Insights into RNAi-based antiviral immunity in Lepidoptera: acute and persistent infections in Bombyx mori and Trichoplusia ni cell lines.

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.

Infection studies of nontarget mammalian cell lines with Bombyx mori macula-like virus.

Bombyx mori-derived cell lines are generally used for Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculovirus expression vector system (BEVS). However, almost all of the B. mori-derived cell lines are persistently infected with Bombyx mori macula-like virus (BmMLV). In this study, nontarget mammalian cell lines were exposed to BmMLV, and their susceptibility was investigated. Real-time PCR showed that viral RNA in virus-inoculated nine mammalian cell lines decreased sharply at 7 days postinfection. Also, there was no significant effect on cell viability of mammalian cells after inoculation with BmMLV. These findings indicate that mammalian cell lines used in this study are not permissive to BmMLV, and BmMLV contamination might not affect the safety aspect of BmNPV-based BEVS.

Infection study of Bombyx mori macula-like virus (BmMLV) using a BmMLV-negative cell line and an infectious cDNA clone.

Previously, a novel macula-like virus was identified from Bombyx mori cultured cell line BmN and termed B. mori macula-like virus (BmMLV). BmMLV encodes a 6.5-kb-long positive, single-strand RNA genome, which contains putative RNA-dependent RNA polymerase (RdRp), coat protein (cp) and p15 genes. In this study, CP expression in several B. mori-derived cell lines was examined by using the CP antibody. Surprisingly, Western blot analysis revealed that all of the cell lines tested have already been infected with BmMLV. To perform reverse genetic studies in BmMLV, a new BmMLV-negative cell line, designated as BmVF from the embryos of B. mori was established. Infection studies showed that BmVF cells were permissive to BmMLV persistent infection. In addition, a full-length infectious cDNA clone of BmMLV, termed pHMLV was developed. Upon transfection of pHMLV into BmMLV-negative BmVF cells, viral CP was detected in both cells and conditioned medium. When the cDNA-derived virus in conditioned medium was inoculated onto BmVF cells, efficient propagation of BmMLV was observed. Collectively, these results indicate that the new BmMLV-negative cell line and the infectious cDNA clone of BmMLV will be useful for elucidation of the mechanism of BmMLV replication and the functional roles of BmMLV genes.