The overarching goal of this project is to improve our understanding of viral pathogenesis for feline coronaviruses, both FCoV-2 and FCoV-1, to determine the mechanisms underlying viral pathogenesis for feline infectious peritonitis (FIP), which (despite recent advance in antiviral treatment) remains one of the most important infectious diseases of cats. Disease progress for FIP is linked to several ‘hot-spot’ mutation and deletions of the virus spike gene, and domain 0 (D0) is one of the more recently defined ‘hot-spots’. Specific Aim 1. To characterize the entry pathway and spike activation for FCoVs lacking D0. We will perform a series of cell-based and in vitro assays to assess virus entry and membrane fusion for the different version spike that are generated in-cat by a process of internal deletion. We will better define the entry pathways of the different forms of the virus, and work with our collaborators to engineer spike in different conformational forms to assess protease cleavage and receptor interactions. Specific Aim 2 – Phenotypic evaluation of a newly identified FCoV-2s containing deletions of D0, including one linked to a transmission event. In this aim, we will extend our studies to novel FCoV-2s with domain 0 deletions, including three that have undergone preliminary genetic characterization in the Whittaker lab, as well as continue surveillance for new D0-deleted FCoV-2s from clinical samples. We will perform studies to assess cell and tissue distribution of the different forms of the virus in clinical samples. Specific Aim 3 – Evaluation of D0 deletions in FCoV-1 spike. In this aim, we will extend our studies to FCoV-1s with domain 0 deletions based on existing database sequences, as well as continue surveillance for new FCoV-1s with genomic deletions, working extensively with the Goodman lab at Cornell, in particular to evaluate a new virus recently identified from Pakistan.