PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis.

The researchers developed a technique that quickly detects African horse sickness virus in cell culture and tissue samples, increasing testing sensitivity and confirming testing accuracy. The method uses reverse transcriptase polymerase chain reaction (RT-PCR) and was successful at detecting the virus in various equine blood and tissue samples.

Research Overview

The study involved the development and assessment of a nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR)- a powerful molecular technique that is used:

• For detecting African horse sickness virus (AHSV) – a highly infectious, non-contagious, insect-borne disease of equines that is often fatal
• In cell cultures and tissue samples

Findings and Methodology

The research findings were based on the following procedures and results:

• Primers (short strand of RNA or DNA) from genome segment three of AHSV serotype 6 (AHSV-6) were used. The RT-PCR assay resulted in the amplification (creation of multiple copies) of a 890 base pair primary PCR product.
• RNAs from nine vaccine strains of AHSV and several AHSV field isolates were detected by this assay. Central African isolates of AHSV-9 and AHSV-6 were some of the field isolates propagated in cell cultures included in the test.
• The nested RT-PCR could detect as few as six viral particles or 0.1 fg of AHSV RNA.
• A second pair of nested primers was used which created a 240-bp PCR product. This confirmed the integrity of the primary PCR product and increased the sensitivity of the PCR assay by at least 1000-fold.
• The RT-PCR assay was further applied to clinical samples and it resulted in the direct detection of AHSV dsRNA from blood and various tissue samples collected from both experimentally and naturally infected equines.

Specificity of the Method

The RT-PCR test showed a high degree of specificity, and it was found that:

• The primary or the nested PCR products were not amplified from closely related orbiviruses, total nucleic acid extracts from uninfected Vero cells, or unfractionated blood from horses and donkeys that were AHSV-seronegative and virus isolation negative.

Conclusion and Future Application

• The RT-PCR method provides a valuable tool for studying the epidemiology of AHSV and can be recommended for quick diagnosis during an disease outbreak among susceptible equines.