对拉米夫定耐药患者乙型肝炎病毒基因型与病毒载量及其核苷酸序列分析

Analysis of hepatitis B virus genotype,virus loads,pre-core/core promoter region of nucleotide sequence in lamivudine-resistant hepatitis B virus in vivo

目的探讨对拉米夫定耐药患者HBV B、C基因型的病毒载量及其核苷酸序列突变等病毒学特性的差异。方法采用ELISA、实时荧光定量PCR和DNA序列分析的方法,检测53例对拉米夫定耐药患者的HBV基因型、病毒载量及其C、P基因核苷酸序列,并将测序结果与基因库中标准HBV基因型序列进行比较分析。结果53例拉米夫定耐药患者中,B基因型22例,C基因型31例;C基因型的YIDD突变率明显高于B基因型(87.1%比36.4%,P<0.01),且C基因型的病毒载量为(7.71±0.80)lg拷贝/mL,明显高于B基因型的(6.97±0.77)lg拷贝/mL(P<0.01);而B基因型的YVDD突变率和前C区突变(G1896A)明显高于C基因型(分别为63.6%比12.9%、77.3%比32.3%,P值均<0.01)。B、C基因型的核心启动子突变(T1762/A1764)比较,差异无统计学意义(31.8%比35.5%,P>0.05)。前C区/核心启动子的突变影响拉米夫定耐药患者的病毒水平。结论拉米夫定耐药患者HBV B、C基因型的YMDD基序分别以YVDD和YIDD变异为主,且基因型C的病毒载量比B基因型要高;B基因型易出现前C区突变(G1896A)。

Objective To analyze the characteristics of lamivudine-resistant hepatitis B virus （HBV） genotypes B and C. Methods Serum samples were collected from 53 patients carrying lamivudine-resistant HBV. HBV genotype was determined by the enzyme-linked immunosorbent assay （ELISA）, HBV DNA was quantified by real-time fluorescent quantitave polymerase chain reaction （PCR） and the sequences of C gene and P gene were analyzed by sequencing and compared with stardard HBV genomie sequence in Genebank. Results In 53 lamivudine-resistant HBV infected patients, genotype B and genotype C accounted for 41. 5% （22/53） and 58. 5% （31/53）, respectively. The occurrence of reverse transcriptase rt2041 mutations （YIDD） was markedly higher in genotype C group than that in genotype B group （87. 1% vs 36.4%, P 〈 0.01）, whereas the occurrences of rt204V mutations （YVDD） and pre-core mutations were higher in genotype B group than those in genotype C group （63.6% vs 12.9 %, 77.3% vs 32.3%, respectively, both P value 〈 0.01）. However, the viral loads in genotype C group were higher than in genotype B group （（7.71±0.80）lg copy/mL vs （ 6.97±0.77 ） lg copy/mL, P 〈 0.01）. No significant difference in the occurrence of core promoter mutations （T1762/A1764） was observed between both genotype group（genotype C： 35.5% ; genotype B： 31.8%, P 〉 0.05）. The differences were not significant in the levels of HBV DNA between patients with YVDD and YIDD mutation. However, Serum HBV DNA levels in patients carrying HBV with mutations in both the precore region and the core promoter region were higher than those in patients carrying viruses without these mutations. Conclusions The YMDD mutational pattern ih genotype B and genotype C is YVDD and YIDD respectively while the viral load of genotype B HBV is higher than that of genotype C viruses. Genotype B HBV is predisposed to develop precore mutation （G1896A）.