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. 2023 Aug;16(8):1682-1689.
doi: 10.14202/vetworld.2023.1682-1689. Epub 2023 Aug 19.

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan

Nurlan Sandybayev  1 , Vitaliy Strochkov  1 , Vyacheslav Beloussov  2 , Shynggys Orkara  1 , Aidyn Kydyrmanov  3 , Yelizaveta Khan  3 , Zhanat Batanova  4 , Markhabat Kassenov  5
Affiliations

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan

Nurlan Sandybayev et al. Vet World. 2023 Aug.

Abstract

Background and aim: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV).

Materials and methods: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method.

Results: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV.

Conclusion: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.

Keywords: equine influenza; hemagglutinin; horses; primers; probe; real-time polymerase chain reaction assay; virus.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure-1
Figure-1
Amplification curves of 10-fold serial dilutions of genomic RNA (5–0.00005 ng/μL per reaction) Equine influenza virus H3.
Figure-2
Figure-2
Determination of the specificity of the real-time polymerase chain reaction assay. 1- RNA of equine influenza virus (EIV), A/equine/Almaty/24/07 (H3N8); 2- RNA of EIV, A/equine/Almaty/26/07(H3N8); 3- RNA of EIV, A/equine/Almaty/27/07(H3N8); 4- RNA of EIV, A/equine/South Kazakhstan/236/12(H3N8); 5- RNA of AIV, H5; 6- RNA of AIV, H9; 7- DNA of Rhodococcus equi; and 8- DNA of Streptococcus equi.
Figure-3
Figure-3
Phylogenetic analysis of the hemagglutinin of the equine influenza virus isolates from the North-Kazakhstan region.
See this image and copyright information in PMC

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