【作者】 闫虹霞；

【导师】 张勇；

【作者基本信息】 中国疾病预防控制中心 ， 流行病与卫生统计学， 2009， 硕士

【摘要】 目的:了解河北正定地区自然人群中乙型肝炎病毒(HBV)基因型、血清型分布情况及基因型与血清型的关系;了解当地自然人群中HBV S基因α抗原决定簇氨基酸变异以及C基因A1762T/G1764A双位点变异和G1896A变异情况;初步探讨HBVα抗原决定簇氨基酸变异与实施HBV计划免疫之间的关系。了解当地HBsAg家庭聚集情况,利用分子生物学特征进一步描述当地人群感染HBV家庭聚集特点。方法:2005年于河北正定地区以家庭为单位整群采集血清样品,采用固相放射免疫法(SPRIA)进行乙肝五项感染指标检测,选择其中表面抗原(HBsAg)阳性的血清,经过QIAamp DNA Blood Mini Kit提取HBV DNA,采用巢式PCR扩增S基因区段,C基因区段,进行序列测定。利用生物信息学软件Lasergene 6.0将其与Genebank中A-H各基因型标准序列进行对比分析,确定HBV的基因型并通过核苷酸序列推测血清型。利用软件将核苷酸序列转换为氨基酸序列,进行HBVα抗原决定簇氨基酸位点变异分析,并对HBV C基因中CP和BCP常见突变A1762T/G1764A、G1896A突变进行描述和分析。对户籍明确人群以HBsAg为指标进行负二项分布拟合优度检验分析当地HBsAg的家庭聚集情况;利用MEGA4.0.1软件对样本S基因测序结果与C基因测序结果分别进行核苷酸同源性比对分析,使用邻居连接方法(Neighbor-Joining法)中p-distance统计方法构建并绘制系统发生树,bootstrap 1000次进行验证,分析同一家庭成员核苷酸相似性。用SAS 8.0整理分析实验结果,以X~2检验及fisher确切概率法进行统计学分析,以P＜0.05为有统计学意义。结果:本研究11478份血清样品中,共检测出HBsAg阳性血清549例,扩增出HBV S区基因序列并且成功测序434例,其中HBV B型基因11例,对应血清型为adw(90.9%)和ayw(9.1%);C型基因417例,对应的血清型为adr(95.2%),adw(3.6%),ayr(1.0%)和ayw(0.2%);D型基因6例,为ayw(66.7%)和adr(33.3%)血清型。当地流行HBV的α抗原决定簇氨基酸突变率为6.7%,HBV计划免疫后出生人群中126、143位氨基酸突变率显著高于计划免疫前出生人群。扩增出HBV C区基因序列并成功测序393例,基本核心启动子BCP A1762T/G1764A突变率40.5%;前C区G1896A的突变率为18.1%;HBeAg的表达与否与BCP双突变(P=0.0204)或前C区G1896A(P=0.0003)突变有明显相关性。收集户籍明确村民血样10011份,涉及家庭3619个,其中HBsAg阳性者533人,分布于455个家庭。河北正定地区HBsAg家庭聚集性不符合负二项分布(X~2=6.75,P＜0.05),聚集指数P=0.64;HBV C基因系统进化分析结果提示研究家庭中53.3%的HBsAg聚集家庭存在HBV家庭内传播。结论:河北正定地区HBV基因型以C型为主,有少量B型和D型;血清型以adr型为主,同时有adw型,ayr型和ayw型;HBV不同基因型与血清型存在一定的相关性:自然人群中存在多种HBV S区α抗原决定簇突变,其中126、143位氨基酸突变可能为与疫苗相关突变;HBV A1762T/G1764A双位点变异和G1896A变异可能与HBeAg表达有关。尚不能认为当地HBsAg阳性者分布有家庭聚集性,HBV分子生物学证据进一步提示当地可能存在HBV母婴传播、夫妻间性传播及父子间水平传播等多种家庭内传播途径。更多还原

【Abstract】 Objective: To determine the distribution of Hepatitis B virus (HBV) genotypes and serotypes in community-based Zhengding area, Hebei province and evaluate the relationship between HBV genotypes and serotypes; analyze the spectra of mutations of the a determinant of HBV, study the relationship between the mutation and the vaccination; analyze the mutations of A1762T/G1764A and G1896A in the population. To describe the households clustering of HBsAg in Zhengding area, Hebei province by HBV epidemiology and molecular evidence.Methods: 11478 people’s sera were collected by family survey and tested by SPRIA with kits in 2005 from Zhengding area, Hebei province, people’s sera with positive HBsAg were collected and the S gene and C gene were amplified by PCR. After sequencing and clastwaling with the standard genotype sequences, the HBV genotype, serotype, the mutant sites of HBVαdeterminant and the A1762T/G1764A and G1896A mutations were detemined. 10011 people’s sera from 3619 familys were collected and tested by SPRIA with kits. Negative-binomial distribution test were employed to fit distribution of the HBsAg. The Neighbor-Joining method was employed and the p-distance modle of MEGA 4.0 soft was selected to identify HBV transmission pairs with HBV S gene and C gene data. Statistical analyses were performed using Fisher exact test and X~2 test with SAS 8.1 software, P<0.05 was considered significant.Result: The study acquired 434 HBV S gene sequences and 393 HBV C gene sequences from 549 HBsAg positive samples eventually. The result of the study demonstrated that the B,C,D genotype have been detected in china and C is the major genotypes. Genotype C was 417 with serotype adr (95.2%), adw (3.6%), ayr (1.0%) and ayw (0.2%) ; Genotype B was 11 with adw (90.9%) and ayw (9.1%) and Genotype D was 6 with serotype ayw (66.7%) and adr (33.3%) . The mutation rate of the HBV a dominant was 6.7% in the community-based population and 9.4% in the population who born after the HBV immunization programe was carried out in this area. The 126、143 amino acid mutant rate is significant different between the two age groups. The BCP A1762T/G1764A and G1896A mutation rates are 40.5%, 18.1%. There is relationship between the HBeAg and the A1762T/G1764A mutation (P=0.0204) and the G1896A mutation (P=0.0003). 10011 sera samples from 3619 familys with definite household register were collected, the households clustering of HBsAg in Zhengding area is not fit the Negative-binomial distribution (X~2=6.75,P<0.05) . The clusting index is 0.64; Various kinds of HBV intrafamily transmission are indentified by the epidemiology and molecular evidence.Conclusion: The result of the study demonstrate that the B、C、D genotype and ayr、ayw、adr and adw serotype of HBV have been detected in Zhengding area,Hebei province. The C is the major genotypes and the adr is the major serotype. Genotype C with adr serotype, genotype B with adw and genotype D with ayw have the relationship. There are kinds of HBVαdeterminant mutations and 126、134 amino acid mutations are vaccine-associated mutation.A1762T/G1764A two-site mutation and G1896A mutation are the mostly common mutations in the HBV C gene region. There are virous kinds of intra-family transmission pattern in the area but the households cluster of positive serum of HBsAg does not exist.更多还原