Vaginal and rectal transmission models of SIV/SHIV in rhesus macaques offer potential insights into the mechanisms by which systemic viral acquisition is established after mucosal exposure to HIV during sexual transmission. The nature of target cells infected and the sites of transmission after vaginal challenge remain only partially defined, and even less is known about early events of rectal transmission. To gain insights into the initial targets of infection, we have developed a single-round dual reporter system that can specifically identify the cells infected by the challenge inoculum. Further, we have recently reported that macaque vaginal challenge with a mixture of the dual reporter vector and replication competent SIV enables us to identify small, early foci of SIV replication 48 hours post-challenge. As illustrated in preliminary data included in the application, we have also been able to adapt the single-round dual reporter system to identify the first cells infected after atraumatic rectal challenge. Interestingly, our studies of vaginal and rectal early transmission with the a single-round dual reporter pseudotyped with M-tropic JRFL and the T-tropic SIVmac239 48 hours post-challenge all show a similar preference for the same early target cells, with the majority of cells infected being Th17 cells, and the immature DC being a minor population. This observation suggests that the available target cells to initiate mucosal acquisition may be limited. To extend these insights, we will examine how different envelope proteins and the SIV protein Vpx influence early tropism. We know that later during pathogenesis and general immune activation, multiple cell types can be infected and depleted after infection becomes systemic. Therefore, we will determine early changes in the SIV/SHIV target cells by examining the kinetics of viral spread as the initial foci of infection expands from 2 to 4 days. Finally, as early as 48 hours post-vaginal challenge with SIVmac239, we observe evidence of host responses to mucosal infection including apoptotic infected cells, lysed infected cells, and phagocytosed infected cells. Likewise, preliminary RNA-Seq analysis revealed changes in gene expression associated with the 48 hour foci of infection. By combining microscopic analyses of tissue sections and detecting the cells associated with host gene expression changes using fluorescent antibodies and RNA probes, we will be able to visualize the first wave of host responses to the virus. We will define the who, where, and when of which cells are infected, which cells are generating virus specific alarms, and which cells are responding to these alarms. Collectively, completion of these studies will result in a great increase in our understanding of the cascade of the earliest events of vaginal and rectal transmission including the location and phenotype of infected cells and the innate host responses to infection within the first few days of infection. A better understanding of the earliest events of mucosal transmission has the potential to advance efforts in HIV prevention science. Knowledge of the cell type and location of the earliest targets of transmission will reveal where the intervention must be targeted for maximal impact.