AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patien...AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012.None of the patients received antiviral therapy.HBV DNA from serum,was quantified by real-time PCR.The HBV genotype was determined by direct sequencing of the S gene.We used the Simpleprobe ultrasensitivequantitative method to detect PC/BCP mutants in each patient.We compared the strain number,percentage,and the changes in PC/BCP mutants in different phases,and analyzed the relationship between PC/BCP mutants and HBe Ag by multiple linear regression and logistic regression.RESULTS:Patients with HBV infection(n = 191) were assigned to groups by phase:Immune tolerance(IT) = 55,Immune clearance(IC) = 67,Low-replicative(LR) = 49,and HBeA g-negative hepatitis(ENH) = 20.Of the patients(male,112; female,79) enrolled,122 were HBe Ag-positive and 69 were HBe Ag-negative.The median age was 33 years(range:18-78 years).PC and BCP mutation detection rates were 84.82%(162/191) and 96.86%(185/191),respectively.In five HBe Ag-negative cases,we detected double mutation G1896A/G1899 A.The logarithm value of PC mutant quantities(log10 PC) significantly differed in IT,IC,and LR phases,as well as in the ENH phase(F = 49.350,P < 0.001).The logarithm value of BCP mutant quantities(log10 BCP) also differed during the four phases(F = 25.530,P < 0.001).Log10 PC and log10 BCP values were high in the IT and IC phases,decreased in the LR phase,and increased in the ENH phase,although the absolute value at this point remained lower than that in the IT and IC phases.PC mutant quantity per total viral load(PC%) and BCP mutant quantity per total viral load(BCP%) differed between phases(F = 20.040,P < 0.001; F = 10.830,P < 0.001),with PC% and BCP% gradually increasing in successive phases.HBeA g titers negatively correlated with PC%(Spearman's rho =-0.354,P < 0.001) and BCP%(Spearman's rho =-0.395,P < 0.001).The negative correlation between PC% and HBeA g status was significant(B =-5.281,P = 0.001),but there was no such correlation between BCP% and HBeA g status(B =-0.523,P = 0.552).CONCLUSION:PC/BCP mutants become predominant in a dynamic and continuous process.Log10 PC,log10 BCP,PC% and BCP% might be combined to evaluate disease progression.PC% determines HBeA g status.展开更多
Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its geno...Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.展开更多
AIM:To construct eukaryotic expression plasmids of full-length Hepatitis B Virus(HBV) genotype C genome,which contain lamivudine-resistant mutants(YIDD,YVDD) or wild-type strain(YMDD) ,and to observe the expression of...AIM:To construct eukaryotic expression plasmids of full-length Hepatitis B Virus(HBV) genotype C genome,which contain lamivudine-resistant mutants(YIDD,YVDD) or wild-type strain(YMDD) ,and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen(HBsAg) and hepatitis B e antigen(HBeAg) ] of the recombinant plasmids in HepG2 cells. METHODS:Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD,pMD18T-HBV/YVDD and pMD18T-HBV/YMDD,using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1(+) ,between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion,and DNA sequence analysis,the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection,the levels of intracellular viral DNA replication were detected by real-time PCR,and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS:Restriction endonuclease digestion and DNA sequence analysis confirmed that the threerecombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells,high levels of intracellular viral DNA replication were observed,and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION:Eukaryotic expression plasmids pcDNA3.1(+) -HBV/YIDD,pcDNA3.1(+) -HBV/YVDD or pcDNA3.1(+) -HBV/YMDD,which contained HBV genotype C full-length genome,were successfully constructed. After transfection into HepG2 cells,the recombinant plasmids efficiently expressed HBV DNA,HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.展开更多
The Amazon region is considered to be a high endemic area for Hepatitis B Virus (HBV) infections, Rondônia state having the highest prevalence. The aim of this study was to identify molecular genotypes and m...The Amazon region is considered to be a high endemic area for Hepatitis B Virus (HBV) infections, Rondônia state having the highest prevalence. The aim of this study was to identify molecular genotypes and mutations in the S gene region of HBV viral genomes from 20 patients using a DNA microarray. Results: Serological tests showed that 88% of patients were HBeAg negative, 82% had anti-HBe antibodies and 33% were co-infected with Hepatitis Delta Virus. Sixteen percent of the patients were considered cirrhotic, and 11% have been transfused. The microarray technique identified the genotypes A (4 patients), D (7 patients) and F (7 patients) in 18 samples. Mutations were detected in all 3 genotypes and, overall, A159G, which has been associated with a reduced antigenicity of the virus, was detected most frequently. In genotype A, G119E was the most frequently detected mutation followed by mutations A159G, F134Y, W172C, Y161F and T143S. A159G was detected in all genotype D and F samples followed by mutations T143S, Y161F, N131T, T114S and G119E in genotype D and mutations T143S, Y161F, N131T, T114S and G119E in genotype F. Conclusion: The analysis of mutations repartition among genotypes suggests that some of them are preferentially or exclusively associated with genotype A, D or F. This type of tool is adapted for clinical and therapy monitoring of patient as well as for molecular epidemiology research on HBV.展开更多
目的探讨慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者前核心区(precore,PC)和基本核心启动子(basalcore promoter,BCP)变异特点以及变异株血清乙肝病毒e抗原(hepatitis B e antigen,HBeAg)、乙肝病毒表面抗原(hepatitis B surface a...目的探讨慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者前核心区(precore,PC)和基本核心启动子(basalcore promoter,BCP)变异特点以及变异株血清乙肝病毒e抗原(hepatitis B e antigen,HBeAg)、乙肝病毒表面抗原(hepatitis B surface antigen,HBsAg)、乙肝病毒脱氧核酸(HBV-DNA)检测的临床意义。方法采集138例慢性HBV感染者清晨空腹血,以免疫化学发光法检测血清HBeAg、HBsAg,并同时采用磁珠法检测HBV-DNA,应用半巢式聚合酶链反应检测PC/BCP区基因突变。结果 101例未经抗病毒治疗的慢性HBV感染者中,HBeAg阴性组PC、BCP、PC+BCP变异率高于HBeAg阳性组,差异有统计学意义(P<0.05)。37例服用核苷类药物一年以上组PC、BCP、PC+BCP检出率要高于未经抗病毒治疗的HBeAg阳性HBV感染者组(P<0.05)。HBeAg阴性HBV感染患者其PC、BCP和PC+BCP的变异率随着病程的增加而增加(P<0.01)。PC、BCP和PC+BCP变异株组与野生株比较,慢性HBV感染者HBeAg含量减少(P<0.05),DNA复制活跃、HBsAg含量增加(P<0.01)。结论变异的发生使HBeAg阴性的慢性HBV感染患者越来越多,服用核苷类抗病毒药物和长的疾病病程可能是变异相关因素。对于HBeAg阴转的慢性HBV感染患者观察DNA、HBsAg含量,可了解体内病毒是否确实已被免疫清除。展开更多
基金Supported by National Science and Technology Major Project of China,No.2012ZX10002007-001-002 and No.2013ZX10002001(to Zhang JM)the National Natural Science Foundation of China,No.81271833 and No.81471933(to Zhang JM)+1 种基金the Science and Technology Plan Project of Taizhou,Zhejiang province,No.1402ky19(to Tu WH and Hou W)the Scientific Research Project of Taizhou University,Zhejiang province,No:2014PY054(to Tu WH and Hou W)
文摘AIM:To investigate precore/basal core promoter(PC/BCP) mutants throughout hepatitis B virus(HBV) infection and to determine their relationship to hepatitis B early antigen(HBeA g) titers.METHODS:We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012.None of the patients received antiviral therapy.HBV DNA from serum,was quantified by real-time PCR.The HBV genotype was determined by direct sequencing of the S gene.We used the Simpleprobe ultrasensitivequantitative method to detect PC/BCP mutants in each patient.We compared the strain number,percentage,and the changes in PC/BCP mutants in different phases,and analyzed the relationship between PC/BCP mutants and HBe Ag by multiple linear regression and logistic regression.RESULTS:Patients with HBV infection(n = 191) were assigned to groups by phase:Immune tolerance(IT) = 55,Immune clearance(IC) = 67,Low-replicative(LR) = 49,and HBeA g-negative hepatitis(ENH) = 20.Of the patients(male,112; female,79) enrolled,122 were HBe Ag-positive and 69 were HBe Ag-negative.The median age was 33 years(range:18-78 years).PC and BCP mutation detection rates were 84.82%(162/191) and 96.86%(185/191),respectively.In five HBe Ag-negative cases,we detected double mutation G1896A/G1899 A.The logarithm value of PC mutant quantities(log10 PC) significantly differed in IT,IC,and LR phases,as well as in the ENH phase(F = 49.350,P < 0.001).The logarithm value of BCP mutant quantities(log10 BCP) also differed during the four phases(F = 25.530,P < 0.001).Log10 PC and log10 BCP values were high in the IT and IC phases,decreased in the LR phase,and increased in the ENH phase,although the absolute value at this point remained lower than that in the IT and IC phases.PC mutant quantity per total viral load(PC%) and BCP mutant quantity per total viral load(BCP%) differed between phases(F = 20.040,P < 0.001; F = 10.830,P < 0.001),with PC% and BCP% gradually increasing in successive phases.HBeA g titers negatively correlated with PC%(Spearman's rho =-0.354,P < 0.001) and BCP%(Spearman's rho =-0.395,P < 0.001).The negative correlation between PC% and HBeA g status was significant(B =-5.281,P = 0.001),but there was no such correlation between BCP% and HBeA g status(B =-0.523,P = 0.552).CONCLUSION:PC/BCP mutants become predominant in a dynamic and continuous process.Log10 PC,log10 BCP,PC% and BCP% might be combined to evaluate disease progression.PC% determines HBeA g status.
文摘Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.
基金The PhD Foundation of Education Ministry, China, No. 20050226002the Doctor Foundation of Harbin Medical University+1 种基金 The Youth Foundation of Heilongjiang Province, No. QC06C061The Foundation of Education Department, Heilongjiang Province, No. 11521089
文摘AIM:To construct eukaryotic expression plasmids of full-length Hepatitis B Virus(HBV) genotype C genome,which contain lamivudine-resistant mutants(YIDD,YVDD) or wild-type strain(YMDD) ,and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen(HBsAg) and hepatitis B e antigen(HBeAg) ] of the recombinant plasmids in HepG2 cells. METHODS:Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD,pMD18T-HBV/YVDD and pMD18T-HBV/YMDD,using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1(+) ,between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion,and DNA sequence analysis,the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection,the levels of intracellular viral DNA replication were detected by real-time PCR,and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS:Restriction endonuclease digestion and DNA sequence analysis confirmed that the threerecombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells,high levels of intracellular viral DNA replication were observed,and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION:Eukaryotic expression plasmids pcDNA3.1(+) -HBV/YIDD,pcDNA3.1(+) -HBV/YVDD or pcDNA3.1(+) -HBV/YMDD,which contained HBV genotype C full-length genome,were successfully constructed. After transfection into HepG2 cells,the recombinant plasmids efficiently expressed HBV DNA,HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.
文摘The Amazon region is considered to be a high endemic area for Hepatitis B Virus (HBV) infections, Rondônia state having the highest prevalence. The aim of this study was to identify molecular genotypes and mutations in the S gene region of HBV viral genomes from 20 patients using a DNA microarray. Results: Serological tests showed that 88% of patients were HBeAg negative, 82% had anti-HBe antibodies and 33% were co-infected with Hepatitis Delta Virus. Sixteen percent of the patients were considered cirrhotic, and 11% have been transfused. The microarray technique identified the genotypes A (4 patients), D (7 patients) and F (7 patients) in 18 samples. Mutations were detected in all 3 genotypes and, overall, A159G, which has been associated with a reduced antigenicity of the virus, was detected most frequently. In genotype A, G119E was the most frequently detected mutation followed by mutations A159G, F134Y, W172C, Y161F and T143S. A159G was detected in all genotype D and F samples followed by mutations T143S, Y161F, N131T, T114S and G119E in genotype D and mutations T143S, Y161F, N131T, T114S and G119E in genotype F. Conclusion: The analysis of mutations repartition among genotypes suggests that some of them are preferentially or exclusively associated with genotype A, D or F. This type of tool is adapted for clinical and therapy monitoring of patient as well as for molecular epidemiology research on HBV.
文摘目的探讨慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者前核心区(precore,PC)和基本核心启动子(basalcore promoter,BCP)变异特点以及变异株血清乙肝病毒e抗原(hepatitis B e antigen,HBeAg)、乙肝病毒表面抗原(hepatitis B surface antigen,HBsAg)、乙肝病毒脱氧核酸(HBV-DNA)检测的临床意义。方法采集138例慢性HBV感染者清晨空腹血,以免疫化学发光法检测血清HBeAg、HBsAg,并同时采用磁珠法检测HBV-DNA,应用半巢式聚合酶链反应检测PC/BCP区基因突变。结果 101例未经抗病毒治疗的慢性HBV感染者中,HBeAg阴性组PC、BCP、PC+BCP变异率高于HBeAg阳性组,差异有统计学意义(P<0.05)。37例服用核苷类药物一年以上组PC、BCP、PC+BCP检出率要高于未经抗病毒治疗的HBeAg阳性HBV感染者组(P<0.05)。HBeAg阴性HBV感染患者其PC、BCP和PC+BCP的变异率随着病程的增加而增加(P<0.01)。PC、BCP和PC+BCP变异株组与野生株比较,慢性HBV感染者HBeAg含量减少(P<0.05),DNA复制活跃、HBsAg含量增加(P<0.01)。结论变异的发生使HBeAg阴性的慢性HBV感染患者越来越多,服用核苷类抗病毒药物和长的疾病病程可能是变异相关因素。对于HBeAg阴转的慢性HBV感染患者观察DNA、HBsAg含量,可了解体内病毒是否确实已被免疫清除。
