儿童乙型肝炎病毒携带者HBV基因型分析

目的检测温州地区儿童乙型肝炎病毒携带者HBV基因型和Ba／Bj亚型的分布，及其与病毒复制水平的关系。方法采用实时荧光PCR技术进行HBV基因分型和HBVDNA定量，对鉴定为B基因型标本，采用PCR-RFLP结合PCR产物直接序列测定其基因亚型（Ba／Bj）。结果温州地区儿童乙型肝炎病毒携带者HBV基因型B、C、B＋C混合型和D型分别占43．92％、53．44％、1．59％和1．06％；C基因型的HBVDNA含量与B基因型之间差异无统计学意义（P2〉0．05）；83例B基因型HBV均为Ba亚型，未发现Bj亚型。结论温州地区儿童乙型肝炎病毒携带者HBV基因型主要为c基因型和B基因型，B基因型HBV主要为Ba亚型。     

中国4城市慢性乙型肝炎病毒感染者中B基因亚型分布

目的了解北京、石家庄、温州和深圳4城市慢性乙型肝炎（乙肝）病毒（HBV）感染者中B基因亚型（Ba和Bj）的分布情况。方法应用聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP），对北京（18例）、石家庄（22例）、温州（34例）、深圳（27例）市101例乙肝患者血清标本，经多对型特异性引物巢式PCR鉴定为B基因型慢性HBV感染者的血清标本进行B基因亚型分析，并对部分血清标本以PCR产物直接测序法确定其亚型，以验证PCR-RFLP的准确性。结果经PCR-RFLP分析101例均为Ba亚型，未发现Bj亚型，其中30例经测序分析也证实为Ba亚型。结论4城市的HBV／B主要为Ba亚型。     

兰州地区乙型肝炎病毒相关肝癌患者中病毒基因型分布特征

目的 了解中国兰州地区HBV相关肝癌患者中病毒基因型分布特征,探讨HBV基因型及其亚型与HCC之间的相关性.方法 筛选兰州地区慢性HBV感染者包括慢性乙型肝炎组(CHB组)、乙型肝炎肝硬化(LC组)和乙型肝炎相关性HCC(HCC组)共142份血清样本,其中HBeAg阳性62份,HBeAg阴性80份.对各组进行HBV DNA的提取及HBV基因组S基因的PCR扩增和测序.将测序后得到的前S/S基因序列引入NCBI的在线病毒基因分型工具来确定HBV基因型.然后采用MEGA4软件构建系统进化树进行HBV基因亚型分析.结果 慢性乙型肝炎组C基因型19份,其中C1亚型9份,C2亚型10份,B基因型17份,均为B2亚型,B型C型混合型2份,D基因型6份,均为D2亚型;乙型肝炎肝硬化组C基因型26份,其中C1亚型9份,C2亚型17份,B基因型8份,均为B2亚型,D基因型2份,均为D2亚型;乙型肝炎相关性HCC组C基因型23份,C1亚型5份,C2亚型18份,B基因型6份,均为B2亚型,B型C型混合型1份,D基因型2份,均为D2亚型.HBV基因C2亚型在慢性乙型肝炎组、乙型肝炎肝硬化组、乙型肝炎相关性HCC组中的分布差异有统计学意义(x2=10.498,P均＜0.05).结论 兰州地区HBV感染者中基因型有B型、C型、D型及B型C型混合型.其中B基因型均为B2亚型,C基因型中检出C1、C2亚型,D基因型均为D2亚型.HCC患者和LC患者的C型检出率均显著高于CHB组,C基因型与较为严重的肝病(LC和HCC)有关.     

四川省两县区乙型肝炎病毒基因型、血清亚型分析

[目的]了解四川省两县区乙型肝炎病毒的基因型和血清型. [方法]从HBsAg、HBeAg均阳性的血清中提取病毒DNA,经多聚酶链反应和S基因核苷酸片段测序后,用进化树和推导氨基酸序列确定基因型和血清亚型.[结果]49份血清标本中,含w亚决定簇的均为B基因型,含r亚决定簇的均为C基因型;其中B基因型/adw2血清亚型31份,B基因型/aywl血清亚型1份,B基因型/adw3血清亚型1份,C基因型/adrq+血清亚型14份,C基因型/ayr血清亚型2份. [结论]两县区乙型肝炎病毒以B基因型/adw2血清亚型和C基因型/adrq+血清亚型为主,B基因型/adw2血清亚型占优势;同时存在少见的B基因型/adw3血清亚型.     

感染乙型肝炎病毒不同基因型和亚型患者的临床特点分析

目的明确乙肝病毒不同基因型和亚型在国内的分布及其与病情的相关性。方法收集来自国内8个地区884例慢性乙型肝炎病毒感染者的血清和临床资料,用PCR-RFLP方法鉴定病毒基因型和亚型,分析不同基因(亚)型与病情间的相关性。结果乙肝病毒B(20.77%)和C (78.22%)基因型为最常见,仅1例为D基因型;所有B基因型均为Ba亚型,C基因型中仅发现C2和C1亚型。北方地区患者多感染C基因型(83.62%)/C2亚型(90.32%)。B基因型患者的年龄明显小于C基因型患者(P<0.000)。不同年龄段B基因型和C基因型比较发现,感染B基因型的患者在30岁以下、30～50岁和50岁以上年龄段中所占比例逐渐下降,感染C基因型的患者比例呈现相反趋势(P=0.000)。C1和C2亚型患者的年龄相近。各基因型在肝硬化、慢乙肝和无症状携带者中的分布无差别。B和C基因型(C1和C2亚型)患者的病毒量之间无差别。B基因型患者的肝脏炎症和纤维化评分明显低于C型患者(P<0.05)。结论北方地区以C基因型和C2亚型为主。B基因型患者的年龄明显小于C基因型。B和C基因型(C1和C2亚型)患者的肝脏炎症和病毒量之间无差别。B基因型患者的肝脏炎症和纤维化评分明显小于C型患者。     

乙型肝炎病毒B基因亚型(Ba,Bj)半巢式PCR检测方法的建立

目的 建立一种灵敏特异且简便易行的乙型肝炎病毒(HBV)B基因Ba和Bj亚型分型方法.方法 用DNAStar软件比较分析GenBank中登录的B基因型HBV(HBV/B)和C基因型HBV(HBV/C)的基因序列,分别设计出HBV/B型特异性引物HB及Ba和Bj基因亚型特异性引物BA和BJ.第一轮PCR反应以HBV/B型特异性引物HB作为上游引物,以日本学者Sugauchi等设计的引物HBAS-4V作为下游引物(外引物);第二轮PCR反应同样以HB作为上游引物,亚型特异性引物BA和BJ作为下游引物(内引物),在同一个反应管中进行.根据PCR产物片段的大小判定B基因亚型.随机选取实验室内经型特异性引物PCR法鉴定为HBV/B(56份)及经preS/S区序列测定证实为HBV/C(15份)的HBV慢性感染者血清标本共71份,以研究设计的引物进行半巢式PCR反应,检测Ba和Bj基因亚型,并以载有HBV Bj亚型基因组的质粒作为Bj亚型的阳性对照.然后随机选取15份B型样本的第一轮PCR产物直接测序,利用Blast和DNAStar软件将测序结果与GenBank中登录的Ba及Bj亚型序列进行同源性分析,验证该半巢式PCR法的准确性.结果 56份HBV/B血清标本经该半巢式PCR法检测,均为Ba亚型,随机选取的15份B型PCR产物直接测序,结果均为Ba亚型,与研究中应用的半巢式PCR方法检测结果一致.15份HBV/C的血清标本检测结果均为阴性.结论 建立了HBV Ba和Bj亚型半巢式PCR法.该方法灵敏、特异、简便、易行,可用于临床和流行病学大样本检测.     

实时荧光PCR法检测乙型肝炎病毒基因型

目的:建立实时荧光PCR法检测乙型肝炎病毒(HBV)基因型。方法:根据乙肝病毒基因序列选取保守区域设计引物,结合不同基因型的差异设计各型的特异荧光探针检测乙型肝炎基因B、C型。结果:200例HBV感染者中基因B型70例占35%,基因C型120例占60%,基因B,C混合型10例占5%,未捡出基因型A、D、E、F,分别取B、C型基因标本10例进行测序结果一致。结论:实时荧光PCR检测乙型肝炎基因型操作简单、快速、适合临床常规检测。     

杭州地区乙型肝炎病毒基因型分析

目的:建立多重荧光PCR技术检测乙肝病毒(HBV)基因型,并了解杭州地区乙型肝炎病毒感染者基因型分布特点。方法:首先优化TaqM an探针多重荧光PCR的反应体系,然后采用该法检测杭州地区150例慢性乙肝患者的HBV基因型,并用测序方法进行确认。结果:B基因型65例(43.3%),C基因型53例(35.3%),B+C混合型24例(16%),未分型8例(5.3%)。结论:杭州地区乙型肝炎病毒感染者基因型以B、C型为主,B、C混合型感染比例较其他地区高,TaqM an探针多重荧光PCR简单经济,可以应用于大规模HBV基因型研究。     

隐匿性乙型肝炎病毒感染可通过密切接触传播

目的探索一名出生时乙肝疫苗免疫成功后仍感染乙肝病毒的小孩的感染来源。方法采集小孩及其父母的血标本进行HBV血清学标志物和病毒载量检测,使用酚氯仿法提取HBV DNA,PCR扩增HBV S基因,将扩增产物进行克隆并测序,利用Mega 5.0和Bioedit 7.0对测序结果进行分析。结果父亲和母亲均为隐匿性乙型肝炎病毒感染,父亲、母亲、儿子的PCR产物分别获得12、11和9个克隆株。父亲克隆株的血清型为adrq+,ayw1,ayw,ayr,基因型为C2、B、B/C;母亲克隆株的血清型为ayw1,adw2,adwq+,基因型为C5、B、B/C、C/G;儿子克隆株的血清型为adrq+,基因型仅为C2。S基因序列进化发生树显示,儿子的所有克隆序列与父亲大多数的克隆序列聚集在一起,而与母亲的克隆序列没有聚集。来自儿子和父亲的S基因克隆序列有相同的氨基酸突变模式(T118K、T123N、G145A)。结论儿子乙型肝炎病毒感染的来源是父亲,提示隐匿性乙型肝炎病毒感染可通过家庭密切接触传播。     

广西那坡县发现乙肝病毒新基因亚型D11

目的 2011年,在广西那坡县发现一例HBsAg阳性携带者,其所携带的HBV利用S基因序列分型法无法分型,本研究意在用全基因组分型法对其进行分型。方法为便于前后对比,除2011年血清标本外,还收集该携带者2018年的血清标本。检测前后2份标本HBV血清标志物和病毒载量,使用酚氯仿法提取HBV DNA,巢式PCR扩增HBV全基因组,将扩增产物进行克隆并测序。利用软件Mega 6.0、 Bioedit 7.0对测序结果进行分析。结果 2011年和2018年的2份标本HBsAg均为阳性, 2份标本的PCR产物分别获得15个克隆株。对获得的克隆株进行生物信息学分析发现, 2011年该携带者体内乙肝病毒基因型为B型、 D型以及未知基因型; 2018年该携带者体内乙肝病毒基因型仅为D型。30个克隆株中有23个单独聚集成簇, bootstrap值为100%,与D1-D10亚型间核苷酸的差异4%。结论该携带者体内的HBV基因型为新基因亚型D11。     

Slave Trade and Hepatitis B Virus Genotypes and Subgenotypes in Haiti and?Africa

In Haiti, >90% of the population descended from African slaves. Of 7,147 Haitian pregnant women sampled, 44% of hepatitis B virus (HBV) infections were caused by genotype A1, which today is found mainly in eastern Africa. Twenty percent belong to a rare subgenotype, A5, which has been found only in the former Bight of Benin, a former primary slave trading post. Haitian A subgenotypes appear to have separated early from the African subgenotypes; the most prevalent genotype and subgenotype in West Africa today (E and A3, respectively) are rare in Haiti. This difference indicates that the dominant subgenotypes in Africa emerged in the general population only after the slave trade and explains the low genetic diversity of genotype E. The high prevalence of HBV genotype E in much of Africa further suggests that HBV hyperendemicity is a recent phenomenon, probably resulting from extensive use of unsafe needles.     

Reconstruction of the epidemic history of hepatitis B virus genotype D in Albania

Despite a recent decrease in the prevalence of HBsAg in the general population, Albania is still highly endemic for HBV infection. Genotype D is the most prevalent HBV strain in the Mediterranean area. We studied the prevalence and distribution of HBV genotypes and subgenotypes in a total of 73 HBsAg-positive patients living in Albania, and reconstructed the epidemiological history of the most prevalent HBV D subgenotype using a "phylodynamic" framework. A time-scaled genealogy of the Albanian patients' and reference P gene sequences with known sampling dates was reconstructed using an MCMC Bayesian approach that allows population growth to be estimated on the basis of coalescent theory. All of the Albanian subjects were infected with the HBV D genotype, and a percentage varying from 44.4% to 100% (depending on the ethnic or risk group) were infected with subgenotype D2, the most prevalent in the study population (72.4%). The other subgenotypes present in a minority of subjects were D1 (13.8%) and D3 (13.8%). The Bayesian skyline plot population dynamics analysis showed that genotype D2 entered the Albanian population in the late 1960s, and that the effective number of infections grew gradually until the second half of the 1980s and more rapidly until the mid-1990s, when it reached a plateau that still persists today. Our data suggest that political and socio-economic factors played an important role in determining the rapid spread of HBV infection in Albania.     

HBV基因型和肝细胞癌的研究现状

目前HBV可分为A～H8种基因型,基因型可分出亚型,HBV基因型及基因亚型呈现显著的地理区域性分布。感染基因型C比B的患者有更高的基因变异率、更高的HBV DNA滴度、更严重的肝损害、更低的抗病毒应答率,HBV基因型与肝细胞癌（HCC）相关。     

Molecular epidemiology of hepatitis B virus in Misiones,Argentina

Hepatitis B virus (HBV) infection is a major public health problem worldwide. The aims of this study were to describe the molecular epidemiology of HBV in the Province of Misiones, Argentina and estimate the phylodynamic of the main groups in a Bayesian coalescent framework. To this end, partial or complete genome sequences were obtained from 52 blood donor candidates.The phylogenetic analysis based on partial sequences of S/P region showed a predominance of genotype D (65.4%), followed by genotype F (30.8%) and genotype A as a minority (3.8%). At subgenotype level, the circulation of subgenotypes D3 (42.3%), D2 (13.5%), F1b (11.5%) and F4 (9.6%) was mainly identified.The Bayesian coalescent analysis of 29 complete genome sequences for the main groups revealed that the subgenotypes D2 and D3 had several introductions to the region, with ancestors dating back from 1921 to 1969 and diversification events until the late ‘70s. The genotype F in Misiones has a more recent history; subgenotype F4 isolates were intermixed with sequences from Argentina and neighboring countries and only one significant cluster dated back in 1994 was observed. Subgenotype F1b isolates exhibited low genetic distance and formed a closely related monophyletic cluster, suggesting a very recent introduction.In conclusion, the phylogenetic and coalescent analyses showed that the European genotype D has a higher circulation, a longer history of diversification and may be responsible for the largest proportion of chronic HBV infections in the Province of Misiones. Genotype F, especially subgenotype F1b, had a more recent introduction and its diversification in the last 20 years might be related to its involvement in new transmission events.     

Hepatitis B virus infection from an evolutionary point of view: How viral, host, and environmental factors shape genotypes and subgenotypes

Hepatitis B virus (HBV) has an overwhelming distribution in the world and causes important human health problems. It has infected one-third of the global population and more than 350 million people are chronic carriers. Several aspects of HBV infection confer adaptive advantages that lead to a highly efficient dissemination of the virus through different routes of transmission. HBV genotypes and subgenotypes have been associated with differences in clinical and virological characteristics, indicating that they may play a role in the virus–host relationship. In particular, a clear association between genotype A and chronic outcomes in both children and adults depending on the subgenotype involved, and between genotype C and a higher risk of complications from HBV infection, has been demonstrated. Interestingly, subgenotype A2 and genotype C are respectively likely to predominate in high-risk groups for sexual transmission and in areas where perinatal transmission is the major mode of HBV dissemination. An evolutionary approach to HBV infection, based on the principles of natural selection, may offer explanations for how modes of transmission may favor some genotypes and subgenotypes over others and, ultimately, influence HBV virulence.     

江苏省2012-2014年柯萨奇病毒A组16型分子流行病学特征分析

目的 了解江苏省2012-2014年柯萨奇病毒A组16型（coxsackievirus A16,CA16）流行毒株基因型概况。方法 收集江苏省2012-2014年儿童手足口病标本并送样,进行病毒分离培养获取相应毒株,用CA16特异性引物通过反转录聚合酶链反应技术进行VP1区基因片段扩增和测序,利用生物信息学软件对序列进行分析,并与CA16参比株序列进行同源性比较并构建基因进化树。结果 分离得到82株CA16毒株,测序结果显示全部属于基因亚型B1,VP1基因分析显示其中9株毒株序列的基因亚型为B1a,另外73株毒株序列的基因亚型为B1b。CA16分离株VP1区核苷酸和氨基酸同源性分别为87.8%～100.0%和97.5%～100.0%;与CA16国际标准株G10核苷酸和氨基酸同源性分别为75.2%～78.2%和90.5%～91.9%。结论 江苏省2012-2014年分离获得CA16毒株属于基因亚型B1,以B1a和B1b两个分支共同进化和流行,其中又以B1b为优势亚型。     

Genotyping and molecular characterization of hepatitis B virus in liver disease patients in Kenya

Hepatitis B virus (HBV) genotypes are important in both the clinical manifestation of disease and treatment response. Although Kenya belongs to the African Region (AFR-E) characterized by high mortality and hyperendemicity of HBV, there is a paucity of HBV genotyping data. The aim of this study was to molecularly characterize the basic core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolated from 61 HBsAg-positive liver disease patients attending Kenyatta National Hospital in Nairobi. HBsAg, HBeAg and viral loads were determined. HBV DNA was amplified and sequenced from 58/61 patients. In addition to the complete genome of two isolates, the BCP/PC and the complete S regions of 43 and 38 isolates, respectively were sequenced. Following phylogenetic analysis of the S region, 38 isolates clustered with subgenotype A1, whereas two isolates clustered with genotype D, one with subgenotype D1 and another as an outlier of the clade containing subgenotype D6 and the D/E recombinant. When the complete genome of the latter isolate was sequenced it clustered with D6. The majority of isolates belonged to serological subtype adw2 and only four to ayw2. Three distinct groups of subgenotype A1, distinguished by different amino acid motifs, circulate in Kenya: two in the African cluster and a monophyletic clade in the “Asian” cluster. HBeAg-negativity was a result of G1896A in genotype D isolates, whereas in subgenotype A1, the HBeAg-negativity was a result of mutations in the Kozak region (1809–1812) or precore start codon (1814–1816). Mutations at positions 1762 and 1764 occurred more frequently in HCC patients (p < 0.05). In conclusion, subgenotypes A1, D1 and D6 circulate in liver disease patients in Kenya, with A1 predominating.     

中国流行的麻疹病毒基因型和亚型趋势分析

目的分析中国（未包括香港、澳门特别行政区和台湾地区）1993～2006年本土麻疹病毒基因型和基因亚型的流行趋势。方法依据全国麻疹实验室网络监测数据库、中国疾病预防控制中心病毒病预防控制所国家麻疹实验室病毒学监测数据库,分析中国麻疹病毒分子流行病学资料。结果1993～2006年,从29个省（自治区、直辖市,下同）分离到748株麻疹病毒,其中743株为H1基因型,1株为H2基因型,1株为A基因型,3株为疫苗相关A基因型。在H1基因型中,684株为H1a基因亚型,50株为H1b基因亚型,9株为H1c基因亚型。从29个省分离到H1a基因亚型（西藏、湖北省未开展）,H1b基因亚型只从10个省分离到,H1c基因亚型在1993～1994年从4个省分离到。自2000年以来,H1a基因亚型逐年成为优势流行亚型,并呈上升趋势;H1b基因亚型逐年转为弱势,其传播于2006年被阻断;而H1c基因亚型的流行自1995年已经消失。结论通过对1993～2006年中国29个省流行的麻疹病毒分子流行病学的系统研究,阐明了在麻疹控制和加速控制阶段中国流行的麻疹野病毒的基因变异规律,以及病毒基因型别在地域和年代上的分布,证实H1基因型是中国近14年麻疹病毒流行的绝对优势本土基因型,其中H1a逐渐成为中国近几年流行的绝对优势基因亚型。     

Molecular epidemiology of hepatitis B virus in Paraguay

Hepatitis B virus (HBV) infection is a leading cause of severe chronic liver disease worldwide. The HBV epidemiology in Latin American countries is complex and the data is still scanty and fragmentary. The aim of this study was to investigate the distribution of HBV genotypes in Paraguay and to estimate the viral population dynamic and spread pattern of the main phylogenetic group. To this end, partial and complete genome sequences were obtained from 60 blood donor candidates and analysed by phylogenetic and Bayesian phylodynamic approaches.The phylogenetic analysis based on sequences of partial Polymerase/Pre-S1 overlapping region showed a predominance of the Native American subgenotype F4 (81.7%), the presence of the European subgenotypes A2 (1.7%) and D3 (8.3%), the African subgenotype A1 (3, 5%) and the Asian subgenotypes B2 (1.7%) and C2 (1.7%). The distribution of HBV genotypes was in accordance with the ethnic composition of the population.The phylogeographic analysis of subgenotype F4 complete genomes suggests that this lineage emerged and spread in the last 300 years. Paraguay was the most probable location of the common ancestor. The lineage diverged into two main clades and spread to neighbor regions, mainly Bolivia and Northwest Argentina, and Buenos Aires. The phylogeny showed a scanty geographical structure and a complex migratory pattern.In conclusion, the HBV genotypes circulating in Paraguay reflect the ethnic origin of the population. The distribution of genotypes and the phylogeographic reconstruction showed the impact of both global and local migrations in shaping the HBV molecular epidemiology in the region.