HBV CccDNA Mutation Characteristics In Pbmcs From Drug-resisted Chronic Hepatitis B Patients

HBV can be detected in peripheral blood mononuclear cells(PBMCs), or other organs outside the liver in chronic hepatitis B patients; viral proteins have been detected in those organs and it is believed that HBV can actively replicate in cells other than liver cells. Covalently closed circular DNA (cccDNA),the primary template for viral replication, is a key factor for persistent infection. The virologic characteristics are fundamentals for the outcome of infections and the efficacy of antiviral treatment. HBV cccDNA, though small in amount, remains permanently when antiviral treatment terminates due to a low level of detectable HBV DNA; cccDNA is the major source responsible for re-attacks in chronic hepatitis B infection. Consequently, test for cccDNA is an important approach to understand viral replication in vivo; PCR, and rolling circle amplification (RCA) methods have been established and improved worldwide to detect and quantitate cccDNA.In recent years, the extensive use of nucleotide analogue has brought about the intractable problem of drug resistance, which is the main cause for treatment failure. However, reports on mutation features and replication potentials of PBMCs cccDNA from drug resisted chronic hepatitis B patients are limited. This study first compared the drug resistance related mutations in RT domain between serum HBV DNA and cccDNA in PBMCs, and then examined the replication capacity of PBMCs’cccDNA. The results would strongly be in favor of understanding the mechanism of drug-associated resistance and infection outside the liver.Objectives:The purpose of this study was to analyze the characteristics of gene mutations in HBV cccDNA in PBMCs from drug resisted chronic hepatitis B patients and test its replicative and expressive activityMethods:30chronic hepatitis B patients were included in this study, all of them experienced antivirus treatment for over six months and drug-resistant strains in serum were detected. Whole blood used to segregate PBMCs and serum used to analyze drug resistance were sampled at the same time point from each patient. After cccDNA were extracted from PBMCs, Plasmid-SafeTM ATP-dependent Dnase (PSAD) digestion, rolling circle amplification and PCR unique in prime spanning the gap were applied to amplify the RT domain of cccDNA; drug resistance related mutations at sites rtM204L, rtM204V, rtM236T, rtA181V, rt184, rt202, rt250were analyzed. Additionally, all amplicons containing the full-length HBV cccDNA were cloned into pGEM-Teasy plasmid; after being purified by BspQ Ⅰ/Sca Ⅰ enzyme, the1.0X HBV cccDNA genomes were transfected into HepG2cells. HBsAg and HBV core particles were measured72hours later to analyze the natural replicative capacity of PBMCs’HBV cccDNA.Results:HBV cccDNA had been detected in16patients out of30; the detection rate is53.3%; and the incidence of cccDNA in PBMCs is independent to serum levels of HBV DNA, HBeAg, and ALT. For the16cases of cccDNA, Genotype B accounted for31.3%(5cases) and genotype C accounted for68.8%C(11cases), which consisted with the results from serum analysis. However, the HBV cccDNA isolated from PBMCs were all wild-type and contained no drug resistance mutation; the results differed from that of serum analysis. The replicative capacity was analyzed from six full-length clones that obtained from three of four chronic hepatitis B patients; HBsAg were determined by ELISA and HBV core particles were determined by real-time PCR, the results showed that the clones are able to replicate.Conclusions:Wild-type HBV strains still predominated in chronic hepatitis B patients’ PBMCs when serum HBV DNA conferred drug resisted mutation after a long term of NA treatment. HBV cccDNA segregated from PBMCs could successfully transfected into cells in vitro and complete a full replication circle, implying that PBMCs can serve as the reservior for wild-type HBV strains in the form of stabilized cccDNA.