DETECTION OF AFRICAN HORSESICKNESS VIRUS BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION (RT-PCR) USING PRIMERS FOR SEGMENT 5 (NS1 GENE)

The reverse transcription followed by the polymerase chain reaction (R T-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 se gment 5 (NS1 gene). Total RNA which contains both messenger RNA and ge nomic dsRNA was extracted by the acid guanidinium-phenol-chloroform me thod from the AHSV infected Vero cells and was used as templates to op timize the RT-PCR. A pair of primer (NP2-NP32) amplified the product o f the expected size from all serotypes of attenuated AHSV when four pa irs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected f rom 1 to 2 cells of the cell monolayer infected with 10(6) TCID50 of A HSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.