SPECIFIC DETECTION OF POSITIVE AND NEGATIVE STRANDED HEPATITIS-C VIRAL-RNA USING CHEMICAL RNA MODIFICATION

Since hepatitis C virus (HCV), a major causative agent of posttranfusi onal. non-A, non-B hepatitis, is a positive stranded RNA virus, it is supposed to replicate via a negative RNA strand. Although strand speci fic reverse transcription-polymerase chain reaction (RT-PCR) method wa s recently developed to detect each strand of HCV RNA, the specificity of the strategy has remained to be determined. In this study, using i n vitro transcribed positive and negative stranded HCV RNAs mixed with hepatic cellular RNA from normal liver, we found that this strategy d id not distinguish between the two RNA strands, but that chemical modi fication of RNA samples at the 3' end followed by strand specific RT-P CR made specific detection possible. Liver tissues, sera and periphera l blood mononuclear cells (PBMC) from ten patients with chronic HCV in fection were analyzed with the novel strategy of RT-PCR combined with RNA modification. Positive and negative strands of HCV RNA were detect ed in liver tissues of ten (100%) and nine (90%) cases, respectively. Negative RNA strand was detected also in sera of five cases (50%), pos itive strand being detected in nine cases (90%). In PBMC, positive str and of HCV RNA was detected in eight cases (80%), whereas negative str and in only one case (10%), suggesting that HCV has much less cellular tropism to PBMC than to hepatocytes.