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

• Published in: Parasites & vectors Vol. 16; no. 1; p. 186
• Main Authors: de Vries, Ellen M, Cogan, Noel O I, Gubala, Aneta J, Rodoni, Brendan C, Lynch, Stacey E
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 06-06-2023; BioMed Central; BMC
• Subjects: Acids; Amplification; Analysis; Anthropogenic factors; Aquatic insects; Arboviruses; Bioinformatics; Cell culture; Chromosomes; Disease transmission; Diseases; Distribution; DNA sequencing; Epidemiology; Genetic aspects; Genomes; Genomics; Genotyping; Glycoproteins; Haplotypes; Human influences; Identification and classification; Laboratory methods; Locating; Methods; Microbiological strains; Mosquitoes; Mutation; Nanopore; Natural environment; Nucleotide sequencing; Nucleotides; Pipeline design; Public health; Risk factors; Rivers; RNA polymerase; RNA viruses; Ross River virus; Single nucleotide polymorphisms; Single-nucleotide polymorphism; SNP analysis; Strains (organisms); Temporal variations; Tiled amplicon sequencing; Tracking; Variable region; Vector-borne diseases; Virus research; Viruses; West Nile virus; Wildlife; Workflow; New Zealand; Australia; Papua New Guinea; Queensland Australia; United States > US; Western Australia Australia; Nanopore; Ross River virus; Bioinformatics; Tiled amplicon sequencing; SNP analysis
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-023-05734-z

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.