First reported evidence of homologous recombination in Ross River virus, sampled in Western Australian mosquitoes.

Homologous recombination contributes to the evolution of many RNA viruses and has been described in several alphaviruses. Ross River virus (RRV) is the most medically important endemic alphavirus in Australia and exists as four distinct genotypes (G1 -G4). During our previous genome-scale phylogenetic analysis of RRV in Western Australia two distinct genome sequences (AN572.1 and AN572.2) were generated by de novo assembly from sequencing reads derived from a single mosquito-pool isolate (AN572). The homogenate comprised Aedes normanensis mosquitoes, collected in northern Western Australia in 1984 - a period of defined G2 and G3 co-circulation. Despite their common origin, AN572.1 clustered within G3 and AN572.2 within G2. Here, a curated dataset of 108 complete RRV genomes sampled between 1959 and 2018 was screened for evidence of recombination using RDP4 and phylogenetic approaches. Both AN572.1 and AN572.2 were defined as recombinants in this analysis, with an approximately 1.2kb recombinant fragment identified within the E1 region of both sequences. Whether this recombination occurred in vivo or in vitro is not currently known. This is the first recorded evidence of natural recombination in RRV. Data Summary The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. The sequences analysed in this investigation were published previously in NCBI GenBank. The accession numbers for the 108 taxa dataset are GQ433354-60, HM234643, KY302801, MH987779-81, MN038196 – MN038289, MW147673, MW238766.