African horse sickness virus (AHSV) is a member of the Orbivirus genus within the Reoviridae family and the aetiological agent for African horse sickness (AHS). It is an arthropod-borne virus transmitted by biting midges belonging to various Culicoides spp. Nine antigenically distinct AHSV (AHSV 1-9) serotypes have been identified and all of them are endemic to South Africa. The AHSV genome comprises of ten double stranded RNA segments and is replicated by an RNA-dependent-RNA polymerase which lacks proof-reading abilities and is therefore prone to mutations. The virus could potentially undergo genetic recombination and reassortment during the co-infection of the same host cells by at least two different virus strains of the same species. Since it is an arbovirus, we proposed that the virus would evolve slower than other RNA viruses that require a single host for replication, due to limitations imposed by its ability to successfully infect and replicate in both insect vector and vertebrate hosts. The focus of this study was to investigate the evolutionary dynamics of AHSV over a period of more than 60 years. The effects of selection, substitution rate and recombination over time were determined. African horse sickness virus isolates from the 1960’s to 2014, archived at the OIE World Reference Centre for AHSV and BTV at the ARC-OVI were propagated in cell cultures, double stranded RNA (dsRNA) was extracted and reverse transcribed to cDNA. The amplified cDNA was submitted for Illumina Next Generation Sequencing and full length genome consensus sequences were generated. The assembled genomes were deposited in GenBank and used in the subsequent bioinformatics analyses. These included phylogenetic analyses, determining substitution rates, selection pressure, intragenic recombination, and reassortment.
All ten segments were predicted to be under purifying selection pressure (dN/dS < 1), while four of the segments included selected sites under positive selection pressure (0.1 significance level). The Bayesian coalescent estimates of mean substitution rates for segments-2, -6, -7, and -9 were the highest of the AHSV genome segments, between 1.75 x10-4 and 6.4 x 10-4 substitutions/ site/ year. The recorded substitutions rates were similar to that (0.52 x 10-4 and 6.9 x 10-4 substitutions/ site/ year) observed in BTV. Estimated substitution rates ranged between 1.5 – 6.4 x10-4 substitutions/ site/ year over the whole genome. The calculated time to most recent common ancestor, differs significantly among the genome sequences, indicating the influence of reassortment or recombination on the genomes. Using RDP4, Bootscan and Simplot programs, intragenic recombination events were predicted in seg-1, seg-6, seg-7 and seg-10. These included both single and double cross-over events. Genetic recombination appears to be less frequent than in bluetongue virus (BTV). Widespread reassortment events were detected, including between vaccine strains and wild type viruses. Similar to BTV, all ten segments were predicted to evolve under strong purifying selection with selected sites under positive selection. The high percentage sequence identity within serotypes reflects the strong selective constraint imposed on arboviruses by the necessity to replicate in both vertebrate host and vector species. This study also reports on a widespread genomic reassortment of African horse sickness virus, including between wild-type and vaccine viruses and provides the first evidence of intragenic recombination.