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

Dengue virus serotype 2 (DENV-2) is a mosquito-borne disease in the family Flaviviridae. It has been 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 the 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 lead to the host acquiring 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 virus in 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 cvcDNA preparation derived from the DENV-2 infected in C6/36 insect cells and subjected to rolling circle amplification (RCA) significantly reduced DENV-2 replication when applied to naive C6/36 cells prior to the DENV-2 challenge. This preliminary work lays the groundwork for further studies using the C6/36 cell model to screen and characterize protective EVE against insect and shrimp viruses.