Real Time RT-PCR Assays for Detection and Typing of African Horse Sickness Virus

Abstract

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.

Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques

“…Samples that gave different results in the Luminex and ELISA tests were also analysed using an OIE‐recommended AHSV SNT (OIE, ). In this study, the SNT was performed with ASHV serotypes 2, 7 and 9, which are the most prevalent serotypes in the area (Diouf et al., ; Bachanek‐Bankowska et al., ).…”

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

“…Although these assays accurately confirmed the serotype of historic prototype South African strains of AHSV, they showed considerable variation in determination of the AHSV type of field strains. More recently, TS RT-qPCR assays for the detection and typing of AHSV have been developed and described (Bachanek-Bankowska et al, 2014).…”

Immunoinformatics Prediction of Peptide-Based Vaccine Against African Horse Sickness Virus

“…(VP2) is the major protective antigen of (AHSV) that is responsible for the serotype formation. The majority of neutralizing epitopes are located on (VP2) which can be considered as the main target of immune response to the virus [11][12][13]17,18].…”

“…Polyvalent live attenuated vaccine (LAV) that is used to control the disease in Africa was proposed to be unsafe due to the probability of re-assortment between AHSV serotypes. In addition, serotypes 5 and 9 are normally excluded from the (LAV) formulations as serotype 5 is difficult to attenuate and partially crossreacts with serotype 8 and serotype 9 partially cross-reacts with serotype 6 [1,9,11,12,[19][20][21]. A recent Approach applied is the recombinant technology that was used in the development of recombinant modified vaccinia ankara (MVA-VP2) vaccine which is highly protective against (AHSV), but the scale of it is limited to only two serotypes; (AHSV-4) and (AHSV-9) [5,9,10,13,17,18,22].…”

“…Peptides elicit more desirable manipulation of immune response through the use of the B-cell epitopes which mainly induce antibody production and the T-cell epitopes that induce cellular response and cytokine secretion as cytotoxic T-cells. This approaches regarding the molecular basis of antigen recognition and HLA binding motifs to host class I and class II MHC proteins is highly supported by the immunoinformatics which aids in designing epitopes based vaccine motifs that serve as therapeutic candidates for many infectious diseases [5,7,11,12,[23][24][25][26].…”