Strand-specific detection of Hantaan virus RNA sequences by in vitro DNA amplification

Hantaan virus often causes a fatal human disease, hemorrhagic fever with renal syndrome (HFRS). An assay for strand-specific detection and quantitation 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 transcriptase reaction (RTR) followed by PCR with two common primers was used to distinguish between positive- and negative-stranded RNA. Using this 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 beginning from 3 days after infection. The negative-stranded RNA (genomic), was detected at 2 days after infection which predominated over the transcript RNA. Three days after infection, the amount of viral RNA attained 80% of the maximum amount which was detected by 6 days after infection, while, 4 days after infection the amount of the positive-stranded RNA became over 80% of the maximum amount of 6 days after infection 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.