Abstract
The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.
Highlights
Mosquito cell cultures are used routinely in arbovirology studies to grow viruses and to elucidate aspects of viral infection and replication in mosquitoes
In this study we describe small RNAs involved in RNA silencing that are derived from the genome of the arbovirus dengue virus type-2 (DENV2) in infected Aedes aegypti mosquito cell lines and mosquitoes
We show that C6/36, a mosquito cell line from A. albopictus, appears to process dengue virus type 2 (DENV2) RNA for silencing differently from A. aegypti mosquitoes, revealing that other small RNA pathways in mosquito cells might have a role in antiviral immunity in this cell line and provide insight into using mosquito cell cultures to study the antiviral response to arboviruses in mosquitoes
Summary
Mosquito cell cultures are used routinely in arbovirology studies to grow viruses and to elucidate aspects of viral infection and replication in mosquitoes. Many of these cell lines were established by Peleg and Singh in the 1960’s [1,2]. Since Aedes aegypti is the most important vector for arboviruses such as dengue, we have used another cell line, derived from A. aegypti embryos and known as Aag, in several recent studies [9,10] This cell line was originally established by Peleg in 1968 and was further characterized by Lan and Fallon in 1990 [11]. Much of what we know about RNAi in insects has been elucidated in Drosophila flies and cultured cells
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