SPECIFIC DETECTION OF HEPATITIS-C VIRUS MINUS-STRAND RNA IN HEMATOPOIETIC-CELLS

The presence of hepatitis C virus (HCV) negative strand RNA in extrahe patic compartments based on PCR detection assays has been suggested in many reports with very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n = 20) and extrahepatic (sera, n = 32; PBMC , n = 26 and fresh bone marrow cells, n = 8) compartments from infecte d patients with three different reverse transcriptase (RT)-PCR-based a ssays using primers located in the 5' noncoding region, with or withou t a rag sequence, or in the nucleocapsid (CAP). Samples were selected to display different viral loads (10(5)-3 x 10(7) genomic equivalent/m l or gram) and viral genotypes (n = 5). Using synthetic as well as bio logical templates, we could document extensive artifactual detection o f negative strand RNA, due to self priming and mispriming events, when either 5' noncoding region primer pair was used, whereas both artifac ts were dramatically reduced (mispriming) or eliminated (self-priming) using CAP-based RT-PCR assay. Mispriming artifacts facts were directl y correlated to the titer of positive strand RNA present in the sample . Using the CAP-PCR assay, the presence of HCV negative strand RNA was found in 75% of livers (16:20) and only 8% of PBMC, independent of th e genotype involved, but could not he documented in sera (0:32) and fr esh bone marrow cells (0:6). These findings suggest that caution regar ding the type of RT-PCR assay used and the level of HCV positive stran d RNA present in the biological sample analyzed has to be taken to avo id false identification of viral reservoirs. The findings suggest that hematopoietic peripheral cells can support HCV replication, although in a very limited number of carriers.