Mechanistic investigation of flavivirus repression by diverse Wolbachia strains in mosquito cell lines
Schultz, Michaela Jane
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Arboviruses are blood-borne pathogens that threaten half of the world’s population. The recent outbreak of Zika virus (ZIKV) in Brazil has highlighted the importance of developing new strategies to limit virus spread. While vaccines are in development, one way to immediately suppress viral transmission is through biocontrol of mosquito vector. Novel biocontrol strategies utilize microbe – mosquito interactions to inhibit the transmission of pathogens. A powerful tool under investigation is the intracellular bacteria, Wolbachia pipientis, which are maintained in insect populations through maternal transmission. The Wolbachia strain wMel can be trans-infected into mosquitos limiting ZIKV transmission. However, thermal stress can hinder maternal transmission of the wMel strain of Wolbachia. For Wolbachia-based technologies of vector control, it is important to have additional strains with viral suppression capabilities available. We characterized alternative Wolbachia strains in A. albopictus mosquito cell lines and the underlying mechanisms of these interactions. We identified two novel Wolbachia strains with robust arbovirus repression. wAlbB, native to mosquitos blocked 90% of ZIKV growth. More strikingly, wStri, a nonnative symbiont, ablated ZIKV growth in A. albopictus cells below the limit of detection. After showing that ZIKV growth is rescued in wStri infected A. albopictus cells by the pharmacological removal of Wolbachia, we established these cells as an in vitro model for mechanistic studies. Using novel labeling and reporter techniques, we isolate a block in virus growth by Wolbachia at two stages of viral growth, entry and translation. We further show that cholesterol, which can partially rescue viral growth in Wolbachia wStri infected cells, aids in viral entry but does not promote viral growth post entry. Beyond our Wolbachia studies, we further investigated the limited arbovirus growth observed in many A. aegypti cell lines and identified two insect–specific viruses which interfere with arbovirus growth. To address the limited biocontrol tools in C. pipiens mosquitos, we characterized commensal microbiota that may be used as a direct competitor of viruses or as a tool to genetically enhance an antiviral response in the mosquito gut. Together this work expands our understanding of Wolbachia-mediated biocontrol strategies and offers novel resources to suppress arbovirus transmission.