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Arthropod-borne flaviviruses such as yellow fever (YFV), Zika and dengue viruses continue to cause significant human disease globally. These viruses are transmitted by mosquitoes when a female imbibes an infected blood-meal from a viremic vertebrate host and expectorates the virus into a subsequent host. Bamaga virus (BgV) is a flavivirus recently discovered in Culex sitiens subgroup mosquitoes collected from Cape York Peninsula, Australia. This virus phylogenetically clusters with the YFV group, but is potentially restricted in most vertebrates. However, high levels of replication in an opossum cell line (OK) indicate a potential association with marsupials. To ascertain whether BgV could be horizontally transmitted by mosquitoes, the vector competence of two members of the Cx. sitiens subgroup, Cx. annulirostris and Cx. sitiens, for BgV was investigated. Eleven to thirteen days after imbibing an infectious blood-meal, infection rates were 11.3% and 18.8% for Cx. annulirostris and Cx. sitiens, respectively. Cx. annulirostris transmitted the virus at low levels (5.6% had BgV-positive saliva overall); Cx. sitiens did not transmit the virus. When mosquitoes were injected intrathoracially with BgV, the infection and transmission rates were 100% and 82%, respectively, for both species. These results provided evidence for the first time that BgV can be transmitted horizontally by Cx. annulirostris, the primary vector of pathogenic zoonotic flaviviruses in Australia. We also assessed whether BgV could interfere with replication in vitro, and infection and transmission in vivo of super-infecting pathogenic Culex-associated flaviviruses. BgV significantly reduced growth of Murray Valley encephalitis and West Nile (WNV) viruses in vitro. While prior infection with BgV by injection did not inhibit WNV super-infection of Cx. annulirostris, significantly fewer BgV-infected mosquitoes could transmit WNV than mock-injected mosquitoes. Overall, these data contribute to our understanding of flavivirus ecology, modes of transmission by Australian mosquitoes and mechanisms for super-infection interference., Author summary Mosquito-borne flaviviruses include medically significant members such as the dengue viruses, yellow fever virus and Zika virus. These viruses regularly cause outbreaks globally, notably in tropical regions. The ability of mosquitoes to transmit these viruses to vertebrate hosts plays a major role in determining the scale of these outbreaks. It is essential to assess the risk of emergence of flaviviruses in a given region by investigating the vector competence of local mosquitoes for these viruses. Bamaga virus was recently discovered in Australia in Culex mosquitoes and shown to be related to yellow fever virus. In this article, we investigated the potential for Bamaga virus to emerge as an arthropod-borne viral pathogen by assessing the vector competence of Cx. annulirostris and Cx. sitiens mosquitoes for this virus. We showed that Bamaga virus could be detected in the saliva of Cx. annulirostris after an infectious blood-meal, demonstrating that the virus could be horizontally transmitted. In addition, we showed that Bamaga virus could interfere with the replication in vitro and transmission in vivo of the pathogenic flavivirus West Nile virus. These data provide further insight on how interactions between viruses in their vector can influence the efficiency of pathogen transmission.

We describe a virus isolated from Culex annulirostris mosquitoes in Australia. Phylogenetic analysis of its RNA-dependent RNA polymerase sequence and that of other related viruses revealed 6 clades, two of which corresponded wholly or partly with existing genera in the family Nodaviridae. There was greater genetic diversity within the family than previously recognized prompting us to suggest that additional genera should be considered within the family.