery of effective, available, and affordable antiviral treatments has been a focus of veterinary research for more than 10 years and recent advances in antiviral therapies for HIV, hepatitis C virus, ebolavirus and SARS CoV-2 have paved the way for similar advances for FCoV. In Chapter 3, we screened 90 putative antiviral compounds for efficacy and cytotoxicity against FIPV serotype II (WSU-79-1146) using real-time RT-PCR based screens and identified 26 compounds with antiviral activity against FIPV representing differing drug classes and mechanisms of action. Further, based on the success of combinatorial therapy strategies in human patients with HIV or hepatitis C, we strategically combined different antiviral compounds in order to identify additive or synergistic effects (combined anticoronaviral therapy, or CACT). Although we demonstrated additive and/or synergistic effects for several antiviral combinations, ultimately a select few monotherapies demonstrated superior efficacy overall. In Chapter 4, we reapproached antiviral assessment using an improved biological colorimetric assay and compared the antiviral efficacies of GC376, nirmatrelvir, remdesivir, GS-441524, molnupiravir (EIDD-2801), and -D-N4-hydroxycytidine (EIDD-1931) against both serotype I and II FIPV, as monotherapies and CACT. We also determined the pharmacokinetic properties of three antiviral compounds, molnupiravir, GS-441524, and remdesivir in cats in vivo as a step towards establishing dose in an antiviral treatment protocol for clinical use. An understanding of viral cell entry, dissemination and persistence are concepts that are also relevant to viral-vectored gene therapy. As a parallel adjunct study to the coronavirus projects, we created a lentiviral-vectored gene therapy approach to treating cats with chronic renal disease-associated anemia. Chronic renal disease (CRD) is a common disease of aged cats, often associated with clinically significant nonregenerative anemia as a result of reduced renal production of erythropoietin (EPO). Multiple approaches to the management of CRD-associated anemia have been attempted, including the use of gene therapy. In Chapter 5, we designed a series of third-generation lentivirus-based vectors to encode and produce the native feEPO protein in tissue culture experiments and in rodent models in vivo. The vectors were designed to include a pharmacologic safety mechanism through the incorporation of the “suicide gene” HSV-TK. This gene product allows for the pharmacologic termination of the therapeutic effect in the event of supraphysiologic polycythemia. We hypothesized that cells transduced in vitro by a lentiviral vector encoding native feline erythropoietin would express feEPO mRNA and biologically active feEPO protein and that this expression could be terminated by the administration of ganciclovir (GCV, the nontoxic substrate of HSV-TK). The in vitro assays facilitated optimization of the vector for three in vivo studies in rats and genetically modified anemic mice in which we demonstrated a significant elevation of blood packed cell volume in treated animals relative to control animals.