| Hepatitis B virus (HBV) infection remains a major public health problem globally. Morethan350million people suffer from chronic HBV infection. Chronic HBV infection is themajor risk of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). In the early stage ofHBV infection, especially when hepatitis B e antigen (HBeAg) is positive, immuneselection is weak and the virus is mostly wildtype. With the age getting older, immuneselection may increase during HBeAg seroconversion. HBV mutations tend to be graduallygenerated during a chronic immunopathological course after infection and more prevalentas chronic HBV infection progress from asymptomatic hepatitis B surface antigen (HBsAg)carrier (ASC) state to LC or HCC.HBV belongs to the hepadnaviridae, with an incomplete double-stranded DNA genomeof3.2kb. HBV genome contains four overlapping open reading frames that encode thesurface, core, X, and polymerase proteins. A major handicap in the former studies is mostinvestigations published are based on an analysis of subgenomic fragments amplified byPCR. This method does not permit analysis of the complexity of mutations present on asingle genomic molecule even when complete HBV genomes are reconstituted fromsubgenomic fragments by cloning. It is found that in Mainland China genotypes B and Care predominant. And these two genotypes have been shown to differ with other genotypesregard to HBeAg seroconversion, clinical diseases, prognosis, and response to interferontreatment.The wide-type sequence of the whole HBV genome is established with HBVstrains prevalent abroad. Therefore, establishing the gene database and the wide-typesequence of HBV strains prevalent in China is a fundamental work for HBV variationstudies to analyze the associations between HBV mutations with some HBV-relateddiseases.Since the introduction of the PCR for amplification of viral DNA, investigation of thesequence heterogeneity of the HBV genome and its role in pathogenesis and viral proteinexpression has become a focus in hepadnavirus research. HBV mutants cannot synthesizeHBeAg because of mutations in the pre-C region. Variants with mutations in theimmunodominant epitopes of the surface gene were also described and may escapeimmune elimination by neutralizing antibodies. Variants with mutations in all other genesand in regulatory regions of the HBV genome were also identified. But the biological andclinical significance and the possible role in viral pathogenesis of most of these variants arefar from being clear. Furthermore, it is important of functional analysis of virus in vitro.However, for the lack of robust infectable cell culture system，analyses of replicationof clinical HBV isolations are based on the transfection of replicative recombinant HBVDNA into hepatoma cell lines that are able to replicate and secrete HBV virions．However，most methods of the phenotype analysis based on the exchange of a cassette or onsite-directed mutagenesis do not take into account the entire HBV genome. We aim totransfect both widetype and mutated full-length of HBV of C genotype into HepG2andHep3B2.1-7cells，exploring the differences of replication and expression of them.Objectives1.To establish the widetype full-length HBV sequences of genotype B and C which areprevalent in China.2.To determine the association of HBV mutation in the whole genome with CHB，LCand HCC.3.To evaluate whether HBV mutation has any impact on replication and expression inHepG2and Hep3B2.1-7cells.MethodsSix hundred ninety-four ASCs,200CHB patients,180cirrhosis patients, and313HCCpatients with intact data of HBV genotyping, DNA sequencing, and serological parameterswere studied. Nucleotides with the highest frequencies in HBV genotypes B and C from allASCs were treated as wild-type nucleotides. We find193bases with point mutation ratehigher than10%in all the samples of CHB, LC and HCC which were assigned as“hotspots”. Multivariate regression analyses were used to explore the relationship betweenmutations with LC and HCC. We transfected both wide-type and mutant full length ofHBV of C genotype into HepG2and Hep3B2.1-7cells to assess the function of genevariability.Results1. we definited the wide-type full-length sequences of HBV genotype B and C fromHBeAg seropositive ASCs. In all the cases, the HCC patients were older, and hadsignificantly higher proportion of men and genotype C than ASCs and the patients withCHB and LC.2. Compared with ASC and CHB patients, mutations of G1613A,C1653T， A1703G,G1719T,A1762T/G1764A, T1978C,A1979G,C2288A, T2857C,T2943G,C3000T,preS2start codon mutation，preS1deletion could increase the risk of cirrhosis, yetT1758C,A109T,T705C could decrease the risk of cirrhosis in genotype C. Comparedwith ASC, CHB and LC patients，C1653T，A1762T/G1764A, A1846T, G1896A,G1899A,C1969T,G2104A,A2189C,C2354A, A3012C, A3097C, T531C,T636A,T796G,PreS2start codon mutation could increase the risk of HCC, but G1634H, G2011M，A162G,T705C were could decrease the risk of HCC in genotype C.3. Multivariate regression analyses showed that age, male, A1762T/G1764A, G1613A,C3000T, PreS2start codon mutation were independently associated with an increased riskof cirrhosis compared with ASC and CHB patients, yet T705could be a protective factor.Age, A1762T/G1764A，G1896A，G1899A, pres2start codon mutation were independentlyassociated with HCC compared with those without HCC.4. Most Haplotypic carriages with two or more HBV mutations were significantlyassociated with HCC. G1896A，G1899Aand PreS2start codon mutation were specific forHCC. A1762T/G1764A had a moderate sensitivity and specificity for HCC.5. Different combination of HBV mutation had different impact on HBsAg expressionand DNA replication in HepG2and Hep3B2.1-7cells transfected of the whole genome.The mutated HBV-mt-4(combined mutation: T1674C-T1753G-A1762T/G1764A-C1766T-G1896A-T3046C-A3150G-G3191A-T126C-preS2deletion (nt.1-55)-pres2startcodon mutation-A162G-C928T-G957A-C1110T-C1155T-C1306A-C1485T-C1491T-T2426A-T2498C-G2511A-A2574G-A2627G）showed the lowest HBsAg level. Itconcluded combined mutations (T3046C-A3150G-G3191A)more than the others. Themutated HBV-mt-5（combined mutation: T1674G-A1762T/G1764A-G1896A-C3116T-preS2start codon-A162G-A895T-G915A-T929A-C940A-A984G-A993G-C1075T-A1221T-G1230C-T1290G-G1317A-A1319C-C1347A-G1386A-C1480A-T1556A-C2444T-A2571G-A2590T-A2631G-C2651T-C2660T-G2699A-C2732T-G2741A-T2768G. It contained combined mutations A895T-T929A-C940A-C1075T-G1230C-C1480A-T1556A-C2444T-A2574G-A2590T-2651T-C2660T-G2699A-C2732T-G2741A-T2768G (Q299K-L310Q-Q314K-R410Q-K494T-V519G-S815L-L858S-M864L-S884F-T887I-R900H-S911L-G914D-L923R) rather than C928T-G957A-T2426A-T2498C (L310Q-L319I-I809N-L833P) compared with the others，but thethereis no statistical significance compared with others on DNA replication. DNA.HBeAg synthesis appeared to be prevented in all the mutated HBV replicating cells except for the widetype–HBV strains.Conclusions1.1762T/G1764A,G1613A,C3000T, PreS2start codon mutation are independentlyassociated with an increased risk of cirrhosis, yet T705could be a protective factorcompared with ASC and CHB patients.Age, male A1762T/G1764A、 G1896A, G1899A,pres2start codon mutation were independently associated with HCC compared withthose without HCC. A1762T/G1764A, G1613A, C3000T,PreS2start codon mutationwere independently associated with an increased risk of cirrhosis.compared with ASCand CHB patients, yet T705could be a protective factor. Age, male, A1762T/G1764A,G1896A, G1899A, pres2start codon mutation. C3000T and T705are novel factorsindependently associated with cirrhosis.2. Different combination of HBV mutation has different impact on HBsAg expressionand DNA replication. High HBsAg expression may be associated with the combinedmutation of T3046C-A3150G-G3191A in pres region which may impact the intergrationof the fuctional sites. The mutated HBVgenomes carried the nucleotide mutationG1896A, introducing a stop signal at codon28within the precore regionand preventingthe HBeAg synthesis which may reduce the DNA replication either. |
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