| HBV (Hepatitis B virus, HBV) infection is an important public healthissue, especialy in China. Because of the virus replication depend on thereverse transcription using a viral polymerase lacking proof-reading ability,this results in the emergence of mutant viruses that can be selected out byhost immunity, viral therapeutic agent and vaccine immunation. Some keymutations also correlated with the virus load, the severity and progress ofdisease. The stress selected mutations mainly located at the immuneepitope, targets of antiviral agents or the regulation section of virusreplication. Basal core promoter region, precore region and the adeterminant region of S gene possessed a higher mutation ratio, and alsobeen the hotspot in HBV mutation analysis.In this study, a larger sample size of serums from chronic HBVinfection children patients and related adult patients were used for mutationanalysis. Briefly, the correlation between mutations(including BCP/precore,core gene and S genes) and clinical characters(such as HBeAg status,immunodominant epitopes, HBV genotype, virus DNA load and ALT level)were analyzed in our study. The main results and conclusions weresummarized as following:1. The main genotype of HBV isoated from Children and adultschronic patients were genotype B(75%). All of the vaccinated children whofailed to be protected by hepatitis B vaccine were borned after2002, agesvaried from6monthes to12years. Adult patients were born before1992, ages varied from20to60years.2. Mutation profiles of BCP/PC regionsMutation ratio of seven sites, including nt1752and nt1753, nt1762，nt1764, nt1846, nt1896, nt1899was analyzed, and a significant differencewas found between e antigen positive and negative children patients groups.By comparison, frequencies of these e antigen seroconversion relatedmutations were significantly lower in e antigen negative HBV infectedchildren patients than that in e antigen negative HBV infected adultpatients(1896,41.1%vs91.67%;1846,17.8%vs37.5%;1899,7.8%vs20.8%), and mutation count of precore and core promoter regions ofsamples from e angtigen negative children patients is also obviously lowerthan in e antigen negative adult patients, prompting the possibility thatmutations in precore/core promoter region may not be the main reasons forthe early e antigen seroconversion process. Correlation analysis provedquantities of precore and core promoter region mutations were positivecorrelated with e antigen seroconversion ratio (R2=0.9088,0.9621forchildren and adult HBV infected patients, respectively). Correlationsbetween different mutation types and clinical characters were investigated,we found that1762/1764double mutation was associated with lower viralload and higher ALT level in HBeAg positive children patients. Themutation profile of BCP/PC regions were correlated with virus genotype.Among the11mutation sites in BCP/PC regions that showed a obvisoulydifferent distribution in genotype B and C, mutation of10sites justapperaed in genotype C, but the nt1752mutation only appeared ingenotype B. Combined mutation G1721A/A1775G/T1858C only appearedin genotype C children patients, and associated with lower age and higherDNA titer. 3. Mutation profiles of Core region.The frequency of amino acid substitutions and average number ofamino acid substitutions were much lower in Chronic HBV infectedchildren patients than in adult patients. The hotspot mutation types mainlyincluding I97L, S87G, P5T, V13A, L60V, P130T, P135Q, which located inB-cell epitopes(core74-89, core130-138) and Th epitopes(core1-20, core50-59). Few mutation located at CTL epitopes. Amino acid substitution incore region also correlated with the e antigen status, substitution ratio ofY38H, S49T, E77D, S87G, I97L, E113, P135Q and R151Q were higher ine antigen negative patients. No positive selection site was discovered incore regions in children patients, but five sites including P5, V13, S87, I97and L130showed a positive selection characters in adult patients. Positiveselection was not appeared in HBeAg positive patients, but in HBeAgnegative patients, nine amino acid sites(P5, V13, G74, A84, S87, I97, L130,P135, R151) showed a positive selection characters.8of9positiveselection sites were located at B cell epitopes(core74-89) and Thepitopes(core1-20). The amino acid substitution in core region wereassociated with higher ALT level and lower DNA titers in e antigen positivechronic infected children patients, but not in e antigen negative patients.4. Mutation profiles of S region.The prevalence of a determinant mutants was higher in vaccinatedchildren with breakthrough infection(32.9%) than in unvaccinated childrenpatients(10%) and adult patients(22.9%). Similar characters were also beenobserved in MHR regions. Hot substitution types in a determine region andMHR region including I110L、K122R、T126A、I126T、P127T、Q129R/H、M133L and G145R. The substitution of G145R, D144A and Q129R onlyobserved in vaccine immunized children patients, that may mediate vaccine escape mutation charaters. Most of amino acid substitution sites thatoccurred in epitope in vaccine immunized children patients located at Bcell epitope(52.8%), the aa substitution site of adult patients major locatedat CTL epitope(64.6%), while the substitution of nonvaccined childrenpatient were distributed on the S region. Among the CTL epitopes and Thepitopes, aa21, aa40, aa44, aa210possessed higher substitution ratio inadult patients, lower substitution ratio in vaccine immunized childrenpatients, while undetected in unvaccinated children patients. The resultsprompt the aa substitution in the four sits correlated with immune selectionpressures. The CTL epitopes S207-216and Th epitopes S37-51possessedhighest substitution ratio in adult patients and children patients, promptingthey were the major region correlated with immune selective pressure. Theamino acid substitution ratio of S region in different genotypes were alsodivergence, genotype C patients possessed higher substitution ratio thangenotype B patients. This divergences were obviously in CTL epitope, notin Th and B-cell epitope, MHR and a determinant region. The amino acidsubstitution ratio and average number of amino acid substitutions of Sregion in HBeAg negative patients were obviously higher than that ofHBeAg positive patients. This divergences were obviously in Th and CTLepitopes, not in B cell epitope, MHR and a determinant region. Substitutionof L21S, N40S and G44E were higher in HBeAg negative patient than inHBeAg positive patients. The mutation ratio of MHR and a determinantregion were associated with lower DNA titer and abnormal ALT level invaccine immunized children patients. In vaccinated children patients,mutation in B cell epitopes were associated with lower DNA titer(P<0.001),in adult patients, the lower DNA titer just correlated with mutation ratio ofCTL epitope. In HBeAg negative patients, the majorty substitution type ofS regions was non-synonymous substitution, while in HBeAg positive patients, the majorty substitution type was synonymous substitutions, thatproved HBeAg negative patients subjected to a much stronger positiveimmune selective pressure. No positive selection site was detected inunvaccinated children patients;5positive selection sites were detected invaccined children patients, mainly located at a determinant region andnon-immune epitope area, correlated with the vaccine escaped characters;In adult patients,12positive selection sites were detected, and mainlylocated at immune epitope area. The substitutions of amino acid probablywere the consequence of the long term host immune system screening. |
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