Annually, more than two million new HIV infections occur worldwide, with the largest proportion of new infections occurring in women through heterosexual intercourse. Considerable attention has been given to understanding the initial events following viral acquisition in the female reproductive tract, however infection throughout the upper reproductive tract has not been reported. Infection is initiated after virions cross the mucus barriers coating the reproductive tract and the epithelial layers to access target cells. The process of HIV-1 transmission has been thought to occur at specific sites. A focus has been on vulnerability of simple columnar epithelium covered endocervical tissue as being especially susceptible. Recent findings by our research team have explored the entirety of the FRT and bring this into question. We find that all areas where virus can cross mucosal barriers are able to be infected with the transmitting inoculum. These findings were made possible by the development of a novel dual reporter lentiviral vector which allows identification of infected tissue at a macroscopic level by luciferase expression as well as at the single cell level by fluorescent reporter expression. Because mucosal barriers are generally effective that spread of infection is an inefficient process. Therefore, it is the goal of this proposal to further elucidat the mechanisms by which transmission occurs, with a specific focus on the upper female reproductive tract. Ex vivo human tissue models and the in vivo Rhesus Macaque SIV challenge model will be used to study the target cells and early infection events after HIV or SIV acquisition. This analysis will use fluorescence microscopy analysis of healthy and infected tissues to describe the immunological environment and how it changes after viral challenge. The experiments proposed in Aim 1 will establish foundational knowledge of the immune cells present in human and monkey ovarian tissue where infection can occur, and compare this to uterine tissue where we have not found evidence of infection. These studies will lead to a better understanding of where preventative antiviral mechanisms such as vaccines need to be poised to act. The outcomes should provide a framework for understanding the vulnerability of ovarian tissue to HIV transmission in women. In Aim 2, the process of viral spread and the feasibility of ovarian tissue supporting sustained infection will be examined using replicating virus infection alongside reporter virus. In Aim 3, the monkey challenge model will be used to determine the effect of changes in viral envelope from chronic to transmitted/founder types which have been implicated as being uniquely suited for initiating infection. In summary, these studies seek to provide a better understanding of the process of viral transmission, specific to the upper FRT, and the importance of target cells and viral envelope determinants to initiate infection.