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 posttransfusional non-A, non-B hepatitis, is a positive stranded RNA virus, it is supposed to replicate via a negative RNA strand. Although strand specific reverse transcription-polymerase chain reaction (RT-PCR) method was recently developed to detect each strand of HCV RNA, the specificity of the strategy has remained to be determined. In this study, using in vitro transcribed positive and negative stranded HCV RNAs mixed with hepatic cellular RNA from normal liver, we found that this strategy did not distinguish between the two RNA strands, but that chemical modification of RNA samples at the 3' end followed by strand specific RT-PCR made specific detection possible. Liver tissues, sera and peripheral blood mononuclear cells (PBMC) from ten patients with chronic HCV infection were analyzed with the novel strategy of RT-PCR combined with RNA modification. Positive and negative strands of HCV RNA were detected in liver tissues of ten (100%) and nine (90%) cases, respectively. Negative RNA strand was detected also in sera of five cases (50%), positive strand being detected in nine cases (90%). In PBMC, positive strand of HCV RNA was detected in eight cases (80%), whereas negative strand in only one case (10%), suggesting that HCV has much less cellular tropism to PBMC than to hepatocytes.
Gunji
T
T
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Kato
N
N
Hijikata
M
M
Hayashi
K
K
Saitoh
S
S
Shimotohno
K
K
D000328:Adult; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D001894:Borohydrides / Q000494:pharmacology; D002908:Chronic Disease; D017931:DNA Primers; D005260:Female; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000382:microbiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008099:Liver / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010504:Periodic Acid / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase
D016375:Antisense Elements (Genetics); D001894:Borohydrides; D017931:DNA Primers; D012367:RNA, Viral; D010504:Periodic Acid; C025364:sodium borohydride; D012194:RNA-Directed DNA Polymerase