A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes

Abstract

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen.We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47?6 % and 71?4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

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

“…Sequence variation among isolates of one serotype has been found to be lower when there is less time separation (1 or 2 years) between isolation and then it seems to be more related to geographic separation (Koekemoer et al, 2003). The prototype strains were all isolated during 1961-1963 and low passage stocks were used to obtain the genome segment 2 nucleotide sequences (Potgieter et al, 2003) that were used to design the probes. Except for the serotype 1 isolates, all the field viruses that were used showed decreased peak T m values when tested.…”

“…The genome segments 2 sequences of the prototype viruses (Potgieter et al, 2003) were used for the design of the hybridization probe sets. When dsRNA from these reference viruses were tested, increased fluorescence signals, that indicate cDNA amplification and probe hybridization, were serotype-specific in all cases.…”

“…Genome segment 2 codes for VP2 and even before large scale nucleotide sequencing had been carried out, it was shown that this part of the genome is highly divergent among the nine serotypes of AHSV (Bremer et al, 1990). This has since been confirmed after sequencing of genome segment 2 of all the serotypes (Potgieter et al, 2003). A logical conclusion was to develop serotype-specific molecular tests that identify nucleotide sequences on genome segment 2.…”

“…The reference strain viruses that were used have been described by Potgieter et al (2003). The field viruses are listed in Table 3 and were all isolated by inoculation of equine blood or homogenized organ samples into suckling mouse brains followed by three passages on BHK cell cultures.…”

Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus

“…AHSV type-specific real-time RT-PCR (TS RTqPCR) assays offer the potential for more rapid determination of the AHSV type involved in such outbreaks. AHSV TS assays that target the portion of the L2 gene encoding the major neutralization determinants of AHSV (Kanai et al, 2014;Potgieter et al, 2003;Vreede and Huismans, 1994;Burrage et al, 1993) are well suited for accurate, rapid serotype determination. Direct TS RT-PCR assays have been developed (Sailleau et al, 2000); however these assays are cumbersome and time consuming as they require the use of gels for assay confirmation.…”