乙型肝炎病毒基因型分型及流行病学研究
摘要
乙型肝炎病毒(HBV)是一种DNA病毒,它是引起多种肝炎的最常见的病毒之一。为不完全闭合,环状双链DNA。依据HBV全基因序列同源性≥92%或S基因序列同源性≥96%将HBV的基因型分为A-H 8种基因型。HBV基因型呈一定的地理区域分布,基因型反映了HBV自然感染史发生的变异特点,是病毒变异进化的结果,有研究表明基因型与HBV的感染途径和疾病进程相关,并可能与治疗的耐药性相关。
本文建立了一种简单易行的HBV基因型分型方法-多对型特异型引物巢式PCR的方法,对HBV的基因型进行分型的研究。经过使用PCR-RFLP方法的验证,可知本方法简单、准确、特异性强。
应用本方法对吉林省的194份HBV阳性患者的血清进行基因型分型,并进行HBV的基因型与临床流行病学的相关研究。结果,在吉林省HBV的基因型中以C基因型为主172例,占患者总数的88.7%;,BC混合型6例,占患者总数的3.1%,B基因型为16例,占患者总数的8.2%;未发现HBV其它的基因型。通过临床流行病学的研究,发现C基因型常见于较重的肝病如慢性肝炎、肝硬化和肝癌等,且相对于B基因型来说,C基因型预后较差。
Hepatitis B virus (HBV) infection is a global health problem, andmore than 2 billion people were infected with HBV, the chronic HBVinfectors are more than 35 million. China has a very high incidence ofhepatitis abut 60 million people by infected. 50% hepatocirrhosis and70-90% liver cancer come on because of the chronic HBV. There are about7-30% HBV carriers infected aberrance of HBV.
HBV genotype has any referenced worth on HBV epidemiology,genetics and diagnoses, prognosis.
An HBV genotype has been classified as sequences which have asimilarity over the whole genome of abut ≥ 92% or S genome of abut ≥96%. HBV is classified into genotypes A-H, which is important for clinicaland etiological investigation.
To establish a simple and practicable method to identify genotypes ofhepatitis B virus, ten primers sets wer designed for six genotypes accordingto genotyped-specific sequence, and used separately for PCR. The genotypeof HBV was identified according to the positive result of PCR. There wasno difference in the genotyping result of by the PCR-RFLP mothed.
To understand the distribution of hepatitis B virus genotype in Jilinprovince and its clinical investigation, Based on nest-PCR usingtype-specific primers for the determination of genotype from 194 share ofpositive blood serum in Jilin province.
The major genotype in Jilin province was genotype C, and thegenotype BC was a definite proportion.The genotype B was 16 samples , Cwas 172 samples , BC was 6 samples and did not fund others genotypes, theprevalence of genotype B was 8.2%, genotype C was 88.7% and that ofgenotype BC was 3.1%. No other genotypes (such as genotype A, E, F, G,H ) were found.We using restriction fragment length polymorphism (RFLP)-PCR tocheck up the truth of results, we found the results are true and credibility.The prevalence of genotype C showed an increasing trend in chronichepatitis, liver cirrhosis and hepatocellular carcinoma group;in contrast ,the prevalence B showed an opposite trend , although no statisticallysignificant different was observed . The HBeAg positive rate was higher,and the anti-HBe positive rate was lower in patients with chronic genotypeC infection than in genotype B.The prevalence of genotype C contributes to the former group'sprogressive liver disease and showed poor clinical outcomes.
本文建立了一种简单易行的HBV基因型分型方法-多对型特异型引物巢式PCR的方法,对HBV的基因型进行分型的研究。经过使用PCR-RFLP方法的验证,可知本方法简单、准确、特异性强。
应用本方法对吉林省的194份HBV阳性患者的血清进行基因型分型,并进行HBV的基因型与临床流行病学的相关研究。结果,在吉林省HBV的基因型中以C基因型为主172例,占患者总数的88.7%;,BC混合型6例,占患者总数的3.1%,B基因型为16例,占患者总数的8.2%;未发现HBV其它的基因型。通过临床流行病学的研究,发现C基因型常见于较重的肝病如慢性肝炎、肝硬化和肝癌等,且相对于B基因型来说,C基因型预后较差。
Hepatitis B virus (HBV) infection is a global health problem, andmore than 2 billion people were infected with HBV, the chronic HBVinfectors are more than 35 million. China has a very high incidence ofhepatitis abut 60 million people by infected. 50% hepatocirrhosis and70-90% liver cancer come on because of the chronic HBV. There are about7-30% HBV carriers infected aberrance of HBV.
HBV genotype has any referenced worth on HBV epidemiology,genetics and diagnoses, prognosis.
An HBV genotype has been classified as sequences which have asimilarity over the whole genome of abut ≥ 92% or S genome of abut ≥96%. HBV is classified into genotypes A-H, which is important for clinicaland etiological investigation.
To establish a simple and practicable method to identify genotypes ofhepatitis B virus, ten primers sets wer designed for six genotypes accordingto genotyped-specific sequence, and used separately for PCR. The genotypeof HBV was identified according to the positive result of PCR. There wasno difference in the genotyping result of by the PCR-RFLP mothed.
To understand the distribution of hepatitis B virus genotype in Jilinprovince and its clinical investigation, Based on nest-PCR usingtype-specific primers for the determination of genotype from 194 share ofpositive blood serum in Jilin province.
The major genotype in Jilin province was genotype C, and thegenotype BC was a definite proportion.The genotype B was 16 samples , Cwas 172 samples , BC was 6 samples and did not fund others genotypes, theprevalence of genotype B was 8.2%, genotype C was 88.7% and that ofgenotype BC was 3.1%. No other genotypes (such as genotype A, E, F, G,H ) were found.We using restriction fragment length polymorphism (RFLP)-PCR tocheck up the truth of results, we found the results are true and credibility.The prevalence of genotype C showed an increasing trend in chronichepatitis, liver cirrhosis and hepatocellular carcinoma group;in contrast ,the prevalence B showed an opposite trend , although no statisticallysignificant different was observed . The HBeAg positive rate was higher,and the anti-HBe positive rate was lower in patients with chronic genotypeC infection than in genotype B.The prevalence of genotype C contributes to the former group'sprogressive liver disease and showed poor clinical outcomes.
引文
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[2] Funk ML, Rosenberg DM, Lok ASF. World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants.J Viral Hep.2002,9:52-61.
[3] 戴志澄, 齐国明.血清流行病学调查,中国病毒性肝炎. 1997:39-58.
[4] 季晓辉, 张建瑶主编. 医学免疫学与医学微生物学. 科学出版 社.7-03-009469-7.
[5] Karin Kidd-Ljunggren , Yuzo Miyakawa ,Alistair H. Kidd。 Genetic variability in hepatitis B viruses . Journal of General Virology. 2002,83: 1267–1280.
[6] Okamoto H, Tsuda F, Sakugawa H,et al.Typing hepatitis B virus by homology in sequence: comarison of surface subtype. J Gen Virol, 1988,69:2575-2583
[7] Norder H, Courouce AM, Magnius LO, et al. Complete gonomes phylogenetic relatedness and structural proteins of six strains of the hepatitis B virus, which nepresent two new genotypes. Virology, 1994,198:489-503.
[8] Stuyver L, De Gendt S, Van Geyt C, et al. A new genotype of hepatitis B virus : complete genome and phylogenetic relatedness. Gen Virol,2000,81:167-174.
[9] Arauz Ruiz P, Norder H, Robertson BH, et, al. Genotype H: a new Amerindian genotype B virus revealed in Central America. Gen Virol, 2002,83(Pt 8):2059-2073.
[10] Okamoto H,Tsuda F,Tanaka T et al.Typing hepatitis B virus by homology in nucleotide sequence:comparison of surface subtype. Gen Virol,1988,69:2575
[11] Norder H,Hammas B,Lofdahl S et al.Comparison of the amino acid sequence of nine different serotypes of hepatitis B surface antigen genomic classification of corresponding hepatitis B virus strains. Gen Virol,1992,73:1201
[12] Stu yver l , De Gendt S , Van Geyt C , et al . A new genotype of hepatitis B virus : complete genome and phylogenetic relatedness. Gen Virol , 2000 , 81 : 67 -74.
[13] Arauz 2Puiz P , Norder H , Robertson BH. et al . Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America . Clin Microbiol , 2002 , 83 (pt8) : 2059 -2073.
[14] Okmoto H, Tsuda F, Saku gawa H, et al. Typing hepatitis Bvirus by homology iin nucleotide sequence: comparison of sueface subtype . Gen Virol, 1988,69:2575-2583
[15] Kao JH, Chen PJ, Lai MY, et al. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology, 2000, 118: 554-559.
[16] Sugauchi F, Orito E, Ichida T, et al. Hepatitis B virus of genotype B with or without recombination with genotype Cover the precore region plus the core gene. J Virol 2002;76: 5985–5992
[17] Lindh M, Gustavson C, Mardberg K, et al. Mutation of nucleotide 1,762 in the core promoter region during hepatitis B virus seroconversion and its relation to liver damage in hepatitis B e antigen carriers. J Med Virol, 1998, 55(3): 185-190.
[18] Dumpis U, Mendy M, Hill A, et al. Prevalence of HBV core promoter/precore/core mutations in Gambian chronic carries. J Med Virl,2001,65(4)∶664.
[19] Lopez CB,Fernandez-Sesma A, Schulman JL,et al. Myeloid dendritic cells stimulate both TH1 and TH2 immune responses depending on the nature of the antigen.J Interferon Cytokine Res,2001,21(9):763-773.
[20] Laport, M. S., A. C. de Castro, A. Villardo, J. A. Lemos, M. C. Bastos, and M. Giambiagi-deMarval. 2001. Expression of the major heat shock proteins DnaK and GroEL in Streptococcus pyogenes:a comparison to Enterococcus faecalis and Staphylococcus aureus. Curr. Microbiol. 42:264-268.
[21] Ohashi T, Hanabuchi S, Kato H, et al. J Virol, 2000, 74(20) :9610-9616.
[21] XIA Guoliang,Omana V Nainan,JIA Zhiyuan,et al.Characterization and distribution of hepatitis B virus genotypes and subtypes in 4 provinces of China. Chin J Epidemiol,2001,22(5):348-351.
[22] XIA Guoliang,Omana V Nainan,JIA Zhiyuan,et al.Characterization and distribution of hepatitis B virus genotypes and subtypes in 4 provinces of China .Chin J Epidemiol,2001,22(5):348-351.
[23] Tsubota A, Arase Y, Ren F, T. Genotype may correlate with liver carcinogenesis and tumor characteristics in cirrhotic patients infected with hepatitis B virus subtype adw. J Med Virol, 2001, 65:257-265.
[24] Chan HL, Tsang SW, Liew CT, et al. Viral genotype and hepatitis B virus DNA levels are correlated with histological liver damage in HBeAg-negative chronic hepatitis B virus infection. Am J Gastroenterol, 2002, 97:406-412.
[25] Ding X, Mizokami M, Ge X, et al. Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases in Nannning, southern China. Hepatol Res, 2002, 22:37-44.
[26] 许军,王齐欣,蒋栋,等. 乙型肝炎病毒基因型与病情轻重的关系.中华肝脏病杂志, 2003, 11:11-13.
[27] Lindh M, Andersson AS. Gusdal A. et al. Genotypes, nt 1858 variants, and geographiorigin of hepatitis B virus large scale analysis using a new genotyping method. J Infect Dis. 1997,175:1285-1293.
[28] Lindh M, Hannoun C, Dhillon AP, et al. Core promoter mutations and genotypes in relation to viral replication and liver damage in East Asian hepatitis B virus carrier. J Infect Dis, 1999,179:775-782.
[29] Mizokamt M, Nakano T, Orito E. et al. Serological detection of hepatitis virus genotype assignment using retrictionfragment length polymorphism patterns. FERS Lett 1999,450:66-71.
[30] 李淑虹,岳亚飞. 外周血单个核细胞在乙肝病毒传播中的作用. 国外医学. 妇幼保健分册. 2004,15(5):296-297
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