Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are key arbovirus vectors of human and animal health relevance. Understanding the biological and molecular factors influencing arbovirus transmission is essential for informing effective mitigation strategies. Culicoides brevitarsis Kieffer is of particular importance due to its close relationship to cattle, high population abundance in suitable sites, and demonstrated vector capacity for arboviruses. However, limited genomic resources have constrained investigation into its molecular and evolutionary biology, necessitating a chromosome-level genome assembly and comparative genomic analyses. A chromosome-level genome assembly of C. brevitarsis was generated using Oxford Nanopore long reads, polished with Illumina short reads, and scaffolded using Hi-C sequencing, followed by genome annotation, comparative dipteran genomics, immune gene characterisation, and evolutionary analyses of vector competence–associated genes. A 129.5 Mb genome was assembled into three chromosomes with high contiguity (N50 = 43 Mb, L50 = 2) and high completeness (98

Bluetongue virus (BTV) is a complex, transboundary arboviral pathogen that circulates globally in ruminant populations. Bluetongue disease is a World Organisation for Animal Health listed disease affecting economically important farmed ruminants. BTV is a dsRNA virus from the Sedoreoviridae family that has a segmented genome. Classification of BTV traditionally focussed on antigenic characteristics. More recently, however, Sanger sequencing methods have been adopted for typing based on specific genome segment analysis and targeted whole-genome sequencing (WGS) is being more widely applied. Despite these advances, standardised approaches for analysing genomic sequencing data to better define individual BTV strains are lacking. New perspectives are needed to update and unify the current array of approaches surrounding BTV genetic characterisation nomenclature. A transition from single-segment-based serotyping and genotype descriptors to adopting WGS-derived descriptors that better describe a detected virus rather than an individual segment is required. This change will improve the ability of researchers to understand and communicate virus movement pathways with improved granularity. This review paper aims to condense the extensive literature on BTV characterisation, genomic sequencing and virus transmission from an Australian perspective. Recommendations are made to assist with clarity and uniformity around virus characterisation, including genomic definitions and more standardised BTV nomenclature.

Around 4500 Ross River virus (RRV) human cases are reported in Australia annually. To date, there is no registered nor licenced vaccine to protect against RRV disease. Identifying and substituting preferred with less-preferred codons and dinucleotides is a recognised strategy to attenuate viruses and may prove useful to vaccine development efforts for RRV and other related viruses. Here, we used bioinformatic approaches aimed at assessing evidence of codon usage and dinucleotide bias in 55 RRV whole genomes sampled from humans (Homo sapiens), macropods (Notomacropus agilis), and the Aedes vigilax mosquito. Our results indicate that RRV undergoes positive and negative codon usage bias with natural selection as the major force driving RRV codon usage patterns. RRV displays a bias towards codons with an A or C at the 3rd position while H. sapiens displays a G or C and N. agilis and Ae. vigilax both show bias towards codons with an A or U at the same 3rd position. RRVs codon usage patterns are coincidental to those displayed by common vertebrate hosts and antagonistic to patterns of Ae. vigilax. The coincidental bias identified suggests vertebrate host gene expression greatly influences RRV evolution. In addition, we show that the UG dinucleotides in RRV are overrepresented at all three codon sites, while CA dinucleotides are only overrepresented at codon sites 1-2 and 2-3. These over and under-representations can be exploited to develop attenuated RRV RNA vaccines. The approach utilised here could also be used to develop vaccines for other alphaviruses of global importance.

Graphical abstract A metatranscriptomics study of a dominant bluetongue virus vector, Culicoides brevitarsis from Australia, identified 10 complete/near-complete viral genomes including 7 novel species.

The current panzootic of high pathogenicity avian influenza virus H5N1 demonstrates how viral incursions can have major ramifications for wildlife and domestic animals. Herein, we describe the recent incursion into Australia of two low pathogenicity avian influenza virus subtypes, H4 and H10, that exhibited contrasting evolutionary dynamics. Viruses detected from national surveillance and disease investigations between 2020 and 2022 revealed 27 genomes, 24 of which have at least one segment more closely related to Eurasian or North American avian influenza lineages than those already circulating in Australia. Phylogenetic analysis revealed that H4 viruses circulating in shorebirds represent a recent incursion from Asia that is distinct from those circulating concurrently in Australian waterfowl. Analysis of the internal segments further demonstrates exclusive, persistent circulation in shorebirds. This contrasts with H10, where a novel lineage has emerged in wild waterfowl, poultry, and captive birds across Australia and has likely replaced previously circulating H10 lineages through competitive exclusion. Elucidating different dynamics for avian influenza incursions supports effective disease risk identification and communication that better informs disease preparedness and response.

Ross River virus (RRV) and Barmah Forest virus (BFV) are arthritogenic arthropod-borne viruses (arboviruses) that exhibit generalist host associations and share distributions in Australia and Papua New Guinea (PNG). Using stochastic mapping and discrete-trait phylogenetic analyses, we profiled the independent evolution of RRV and BFV signature mutations. Analysis of 186 RRV and 88 BFV genomes demonstrated their viral evolution trajectories have involved repeated selection of mutations, particularly in the nonstructural protein 1 (nsP1) and envelope 3 (E3) genes suggesting convergent evolution. Convergent mutations in the nsP1 genes of RRV (residues 248 and 441) and BFV (residues 297 and 447) may be involved with catalytic enzyme mechanisms and host membrane interactions during viral RNA replication and capping. Convergent E3 mutations (RRV site 59 and BFV site 57) may be associated with enzymatic furin activity and cleavage of E3 from protein precursors assisting viral maturation and infectivity. Given their requirement to replicate in disparate insect and vertebrate hosts, convergent evolution in RRV and BFV may represent a dynamic link between their requirement to selectively 'fine-tune' intracellular host interactions and viral replicative enzymatic processes. Despite evidence of evolutionary convergence, selection pressure analyses did not reveal any RRV or BFV amino acid sites under strong positive selection and only weak positive selection for nonstructural protein sites. These findings may indicate that their alphavirus ancestors were subject to positive selection events which predisposed ongoing pervasive convergent evolution, and this largely supports continued purifying selection in RRV and BFV populations during their replication in mosquito and vertebrate hosts.

Few insect-specific alphaviruses (ISA) have been discovered, with even fewer culturable to facilitate full characterisation. Here, we report the recovery of an infectious clone of Yada Yada virus (YYV)—a virus previously only detected by metagenomic sequencing of mosquito homogenates. Using the infectious clone, we confirmed the inability of YYV to replicate in vertebrate cells in vitro, with replication limited to only Aedes mosquito-derived cell lines. We further produced and characterised the first monoclonal antibodies (mAbs) to ISAs. Through successful replacement of the structural proteins of YYV with those of other ISAs, Eilat virus, Agua Salud (ASALV), Taï Forest (TALV) and Mwinilunga alphaviruses (MWAV), we established that a replication block for in vitro culture of TALV and MWAV in mosquito cells does not exist at virus entry. Unexpectedly, ASALV structural proteins were recognised by cross-reactive mAbs made to chikungunya (CHIKV) and Ross River viruses (RRV), suggesting a potential antigenic link between ASALV and pathogenic alphaviruses. The YYV genetic backbone was also investigated to generate chimeras displaying the structural proteins of various pathogenic vertebrate-infecting alphaviruses including CHIKV, RRV, Barmah Forest, Sindbis and Mayaro viruses. These chimeras retained the antigenic properties of the parental viruses and did not replicate in vertebrate cells, demonstrating the potential of the YYV platform for vaccine and diagnostic antigen production.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans , is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus . Spatial scanning statistics revealed overlap between clusters of M. ulcerans -positive Ae. notoscriptus , M. ulcerans -positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

The Australian Biosecurity Genomic Database (ABGD) is a curated collection of reference viral genome sequences based on the Australian National Notifiable Disease List of Terrestrial Animals. It was created to facilitate the screening of high-throughput sequencing (HTS) data for the potential presence of viruses associated with notifiable disease. The database includes a single verified sequence (the exemplar species sequence, where relevant) for each of the 60 virus species across 21 viral families that are associated with or cause these notifiable diseases, as recognized by the World Organisation for Animal Health. The open-source ABGD on GitHub provides usage guidance documents and is intended to support building a culture in Australian HTS communities that promotes the use of quality-assured, standardized, and verified databases for Australia's national biosecurity interests. Future expansion of the database will include the addition of more strains or subtypes for highly variable viruses, viruses causing diseases of aquatic animals, and genomes of other types of pathogens associated with notifiable diseases, such as bacteria. Database URL: https://github.com/ausbiopathgenDB/AustralianBiosecurityGenomicDatabase.

Ross River virus (RRV) is Australia’s most common and widespread mosquito-transmitted arbovirus and is of significant public health concern. With increasing anthropogenic impacts on wildlife and mosquito populations, it is important that we understand how RRV circulates in its endemic hotspots to determine where public health efforts should be directed. Current surveillance methods are effective in locating the virus but do not provide data on the circulation of the virus and its strains within the environment. This study examined the ability to identify single nucleotide polymorphisms (SNPs) within the variable E2/E3 region by generating full-length haplotypes from a range of mosquito trap-derived samples. A novel tiled primer amplification workflow for amplifying RRV was developed with analysis using Oxford Nanopore Technology’s MinION and a custom ARTIC/InterARTIC bioinformatic protocol. By creating a range of amplicons across the whole genome, fine-scale SNP analysis was enabled by specifically targeting the variable region that was amplified as a single fragment and established haplotypes that informed spatial-temporal variation of RRV in the study site in Victoria. A bioinformatic and laboratory pipeline was successfully designed and implemented on mosquito whole trap homogenates. Resulting data showed that genotyping could be conducted in real time and that whole trap consensus of the viruses (with major SNPs) could be determined in a timely manner. Minor variants were successfully detected from the variable E2/E3 region of RRV, which allowed haplotype determination within complex mosquito homogenate samples. The novel bioinformatic and wet laboratory methods developed here will enable fast detection and characterisation of RRV isolates. The concepts presented in this body of work are transferable to other viruses that exist as quasispecies in samples. The ability to detect minor SNPs, and thus haplotype strains, is critically important for understanding the epidemiology of viruses their natural environment.

AbstractIn temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused byMycobacterium ulcerans. Native possums are the major local environmental reservoir ofM. ulceransas they not only develop Buruli lesions but they also shedM. ulceransin their excreta. However the way humans acquireM. ulceransfrom possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes inM. ulceranstransmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and fourad hocfield surveys across an area of 350km2on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns ofM. ulcerans-positive mosquito occurrence withM. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstructM. ulceransgenomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period.Culex molestusandAedes notoscriptuswere the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association betweenM. ulceransandAe. notoscriptus(p<0.0001) with a maximum likelihood estimate (MLE) of 5.88M. ulceranspositive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters ofM. ulcerans-positiveAe. notoscriptus,M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fedAe. notoscriptusshowed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed sharedM. ulceransgenome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate thatAe. notoscriptuslikely transmitM. ulceransin southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

AbstractIn recent years reported cases of Buruli ulcer (BU), caused byMycobacterium ulcerans(MU), have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how MU circulates in the environment and transmits to humans we analyzed environmental samples collected from 115 properties of recent BU cases and from 115 postcode-matched control properties, for the presence of MU. Environmental factors associated with increased odds of MU presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums (Pseudocheirus peregrinus) and overhead powerlines. However, only powerlines and the absence of the native plantMelaleuca lanceolatawere associated with BU case properties. Samples positive for MU were more likely to be found at case properties and were associated with detections of MU in ringtail possum feces, supporting the hypothesis that MU is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with MU positive properties versus case properties indicates a strong human behavioral component or the influence of other environmental factors in disease acquisition that requires further study.Article Summary LinePossums, powerlines, and native vegetation are associated with the presence ofMycobacterium ulceransin residential properties in Victoria, Australia.

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north.

In recent years reported cases of Buruli ulcer, caused by Mycobacterium ulcerans, have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how M. ulcerans circulates in the environment and transmits to humans we analyzed environmental samples collected from 115 properties of recent Buruli ulcer cases and from 115 postcode-matched control properties, for the presence of M. ulcerans. Environmental factors associated with increased odds of M. ulcerans presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums (Pseudocheirus peregrinus) and overhead powerlines. However, only overhead powerlines and the absence of the native plant Melaleuca lanceolata were associated with Buruli ulcer case properties. Samples positive for M. ulcerans were more likely to be found at case properties and were associated with detections of M. ulcerans in ringtail possum feces, supporting the hypothesis that M. ulcerans is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with M. ulcerans positive properties versus case properties indicates the involvement of human behavior or the influence of other environmental factors in disease acquisition that requires further study.

Dispersal is a critical parameter for successful pest control measures as it determines the rate of movement across target control areas and influences the risk of human exposure. We used a fine-scale spatial population genomic approach to investigate the dispersal ecology and population structure of Aedes notoscriptus, an important disease transmitting mosquito at the Mornington Peninsula, Australia. We sampled and reared Ae. notoscriptus eggs at two time points from 170 traps up to 5 km apart and generated genomic data from 240 individuals. We also produced a draft genome assembly from a laboratory colony established from mosquitoes sampled near the study area. We found low genetic structure (F-st) and high coancestry throughout the study region. Using genetic data to identify close kin dyads, we found that mosquitoes had moved distances of >1 km within a generation, which is further than previously recorded. A spatial autocorrelation analysis of genetic distances indicated genetic similarity at >1 km separation, a tenfold higher distance than for a comparable population of Ae. aegypti, from Cairns, Australia. These findings point to high mobility of Ae. notoscriptus, highlighting challenges of localised intervention strategies. Further sampling within the same area 6 and 12 months after initial sampling showed that egg-counts were relatively consistent across time, and that spatial variation in egg-counts covaried with spatial variation in Wright's neighbourhood size (NS). As NS increases linearly with population density, egg-counts may be useful for estimating relative density in Ae. notoscriptus. The results highlight the importance of acquiring species-specific data when planning control measures.

Surveillance programs are essential for the prevention and control of mosquito-borne arboviruses that cause serious human and animal diseases. Viral metatranscriptomic sequencing can enhance surveillance by enabling untargeted, high-throughput arbovirus detection. We used metatranscriptomic sequencing to screen field-collected mosquitoes for arboviruses to better understand how metatranscriptomics can be utilised in routine surveillance. Following a significant flood event in 2016, more than 56,000 mosquitoes were collected over seven weeks from field traps set up in Victoria, Australia. The traps were split into samples of 1000 mosquitoes or less and sequenced on the Illumina HiSeq. Five arboviruses relevant to public health (Ross River virus, Sindbis virus, Trubanaman virus, Umatilla virus, and Wongorr virus) were detected a total of 33 times in the metatranscriptomic data, with 94% confirmed using reverse transcription quantitative PCR (RT-qPCR). Analysis of metatranscriptomic cytochrome oxidase I (COI) sequences enabled the detection of 12 mosquito and two biting midge species. Screening of the same traps by an established public health arbovirus surveillance program corroborated the metatranscriptomic arbovirus and mosquito species detections. Assembly of genome sequences from the metatranscriptomic data also led to the detection of 51 insect-specific viruses, both known and previously undescribed, and allowed phylogenetic comparison to past strains. We have demonstrated how metatranscriptomics can enhance surveillance by enabling untargeted arbovirus detection, providing genomic epidemiological data, and simultaneously identifying vector species from large, unsorted mosquito traps.

Outbreaks of avian influenza virus (AIV) from wild waterfowl into the poultry industry is of upmost significance and is an ongoing and constant threat to the industry. Accurate surveillance of AIV in wild waterfowl is critical in understanding viral diversity in the natural reservoir. Current surveillance methods for AIV involve collection of samples and transportation to a laboratory for molecular diagnostics. Processing of samples using this approach takes more than three days and may limit testing locations to those with practical access to laboratories. In potential outbreak situations, response times are critical, and delays have implications in terms of the spread of the virus that leads to increased economic cost. This study used nanopore sequencing technology for in-field sequencing and subtype characterisation of AIV strains collected from wild bird faeces and poultry. A custom in-field virus screening and sequencing protocol, including a targeted offline bioinformatic pipeline, was developed to accurately subtype AIV. Due to the lack of optimal diagnostic MinION packages for Australian AIV strains the bioinformatic pipeline was specifically targeted to confidently subtype local strains. The method presented eliminates the transportation of samples, dependence on internet access and delivers critical diagnostic information in a timely manner.

BACKGROUND:Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia's coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus, which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax. We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax, with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps.METHODS:Trapping was performed during the 2017-2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to.RESULTS:Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax, with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia.CONCLUSIONS:Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus, making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species.

African Swine Fever (ASF) has been spreading in numerous southeast Asian countries since a major incursion in mainland China in 2018. Timor-Leste confirmed an outbreak of ASF in September 2019 which resulted in high mortalities in affected pigs. Pigs in Timor-Leste are the second most common type of livestock kept by villagers and represent a traditionally important source of income and prestige for householders. In order to understand the extent of ASF infected villages in Timor-Leste a prevalence survey was designed and conducted in November-December 2019. Timor-Leste has limited laboratory facilities and access to qPCR diagnostic tests. Therefore, a loop mediated isothermal amplification (LAMP) assay was used to detect ASF positive blood samples collected during the prevalence survey. The LAMP assay was proven to be a robust, highly specific and sensitive laboratory test for ASF suitable for use in the field and where there are limited laboratory facilities. The results of the prevalence survey allowed the extent of the ASF incursion to be delineated and the introduction of a disease response strategy to limit the spread of ASF and assist in the recovery of the pig population in Timor-Leste.

After publication, the authors were made aware of a short sequence on GenBank (JQ749729.1)with high similarity to Yada Yada virus (YYV).The 331-bp sequence is part of the nonstructural polyprotein (NSP4) and shares 97% nucleotide identity and 99.1% amino acid identity with YYV.This sequence was detected in an Aedes notoscriptus mosquito collected in North Melbourne, Australia, in 2011.The similarity of this sequence to YYV suggests that the virus from which it originated is part of the mosquito-specific alphavirus complex, which consists of viruses that have been detected only in Anopheles and Culex mosquitoes.