乙肝病毒基因型与应答反应关系及逆转录酶和全基因组变异位点分析
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摘要
乙型肝炎病毒(hepatitis B virus, HBV)感染呈世界范围广泛流行,全球仅慢性HBV携带者就有近四亿人。如果不经过恰当治疗的话,至少有6千万人会过早地因肝病而死亡。根据HBV全基因序列异质性≥8%的界线,可将HBV分为A～H八种不同的基因型。这些基因型反映了HBV自然感染时发生变异的特点,是病毒变异进化的结果。现有研究表明HBV基因型与疾病的临床表现、治疗效果及预后有密切的联系。
HBV具有独特的生活周期,由于其复制需通过逆转录机制,且其逆转录酶缺乏3’→5’外切核酸酶活性,故HBV变异较为常见。并且,在内源性(宿主免疫清除)和外源性(疫苗和抗病毒药物)的选择性压力作用下,更容易选择出逃避突变株。
核苷(酸)类似物是目前临床上治疗HBV感染的重要抗病毒药物,此类药物通过直接抑制HBV DNA复制,进而改善肝脏组织学病变,延缓乙肝病情进展,减少合并症的发生。但长期治疗发生病毒耐药是此类药物面临的共同问题。对抗病毒药物耐药可导致病毒学反弹及病情恶化。因此,具有强效、低耐药及无交叉耐药的抗HBV药物对于预防肝病的发展具有十分重要的意义。阿德福韦(ADV)是基本符合上述3个条件的药物。
本项研究针对我院ADVⅢ期临床5个项目238例慢性乙肝患者,所有患者均为HBeAg阳性,阿德福韦酯每天10mg,疗程48周(具体方案详见相关章节)。我们首先对所有HBV标本进行基因分型,了解本地区基因型的分布状况;然后分析ADV治疗慢性乙肝患者,不同基因型在治疗过程中病毒学、生化学指标等方面反应的差异;最后对临床耐药毒株进行HBV DNA逆转录酶(RT)区及全基因组测序分析,筛选耐药毒株的变异位点,从分子层面解析HBV耐药的机制。
在第一部份研究内容中我们首次通过对GenBank中所有S基因900余条核苷酸序列进行比对分析,找出了各基因型的型特异核苷酸,并根据GenBank中HBV 12株G型、11株H型序列自行设计扩增G型、H型的型特异引物(国内目前没有HBV G型、H型的报道),采用A～H基因型特异性引物-PCR分型法成功对218株HBV进行分型,结合S区测序,型特异核苷酸分析对余下20株也完成了分型。结果表明:B基因型159例、C型69例、B+C和B+D混合型分别有6例和4例。B型、C型、B+C和B+D混合型检出率分别为66.8%、28.9%、2.5%和1.6%。本地区基因型分布以B型和C型为主,少量D基因型以混合感染方式存在,未检出A、E、F、、G和H基因型。
对其中3株HBV进行全基因组序列分析显示:No.21和No.55 nt1838位均为a,按Ba与Bj定义划分No.21和No.55属于Ba亚型。进一步作序列比对分析显示No.21、No.55与Ba亚型差异最小(≤0.16%),属于Ba亚型,No.124与C基因型差异最小(≤2.0%),属于C基因型。这两种方法的划分结果与型特异引物PCR法相一致。
对41株B基因型pol酶RT区核苷酸、氨基酸序列分析显示其中40株某些氨基酸位点与Bj(日本亚型rt153R、rt207V和rt272C)亚型高度一致,而与Ba(亚州亚型rt153W、rt207M、rt272W)亚型不相符。由于RT区负责HBV前基因组RNA逆转录为负链DNA并指导正链DNA的合成,在进化上相对保守,说明本地区HBV病毒株与Bj亚型具有遗传学同源性,同时又具有与该地区之外的其它Ba亚型不相同的遗传学特性,据此,我们认为可以从Ba亚型进一步细分出Bac亚亚型,c代表重庆地区首次分离到的该型病毒株。
在第二部份研究内容中我们在基因分型基础上以回顾性分析的方式研究了阿德福韦酯Ⅲ期临床4个项目177例(B基因型102例,C基因型65例,10例为B+C和B+D混合型)慢性乙肝患者不同基因型对抗病毒治疗患者血清生化指标及病毒学指标的影响。结果显示:治疗48周时基因型B组和基因型C组HBV DNA下降均值分别为3.6log10copies/ml和3.1log10copies/ml,差异具有显著性(P < 0.05),而第12周、24周时两组差异无显著性(P >0.05)。治疗48周时基因型B组和基因型C组分别有43例(42.2%)和22例(33.8%)出现血清HBV DNA阴转,差异具有显著性(P < 0.05);两组HBeAg阴转率和抗HBe血清转换率差异无显著性(P > 0.05)。各时间段两组ALT复常率无显著性差异(P > 0.05)。结果表明阿德福韦酯治疗慢性乙肝48周时,部份病毒学指标(如血清HBV DNA下降均值和HBV DNA阴转率)基因型B可能优于基因型C。但由于阿德福韦酯抗病毒作用起效较缓慢,因此,需要更长的观察时间来证实这一现象。
在第三部份研究内容中根据HBV DNA值筛选出原发性耐药毒株21株及继发性耐药毒株9株。对HBV pol酶RT区进行PCR扩增、测序、核苷酸及氨基酸序列比对分析,我们首次发现多态性位点rtN118H、rtM271L和rtY141F,其发生率分别为:23.8%、14.3%和14.3%。未发现rtN236T和A181V/T突变。1例患者出现1个RT区C保守区氨基酸变异(rtM207V);6例患者出现9个HBV RT非保守区氨基酸变异。我们还成功构建了3条耐药毒株治疗前全基因序列,分析发现X基因、Core/e基因及BCP区均有重要位点发生变异。上述研究提示阿德福韦酯耐药株的氨基酸变异不仅见于RT B区181和D区236位点, C区207位点、RT非保守区和调控区也可发生多个变异,从而对HBV的耐药性产生影响。
Hepatitis B virus (HBV) infection is a global health problem, and more than 400 million people in the world are chronic carriers of the virus. The clinical manifestations of HBV infection range from acute self-limiting infection, inactive carrier state, fulminant hepatic failure and chronic hepatitis with progress to liver cirrhosis, and hepatocellular carcinoma (HCC). Based on an inter-group divergence of 8% or more in the complete genome nucleotide sequence, HBV has been classified into A～H eight genotypes. HBV genotypes exhibit distinct geographical distributions. It’s well known that HBV genotypes might influence the severity of liver disease, mutation patterns in precore and core promoter regions, and response to antiviral treatment.
In the first part HBV strains from 238 HbeAg-positive chronic hepatitis B(CHB) patients in five adefovir dipivoxil(ADV) phaseⅢclinical trials who received ADV 10mg per day for one year had been genotyped. 218 out of 238 HBV strains were genotyped by type-specific primer PCR, and the other 20 strains were genotyped with type-specific nucleotide analysis. Only three genotypes (genotype B, C and D) were observed in Chongqing, China. 159 (66.8%) cases were genotype B, 69(29.0%) cases were genotype C, 6(2.5%) cases were mixtures of genotype B and C, and 4(1.7%) cases were mixtures of genotype B and D. Genotype B and C were the main genotypes in Chongqing municipality, genotype A, E, F, G and H were not observed.
The whole genome sequence of three samples (No.21, No.55 and No,124) were analyzed. The nucleotides sequence variation between No.21, No.55 and genotype Ba were less than 0.16%, and the sequence variation between No.124 and genotype C were less than 2.0%. Nucleotides at 1838 were A in both No.21 and No.55. Both results suggested that No.21, No.55 belonged to genotype Ba . These results conformed to the type-specific primer PCR methods.
We analyzed the nucleotides and the amino acids sequences of retrotranscriptase of 41 HBV strains. Three sites of amino acids of 40 strains were identical with the subgenotype Bj(rt153R, rt207V and rt272C), while subgenotype Ba had different amino acids at these sites(rt153W, rt207M and rt272W, respectively). Retrotranscriptase is relatively conserved in the virus evolution process. This results suggested HBV strains in Chongqing area had the similar genetic background with subgenotype Bj, however, the Chongqing strains also owned its unique characteristics from other subgenotype Ba, although it belonged to Ba. Based on these observations, we think that subgenotype Ba can be further divided into sub-subgenotype Bac, c stands for Chongqing area where Bac is first reported.
In the second part, we investigated the role of HBV genotypes on the response to ADV antiviral therapy. Data of 177 HBV samples with known genotypes from HBeAg-positive CHB patients treated with ADV had been retrospectively analyzed. The clinical data include: serum HBV DNA seroclearance, mean HBV DNA reduction, HBeAg loss, anti-HBe seroconversion and serum ALT. The number of genotype B and genotype C were 102 and 65 cases, respectively. Baseline data of both groups were comparable. The mean HBV DNA reduction in patients with genotype B and genotype C at the treatment of 12, 24 and 48 weeks were 2.2log、2.1log (P>0.05), 2.7log、2.4log(P>0.05) and 3.6log、3.1log(P<0.05), respectively. At the end of therapy (48 weeks), 43(42.2%) patients and 22(33.8%) patients with genotype B and genotype C infection had achieved HBV DNA seroclearance (P<0.05), respectively. Our results suggested that HBV genotype B seemed to have a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than genotype C. But longer terms of ADV treatment are needed to verify this conclusion.
Third part: Based on the HBV DNA values, 30 HBV ADV-resistant strains (including: 21 primary resistant strains and 9 secondary resistant strains) had been selected out. After PCR amplification of the retrotranscriptase region and the nucleotide, amino acid alignment, we first found that polymorphism sites of rtN118H, rtM271L and rtY141F occurred with frequency of 23.8%, 14.3% and 14.3%, respectively. One patients with chronic hepatitis B had one amino acid variation in the RT C conserved domain,Six patients had nine amino acids variation in the RT none-conserved domain. We further constructed and analyzed the whole HBV genome sequence of three secondary resistant strains, and found that regulatory and structure elements such as X gene, BCP region and core/e gene also had important variations.
In short, our results suggested that ADV-resistant HBV strains may harbor several amino acid variances.not only in the conserved regions of RT, but also in the none-conserved and the regulatory regions.
HBV具有独特的生活周期,由于其复制需通过逆转录机制,且其逆转录酶缺乏3’→5’外切核酸酶活性,故HBV变异较为常见。并且,在内源性(宿主免疫清除)和外源性(疫苗和抗病毒药物)的选择性压力作用下,更容易选择出逃避突变株。
核苷(酸)类似物是目前临床上治疗HBV感染的重要抗病毒药物,此类药物通过直接抑制HBV DNA复制,进而改善肝脏组织学病变,延缓乙肝病情进展,减少合并症的发生。但长期治疗发生病毒耐药是此类药物面临的共同问题。对抗病毒药物耐药可导致病毒学反弹及病情恶化。因此,具有强效、低耐药及无交叉耐药的抗HBV药物对于预防肝病的发展具有十分重要的意义。阿德福韦(ADV)是基本符合上述3个条件的药物。
本项研究针对我院ADVⅢ期临床5个项目238例慢性乙肝患者,所有患者均为HBeAg阳性,阿德福韦酯每天10mg,疗程48周(具体方案详见相关章节)。我们首先对所有HBV标本进行基因分型,了解本地区基因型的分布状况;然后分析ADV治疗慢性乙肝患者,不同基因型在治疗过程中病毒学、生化学指标等方面反应的差异;最后对临床耐药毒株进行HBV DNA逆转录酶(RT)区及全基因组测序分析,筛选耐药毒株的变异位点,从分子层面解析HBV耐药的机制。
在第一部份研究内容中我们首次通过对GenBank中所有S基因900余条核苷酸序列进行比对分析,找出了各基因型的型特异核苷酸,并根据GenBank中HBV 12株G型、11株H型序列自行设计扩增G型、H型的型特异引物(国内目前没有HBV G型、H型的报道),采用A～H基因型特异性引物-PCR分型法成功对218株HBV进行分型,结合S区测序,型特异核苷酸分析对余下20株也完成了分型。结果表明:B基因型159例、C型69例、B+C和B+D混合型分别有6例和4例。B型、C型、B+C和B+D混合型检出率分别为66.8%、28.9%、2.5%和1.6%。本地区基因型分布以B型和C型为主,少量D基因型以混合感染方式存在,未检出A、E、F、、G和H基因型。
对其中3株HBV进行全基因组序列分析显示:No.21和No.55 nt1838位均为a,按Ba与Bj定义划分No.21和No.55属于Ba亚型。进一步作序列比对分析显示No.21、No.55与Ba亚型差异最小(≤0.16%),属于Ba亚型,No.124与C基因型差异最小(≤2.0%),属于C基因型。这两种方法的划分结果与型特异引物PCR法相一致。
对41株B基因型pol酶RT区核苷酸、氨基酸序列分析显示其中40株某些氨基酸位点与Bj(日本亚型rt153R、rt207V和rt272C)亚型高度一致,而与Ba(亚州亚型rt153W、rt207M、rt272W)亚型不相符。由于RT区负责HBV前基因组RNA逆转录为负链DNA并指导正链DNA的合成,在进化上相对保守,说明本地区HBV病毒株与Bj亚型具有遗传学同源性,同时又具有与该地区之外的其它Ba亚型不相同的遗传学特性,据此,我们认为可以从Ba亚型进一步细分出Bac亚亚型,c代表重庆地区首次分离到的该型病毒株。
在第二部份研究内容中我们在基因分型基础上以回顾性分析的方式研究了阿德福韦酯Ⅲ期临床4个项目177例(B基因型102例,C基因型65例,10例为B+C和B+D混合型)慢性乙肝患者不同基因型对抗病毒治疗患者血清生化指标及病毒学指标的影响。结果显示:治疗48周时基因型B组和基因型C组HBV DNA下降均值分别为3.6log10copies/ml和3.1log10copies/ml,差异具有显著性(P < 0.05),而第12周、24周时两组差异无显著性(P >0.05)。治疗48周时基因型B组和基因型C组分别有43例(42.2%)和22例(33.8%)出现血清HBV DNA阴转,差异具有显著性(P < 0.05);两组HBeAg阴转率和抗HBe血清转换率差异无显著性(P > 0.05)。各时间段两组ALT复常率无显著性差异(P > 0.05)。结果表明阿德福韦酯治疗慢性乙肝48周时,部份病毒学指标(如血清HBV DNA下降均值和HBV DNA阴转率)基因型B可能优于基因型C。但由于阿德福韦酯抗病毒作用起效较缓慢,因此,需要更长的观察时间来证实这一现象。
在第三部份研究内容中根据HBV DNA值筛选出原发性耐药毒株21株及继发性耐药毒株9株。对HBV pol酶RT区进行PCR扩增、测序、核苷酸及氨基酸序列比对分析,我们首次发现多态性位点rtN118H、rtM271L和rtY141F,其发生率分别为:23.8%、14.3%和14.3%。未发现rtN236T和A181V/T突变。1例患者出现1个RT区C保守区氨基酸变异(rtM207V);6例患者出现9个HBV RT非保守区氨基酸变异。我们还成功构建了3条耐药毒株治疗前全基因序列,分析发现X基因、Core/e基因及BCP区均有重要位点发生变异。上述研究提示阿德福韦酯耐药株的氨基酸变异不仅见于RT B区181和D区236位点, C区207位点、RT非保守区和调控区也可发生多个变异,从而对HBV的耐药性产生影响。
Hepatitis B virus (HBV) infection is a global health problem, and more than 400 million people in the world are chronic carriers of the virus. The clinical manifestations of HBV infection range from acute self-limiting infection, inactive carrier state, fulminant hepatic failure and chronic hepatitis with progress to liver cirrhosis, and hepatocellular carcinoma (HCC). Based on an inter-group divergence of 8% or more in the complete genome nucleotide sequence, HBV has been classified into A～H eight genotypes. HBV genotypes exhibit distinct geographical distributions. It’s well known that HBV genotypes might influence the severity of liver disease, mutation patterns in precore and core promoter regions, and response to antiviral treatment.
In the first part HBV strains from 238 HbeAg-positive chronic hepatitis B(CHB) patients in five adefovir dipivoxil(ADV) phaseⅢclinical trials who received ADV 10mg per day for one year had been genotyped. 218 out of 238 HBV strains were genotyped by type-specific primer PCR, and the other 20 strains were genotyped with type-specific nucleotide analysis. Only three genotypes (genotype B, C and D) were observed in Chongqing, China. 159 (66.8%) cases were genotype B, 69(29.0%) cases were genotype C, 6(2.5%) cases were mixtures of genotype B and C, and 4(1.7%) cases were mixtures of genotype B and D. Genotype B and C were the main genotypes in Chongqing municipality, genotype A, E, F, G and H were not observed.
The whole genome sequence of three samples (No.21, No.55 and No,124) were analyzed. The nucleotides sequence variation between No.21, No.55 and genotype Ba were less than 0.16%, and the sequence variation between No.124 and genotype C were less than 2.0%. Nucleotides at 1838 were A in both No.21 and No.55. Both results suggested that No.21, No.55 belonged to genotype Ba . These results conformed to the type-specific primer PCR methods.
We analyzed the nucleotides and the amino acids sequences of retrotranscriptase of 41 HBV strains. Three sites of amino acids of 40 strains were identical with the subgenotype Bj(rt153R, rt207V and rt272C), while subgenotype Ba had different amino acids at these sites(rt153W, rt207M and rt272W, respectively). Retrotranscriptase is relatively conserved in the virus evolution process. This results suggested HBV strains in Chongqing area had the similar genetic background with subgenotype Bj, however, the Chongqing strains also owned its unique characteristics from other subgenotype Ba, although it belonged to Ba. Based on these observations, we think that subgenotype Ba can be further divided into sub-subgenotype Bac, c stands for Chongqing area where Bac is first reported.
In the second part, we investigated the role of HBV genotypes on the response to ADV antiviral therapy. Data of 177 HBV samples with known genotypes from HBeAg-positive CHB patients treated with ADV had been retrospectively analyzed. The clinical data include: serum HBV DNA seroclearance, mean HBV DNA reduction, HBeAg loss, anti-HBe seroconversion and serum ALT. The number of genotype B and genotype C were 102 and 65 cases, respectively. Baseline data of both groups were comparable. The mean HBV DNA reduction in patients with genotype B and genotype C at the treatment of 12, 24 and 48 weeks were 2.2log、2.1log (P>0.05), 2.7log、2.4log(P>0.05) and 3.6log、3.1log(P<0.05), respectively. At the end of therapy (48 weeks), 43(42.2%) patients and 22(33.8%) patients with genotype B and genotype C infection had achieved HBV DNA seroclearance (P<0.05), respectively. Our results suggested that HBV genotype B seemed to have a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than genotype C. But longer terms of ADV treatment are needed to verify this conclusion.
Third part: Based on the HBV DNA values, 30 HBV ADV-resistant strains (including: 21 primary resistant strains and 9 secondary resistant strains) had been selected out. After PCR amplification of the retrotranscriptase region and the nucleotide, amino acid alignment, we first found that polymorphism sites of rtN118H, rtM271L and rtY141F occurred with frequency of 23.8%, 14.3% and 14.3%, respectively. One patients with chronic hepatitis B had one amino acid variation in the RT C conserved domain,Six patients had nine amino acids variation in the RT none-conserved domain. We further constructed and analyzed the whole HBV genome sequence of three secondary resistant strains, and found that regulatory and structure elements such as X gene, BCP region and core/e gene also had important variations.
In short, our results suggested that ADV-resistant HBV strains may harbor several amino acid variances.not only in the conserved regions of RT, but also in the none-conserved and the regulatory regions.
引文
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