Abstract
Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.
Highlights
Mosquitoes serve as primary vectors for the vast majority of arthropod-borneviruses, which pose a global health threat to humans and other vertebrates
We investigated the presence of insect-specific viruses (ISVs) in the uninfected CT, U4.4, Aag2, C6/36 and AP-61 cells by de novo assembly of small RNA reads, using a pipeline previously described by Carissimo et al [48,49]
As contigs were found for Phasi Charoen-like virus (PCLV), Calbertado virus (CLBOV), Flock House virus (FHV), and Culex narnavirus 1 (CxNV1) by de novo assembly of small RNAs from CT cells (Table 2), we investigated the small RNA profiles to these viruses
Summary
Mosquitoes serve as primary vectors for the vast majority of arthropod-borne (arbo)viruses, which pose a global health threat to humans and other vertebrates. With the introduction of next-generation sequencing technologies and metagenomics into the field of virology, it becomes increasingly clear that many insects and insect cell lines, including mosquitoes, carry persistently infecting insect-specific viruses (ISVs) [1,2,3,4]. The presence of ISVs in mosquitoes and mosquito cell lines can interfere with the infection and replication of arboviruses [5,6,7,8,9,10,11] and may thereby affect. It is important to investigate the presence of ISVs in both cell culture systems and mosquito colonies used for experiments. MiRNAs have a length of ~22–23 nts and are produced by the ribonucleases
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