Endosymbionts and viromes: interplays between Wolbachia pipientis and the virome of Aedes mosquitoes

Abstract

The yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus are the two most significant vectors of arthropod-borne viruses including dengue, yellow fever, Zika and Chikungunya viruses. Considerable progress has been made towards determining mosquito host factors controlling competence for transmission of arboviruses. Recent studies have demonstrated that insect-specific viruses (ISVs) can modulate refractoriness of Ae. aegypti and Ae. albopictus to arbovirus infection in vitro and in vivo. While research is ongoing to implicate ISVs as potential pro or antiviral host factors, we have limited understanding of the composition and diversity of viruses associated with these mosquitoes.One promising strategy for the control of arboviruses in these mosquitoes is the stable transinfection of the endosymbiotic bacterium Wolbachia pipientis into Ae. aegypti and Ae. albopictus mosquitoes. Wolbachia has been demonstrated to restrict a range of RNA viruses in Drosophila melanogaster and both arboviruses and ISVs in Ae. aegypti and Ae. albopictus, suggesting complex multi-factorial contributions of the virome and bacterial composition to mosquito vector competence.To unpack these complex interactions, we first set out to uncover previously unknown virome diversity in the mosquitoes using a metagenomics survey approach through the analysis of ~3000 published RNA-Seq libraries from wild-caught, laboratory colonies and cell line samples. We demonstrate that common laboratory colonies and cell lines harbour numerous novel mononegaviruses, orthomyxoviruses, and negeviruses. Additionally, we analysed ~100 individual mosquito transcriptomes and demonstrate that inter-host variation of ISVs exists between mosquitoes as well as heterogeneity between different laboratory strains. The results of which providing a resource for the further assessment of the ecology, evolution and interaction of ISVs with their mosquito hosts and arboviruses they transmit.One virus uncovered from these analyses was Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales. AeAV was present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. We experimentally demonstrated the host range of AeAV as being infectious to Aedes cells, but not to three mammalian cell lines. Reanalysis of a small RNA library of Ae. aegypti cells coinfected with AeAV and Wolbachia indicated an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces dengue virus replication in vitro.In addition to the interactions between AeAV, Ae. aegypti and Wolbachia, we also explored the effect of Wolbachia infection on Aedes albopictus densovirus (AalDNV-1), a single-stranded DNA virus. We demonstrated that Wolbachia enhances AalDNV-1 in Aedes cell lines in a density-dependent manner. Further, we found that in Wolbachia-infected Aag2 cells greater absolute abundance of virus-derived short interfering RNAs was produced compared to uninfected cells. Additionally, we found the production of virus-derived PIWI-like RNAs (vpiRNA) in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA U1 bias signature, whereas the typical “ping-pong” signature (U1 - A10) was evident in vpiRNAs from the cytoplasmic fractions. These findings are the first report of the density-dependent enhancement of a DNA virus by Wolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time. The results from both of these studies add to previous evidence indicating Wolbachia does not restrict a range of negative-strand RNA viruses or DNA viruses.Finally, using the pipeline described for the data-mining of ISVs associated with Aedes mosquitoes, we explored the ability to uncover further host associations of divergent flaviviruses. Most flaviviruses are disease-causing pathogens of vertebrates maintained between mosquitoes or ticks and vertebrate hosts. However poor sampling of flaviviruses outside the vector-borne flaviviruses has presented a narrow understanding of flavivirus diversity and evolution. In the final study presented here, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum-likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. Also, we identified variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNAi response. Taken together the evidence suggests Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. These data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expanding our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.