Gammaherpesviruses are a clinically significant cause of cancer and are primarily transmitted via saliva. However, the specific sites of viral replication in oral tissues resulting in salivary shedding are poorly understood. Rhesus macaques (RM) are naturally infected with three gammaherpesviruses: retroperitoneal fibromatosis herpesvirus (RFHV), an ortholog of KSHV, rhesus lymphocryptovirus (RLCV), closely related to EBV, and rhesus rhadinovirus (RRV). Rhesus macaques have been used as models of gammaherpesvirus-associated malignancies in the context of SHIV/SIV infection and offer an opportunity to study oral biology of gammaherpesviruses in greater detail.
Oral fluid and oral tissues from 30 RM experimentally infected with SIV or not were collected during necropsy. These included buccal and gingival tissue, parotid, submandibular and sublingual salivary glands, submandibular lymph nodes, adenoid, palatine and inguinal tonsil, soft palate and tongue. DNA was extracted and tested by qPCR for RRV, RLCV, and RFHV viral load. In situ hybridization targeting viral DNA was performed, for all 3 viruses, in all tissue types and highly positive tissues were used to phenotype the cells harboring viral DNA.
Rhesus gammaherpesviruses were detected in the oral fluid and oral tissues of all 30 animals examined; many were positive for more than one virus. By qPCR, the highest levels of RFHV were identified in gingiva, tongue, and submandibular lymph nodes while the highest levels of RLCV and RRV were detected in adenoid and palatine tonsil. Using ISH, most infection events for all three viruses were visualized in lymphoid tissues including lymph node and palatine tonsil. Multiplexing ISH with antibody-based phenotyping revealed a broad range of infected cell types including B- and T-lymphocytes, fibroblasts, epithelial cells, and NK-cells. Certain infected cell types, especially for RFHV, remain unidentified and phenotyping experiments are ongoing.
This is the first study examining RRV, RLCV, and RFHV viral load in rhesus oral tissues and oral fluid and may provide insights into human gammaherpesvirus biology within the oral compartment.