Development of Reverse Genetics for Feline Infectious Peritonitis Virus and Its Use in Mechanistic Study of Antivirals Against Coronavirus Infection

Abstract

Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses within the order Nidovirales, family Coronaviridae that infect humans and a wide range of mammals, exhibiting significant cross-species transmission potential. While most human CoVs cause mild respiratory illness, the emergence of the zoonotic SARS-CoV, MERS-CoV, and SARS-CoV-2 has highlighted their pandemic threat and underscored the urgent need for effective antiviral therapeutics. Despite the discovery of numerous compounds with anti-CoV activity, only a few, such as Paxlovid (a combination of nirmatrelvir and ritonavir), remdesivir, and molnupiravir, have been approved by the Food and Drug Administration. The continuous emergence of highly pathogenic CoVs, combined with their high mutation rates, demands robust experimental platforms to accelerate antiviral drug discovery and elucidate viral pathogenic mechanisms. Reverse genetics is a powerful approach that enables manipulation of the viral RNA genome, facilitating research on antiviral screening, vaccine development, virulence gene identification, and pathogenesis studies. In particular, replicon systems, non-infectious viral constructs lacking structural genes, enable safe and high-throughput antiviral testing under BSL-2 conditions.

This thesis used feline infectious peritonitis coronavirus (FIPV) as a model coronavirus, as it shares structural and physicochemical similarities with highly pathogenic human CoVs but can be handled under biosafety level 2 (BSL-2) containment. The overarching goal was to develop a reverse genetics system for FIPV and use it to investigate the mechanisms of novel antiviral candidates, thereby providing a platform for broad-spectrum anti-CoV drug discovery. The thesis consists of four chapters:

Chapter 1 reviewed current knowledge of coronaviruses, their reverse genetics systems, and antiviral strategies, emphasizing the importance of identifying new therapeutic targets and specific antiviral compounds.

Chapter 2 aimed to establish a reverse genetics system for FIPV. Using an in vitro ligation approach, we assembled a full-length cDNA clone of the FIPV WSU79-1146 strain (rFIPV-WT) and generated recombinant reporter viruses (rFIPV-msfGFP and rFIPV-Rluc) by inserting superfolder GFP (msfGFP) or Renilla luciferase (Rluc) reporter genes. Furthermore, we constructed replicons (repFIPV-msfGFP and repFIPV-Rluc) by replacing FIPV structural protein genes with reporter genes, thereby abolishing the production of infectious particles. These recombinant tools exhibited high sensitivity in quantifying antiviral efficacy and allowed the evaluation of drug-resistance mutations. This chapter provided a versatile molecular platform to investigate viral replication mechanisms and enabled safe, efficient antiviral compound screening under BSL-2 conditions.

Chapter 3 investigated the antiviral mechanism of KR-206238, a novel compound that inhibits coronavirus replication. I first demonstrated its antiviral activity in FIPV-infected CRFK cells, where it acted at the early stage of infection. To test whether KR-206238 targets viral entry or genome replication, I performed pseudovirus and replicon assays, but it showed no inhibitor effect in either system, suggesting an indirect mechanism. I hypothesized that KR-206238 exerts its antiviral effect by modulating host cellular pathways. Untargeted metabolomics revealed that KR-206238 downregulated the biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in both infected and uninfected cells. Supplementation with exogenous PC or PE reversed its antiviral activity, supporting the hypothesis that KR-206238 disturbs PC/PE anabolism. Immunofluorescence analysis showed that KR-206238 altered the morphology and distribution of FIPV replication centers, suggesting that it may inhibit viral replication by interfering with the proper formation of these structures. Additionally, KR-206238 exhibited broad-spectrum activity against other positive-sense RNA viruses, including human coronaviruses, flaviviruses, and picornaviruses. These findings identified KR-206238 as a promising broad-spectrum antiviral candidate and implicate PC/PE lipid metabolism as a novel host pathway critical for coronavirus replication.

Chapter 4 explored the potential antiviral mechanism of emodepside, an anthelmintic drug currently used for cats, hypothesized to exert antiviral effects on coronavirus infection. Emodepside significantly inhibited FIPV at an early stage of the viral life cycle. Different from KR-206238, emodepside inhibited both pseudovirus entry and replicon replication, suggesting a dual mode of action targeting entry and genome replication. Transcriptome sequencing revealed that FIPV infection induced cellular antiviral pathways in CRFK cells, which were reversed by emodepside treatment; however, emodepside alone did not induce these pathways, suggesting that its antiviral activity was not mediated by host immune activation. To identify potential viral targets, FIPV was serially passaged under increasing emodepside pressure, generating resistant strains. Whole-genome sequencing of resistant strains identified multiple mutations in the Nsp4 region, which were introduced into the viral genome using the reverse genetics system to generate recombinant viruses with resistance. This genetic validation implicated Nsp4 as one of the antiviral targets of emodepside. Since Nsp4 is not directly involved in viral entry, these results suggest that emodepside may inhibit entry through an additional mechanism, warranting further investigation.

In conclusion, this study established a robust reverse genetics system for FIPV and applied it to elucidate the mechanisms of two novel antiviral candidates. KR-206238 was found to inhibit coronavirus replication indirectly by disrupting PC/PE lipid metabolism, while emodepside directly targeted both viral entry and genome replication, with Nsp4 identified as a putative viral target. Together, these findings not only provide novel mechanistic insights into coronavirus-host interactions but also identify promising therapeutic targets for the development of broad-spectrum antivirals against coronaviruses and other positive-sense RNA viruses.

Date of Award	14 May 2026
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Nikolaus OSTERRIEDER (Supervisor), Yun Young GO (Supervisor) & Akos KENEZ (Supervisor)