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

Abstract

A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell cultures, were detected by this assay. A second pair of nested primers (P3 and P4) was used to produce a 240-bp PCR product. The RT-PCR described below detected as little as 0.1 fg of AHSV RNA, which is equivalent to six viral particles. The nested amplification confirmed the integrity of the primary PCR product and increased the sensitivity of the PCR assay by at least 1000-fold. Application of this RT-PCR assay to clinical samples resulted in direct detection of AHSV dsRNA from blood and a variety of tissue samples collected from equines infected experimentally and naturally. The specificity studies indicated that the primary or the nested PCR products were not amplified from, closely related orbiviruses including, bluetongue virus (BTV) prototypes serotypes 1, 2, 4, 10, 16 and 17; epizootic hemorrhagic disease of deer virus (EHDV) prototypes serotypes 1 and 2; EHDV-318, Sudanese isolates of palyam serogroup of orbiviruses; total nucleic acid extracts from uninfected Vero cells; or unfractionated blood from horses and donkeys that were AHSV-seronegative and virus isolation negative. The RT-PCR provides a valuable tool for study of the epidemiology of AHSV and can be recommended for rapid diagnosis during an outbreak of the disease among susceptible equines.