黑龙江肇东自然人群乙肝病毒分子流行病学初步分析

发布时间：2018-07-25 12:58

【摘要】： 目的:了解黑龙江省肇东市自然人群中乙型肝炎病毒(HBV)基因型、血清亚型分布情况及基因型与血清亚型的关系;了解自然人群中HBV S基因的核苷酸水平变异及氨基酸水平变异情况;分析HBV基因型与临床转归之间的关系。方法:2005年,以黑龙江省肇东市双胜村和先锋村两个农村为研究现场,以全屯居民为研究对象,对全屯居民进行了一次横断面血清流行病学调查,采用固相放射免疫(SPRIA)法检测HBsAg、抗-HBs和抗-HBc三项乙肝病毒感染指标,采用酶联免疫(ELISA)法检测HBeAg和抗-HBe两项感染指标。结合1986年这两个现场的血清流行病学调查资料,综合ALT、B超和临床体检等结果,按照2005年12月10日制定的《慢性乙型肝炎防治指南》的诊断标准,选择可作出明确临床诊断的110例作为研究对象。其中无症状HBV携带者(ASC)86例,慢性乙型肝炎(CH)12例,肝硬化(LC)5例,肝细胞肝癌(HCC)1例,恢复6例。男性60例,女性50例。平均年龄36岁。对从110例研究对象中抽取的血清标本采用巢式PCR法扩增HBV DNA。利用QIAamp DNA Blood Mini Kit,按照试剂盒操作说明书要求从200μl血清中提取HBV DNA,将其作为模板,设计合成2对引物扩增HBV S基因。扩增产物经1%琼脂糖凝胶电泳鉴定后,应用Wizard SV Gel and PCR Clean-Up System试剂盒进行纯化回收。将回收后的产物送生物公司利用ABI 3773自动荧光测序仪测序。利用DNASTAR软件对测序标本的核苷酸序列与从GenBank中获取的HBV A-H各基因型标准序列进行比对分析,绘制基因进化树,确定基因型。由核苷酸序列推导氨基酸序列,确定血清亚型。应用SAS 8.1软件对实验结果进行Fisher确切概率法的统计学处理分析,以P0.05为有统计学意义。结果: 110例血清标本中,97例HBV DNA阳性,阳性率为88.2%。97例标本的基因型分布为:B基因型9例,占9.3%;C型87例,占89.7%;D型1例,占1.0%。血清亚型分布为:adr亚型77例,占79.4%;adw2亚型11例,占11.3%;ayr亚型8例,占8.2%;ayw2亚型1例,占1.0%。9例B基因型标本的血清亚型均为adw2亚型;87例C基因型标本的血清亚型为: adrq+ 75例(占86.2%),adrq- 2例(占2.3%),adw2 2例(占2.3%);ayr 8例(占9.2%);1例D基因型标本的血清亚型为ayw2亚型。用Fisher确切概率法对不同基因型的血清亚型构成进行统计分析,P值为0.0000,有统计学意义。 9例B基因型感染者中,4例(44.4%)为ASC,5例为CH(55.6%);87例C基因型感染者中,3例为恢复者(3.4%),73例为ASC(83.9%),7例为CH(8.1%),3例为LC(3.4%)和1例为HCC(1.2%);1例D基因型感染者为ASC(100%)。Fisher确切概率法对不同基因型的临床转归进行统计分析,P值为0.0288,有统计学意义。本次试验共获得84例HBV S基因540nt完整序列结果。通过对这些核苷酸序列的碱基替换位点以及由核苷酸序列推导出的氨基酸序列的突变位点分析,发现131个位点有碱基替换现象,其中40个位点为同义突变, 89个位点为错义突变,2个位点为无义突变。主要结构同源区(MHR,aa100-160)发现45个位点有碱基替换现象,其中18个位点为同义突变,27个位点为错义突变。错义突变中有2个位点产生了有意义的氨基酸突变—Q129H(1例)和M133L(1例),位于抗原决定簇a的第一个茎环结构内。1例在aa119~120间有三个氨基酸TTE的插入。HBV S基因每100个核苷酸替换的频数为2.19。结论: 1黑龙江肇东市HBV基因型分布以C基因型为主,存在少量的B型和D型。 2黑龙江肇东市HBV血清亚型分布以adr亚型为主(adrq+和adrq-均有,但以adrq+为主),存在少量的adw2、ayr和ayw2亚型。 3 HBV不同基因型的血清亚型的构成不同。B基因型标本的血清亚型均为adw2;C型标本的血清亚型主要为adr,有少量的adw2和ayr;D型标本的血清亚型为ayw2。提示HBV基因型与血清亚型存在一定的相关性。 4 HBV不同基因型的临床转归不同。B基因型感染者中ASC和CH均有,CH与ASC者相比稍多。C基因型感染者中以ASC为主,CH次之,其次为恢复、LC和HCC。提示B基因型感染者较易形成CH,C基因型感染者各种临床转归均有,绝大多数为ASC。 5 HBV S基因在许多位点发现碱基替换现象,但近1/3碱基替换为同义突变。自然人群中存在一定的HBV S基因突变,未发现疫苗相关突变。核苷酸替换的平均频数较低,提示S基因相当保守。
[Abstract]:Objective: to understand the genotype of hepatitis B virus (HBV), the distribution of serum subtypes and the relationship between the genotype and the subtype of serum in the natural population of Zhaodong, Heilongjiang, and to understand the variation of the nucleotide level and the amino acid level of the HBV S gene in the natural population, and to analyze the relationship between the HBV type and the clinical outcome.
Methods: in 2005, two rural areas of Zhaodong city and Xianfeng village in Zhaodong, Heilongjiang Province, were used as the research sites. The residents of whole Tuen Tuen were studied. A cross-sectional sero epidemiological survey was carried out on the residents of Tuen Tuen. The solid phase radioimmunoassay (SPRIA) was used to detect the HBsAg, anti -HBs and anti -HBc hepatitis B virus infection indexes, and the enzyme linked immunization (ELI) was used. SA) was used to detect two indexes of HBeAg and anti -HBe infection. Combined with the data of the sero epidemiological investigation of the two sites in 1986, combined with the results of ALT, B ultrasound and clinical examination, according to the diagnostic criteria of the guide for the prevention and treatment of chronic hepatitis B made in December 10, 2005, 110 cases of clinical diagnosis were selected as the research object. 86 cases of symptomatic HBV carriers (ASC), 12 cases of chronic hepatitis B (CH), 5 cases of cirrhosis (LC), 1 cases of hepatocellular carcinoma (HCC), 6 cases, 60 males and 50 females. The average age was 36 years old. The serum samples extracted from 110 subjects were amplified by the nested PCR method for HBV DNA. using QIAamp DNA Blood Mini, according to the kit operation instructions HBV DNA was extracted from the serum of 200 mu L, which was used as a template to design and synthesize 2 pairs of primers to amplify HBV S gene. The amplified product was identified by 1% agarose gel electrophoresis and purified and recovered by Wizard SV Gel and PCR Clean-Up System kit. The ASTAR software compares the nucleotide sequence of the sequenced specimens with the standard sequence of HBV A-H genotypes obtained from the GenBank, draws the gene evolution tree and determines the genotype. The amino acid sequence is deduced from the nucleotide sequence and the serum subtype is determined. The statistical processing score of the exact probability method of Fisher in the experimental fruit is carried out by the SAS 8.1 software. In the case of P0.05, there is a statistical significance.
Results: of the 110 serum specimens, 97 cases of HBV DNA were positive, and the positive rate was the genotype distribution of 88.2%.97 samples: 9 cases of B genotype, 9.3% in C type, 89.7% in C type and 1 in D type, which accounted for 77 cases of adr subtype, 79.4%; adw2 subtype 11, Ayr subtype 8, 8.2%; ayw2 subtype 8.2%; ayw2 subtype 1 cases accounted for 1.0%.9 cases genetic specimens Serum subtypes were adw2 subtypes; serum subtypes of 87 C genotypes were adrq+ 75 cases (86.2%), 2 cases (2.3%), adw2 2 cases (2.3%), Ayr 8 cases (9.2%), and 1 D genotypes of serum subtype ayw2 subtype. The serum subtypes of different genotypes were statistically analyzed with Fisher exact probability method, P value 0. There were P values 0. There were P values. Statistical significance.
Among the 9 cases of B genotype infection, 4 cases (44.4%) were ASC and 5 were CH (55.6%). 87 cases of C genotype infection, 3 were restorer (3.4%), 73 were ASC (83.9%), 7 was CH (8.1%), LC (3.4%) and HCC were HCC (ASC), and ASC (ASC).Fisher probability method for the clinical outcome of different genotypes, P values were 0.0288, there is a statistical significance.
A total of 84 HBV S gene 540nt sequences were obtained in this experiment. By analyzing the nucleotide substitutions of these nucleotide sequences and the mutation sites of amino acid sequences derived from nucleotide sequences, 131 sites were found to have basic substitutions, of which 40 were synonymous, 89 were missense mutations and 2 loci were used. The main structure homologous region (MHR, aa100-160) found that 45 sites have base substitutions, 18 of which are synonymous and 27 are missense. 2 sites in the missense mutation produce significant amino acid mutations - Q129H (1 cases) and M133L (1 cases), and.1 in the first stem ring structure of the antigen determinant a in a There were three amino acid TTE insertions between a119 and 120. The frequency of HBV S gene substitution was 2.19 per 100 nucleotides.
Conclusion:
The distribution of HBV genotypes in Zhaodong City of Heilongjiang Province was mainly C genotype, with a small number of B and D genotypes.
2 The distribution of HBV serotypes in Zhaodong City of Heilongjiang Province was mainly ADR subtypes (adrq + and adrq - both, but mainly adrq +), with a small number of adw2, Ayr and ayw2 subtypes.
The serum subtypes of different genotypes of different genotypes of 3 HBV were all adw2 in the serum subtypes of.B genotypes; the serum subtypes of C specimens were mainly ADR, a small amount of adw2 and Ayr; the serum subtypes of D type specimens were ayw2. suggesting that there was a certain correlation between the HBV genotypes and the serum subtypes in the D type specimens.
The clinical outcomes of 4 HBV genotypes were both ASC and CH in different.B genotypes. There were a little more.C genotypes in CH and ASC than those with ASC, and the second was the recovery. LC and HCC. suggested that the B genotypes were easier to form.
The 5 HBV S gene found base substitution at many sites, but the near 1/3 base was replaced by a synonymous mutation. There was a certain HBV S gene mutation in the natural population, and no vaccine related mutations were found. The average frequency of the nucleotide substitution was low, suggesting that the S gene was rather conservative.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R181.3

【相似文献】