The Australian backyard mosquito, Aedes notoscriptus, is a highly urbanised pest species that has invaded New Zealand and the USA. Importantly, Ae. notoscriptus has been implicated as a vector of Ross River virus, a common and arthritogenic arbovirus in Australia, and is a laboratory vector of numerous other pathogenic viruses, including West Nile, yellow fever, and Zika viruses. To further explore live viruses harboured by field populations of Ae. notoscriptus and, more specifically, assess the genetic diversity of its virome, we processed 495 pools, comprising a total of 6,674 female Ae. notoscriptus collected across fifteen suburbs in Brisbane, Australia, between January 2018 and May 2019. Nine virus isolates were recovered and characterised by metagenomic sequencing and phylogenetics. The principal viral family represented was Flaviviridae. Known viruses belonging to the genera Flavivirus, Orbivirus, Mesonivirus, and Nelorpivirus were identified together with two novel virus species, including a divergent Thogoto-like orthomyxovirus and an insect-specific flavivirus. Among these, we recovered three Stratford virus (STRV) isolates and an isolate of Wongorr virus (WGRV), which for these viral species is unprecedented for the geographical area of Brisbane. Thus, the documented geographical distribution of STRV and WGRV, both known for their respective medical and veterinary importance, has now been expanded to include this major urban centre. Phylogenies of the remaining five viruses, namely, Casuarina, Ngewotan, the novel Thogoto-like virus, and two new flavivirus species, suggested they are insect-specific viruses. None of these viruses have been previously associated with Ae. notoscriptus or been reported in Brisbane. These findings exemplify the rich genetic diversity and viral abundance within the Ae. notoscriptus virome and further highlight this species as a vector of concern with the potential to transmit viruses impacting human or animal health. Considering it is a common pest and vector in residential areas and is expanding its global distribution, ongoing surveillance, and ecological study of Ae. notoscriptus, together with mapping of its virome and phenotypic characterisation of isolated viruses, is clearly warranted. Immanently, these initiatives are essential for future understanding of both the mosquito virome and the evolution of individual viral species.

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with RNA sequencing is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO₂-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of ≤200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using RNA sequencing generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, demonstrating that different epidemiological, immunological, and evolutionary processes may actively shape the evolution of Australian arboviruses. These results underscore the need for more genomic surveillance data to explore the complex evolutionary pressures acting on arboviruses. Overall, our findings highlight the effectiveness of our methodology, which can be applied broadly to enhance arbovirus surveillance in various ecological contexts and improve understanding of transmission dynamics.

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with metatranscriptomics is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of ≤200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using meta-transcriptomics generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, highlighting long-distance dispersal across the Australian continent and continuous circulation characterised by constant turnover of virus lineages.