The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo.

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.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction.

Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.