Project Summary/Abstract Zika virus, a small positive-stranded RNA virus, causes a remarkable spectrum of infection outcomes, ranging from asymptomatic infection in up to 80% of individuals to microcephaly in congenital Zika syndrome. This variation in human susceptibility was highlighted during the 2015-2016 epidemic of the Americas that resulted in over a million infections and 3,500 cases of microcephaly in Brazil alone. These differences in outcome arise from the complex interplay of exposure, environment, age, Zika virus genetics, and human genetics. The overall goal of my lab is to understand how human genetic diversity regulates susceptibility and severity of infections. Famous examples of genetic differences that profoundly impact susceptibility include sickle cell allele protection against malaria and CCR5 deletion protection against HIV. Such genetic differences can lead to insights on pathogenesis, drug targets (e.g. CCR5 inhibitors), and more personalized care. While genome-wide association studies (GWAS) are a powerful approach to discover common genetic differences that confer disease resistance, there are no published GWAS of human Zika virus infection. However, studies of dizygotic twins with discordant outcomes have demonstrated that cells from the twin exhibiting congenital Zika syndrome are more susceptible to infection in vitro. Therefore, we hypothesize that human genetic differences that regulate Zika infection can be identified through measuring inter-individual variation in cellular infection phenotypes. To facilitate identification of genetic variants that affect cellular infection phenotypes, we developed a rapid single- cell GWAS approach called scHi-HOST (single-cell High-throughput Human in vitrO Susceptibility Testing). scHi- HOST facilitates assignment of each cell to a genotyped individual, phenotyping of each cell for viral entry and burden, and measurement of host transcriptomic response, all from a single scRNA-seq experiment. We propose that coupling cell-based human genetics and functional validation will facilitate discovery of novel pathways and genetic determinants of susceptibility. Specifically, we will 1) identify human genetic variants that confer susceptibility/resistance to cellular Zika virus infection for strains representing the major Zika virus lineages, and 2) experimentally validate genetic variants and causal genes associated with cellular susceptibility to Zika virus using CRISPR-based approaches in diverse cell types. Thus, this project will generate an experimentally validated, high-resolution analysis of how human genetic variants impact cellular infection phenotypes during Zika virus infection. This will serve as a springboard for characterizing identified variants more deeply through future mechanistic studies and examining clinical relevance in human studies. Understanding these differences could lead to new diagnostic approaches in identifying at-risk individuals and novel therapeutic s...