Host reverse transcriptase helps establish and maintain persistent dengue virus infections in C6/36 insect cells

C6/36 cells challenged with Dengue virus serotype 2 (DENV-2 strain NGC) show initial cytopathic effects (CPE) they overcome within 3 split passages and resume normal growth despite persistent infection with DENV-2. We hypothesized that tolerated persistent infections also required persistent host reverse-transcriptase (HRT) activity, and this was subsequently proven using the RT inhibitor AZT (azidothymidine, a nucleoside analogue antiretroviral drug) to treat C6/36 cells challenged with DENV-1. We confirmed this using the RT inhibitor tenofovir disoproxil fumarate (TDF) with C6/36, U4.4cells challenged with DENV-1. However, it has never been determined whether the persistently tolerated infections are also dependent on persistent HRT activity. This brief report reveals that persistent HRT activity is required to maintain persistent DENV-2 infections. Specifically, TDF treatment (0.1 mM) revealed minor toxicity to naive C6/36 cells but led to cell death instead of accommodation upon concomitant challenge with DENV-2, as previously reported for DENV-1. However, TDF treatment of stable, grossly normal C636 cell cultures persistently infected with DENV-2 for up to 30 split passages reverted to CPE, supporting our earlier hypothesis that persistent host RT activity is also required to maintain persistent infections. This is a practical tool for research applications in virology of crustaceans and insects for screening living, grossly healthy, imported animals for listed viral pathogens.