Mutations in feline infectious peritonitis virus nonstructural protein 14/16 methyltransferase attenuate the pathogenicity of the virus in cats

Feline infectious peritonitis virus (FIPV) can cause an immune-mediated disease that is fatal to felines, but there is a lack of clinically effective protection conferred by vaccines. The methyltransferase (MTase) activity of the coronavirus nonstructural proteins nsp14 and nsp16 affects virulence, but there are no studies on the effect of nsp14 and nsp16 mutations affecting enzyme activity on the virulence of FIPV. In this study, we successfully rescued two mutant strains based on the previous infectious clone QS-79, named FIPV QS-79 dnsp14 and dnsp16, by mutating the MTase active sites of nsp14 (N415) and nsp16 (D129). The dnsp14 and dnsp16 exhibited similar syncytium formation ability and growth kinetics as their parental strains <i>in vitro</i>. Moreover, both mutants stimulated increased expression of interferon and cytokines in a macrophage lineage cell line. The dnsp14 and dnsp16 exhibited significantly lower pathogenicity in cats, with a 75% reduction in mortality compared to the wild type. The dnsp14 and dnsp16 were administered to cats at high (10⁵ TCID₅₀) and low (10⁴ TCID₅₀) doses for immunization purposes. The dnsp14 resulted in the production of high neutralizing antibody titers (>1:156) at both doses, whereas the dnsp16 only induced low neutralizing antibody titers (<1:64). And the dnsp14 provided protection to 50% of the cats at both immunization doses, whereas the dnsp16 failed to provide protection to cats of the challenge assays. The above results show that these two mutant strains are less pathogenic in cats, and dnsp14 induces a better humoral immune response and protection than dnsp16.IMPORTANCEFeline infectious peritonitis virus is a significant pathogen that affects felines worldwide, and its high fatality rate has long been a concern in the pet medical industry. Previous studies have indicated that the virus encodes nsp14 and nsp16 methyltransferases, which play a crucial role in viral genome replication and evasion of innate immunity. In this study, we aimed to inhibit the MTase activity by mutating the methyltransferase of feline infectious peritonitis virus nonstructural protein 14/16. This mutation resulted in the construction of a vaccine candidate with reduced virulence, efficient replication in the host, and the ability to provide partial protection from the virulent parental virus. Our findings offer valuable insights for the development of live attenuated vaccines that target the nsp14/nsp16 MTase of feline coronaviruses.