DENGUE MICROEVOLUTIONARY PROCESSES IN WOLBACHIA-INFECTED MOSQUITO CELL LINES

Abstract

Intro: Dengue virus (DENV) is one of the most important arboviral human pathogens with an estimated 25,000 deaths/year worldwide. Biological control of Aedes-borne transmission using the endosymbiotic bacteria Wolbachia has resulted in a reduction of dengue incidence in endemic areas. However, there is a concern that DENV, an RNA virus, could mutate to escape Wolbachia blocking, leading to reduced effectiveness of this intervention. Little is known about the intracellular interactions between the virus and Wolbachia during coinfection of mosquitoes, and any Wolbachia-mediated effect on DENV genetic diversity. Methods: Two DENV strains were used, and infections performed on paired C6/36 mosquito cell lines, one infected with the Wolbachia strain wAlbB and an uninfected, control cell line. Supernatants and cells were sampled at several time points over a period of 6 days. Resulting virus titres were evaluated using qRT-PCR and DENV genetic diversity throughout infection determined using nanopore long-read sequencing. Intracellular localisation between Wolbachia and DENV at several timepoints was determined using transmission electron microscopy (TEM). Findings: Changes in DENV genetic diversity were compared between serotypes and any amino acid changes observed were modelled to determine their functional impact on the virus. We determined whether Wolbachia and DENV colocalise to the endoplasmic reticulum and investigated any subcellular changes during the time course of infection. Cellular structural changes were correlated to changes in viral titre and viral genetic diversity. Discussion: Sampling and evaluation of viral titre, selection pressure, and intracellular interactions throughout the time-course of infection will provide a broad picture of how Wolbachia and DENV interact within the mosquito cell. Understanding the long-term ability of Wolbachia to continue blocking DENV transmission relies on a thorough understanding of underlying cellular interactions and mechanisms. Conclusion: The results shed light on DENV localisation within Wolbachia- infected cells and the fine scale genetic changes in the virus that may occur during infection.