【作者】 黄勇；

【导师】 黄爱龙；

【作者基本信息】 重庆医科大学 ， 内科学， 2014， 博士

【摘要】 乙型肝炎病毒（Hepatitis B virus, HBV）感染是全球普遍的公共卫生问题，我国尤为严重。在HBV感染过程中，由于病毒自身的特殊复制方式，加上机体的免疫选择压力、抗病毒药物以及疫苗诱导等因素的作用下，HBV基因组很容易发生变异，导致免疫逃逸或耐药。同时某些关键位点的突变可能还与病毒的复制水平、疾病的严重程度及进展和预后等相关。HBV各个基因区段均可发生突变，自然发生的突变可分布于HBV整个基因组，而经过压力选择的变异可能主要位于免疫攻击的表位、药物作用的靶位或病毒复制的调控区，其中基本启动子区、前C区和S抗原a决定簇等部位是突变发生率较高的区段，也是HBV基因变异研究的热点。本研究对中国南方地区较大样本量的慢性HBV感染儿童患者以及成人对照患者血清样本，采用直接测序的方法检测了启动子区/前C区、C基因区及S区核苷酸序列的突变情况，并比较分析了C基因区及S区推导氨基酸的突变特征，探讨在分析区段序列的突变与HBeAg状态、免疫表位、HBV基因型、血清DNA载量、ALT水平和年龄等资料间的关系。主要结果及结论如下：1.儿童及成人HBV感染患者均以B基因型为主，约占75.0%，C基因型约占25.0%。2. BCP/PC区突变情况：我们在儿童患者中检出7个与HBeAg状态显著相关的突变位点，其中nt T1753G/C、A1762T、G1764A、A1846T、G1896A、G1899A突变主要见于HBeAg阴性患儿，而nt A1752G突变主要见于HBeAg阳性患儿。在成人患者中检出ntA1846T、G1896A、G1899A、A1762T、G1764A五个与HBeAg阴性状态相关的位点。7个突变位点中，仅发现A1762T/G1764A双突变在HBeAg阳性患者中与低的血清DNA载量和异常ALT水平相关，但在HBeAg阴性患者中无相关。HBeAg阴性患者BCP/PC区平均核苷酸突变数量显著高于HBeAg阳性患者，BCP/PC区突变核苷酸数量与HBeAg阴性样本比率呈正相关（儿童患者R2=0.909,成人患者R2=0.962）。儿童患者BCP/PC区平均核苷酸突变数量显著低于成人患者，年龄越低的儿童，平均核苷酸突变数量也越低，随着患者年龄的增加及感染时间增长，核苷酸突变数量逐渐累积增加。核苷酸A1846T、G1896A、G1899A在HBeAg阴性儿童患者中的突变率显著低于HBeAg阴性成人患者（G1896A突变：41.1%比91.67%；A1846T突变：17.8%比37.5%；G1899A突变：7.8%比20.8%）。BCP/PC区核苷酸突变频率与基因型相关，C基因型患者BCP/前C区平均核苷酸突变数量显著高于B基因型患者。我们筛选到11个与基因型相关的突变位点，其中nt1721等10个位点的突变主要见于C基因型患者，而nt1752突变主要见于B基因型患者。nt G1721A、A1775G、C1856T和T1858C突变主要以联合突变的模式出现。联合突变类型G1721A/A1775G/T1858C仅出现在C基因型儿童患者中，并且与较低的患者年龄和较高的DNA滴度相关。3. C基因区突变情况：慢性HBV感染儿童C基因区氨基酸突变率及平均氨基酸突变数均远低于成人患者。C基因区氨基酸热点突变类型主要有P5T、V13A、L60V、S87G、I97L、P130T、P135Q等，这些突变位点主要位于B细胞表位（Core74-89、Core130-138）和Th表位（Core1-20、Core50-69），CTL表位突变较少。C基因区氨基酸的突变与HBeAg阴性状态显著相关，Y38H、S49T、E77D、S87G、I97L、E113、P135Q、R151Q等八种突变类型在HBeAg阴性儿童及成人患者中的突变率均明显高于HBeAg阳性患者。在HBeAg阳性儿童患者中，发生氨基酸突变的患者ALT水平更高、DNA滴度更低，但在HBeAg阴性儿童患者中则无相关性。儿童患者C基因区核苷酸突变以同义替换为主，本次分析未检出正选择位点；而成人患者同义替换与错义替换相近，检出P5、V13、S87、I97和L130五个正选择位点。C基因区正选择位点更多见于HBeAg阴性患者，我们在HBeAg阳性成人患者中未检测到正选择位点，但在HBeAg阴性成人患者中检测到九个正选择位点（包括：P5、V13、G74、A84、S87、I97、L130、P135、R151），其中88.9%（8/9）的正选择位点位于免疫表位区，以B细胞表位和Th表位为主。4. S区突变情况：疫苗接种免疫保护失败儿童a决定簇氨基酸突变率32.9%，高于未接种疫苗患儿（10%）和成人患者（22.9%）。疫苗接种儿童MHR区氨基酸突变率50.6%，也高于未接种疫苗儿童（30%）和成人患者（39%）。在MHR和a决定簇突变率较高的热点突变类型主要有I110L、K122R、T126A、I126T、P127T、Q129R/H、M133L及G145R等。Q129R、D144A、G145R和N146D仅在疫苗接种组儿童中被检出，可能与疫苗逃逸相关。疫苗接种儿童免疫表位区发生的突变，主要位于B细胞表位（占52.8%），而成人患者主要位于CTL表位（占64.6%），未接种疫苗儿童散在分布于S区。在CTL和Th表位区，aa21、aa40、aa44、aa210在成人患者中突变率较高，在疫苗接种儿童中突变率较低，而在未接种儿童中未见检出，提示这四个位点的突变可能与免疫逃逸相关。突变率最高的CTL和Th表位分别为S207-216、S37-51，这两个表位可能是S抗原承受免疫选择压力的主要区域。S区氨基酸突变与基因型相关。C基因型患者S区氨基酸突变发生率、平均氨基酸突变数均显著高于B基因型患者。该差异主要存在于CTL表位区，而在Th表位、B细胞表位/MHR及a决定簇则无明显差异。aa3、aa53、aa68、aa194及aa210五个位点在C基因型儿童及成人患者中的突变率显著高于B基因型患者（P<0.001）。S区氨基酸突变与HBeAg状态相关。HBeAg阴性患者S区氨基酸突变率及平均氨基酸突变数均显著高于HBeAg阳性患者。在Th表位该差异最显著，在CTL表位也有显著性差异，但在B细胞表位/MHR及a决定簇，差异无显著性意义。位于Th表位和/或CTL表位的L21S、N40S、G44E突变与HBeAg状态最相关，在HBeAg阴性患者中的突变率明显高于HBeAg阳性患者。疫苗接种儿童MHR及a决定簇突变与低的DNA滴度及异常ALT水平相关，与年龄、性别、HBeAg状态及基因型无相关。成人患者MHR及a决定簇突变与上述临床资料间均无相关。在疫苗接种儿童患者中，B细胞表位突变与低的DNA滴度相关(P<0.001)，CTL表位突变与DNA滴度不相关；但在成人患者则刚好相反，CTL表位突变与低的DNA滴度相关(P<0.001)，B细胞表位突变与DNA滴度不相关。HBeAg阴性患者S区核苷酸突变以非同义替换为主，而在HBeAg阳性患者以同义替换为主，HBeAg阴性患者受到的选择压力更强。B基因型患者S区核苷酸突变以非同义替换为主，C基因型患者以同义替换为主，B基因型患者受到更强的选择压力，且随着年龄的逐渐增加，B基因型患者进化速度更快。在S区，未接种疫苗儿童受选择压力较低，未筛选到有显著性意义的正选择位点，氨基酸突变多为自然筛选。疫苗接种儿童受到较弱的选择压力，S区检出5个正选择位点，这些位点主要位于a决定簇及非免疫表位区，突变主要与疫苗逃逸相关。成人患者受到较强的选择压力，S区检出12个正选择位点，这些位点主要位于免疫表位区，氨基酸突变主要与免疫逃逸相关。更多还原

【Abstract】 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.更多还原