A New Strategy For Constructing In Vitro Replication-Competent Replicon Of Hepatitis B Virus And Its Preliminary Application

Hepatitis B virus (HBV) infection represents a global public health problem, with over 360 million chronically infected patients worldwide. For the lack of a robust infectable cell culture system, analyses related to replication of clinical HBV isolations are based on the transfection of replicative recombinant HBV DNA into hepatoma cell lines that are able to replicate and secrete HBV virions. However, until recently, the methods of the phenotype analysis based on the exchange of a cassette or on site-directed mutagenesis do not take into account the entire HBV genome variability. Meanwhile, current methods for constructing 1.1 genome length units work well for drug susceptibility assays, but are not very suitable for research on HBV replication capacity or regulation since a mammalian promoter has to be used to drive pgRNA transcription. Here we described a new strategy for constructing 1.3 genome length units that contain HBV intrinsic promoter necessary for pgRNA transcription and supporting HBV replication in vitro. This strategy may help investigations related to HBV, such as drug resistance assay and replication capability assay for specific HBV strains.Objective: To establish a new strategy for constructing 1.3-genome-length HBV DNA recombinant plasmids supporting HBV replication in vitro that enables the efficient cloning and expression of HBV isolates from patient sera.Methods: 1.Two chronic Hepatitis B patients were included in our study. Overlap extension PCR was used to get the 0.9 and 0.4 unit genome length HBV DNA. Using the SpeⅠsite, we ligated the 0.4 and 0.9 unit and cloned the 1.3 unit into vector pCDNA3.1, and named pHBV1.3-1, pHBV1.3-2 respectively.2. Replicons were transfected into HepG2 cells and allowed to replicate for 5 days, after which intracellular replicative intermediates were extracted and analyzed by Southern blot. We used the digoxigenin-labeled DNA hybridization assays to detect HBV DNA, and detected the probe labeling efficiency.3. pHBV1.3-1 and pHBV1.3-2 are transfected into HepG2 cells, and treated with entecavir drugs (0, 0.5, 2, 10, 100nmol/L) for 4 days. Viral replicative intermediates are purified and subjected to Southern blot analysis. The sensitivity of this method was tested at the same time.Result: 1. Two 1.3-genome-length HBV DNA recombinant plasmids from HBV isolates of 2 patients were constructed.2. Various replicative intermediates - relaxed circular DNA (rcDNA), double-stranded linear DNA (DSL) and single-stranded DNA (ss DNA) - can be detected by Southern Blot clearly.3. Entecavir drug susceptibility assays through our new strategy shown IC50-1=1.8±0.34 nmol/L,IC50-2=1.4±0.27 nmol/L,for pHBV1.3-1/2 respectively.Conclusion: We have developed a new strategy that enables the efficient cloning and expression of HBV isolates from patient sera. Our strategy for constructing HBV 1.3 genome units with several advantages: (1) The ligation of PCR fragments relies on the overlap extension PCR, exempting the process of careful selection for restriction sites and multistep digestion and ligation; (2) The overlap region used in this strategy is a natural structure, and special design for overlap extension reaction is not needed; (3) This method enables the cloning of the whole HBV genome isolated from a given sample, and appears theoretically more appropriate to study complex individual situation. Southern Blot analysis for HBV replication and drug susceptibility assays displayed a result which provides a new methodology to study HBV pathogenesis and drug resistance mechanism.