A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay.
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Date
2014
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Abstract
African horse sickness (AHS) is a viral disease that afflicts all equine species and has a 90%
mortality rate in unvaccinated horses. The disease has a devastating effect on the national
herd of South Africa each year and affects both the sport and racehorse industries, including
the export of prized bloodstock, as well as the rural and subsistence economies that depend
on animal traction. Transmitted by the Culicoides spp. of biting midge, the virus belongs to
the Orbivirus genus of the Reoviridae family with nine known serotypes and ten genome
segments. Segment 2 (which encodes VP2) is responsible for serotype determination while
segment 10 (which encodes NS3) is merely serotype-divergent. Knowledge of the
seroprevalence of the virus is poor. The increasing reluctance of horse owners to use the
registered vaccine due to perceived inefficacy is of concern. As a means to increase
knowledge output in this regard, and potentially provide a service to horse owners, a rapid
serotyping assay is sought based on High Resolution Melt (HRM) analysis. HRM analysis is
a powerful tool that is based on the release of a DNA intercalating dye from polymerase
chain reaction products through gradual and controlled heating. The dye is released at a
specific point that is dependent on the unique sequence of the amplicon. It can thus be used
to distinguish, very sensitively, differences in divergent amplicons. Using a range of freely
available bioinformatics software, such as Clustal X2, Primaclade, Treeview and BLAST
analysis, primers were designed based on segment 2 that sought to differentiate the
individual serotype from previously defined clades based on a pair of segment 10 primers.
Reference and field isolates of the AHS virus were obtained from the National Institute of
Communicable Disease and the Onderstepoort Veterinary Institute, South Africa, and were
propagated on Vero cell monolayers. Total RNA was extracted using guanidine-thiocyanate
and verified as containing AHSV genomic material using primers recognised by the World
Organisation for Animal Health that target the genome segment encoding VP7. Variable
amounts of total RNA did not influence the downstream analysis as individual serotypes were
easily distinguished using HRM despite wide ranging template concentrations. Through
testing the primers designed in the present study, various serotype anomalies were
discovered with regard to the isolates obtained from the Onderstepoort Veterinary Institute.
Serotype-specific primers and the segment 10 primers were used to interpret the serotype
anomalies through High Resolution Melt analysis. Sequencing confirmed the anomalies:
serotype 2 isolates were serotype 6 isolates and a serotype 5 isolate was serotype 8. A
proposed protocol for a rapid serotyping assay was investigated. This involved an initial PCR
to determine into which clade of segment 10 the sample fits. Following this, the serotype was
elucidated for each clade using segment 2 clade-specific primers. These reactions were
performed in the Corbett Rotor-Gene™ 6000 and its in-built software was used. However,
limitations of the software soon became apparent, as it was not able to completely
distinguish the serotypes. Alternate methods were sought and included ScreenClust HRM®
Software, principal component analysis (PCA) and discriminant analysis (DA). The use of
normalised HRM fluorescence curves for PCA and DA was effective in standardising the
template concentrations. These methods were successful in determining the serotype and
rendered results with greater statistical confidence. Finally, a phylogenetic analysis was
performed of all the available AHS virus sequences to determine the degree of possible
genetic drift in the virus that may give rise to a new serotype. Minimal to no genetic drift could
be found comparing sequences from the 1960s to sequences from the 2000s. This study
presents the ability of HRM analysis to recognise and define AHS virus serotype anomalies,
provide a new protocol for the serotyping of the virus with an extensive statistical analysis,
the first for an orbivirus. Furthermore, the protocols described can be extrapolated to other orbiviruses.
Description
Ph. D. University of KwaZulu-Natal, Pietermaritzburg 2014.
Keywords
African horse sickness--South Africa., African horse sickness virus--South Africa., Horses--Diseases--South Africa., Horses--Virus diseases--South Africa., Veterinary diagnostic virology., Theses--Biochemistry.