Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene).
The complete sequence of the major core protein (VP3) gene of African horsesickness virus serotype 4 (AHSV-4; vaccine strain) was determined by analysis of a complete cDNA clone representing segment 3. The RNA was 2,789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (BTV-10) revealed 58% nucleotide similarity. Based on these data, the reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to the specific detection of AHSV using a pair of primers designed for AHSV-4 VP3 gene. Approximately 230 bp of PCR products were amplified by RT-PCR from the total RNA extracts (mRNA and dsRNA) of Vero cells infected with eight serotypes of AHSV. No product was observed analogous to other orbiviruses. The supernatant of the infected cell culture fluid without any RNA purification was also suitable as a template for RT-PCR after being denatured at 94 degrees C for 5 min. The sensitivity of this method was between 10(0) and 10(1) TCID50 when viral RNA from the supernatant of infected cell culture was subjected to RT-PCR. The whole procedure for detecting the virus RNA by RT-PCR could be carried out within 5 h. The RT-PCR with AHSV VP3 gene as a target was found to be a simple, highly specific and sensitive assay for AHSV.
Sakamoto
K
K
Exotic Diseases Research Division, National Institute of Animal Health, Tokyo, Japan.
Punyahotra
R
R
Mizukoshi
N
N
Ueda
S
S
Imagawa
H
H
Sugiura
T
T
Kamada
M
M
Fukusho
A
A
D000356:African Horse Sickness Virus / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014709:Vero Cells
D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral