Circular viral copy DNA of Dengue virus (DENV-2) isolated from infected mosquito cell cultures

Abstract

ABSTRACTDengue virus serotype 2 (DENV-2) is a mosquito-borne disease in the familyFlaviviridae. We have previously shown that DENV-2 can infect C6/36 mosquito cells and cause initial cytopathic effects that dissipate upon serial split-passage to yield persistently infected cultures with normal growth and morphology. In other words, the cell line accommodated persistent DENV-2 infections. It has recently been found that insect viral infections induce production of viral copy DNA (vcDNA) fragments via host reverse transcriptase (RT). The vcDNA occurs in both linear (lvcDNA) and circular (cvcDNA) forms and produces small interfering RNA (siRNA) transcripts that can result in an immediate protective RNA interference (RNAi) response. The vcDNA can also result in host acquisition of endogenous viral elements (EVE) in genomic DNA. Thus, we hypothesized that DENV-2 cvcDNA and DENV-2-EVE would arise in C6/36 insect cells challenged with DENV-2 virusin vitro. Here we describe the successful isolation and characterization of cvcDNA constructs homologous to DENV-2 from laboratory challenges with C6/36 cells. At least 1 of these appeared to arise from a DENV-2-EVE. We also show that a linear vcDNA preparation derived from the DENV-2-EVE sequence by PCR significantly reduced DENV-2 replication when applied to naive C6/36 cells prior to DENV-2 challenge. This is preliminary work designed to lay the groundwork for further studies on use of the C6/36 cell model for screening and characterization of protective EVE against both insect and shrimp viruses.