Generation And Characterization Of Novel Stable Cell Line Producing Hepatitis B Virus Resistant To Nucleoside Or Nucleotide Analogs

Hepatitis B virus, a DNA virus that replicates its genome via an RNA intermediate using reverse transcription, affects 350 to 400 million people and accounts annually for million deaths worldwide from cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Nucleoside and nucleotide analogs (NAs) treatment can suppress viral replication and delay the process of liver disease. However, due to the error-prone nature of reverse transcription, drug-resistant HBV mutants frequently arise during long-term therapy, causing treatment failure and advancing of liver disease. This issue poses a challenge to clinical practice. For detailed studies of drug-resistant variants and for large-scale screenings of potential agents against drug-resistant mutants, a reliable system other than those transient transfected cell models must be developed. However, several cell lines reported foster less overall viral replication and show less protein expression than wild-type HBV, limiting their clinical applications as screening tools. The present study was designed to generate a series of stable cell lines expressing high level of drug-resistant variants and viral proteins.In the first part, we developed eight typies of HBV expression vectors, include wild-type and several drug-resistant genotypies arised most commonly in clinic. After cell transfection and clone selection, a stable cell line producing wild-type hepatitis B virus was identified and characterized. Thus the system for large-scale screening of stable cell line producing hepatitis B virus was established.In the second part, a stable HepG2-based cell line HepG2-HS204V was identified using the system previously established. We focused on the characterization of the novel stable cell line producing hepatitis B virus resistant to lamivudine. High level of intracellular viral replication and similar extracellular DNA secretion were observed when compared to HepG2.2.15. And a ten-fold level of viral protein expression also was characterized. Drug assay revealed a definite lamivudine resistant phenotypic profile and adefovir sensitivity. The integrated HBV genome was detected by Southern blot. Compared with the HepG2.2.15 cell line, it shows a similar level of pgRNA transcription and higher subgenome RNA transcriptional level. Different typies of viral particle were pured by buoyant density separation and identified by HBsAg and HBeAg enzyme immunoassay. The Dane paticle was detected by conventional and immunogold electron microscope.In the third part, two cell clones resistant to adefovir were identified. One producing high level of N236T variant HBV called HepG2-HS236T and the other producing mild level of A181V variant were characterized in the same way. HepG2-HS236T revealed higher levels of intracellular and extracellular virus and expected definite phenotype of lamivudine sensitivity as well as adefovir resistance. The integrated HBV genome was identified by Southern blot and the viral particle was detected by electron microscope. And the A181V stable cell line just produced lower levels of both intracellular and extracellular virus and viral protein compared to HepG2-HS236T.In the fourth part, the stable cell line producing HBV resistant to entecavir also was identified. But unfortunately just lower levels of intracellular viral replication and extracellular virus secretion were observed. Further work for cell clone selection and characterization was undergoing.Together, these novel cell lines with high levels of viral replication and proteins production described here provides a potentially valuable tool for the academic study related to drug resistance and viral protein.