Feline enteric coronavirus infection (FECV) can mutate to become feline infectious peritonitis virus (FIPV) causing FIP in some cats and large cat species which is invariably fatal. FECV is largely restricted to the intestinal tract, while FIPV gains monocyte/macrophage tropism allowing for systemic spread. FIPV spike protein is found on the virion surface and contains the receptor binding domain and fusion peptide and has repeatedly been found important for macrophage tropism. We are proposing a novel approach to identify mutations that may be responsible for both early and late events in FIP pathogenesis and to understand mechanisms leading to monocyte and macrophage tropism. Our rationale for proposing these experiments is to gain a more comprehensive understanding of the critical steps leading to fatal pathogenesis that may lead to novel, early therapeutic interventions.Hyp othesis/Objectives: Our overarching hypothesis is that multiple, distinct mutations within the FIPV spike protein alter its functional properties and allow the virus to gain access to monocytes and macrophages and we propose the following objectives to test our hypothesis. Objective 1: Identify spike mutations leading to monocyte-associated viremia in healthy cats. Objective 2: Determine the mechanism and functional consequences of spike cleavage at the S1/S2 protease cleavage site. Objective 3: Determine the functional consequences and spike mutations responsible for reduced pH dependence of FIPV. Study Design: In objective 1, we will compare the spike gene sequence of FECV collected from feces and blood of healthy cats in which monocyte-associated viremia is detected in order to identify mutations responsible for early events that may lead to monocyte and/or macrophage tropism. In objective 2, we will generate pseudoviruses carrying FECV spike with the highly conserved furin consensus cleavage site and those with mutations in the site that are representative of what we observed in clinical samples from FIP cats. Then, we will compare spike cleavage during entry and egress and we will compare the ability of the pseudoviruses to productively enter primary macrophages. In objective 3, we will quantitate fusion of FECV relative to FIPV as a measure of pH and we will track the fate of viruses in endosomes during infection of primary macrophages. Further, we will identify spike mutations responsible for the reduced pH dependence of FIPV. Preliminary Data: Monocyte-associated viremia has observed been in healthy cats. FECV has a limited ability to enter macrophages and, virions that do enter are unable to productively infect the cells and spread. These results suggest that multiple mutations may be necessary for monocyte/macrophage tropism and for the virus to gain an ability to spread between cells. We have found that a highly conserved furin cleavage site at the S1/S2 domain boundary in FECV spike is consistently mutated in clinical samples of FIP cats which suggests a change in protease specificity that may be critical for pathogenesis. Further, we have found that FIPV has a reduced pH dependence compared to FECV, suggesting that FIPV may be able to escape phagolysosomes in macrophages by fusing early in the endocytic pathway. Expected Results: We expect to identify specific mutations in spike that are important for early and late events in pathogenesis and we expect to understand the contributions and mechanisms of spike cleavage and pH dependence in macrophage tropism. Budget and Timeline: Our total requested project costs are $187,655 for a total duration of three years. Per year requests are: Year 1 $61,129, Year 2 $62,494 and Year 3 $64,032. Potential Impact for Animal Health: Acquisition of monocyte/macrophage tropism is believedto be a critical determinant of FIP pathogenesis and we propose to understand this tropism change mechanistically and further, to identify the mutations responsible. We expect the work proposed here to advance our understanding of both early and late events in FIP pathogenesis and to provide critical information on a diagnostic test currently under development in our lab. Further, we anticipate a potential impact in the development of novel, early therapeutic interventions.