Thus, a long-term study was performed to address the following questions through sequential experiments: (i) does in vivo passage of RT-SHIV lead to higher or more consistent virulence, (ii) does prolonged tenofovir treatment initiated during chronic RT-SHIV infection lead to the emergence of K65R viral mutants, (iii) what are the clinical implications of K65R mutants, and (iv) what is the role of CD8+ cells and continued tenofovir treatment in controlling viremia of K65R RT-SHIV? This evidence concerns the gene CD8A and infection.