African horse sickness (AHS), caused by the African horse sickness virus (AHSV), is an economically important disease of equids. Outbreaks of the disease usually have a devastating effect on the susceptible equine populations. Due to the severity of AHS in horses, the economical impact and ability to spread rapidly, the disease has been listed by the World Organisation for Animal Health (OIE) as a notifiable disease. Nine serotypes of the virus have been identified. Transmission is mediated by biting midges of the genus Culicoides. Despite the fact that a cell culture-attenuated live virus vaccine is commercially available, AHS outbreaks occur annually in endemic regions. Several shortcomings of the vaccine itself and its application have been identified that may explain the occurrence of outbreaks.
In this study, we sought to evaluate the level of genetic divergence between the AHSV reference strains that the current polyvalent vaccine was based on and recent field isolates. The effectivity of the vaccine to induce complete protective immunity against current circulating strains is, thus, an additional concern and warranted further investigation. Similarly, the current serological diagnostic assays to detect virus or antibodies to the virus in the recent field isolates or horse sera respectively, are based on the reference isolates. Towards accomplishing the above mentioned goal, a panel of recent AHSV field strains, isolated between 1994 and 2009, were selected for comparative analysis to the reference strains isolated in the 1960s. The complete, segmented double stranded RNA (dsRNA) genomes of nine recent field isolates of AHSV, representing serotypes 1 to 9, were characterised. Ultra-deep sequencing data of cDNA copies of the genome segments were generated. Consensus nucleotide sequences of each of the ten segments of each isolate were successfully assembled. Sequence data analysis showed that each virus isolate not only contained single serotype of AHSV, but was also free from other contaminating equine dsRNA viruses, such as equine encephalosis virus (EEV). The sequence data also confirmed the serotypes of the virus, as previously determined by virus neutralisation (VN) assays and reverse-transcription polymerase chain reaction (RT-PCR).
Intra-serotype comparative sequence analyses of the corresponding segments of the reference and recent field strains showed a maximum variability of 28% and 9% on nucleotide and amino acid level, respectively. The most variable genome segments were S2 encoding viral protein 2 (VP2), followed by S10 encoding non-structural protein 3 (NS3) and S6 encoding viral protein 5 (VP5). Therefore, these segments of the AHSV-9 reference and recent strains were further investigated. Comparative analysis of VP2 of the AHSV-9 reference strain with those of more recent field strains revealed a number of dissimilar amino acid mutations within, or adjacent to, known epitope-containing regions. In addition, significant changes were observed in the amino terminus. Some of the mutations correlated with altered predicted secondary structure and/or antigenicity profiles. Similar analysis of AHSV-9 VP5 indicated that the region between residue 101 and 201 was variable, although the overall predicted secondary structure appeared to be conserved. Our results indicated that the hydrophobic domain regions 1 (HD-1) and 2 (HD-2) respectively, in S10 (NS3), previously reported to play an important role in the function of NS3, remained conserved.