Version 1: Host reverse transcriptase is required to maintain persistent dengue virus infections in C6/36 insect cells

It was previously shown that C6/36 cells challenged with Dengue virus serotype 2 (DENV-2) show initial cytopathic effects (CPE) but overcome these within 3 split passages to resume normal growth and morphology for up to 30 split-passages, despite persistent infection with DENV-2. Establishment of the tolerated persistent infection was proposed to require action of host reverse-transcriptase (HRT) activity. This was subsequently proven using the RT inhibitor tenofovir disoproxil fumarate (TDF) in C6/36 challenged with DENV-1. However, it was unknown whether the persistent infection was dependent on persistent HRT activity. This brief report describes a test of our hypothesis that persistent HRT activity is required to maintain a persistent infection of DENV-2 by showing that its inhibition would result in a return of CPE followed by C6/36 cell death. TDF treatment (0.1 mM) revealed little toxicity to naive C6/36 cells but resulted in cell death upon concomitant challenge with DENV-2, similar to the previous report for DEN-1. However, TDF treatment of stable, grossly normal C6/36 cell cultures persistently infected with DENV-2 for up to 30 split passages also induced severe CPE and death. The results revealed a key role of host RT, not only in the establishment of persistent infections, but also in their long-term maintenance. Persistent infections in shrimp and insects are sometimes undetectable by routine histological analysis, in situ hybridization, immunohistochemistry or PCR. Our results suggest that treatment of grossly normal insects and crustaceans or their cells with TDF may be a simple approach to induce gross disease or increase viral quantities for purification purposes.