Fine-scale genomic tracking of Ross River virus using nanopore sequencing.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Rapid, in-field deployable, avian influenza virus haemagglutinin characterisation tool using MinION technology.

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.

Genome Sequences of Barmah Forest Virus Strains Isolated from Mosquitoes Trapped in Australian Defence Force Training Areas Reveal Multiple Nucleotide Insertions in the 3' Untranslated Region.

The complete genome sequences of two Barmah Forest virus (BFV) strains isolated from mosquitoes trapped in the Australian Defence Force (ADF) training areas during 2017 and 2018 reveal multiple nucleotide insertions in the 3' untranslated region (UTR) of ADF BFV strains compared with the BFV prototype strain whole-genome sequence in GenBank.

Genome Sequences of Three Ross River Virus Isolates Obtained from the Australian Defence Force.

The complete genome sequences of three Ross River virus (RRV) isolates from infected Australian Defence Force (ADF) personnel and from mosquitoes collected in ADF training areas were determined. Phylogenetic analysis in comparison with all available complete RRV nucleotide sequences from GenBank split these three RRV isolates into two distinct sublineages.

Genomic Characterisation of Vinegar Hill Virus, An Australian Nairovirus Isolated in 1983 from Argas Robertsi Ticks Collected from Cattle Egrets.

This report describes the near complete genomic sequence and subsequent analysis of Vinegar Hill virus (VINHV; tentative member of the genus Orthonairovirus, family Nairoviridae, order Bunyavirales). VINHV is the second nairovirus reported to be isolated on mainland Australia and the first to be sequenced and analysed. Our genetic analysis shows that VINHV belongs to the Dera Ghazi Khan genogroup, a group of viruses previously isolated in other parts of the world including Asia, South Africa, and the USA. We discuss possible routes of entry for nairoviruses into Australia and the need to understand the virome of Australian ticks in the context of new and emerging disease.

Genomic characterisation of three Mapputta group viruses, a serogroup of Australian and Papua New Guinean bunyaviruses associated with human disease.

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.

Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae.

Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large diversity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus; however, the genomes of these two viruses are significantly diverged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

Molecular-beacon multiplex real-time PCR assay for detection of Vibrio cholerae.

A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

Multiplex real-time PCR detection of Vibrio cholerae.

Cholera is an important enteric disease, which is endemic to different regions of the world and has historically been the cause of severe pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the toxigenic strains are causative agents of potentially life-threatening diarrhoea. A multiplex, real-time detection assay was developed targeting four genes characteristic of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin (rtxA), extracellular secretory protein (epsM), mannose-sensitive pili (mshA) and the toxin coregulated pilus (tcpA). The assay was developed on the Cepheid Smart Cycler using SYBR Green I for detection and the products were differentiated based on melting temperature (Tm) analysis. Validation of the assay was achieved by testing against a range of Vibrio and non-Vibrio species. The detection limit of the assay was determined to be 10(3) CFU using cells from pure culture. This assay was also successful at detecting V. cholerae directly from spiked environmental water samples in the order of 10(4) CFU, except from sea water which inhibited the assay. The incorporation of a simple DNA purification step prior to the addition to the PCR increased the sensitivity 10 fold to 10(3) CFU. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays.