STRAND-SPECIFIC DETECTION OF HANTAAN VIRUS-RNA SEQUENCES BY IN-VITRO DNA AMPLIFICATION

Hantaan virus often causes a fatal human disease, hemorrhagic fever wi th renal syndrome (HFRS). An assay for strand-specific detection and q uantitation of Hantaan virus RNA was developed based on the polymerase chain reaction (PCR). A protocol for the efficient detection of both genomic RNA and its transcript is presented, in which a reverse transc riptase reaction (RTR) followed by PCR with two common primers was use d to distinguish between positive- and negative-stranded RNA. Using th is method, the growth characteristics of Hantaan virus, strain 76-118, were studied in Vero E6 cells. Positive-stranded RNA was detected on the next day after infection, and the amount increased remarkably begi nning from 3 days after infection. The negative-stranded RNA (genomic) , was detected at 2 days after infection which predominated over the t ranscript RNA. Three days after infection, the amount of viral RNA att ained 80% of the maximum amount which was detected by 6 days after inf ection, while, 4 days after infection the amount of the positive-stran ded RNA became over 80% of the maximum amount of 6 days after infectio n and reached the amount of viral RNA. Both RNAs reached plateaus at 6 days after infection and after that the amounts of synthesized viral RNA and its transcripts were constant.