The unprecedented outbreak of feline infectious peritonitis (FIP) in Cyprus in 2023 was caused by FCoV-23, a novel
feline/canine coronavirus type-2 (FCoV-2). Our previous work on the structure and function of the FCoV-23 spike protein focused on the impact of novel variants of the virus identified in Cyprus that had a deleted N-terminal domain of spike (domain 0), which markedly increased virus entry and fusion and likely impact viral pathogenesis and FIP. We also identified a previously unrecognized protease cleavage-activation site in FCoV-23 spike domain D (S1’ ). We have now identified pathogenic variants of FCoV-2 from the United States that also exist with an intact or deleted domain 0 (i.e. in both ‘long’ and ‘short’ forms). Notably, our newly characterized FIP-associated domestic FCoV-2s have a mutated S1’ site. We will test these cleavage site mutations to determine effects on infection and disease (as previously with FCoV-1, the most common cause of FIP), examining whether these novel FCoV-2s that have predicted down-regulated or nonfunctional cleavage sites provide a molecular explanation for the rapid-onset FIP seen in these cats. We have also identified S1’ variants with predicted up-regulated cleavage sites, as seen in cer tain canine coronaviruses (CCoV-2s) connected to gastrointestinal disease and virus shedding, and which may act as a viral reservoir for novel FCoV-2s. These will also be tested for altered spike function. Overall , we will test whether modification of S1’ leads to viruses with altered cell entry and membrane fusion proper ties, leading to altered virus pathogenesis and transmission, with the goal of improving our preparedness for novel outbreaks of FIP in cats. When combined with ongoing surveillance the results of this study will allow for rapid diagnosis and management of high-risk FCoVs with altered pathogenic proper ties.