| Chronic infection of hepatitis B virus (HBV) is the main pathogenic factor for development of hepatocellular carcinoma (HCC). Its detection and staging are critical for its monitoring and hence prevention of HCC. Chronic HBV infection is determined conventionally by sero-detections for hepatitis B surface antigen (HBsAg) and/or viral DNA. However, neither of the protocols can show the dynamic expression condition of HBV genome in liver, as the viral RNAs have not been considered conventionally viral seromarkers. There is increasing evidence recently that the analysis of RNA circulating in the blood allows to establish expression profiles. A recent study on RNA extracted from sera of chronically infected individuals have established full-length RNA (fRNA) and truncated RNA(trRNA) as novel markers in HBV serology. fRNA correlated with the markers of replicative infection including HBeAg and viral DNA. In contrast, trRNA were found in the presence as well as in the absence of HBeAg and HBV DNA. Further more, this two markers can also reflect expression status of HBV genome in liver. This not only demonstrates the existence of circulating HBV RNAs, but also provided a possibility for the detection of HBV RNA in sera. Clearly, effects should be made to establish a quantitative detection system to estimate the copy number of the viral transcripts. This will be very helpful for describing the dynamic change of the markers from replicative to non-replicative infection stages, and for monitoring the anti-virus therapy after interfered by anti-virus medicine.In this study, a quantitative detecting system is described which allows us to characterize the circulating viral transcripts in detail. The copy number of fRNA and trRNA, as well as DNA/RNA of X region, Core and X-PreCore were determined, and the presence of each transcript was trial to be associates with definite stages during HBV gene replication.The sensitivity, fidelity and reproducibity of the quantitative assay was further demonstrated by applying it during lamivudine therapy in four patients with chronic HBV infection. Lamivudine inhibits viral replication at the level of progeny DNA synthesis. Specifically, its incorporation into nascent DNA causes chain termination. This applies both to the reverse transcriptase-and the DNA-dependent DNA polymerase activity of the virus. Viral nucleic acids were extracted from serial serum samples taken during a 14-week therapyand analyzed via competitive PCR and RT/PCR. Individual assays targeted the sequence reversely transcribed during genome replication, including the X, core (C) and precore-X (preC-X) gene segment Identical copy numbers (109 copies per ml of serum) were determined for each of these segments before treatment. With the onset of treatment, as expected a decrease in HBV serum DNA levels was observed. However, depending on the assay used, there were significant differences in the apparent copy numbers indicating an overrepresentation of the X segment These results suggest a persistent production and release of nucleocapsids/virions containing predominantly DNA synthesized early during genome replication. In corresponding assays for HBV RNA, the copy number of X segment molecules persisted at about 10 7 copies per ml of serum, two orders of magnitude below the initial X DNA copy level. In contrast, RNA carrying the C or the preC/X segment decreased to copy numbers significantly below the ones determined for X segment molecule values. Both for X and C segments, a coexistence of DNA and RNA at similar levels was observed during the second half of the treatment period. Polyadenylated HBV RNA was determined using anchored oligo(dT) primers targeting poly (A) addition sites downstream of fRNA and within X ORF of trRNA. The respective RNA species were present at 105 copies per ml of serum during most of the treatment period followed by a decrease by a log ten unit. The excess of X segment RNA over fRNA levels suggests a packaging related removal of poly(A) 3'-ends.In conclusion, our study has prov...
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