FIG 4.

Multivirus competition assays to assess the transmission fitness across penile and cervical tissue. Penile and cervical explants were exposed to mixtures of three Env_chronic viruses (I10, K44, and Q0) and three to five Env_acute viruses (B1, B2, B3, B4, B7, B8, B9, B14, B17, B19, and B20) for 3 h. After overnight culture, migrating cells from the tissue were collected and cocultured with CD4⁺ PM1 T cells (MC+PM1). Following 10 days in culture, both tissue and MC+PM1 cocultures were lysed and PCR amplified, and replication of viruses was analyzed by NGS. (a to p) Percent replication of eight different mixtures of Env_chronic and Env_acute viruses in tissues from donor A and donor B and Env_chronic and Env_acute viruses in the corresponding MC+PM1 cocultures. Each graph shows the percentage of sequence reads for donor A and donor B from the NGS analysis mapped to the viruses present in the competitions. (q and r) Side-by-side comparison of Env_chronic and Env_acute virus replication in penile tissue and Env_chronic and Env_acute virus replication in cervical tissue (q) and comparison of transmission fitness of Env_chronic and Env_acute viruses in MC+PM1 derived from penile tissue with transmission fitness of Env_chronic and Env_acute viruses in MC+PM1 from cervical tissue (r) of one competition set (I10, K44, and Q0 and B1, B3, B4, B19, and B20).