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

Abstract

Highlights: Three triplex AHSV TS RT-qPCR assays that can be applied directly to nucleic acid extracted from blood samples collected from AHSV infected horses are described.  Multiplexing of the primers and probes for 9 AHSV serotypes increases assay output.  The use of these assays in conjunction with a previously described group specific AHSV RTqPCR assay with documented diagnostic accuracy can expedite investigation of AHS outbreaks and guide response strategies such as vaccination. | Page AbstractBlood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.

African Horse Sickness Caused by Genome Reassortment and Reversion to Virulence of Live, Attenuated Vaccine Viruses, South Africa, 2004–2014

“…We extracted genomic double-stranded RNA from all AHSV strains evaluated from virus-infected cells by using TRIzol reagent (Life Technologies, Johannesburg, South Africa). We identified and typed AHSV isolates by using group-specific (GS) real-time reverse transcription PCR (rRT-PCR) assays ( 22 ) and type-specific (TS) rRT-PCR assays targeting the gene encoding viral protein (VP) 2 (VP2) ( 23 ). …”

Establishing post‐outbreak freedom from African horse sickness virus in South Africa's surveillance zone

“…The lack of DIVA capability and the group‐specific nature of the screening PCR resulted in the requirement for further diagnostic testing to establish a final case classification. AHSV typing, using a type‐specific RT‐qPCR (Weyer et al, ), and virus isolation (VI), was performed on all suspect samples. The latter assisted in establishing the likelihood of suspect results originating from live virus circulation rather than residual RNA from prior vaccination.…”

A field investigation of an African horse sickness outbreak in the controlled area of South Africa in 2016

“…This test has a median diagnostic sensitivity and specificity of 97.8% and 99.9%, respectively . Positive samples were typed using a combination of three triplex RT-qPCR assays (Weyer et al, 2015).…”