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Culicoides spp., a common biting midge genus, are haematophagous insects that can transmit pathogens to humans and other animals. Some species transmit arboviruses, including bluetongue virus, epizootic haemorrhagic disease virus, African horse sickness virus and Schmallenberg virus to vertebrates, which can be detrimental to livestock and wild animals. Culicoides spp. can also have a diversity of insect-specific viruses (ISVs) that can only be transmitted between insects and others related to known arboviruses. For Culicoides brevitarsis and other Culicoides spp. in Australia, the virome is largely unexplored. We used high-throughput sequencing to characterise the virome of C. brevitarsis collected from Casino, New South Wales, Australia. For virus detection, the total RNA was extracted from pools of C. brevitarsis followed by rRNA depletion and Illumina short-read-based RNA sequencing. The reads were quality-checked, filtered and assembled into contigs, compared with the non-redundant protein and conserved domain databases for viral detection and genome organisation, respectively. The phylogenetic analysis was used to further characterise the viruses. We detected new virus diversity including ten viruses belonging to eight different families with complete or near-complete coding regions. Seven of these were novel virus species belonging to the families: Chuviridae , Orthomyxoviridae , Peribunyaviridae , Qinviridae , Rhabdoviridae and Solemoviridae . In addition, the novel Peribunyaviridae virus should also be considered part of a new genus. Whilst most of the detected viruses grouped into families with viruses that can infect insects, animals or both, the novel species of Solemoviridae was closely related to an economically important plant pathogen, the sugarcane yellow leaf virus. Our quantitative PCR-based screening confirmed the absence of any Wolbachia endosymbiont within the collected samples. Furthermore, we detected fragments of three more virus families known to infect fungi and plants. The detection of potential arboviruses and ISVs in Culicoides spp. is important in understanding virus epidemiology.

Japanese encephalitis virus (JEV) is transmitted by Culex species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. Cx. annulirostris has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian Cx. annulirostris with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian Cx. annulirostris is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.