| BackgroundHepatitis B virus (hepatitis B virus, HBV) infection leads to worldwide epidemic, withvaried epidemic prevalence in different regions. The prevalence of HBV in China hasdeclined since the Expanded Program on Immunization began in1992. Due to the status of9300million carriers and20million chronically infection, treatment for HBV remains aserious challenge. The genome sequence of the hepatitis B virus is highly heterogeneity andwas classified into nine major genotypes (A-I). Different genotype is associated with differentbiological characteristics, response to drugs, disease progression, and clinical outcome.Genotype B and C were the most prevalent genotypes in China. It was a leading cause forhepatic fibrosis, liver failure and hepatocellular carcinoma of HBV patients.Currently, the overall objective for chronic hepatitis B treatment was: Long-termsuppress HBV DNA level as low as possible to reduce inflammation and fibrosis, and delaydisease progression and complications. Nucleos (t) ide analogs are common anti-HBV agents.There are four NAs have been proved for clinical usein china: lamivudine (LAM), entecavir(ETV), tebivudine (LdT) and adefovir dipivoxil (ADV). The tenofovir disoproxil fumarate(TDF) is still under clinical trial in China. Nucleos (t) ide analogs act directly on the HBVreverse transcriptase (RT) and are potent inhibitors of HBV replication. However, they cannot completely eradicate the covalently closed circular DNA (cccDNA) in the liver cells.Therefore, through the long-term use of NAs suppresses viral replication, it also leads totreatment failure due to the selection and flourish of mutant strains in quasi-species underdrug pressure.The cell-line model is a convenient and efficient technology for studies on the infectionhistory, biological characteristics, drug efficacy, treatment regimen, new drugs and vaccines.Application of HBV infection model is limited based on the fact that HBV infects exclusivelyhepatic cells, besides, culturing condition is strict and the cells show slow sensitivity to thevirus. Virus transiently transfected system based on hepatoma cell line cannot support stablelong-term viral replication. This system is not suitable for the standardized experiment, suchas drug sensitivity test. There are several stable cell lines can secrete HBV virons that have been established more than20years, such as the widely used HepG2.2.15cell line. In orderto study the anti-viral resistance mutations, other laboratories established the HBV stable cellline with drug-resistance mutations. There are some problems in such cell-line models: Thefirst, low HBV DNA, low stability and difficult to simulate the natural infection status;second, lacking of genotype B and C strains stable cell lines which are popular in China；finally, the artificial drug-resistant mutant cell lines, which maybe affected the phenotype ofthe whole HBV. It is necessary to isolate viral isolates from the Chinese patients withwild-type or drug-resistance mutations HBV full-length genome sequences of prevalentgenotypes in China and establish corresponding stable cell lines. Such research would benefitlocal evaluation of antiviral drugs and prevention programs.The purpose of this study is to establish popular HBV strain stable cell line in China, thegenotype CHBV strains were isolated from Chinese patients. We got the differentdrug-resistant representative mutations stable cell lines by integrating HBV gene into thegenome of HepG2hepatoma cell lines, and preformed phenotype analysis for the variety ofnucleoside and nucleotide analogues, and provided an ideal cell lines for anti-HBV efficacyand evaluated the new antiviral drugs.Objective(1) Full-length genome of genotype B or C HBV was isolated from the serum of patients withchronic hepatitis B in China, wild type HBV and typical drug-resistant mutation cloneswere picked out to construct the eukaryotic expression vectors of HBV by differentstrategies. To screen and build stable cell lines secrete long-term high level HBV DNA,HBsAg and HBeAg and complete HBV virons.(2) To evaluate the clones of stable cell lines replication capacity by various methods. Toconfirm the stable cell lines drug-resistant phenotypic characteristics, we analyzed fournucleos(t)ides in vitro.Method(1) In this study, HBV DNA were extracted from serum samples that obtained from chronichepatitis B patients in the302Hospital of PLA RT/S gene and precore/core wereamplified by polymerase chain reaction (PCR) and sequenced by direct sequencinganalysis. Phylogenetic tree of the RT/S region sequence was generated by MEGA4software for genotype analysis. Three HBV strains of genotype C (wild-type strains wt;entecavir resistant strains ETVr; multi-drug resistant strainsMDR) was amplifiedinfull-length, sequenced and cloned the full-length HBV genome. According to thefull-length HBV sequences, we designed the primers，and got the1.1-fold and1.38-fold HBV replicons by constructed the pTriEx-mod and pCDNA3.1(+) expression vectorwith the products from PCR, and transfected the HBV replicons into HepG2cell line.Positive cell clones were selected by G418, and amplified the full-length HBV genomicDNA in supernatant by PCR.(2) G418-resistantclonessecreting HBV DNA, HBsAg and HBeAg were sub-cloned bylimiting dilution. Sub-cloned cells with high levels of HBV DNA and antigens wereexpanded and validated by immunohistochemistry, real-time PCR, Southern blotting,electron microscopy (EM), DNA sequencing and phenotypic analysis of drug resistance.(3) The common nucleoside analogues (acid) in China: LAM, ETV, ADV, and TDF wereused to evaluate the various stable cell lines application effect.Results(1) Wild-type1.3-foldreplicon of the genotype B HBV strains and1.1-fold ETVr and MDRvector were constructed and denominated pN31-51-6-1, pTriEX-1.1-63-2,pTriEX-1.1-37-17. Three HBV-replicating cell lines HepG2.51-6-1(wild-type),HepG2.A64(ETV-r) and HepG2.1403F (MDR) were acquired which stably propagatedover29months,44months and29months, respectively. All the cell lines were confirmedby the HBV sequences in culture supernatant and the original HBV vectors. The cell lineshad higher or comparable levels of secreted HBV DNA, HBsAg and HBeAg compared tothat produced by HepG2.2.15cells. The average HBsAg/HBeAg OD values were3.25±0.41/3.13±0.47,1.83±0.78/1.32±0.63,2.24±0.74/1.0~8±0.26, respectively. HBcAgand HBsAg were detectable by immunohistochemistry. Culture supernatant/intracellularviral replication intermediates core particles DNA were4.53×10~5/4.57×10~7copies/ml,4.27×10~5/6.47×10~7copies/ml,1.77×10~5/7.41×10~7copies/ml,2.63×10~5/2.34×10~8copies/ml, respectively. The cccDNA were9.6,22,6.8copies/cell respectively. The HBVreplication intermediates showed three clear belt of rcDNA, dsDNA and ssDNAbySouthern blot analysis. The42nm large and20nm small HBV particles were observed bythe transmission EM.(2) Viral replication in HepG2.51-6-1cells was susceptible to LAM, ADV, ETV and TDF,similar with HepG2.2.15cells. Viral replication in HepG2.A64cellsdecreased>1000-fold susceptibility to LAM and345-fold susceptibility to ETV, butremained sensitive to ADV and TDF compared with that in HepG2.51-6-1cells; viralreplication in HepG2.1403F cells decreased>1000-fold,102-fold,13-fold, and6-foldsusceptibility to LAM, ETV ADV, and TDF, respectively. ConclusionWe established stable cell lines, which were isolated from the popular genotype B and CHBV strains in China. The stable cell line contain different drug resistance constructs,changed the situation of China in this field that cell lines were depend on the genotype DHepG2.2.15cell line. These cell lines provided additional tools for virologic and antiviral studies. |
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