A correlation between capsid protein VP2 and the plaque morphology of African horse sickness virus in cell culture.

The attenuated live virus vaccine that is used in South Africa to protect against African horse sickness infection was developed more than 50 years ago. With the selection of the vaccine strains by cell culture passage, a correlation between the size of plaques formed in monolayer Vero cultures and attenuation of virus virulence in horses was found. The large plaque phenotype was used as an indication of cell culture adaptation and strongly correlated with attenuation of virulence in horses. There was never any investigation into the genetic causes of either the variation in plaque size, or the loss of virulence. An understanding of the underlying mechanisms of attenuation would benefit the production of a safer AHSV vaccine. To this end, the genomes of different strains of two African horse sickness isolates, producing varying plaque sizes, were compared and the differences between them identified. This comparison suggested that proteins VP2, VP3, VP5 and NS3 were most likely involved in the determination of the plaque phenotype. Comparison between genome sequences (obtained from GenBank) of low and high passage strains from two additional serotypes indicated that VP2 was the only protein with amino acid substitutions in all four serotypes. The amino acid substitutions all occurred within the same hydrophilic area, resulting in increased hydrophilicity of VP2 in the large plaque strains.

Serotype-specific detection of African horsesickness virus by real-time PCR and the influence of genetic variations.

Real-time PCR hybridization probe sets were tested for the specific detection of amplified genome segment 2 cDNA from all nine serotypes of African horsesickness virus (AHSV). The hybridization probes were derived from the sequences of genome segments 2 of the nine reference strains of the virus and were designed to have clearly distinguishable peak melting temperatures. Viral dsRNA from each of the serotypes was specifically detected after reverse transcription, real-time PCR and melting curve analysis. The method was used to successfully serotype a range of field isolates, although most of the these showed peak melting temperature shifts. These shifts could be related to nucleotide substitutions in the regions that are targeted by the probes. Sensitivity was demonstrated to be sufficient for use with dsRNA isolated directly from infected organ samples, making it potentially useful as a rapid diagnostic tool.

Investigations on outbreaks of African horse sickness in the surveillance zone in South Africa.

Confirmed outbreaks of African horse sickness (AHS) occurred in the surveillance zone of the Western Cape in 1999 and 2004, both of which led to a two-year suspension on the export of horses. Light trap surveys in the outbreak areas showed that known vector competent Culicoides species, notably C. imicola, were abundant and present in numbers equal to those in the traditional AHS endemic areas. Isolations of AHS virus serotypes 1 and 7, equine encephalosis virus, and bluetongue virus from field-collected C. imicola in the surveillance zone demonstrated that this species was highly competent and could transmit viruses belonging to different serogroups of the Orbivirus genus. Molecular identification of recovered virus isolates indicated that at least two incursions of AHS into the surveillance zone had taken place in 2004. The designation of an AHS-free zone in the Western Cape remains controversial since it can be easily compromised, as evidenced by the two recent outbreaks. In light of the results reported in the present study, the policy of maintaining a large population of unvaccinated horses in the surveillance zone should be reconsidered, as it leaves them vulnerable to infection with AHS virus, which is the most pathogenic of all equine viruses.

African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization.

Since protection against African horsesickness (AHS) is serotype-specific, rapid serotyping of AHSV is crucial to identify the correct vaccine serotype for efficient control of the spread of AHS outbreaks, especially when they occur in non-endemic regions. This paper describes the first one-day serotyping procedure that requires only a single RT-PCR and hybridization and which can identify multiple serotypes in mixed infections in one assay. The same region of genome segment 2 of all nine AHSV serotypes is amplified in a single RT-PCR. A universal primer set, designed to amplify the 5'-terminal 521-553bp of genome segment 2 of all of the nine AHSV serotypes with one reaction, was used to generate serotype-specific probes from dsRNA prepared from infected tissue cultures or organ samples. These probes hybridized serotype-specifically with immobilized genome segment 2 cDNA of the nine AHSV reference serotypes in a checkerboard reverse line blot format. All nine AHSV reference and the seven vaccine strains and field viruses isolated up to 28 years apart could be serotyped accurately within a day. The sensitivity of the method is 1pg dsRNA which is sufficient to serotype AHSV directly from lung and spleen specimens of infected horses.

VP2 gene phylogenetic characterization of field isolates of African horsesickness virus serotype 7 circulating in South Africa during the time of the 1999 African horsesickness outbreak in the Western Cape.

We present the first VP2-gene phylogenetic analysis of African horsesickness (AHS) viruses within a serotype. Thirteen AHSV 7 isolates were obtained from cases that occurred in South Africa during 1998-1999, and three were historical AHSV 7 isolates. The goals were to start a database of isolates of known location and time of isolation and to determine if we could identify the origin of an AHS outbreak in the surveillance area in the Western Cape. We prepared full-length cDNA copies of the VP2-genes of the isolates. Nucleic acid sequence data of a 786 bp region was used to characterize the genetic relationships between the isolates. The nucleic acid identities between the isolates ranged from 95.5 to 100%. Isolates from common geographical regions grouped together. Characterization of field isolates revealed the presence of two AHSV 7 lineages in South Africa during this period. The grouping of the viruses into two clades accurately reflected the geographical groupings of the isolates. The average nucleic acid divergence between the clades was 4.3%. Within the clades the divergence was 0.5 and 0.1%, respectively. The data suggests that the AHS outbreak in the Western Cape could have been an incursion from the Kwazulu Natal Province.