Relation between hepatitis B virus genotypes and gene mutation of basic core promoter in Li nationality

Objective：To investigate the relation between hepatitis B virus（HBV） genotypes and the double mutation of A-to-T nucleotide（nt） 1762 and G-to-A nt 1764 in basic core promoter（BCP T1762/A1764） in patients of the Li nationality. Methods：Subjects were 125 HBV DNA positive patients that belong to the Li nationality on Hainan Island. HBV DNA genotype was determined by real time fluorimetry polymerase chain reaction. BCP T1762/A1764 mutation was performed using the direct sequencing method. Results：The prevalence rates of genotype B, genotype C, genotype D, genotype C and D mixed infection（genotype C＋D） and genotype B and D mixed infection （genotype B ＋C） were 31.20%, 53.60%, 12.00%, 2.40% and 0.80% respectively. Mutation frequencies in patients infected with HBV genotype C（58.21%） were significantly higher than in those infected with other genotypes （P 〈 0.01 ）. The serum viral load of the patients with genotype C（5.74_＋ 1.21） was also higher than that of those with genotype B（P 〈0.01）. Conclusion：The major genotypes in the Li nationality were genotype C and genotype B. The infection of genotype D and mixed infection also occurred in the Li nationality. Genotype C HBV has a higher replication rate, and the different degrees of pathogenecity among HBV genotypes may be related to BCP T1762/ A1764 mutation frequency.

Visualization of Gene Mutation Complicated Pattern of Hepatitis B Virus Based on Cellular Automata

Hepatitis B virus shows instantaneous and high rate mutations in biological experiments, some sorts of which affect the efficiency of virus replication greatly through enhancing or depressing the viral replication, while others have no influence at all. Taking advantage of prominent features of cellular automata, we simulate the effect of hepatitis B virus gene mutation on its replication efficiency. The computer simulation results demonstrate the feasibility of our novel model by comparing with the results of biological experiments.

Effects of HBV gene variations on disease development and antiviral therapy for patients with chronic hepatitis B

Viral variation may change pathogenicity, escape immunity, lead to persistence infection, and cause drug resistance against antiviral therapy. This study was undertaken to investigate the effects of HBV gene variation on the progression of disease and on the efficacy of antiviral therapy for patients with chronic hepatitis B(CHB). METHODS:Hepatitis B virus (HBV) gene mutational sites were detected using gene chip in selected hepatitis B patients. RESULTS:In the patients HBeAg did not show serologic conversion or HBeAg(-)/anti-HBe(+), but their HBV DNA remained positive 24 weeks after α-interferon therapy, which was associated with mutations of nt1896, nt1814, nt1762 and nt1764. In the patients, that HBV DNA levels decreased or were undetectable, but rebounded later after antiviral therapy by lamivudine was associated with mutations of aa528 and(or) aa552(i.e.YMDD mutation), which resulted in lamivudine-resistance. YMDD mutation was prone to occur 52 weeks after lamivudine therapy in some chronic hepatitis B patients (26.4%). Nt1896 mutation was common in most chronic hepatitis B patients (68.5%). Chronic severe hepatitis, cirrhosis, and primary liver carcinoma were related to the mutations of nt1896, nt1762 and nt1764. CONCLUSIONS:HBV gene mutations could aggravate patient's condition and affect the efficacy of antiviral therapy. The regular detection of HBV gene mutation is helpful for identification of disease prognosis and adjustment of therapeutic strategy.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

AIM:To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations. METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods. RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non- cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues. CONCLUSION: HBx deletion, especially the COOH- terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.

PCR restriction fragment length polymorphism in detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine

Objective: To analyse the emergence of YMDD motif(tyrosine-methionine-aspartate-aspartate) variants inpatients with hepatitis B treated with lamivudine.Methods: The amino acid substitution from methio-nine or isoleucine at the YMDD motif at the HBVpolymerase gene is a main mutation resistant to lami-vudine treatment. Generated from a fragment of do-main C of the polymerase gene, patients HBV DNA,which had been positive previously became positive a-gain ever since it had been negative during lamivudi-ne therapy. Variants were detected by cleavage of theproducts of the three PCRs with following enzymes:FokI, SspI, Alw441. The results of PCR-RELP wereanalysed by 8. 4% polypropylene acidemide gel elec-trophoresis. PCR-RFLP assay was compared to di-rect sequencing.Results: HBV DNA was positive again in 33 patientsand positive for one year in 2 patients. YMDD vari-ants were detected in serum 14 of 35 patients, YIDDvariants in 4, YVDD variants in 6, and YI/MDD va-riants in 1; all were in concordance with the resultsof direct sequencing. The samples of other 3 patientsshowed YI/VDD mutations, as shown by direct se-quencing. The results of PCR-RFLP assay of themixed sera of YIDD and YVDD variants were similarto those sera of YI/VDD variants.Conclusion: PCR-RFLP is suitable for rapid detec-tion of YMDD variants of viral polymerase in hepati-tis B virus patients treated with lamivudine.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated

AIM: To investigate the biological impacts of 'hot-spot'mutations on genotype B and C HBV X proteins (HBx).METHODS: Five types of'hot-spot' mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I,I127T+K130M+V131I, or K130M+V131I+F132Y, respectively,were generated by means of site-directed mutagenesis.To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity,and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test.RESULTS: As compared to standard genotype B HBx,mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity,compared to standard genotype C HBx, F132Y and K130M+V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity,while the mutants of I127T and I127T+K130M+V131I showed no differences.CONCLUSION: 'Hot-spot' mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most 'hot-spot' mutations on HBx are genotype B and C differentiated.

Mutations in precore and core promoter region of HBV in patients with hepatic failure

Objective: To clarify the association of hepatitis Bvirus mutants in precore and core promoter regions inpatients with hepatic failure and HBeAg state.Methods: Precore and core promoter regions of 25HBV isolates from the patients with hepatic failurewere analyzed by polymerase chain reaction (PCR)direct sequencing approach.Results: Precore G-to-A^(1896) mutants were identified in16 (64%) of the 25 isolates. The 'hot spot' mutationsat A-to-T^(1762) and G-to-A^(1764) were present together in19 (76%) of the 25 isolates, while C-to-T^(1653) and T-to-C^(1753) existed in a mutually exclusive manner andmore frequently in hepatic failure with liver cirrhosisgroup than in hepatic failure with chronic hepatitisgroup (100% vs 50%). Both A^(1896) and T^(1762)-A^(1764)could be found frequently in HBeAg-positive subjects(77.8% and 88.9%), whereas T^(1653)/C^(1753) was moreprevalent in anti-HBe-positive subjects than inHBeAg-positive subjects (93.8% vs 33.3%).Conclusions: The whole frequency of mutations in pre-core and core promoter gene will become more fre-quent as HBV infection is to be persistent. Mutationto T^(1653)/C^(1753) may be useful as a marker for hepaticfailure. It requires further study whether the mixedinfection of mutants and wilds will develop and affectthe condition of HBeAg in serum along the progres-sion of liver disease.

Mispairing PCR－RFLP：a simple method to detect A_（83） point mutation in HBV preC region

To develop a new method for detecting the A(83) point mutation in HBV preC region, a mispairing primer was designed, which could introduce a Bsu 36I restriction site into the amplified product of A(83) mutant. After polymerase chain reaction(PCR), the amp

Identification and Characterization of Hepatitis B Virus Immune Escape Mutants in Kenya

Hepatitis B Virus (HBV) infections affect about 400 million people globally and cause about 1.4 million deaths annually. The virus displays high levels of genetic variations/mutations, some of which are immune escape mutants. The prevalence of HBV infection in Kenya is high at about 8%. This study aimed at identifying and characterizing HBV immune escape mutants in Kenya. From 547 HBV sequences available in Kenya in NCBI, and HBVdb databases in July 2021, 120 full sequences were retrieved. The S gene sequences at position 1-225, which included the “a” determinant region of the gene were analyzed using various bioinformatics tools such as Bioedit software, and Emboss Cons. The clinical significance was flagged from the search of peer-reviewed journals. Forty-six HBV-positive blood donor samples were obtained from the Kenya National Blood Transfusion Services without personal identifiers, DNA extracted, and sequenced targeting positions 1 to 520 of S genes. Mutations were similarly identified from seventeen sequences after cleaning and analysis. Out of 120 sequences that were extracted from databases and analyzed, 79 different mutations were identified. Fifteen of them were of clinical importance with an occurrence frequency of at least 5% were obtained. The majority (64.6%, n = 51), with S207N and A194V being most dominant, could result in immune escape and reduced HBsAg detection signals while 24.1% (n = 19) could result in immune escape/reduced HBsAg detection signals and high probability of hepatocellular carcinoma. Most likely to occur on the amino acids Alanine, Lysine, Serine, Asparagine, and Valine in decreasing order. The most dominant genotype was found to be Genotype A (N = 10), while four sequences were Genotype D. In contrast to the in-silico studies, the sequences from HBV samples from blood donors did not demonstrate the presence of S207N and A194V mutations and all the genotypes were type A1. Only two (13.3%) samples showed the same mutations of sK122R and sT143S for both in-silico analysis and actual sequenced samples. This study did not identify G145R mutation which is the commonest mutation within the HBsAg immunodominant “a” determinant that is associated with immune escape. The concordance of mutations in “a” determinant region of HBsAg gene among various studies is minimal. The study identified new mutations (sA194Y, sS207, sA194S, sS207I, sP46A, sA194T, sS207I, sP46R, and sT143P) that had not been published before. Four (20%) of the mutations were clinically significant. These included sS207R, sT143S, sC76F and sK122R.

南京地区268例慢性HBV感染者天然耐药突变调查及影响因素分析

目的调查分析南京地区268例慢性乙型肝炎病毒(HBV)感染者天然耐药突变情况,并分析影响天然耐药突变发生的相关因素。方法回顾性分析2019年10月—2022年10月南京地区268例慢性HBV感染者的资料,统计天然耐药发生情况及耐药突变类型,比较发生突变和未发生突变患者临床特征,采用logistic分析影响天然耐药突变发生的因素。结果268例慢性HBV感染患者经基因测序检测,共有9例天然耐药突变,检出率为3.36%(9/268);9例天然耐药突变患者耐药位点分别为rt180M、rt204V、rt204I、rt250V、rt180M、rt204V+rt180M、rt213T、rt236T和rt204I+rt180M,发生突变患者年龄、HBV DNA水平、病程均高于未发生突变患者(P<0.05);Logistic多因素回归分析显示,年龄、HBV DNA水平、病程均是影响慢性HBV感染者发生天然耐药突变的独立危险因素(OR=4.162,4.411,5.766,P<0.05)。结论该地区慢性HBV感染者存在天然耐药突变,年龄、HBV DNA水平、病程均是影响慢性HBV感染者发生天然耐药突变的独立危险因素,应加强防范。

Hepatitis B virus enhancer Ⅰ in chronic carriers resistant to interferon treatment

OBJECTIVE?To study the structural and preliminary functional characterization of the hepatitis B virus(HBV) enhancer Ⅰ in patients with chronic hepatitis B treated with interferon (IFN).?METHODS?The characteristics of the HBV enhancer Ⅰin 12 chronic carrier who were treated with alpha interferon was detected by the methods of molecular biology including PCR, cloning of PCR products, sequencing and cell culture.? RESULTS ?Four of 6 patients cleared viral DNA; all 6 in this group also seroconverted from e antigen to antibody. Prior to therapy, the HBV enhancer Ⅰ region demonstrated many point mutations in all 6 patients who became nonresponders, compared to patients who responded to interferon. The mutated sequences, many of which were within regions of transcription factor binding, were significantly more active than the corresponding wild type sequences in reporter gene assays.?CONCLUSION? These results imply that the mutations found in nonresponders appear to render the virus less sensitive to interferon.

西藏地区CHB抗病毒初治患者乙肝病毒基因分型及基因突变情况的分析

目的 分析西藏地区CHB抗病毒初治患者乙肝病毒基因型及其突变情况。方法 选取2021年12月~2022年12月在我院感染门诊就诊的慢性乙型肝炎(CHB)初始抗病毒治疗患者共48例,采用Sanger测序法检测HBV的9个基因型(即A-I),同时检测HBV核苷类似物耐药相关的22个位点,比较不同基因型组HBV-DNA定量水平、HBeAg阳性率、肝硬化或肝癌发生率、基因突变率的情况,根据基因突变情况指导临床抗病毒药物选择及预测疾病预后。结果HBV基因分型:HBV B基因型2例(4.2%),C基因型33例(68.7%),D基因型13例(27.1%);B型均为HBeAg阴性感染且基因突变率100%,C型与D型HBeAg阳性率分别为78.8%、84.6%,基因突变率为18.2%、30.8%;C型肝硬化或肝癌发生率为9.1%,D型为15.4%。结论 通过CHB患者的HBV基因分型及耐药突变检测分析,为临床抗病毒治疗药物选择提供参考依据,可预测患者的预后。

拉米夫定耐药与HBV YMDD变异关系的研究

探讨拉米夫定耐药与HBVYMDD变异之间关系。对拉米夫定治疗的慢性乙型肝炎进行肝功能、乙肝病毒血清学指标、YMDD耐药基因变异检测。 10 2例慢性乙型肝炎血清中 ,HBVDNA阴性 6 3例 (6 1 8% ) ,39例阳性 ,其中HBVYMDD变异 2 2例 ,变异率为 5 6 4 % ,并与用药时间长短有关 ,9- 17个月和 18- 30个月变异检出率分别为5 1 7%和 70 % ,HBVYMDD变异组ALT为 (5 9 94± 8 6 0 )U/L ,显著高于无HBVYMDD变异组的 (5 0 6 0± 6 12 )U/L。基因芯片技术检测HBVYMDD变异有一定的临床意义 ,YMDD变异是HBV对拉米夫定耐药的原因之一。

拉米夫定耐药慢性乙型肝炎患者HBV P基因区突变序列分析

目的探讨拉米夫定耐药慢性乙型肝炎(CHB)患者HBVP基因逆转录酶区(RT区)序列突变特点。方法收集115例接受拉米夫定治疗的CHB患者的临床资料,采用PCR产物直接测序法检测HBVP基因RT区序列耐药变异。结果入选的115例患者中103例检测到拉米夫定基因型耐药变异,最主要的耐药变异模式为rtL180M+rtM204V和rtM204I,分别占58.3%和22.3%,其他耐药位点包括rtL80V/I、rtT184S和rtA200V,并在5例患者中检测到RT区3个位点的联合变异。结论对拉米夫定治疗患者的耐药检测除了HBVP基因区常见的rtL180和rtM204位点变异外,还应考虑其他位点的联合耐药变异。

HBV前C区基因变异与中医证候的相关性研究

研究慢性乙型肝炎患者HBV前C区基因变异的变化规律与中医证候的相关机制。基因芯片检测慢性乙型肝炎不同证候的HBV前C区基因变异。不同位点变异率(%)与证候相关,肝肾阴虚1764(38%),1896(68%),1899(38%);瘀血阻络1762(93%),1764(90%),1899(50%)。不同证候变异株信号强度差异显著,1762、1764;瘀血阻络>肝肾阴虚>肝郁脾虚;1862:肝肾阴虚>肝郁脾虚;>1896:肝肾阴虚>瘀血阻络>湿热中阻>肝郁脾虚;1899:瘀血阻络>肝肾阴虚>湿热中阻>肝郁脾虚。慢性乙型肝炎HBV前C区基因变异与中医证候相关,为分子证候辨证提供了一定实验依据和研究基础。

乙型肝炎病毒BCP联合点突变与中医证型的关系

目的观察乙型肝炎病毒(hepatitisBvirus,HBV)基本C区启动子(basalcorepromoter,BCP)联合点突变与中医证型的关系。方法收集未经拉米夫定及干扰素治疗的HBVDNA阳性慢性乙型肝炎患者102例,分为湿热中阻型、肝郁脾虚型、肝肾阴虚型、脾肾阳虚型和瘀血阻络型,分别检测血清HBVDNA、肝脏生化指标及BCPnt1762AT和nt1764GA联合点突变。结果实证组的变异株检出率明显高于虚证组,其中湿热中阻型患者变异株的检出率最高。结论实证者可能较易发生BCP联合点突变,其中湿热中阻型患者可能更为显著。

HBeAg阴性慢性乙型肝炎患者乙肝病毒基因变异和分型研究

目的探讨HBeAg阴性的慢性乙肝患者乙肝病毒基因变异情况和分型的关系及其临床意义。方法采用荧光定量聚合酶链反应(PCR)检测乙肝病毒脱氧核糖核酸(HBV-DNA)、实时荧光PCR检测乙肝病毒基因分型、PCR-反向点杂交方法检测乙肝病毒基因变异,并对其关系进行分析。结果在389例HBeAg阴性的慢性乙肝患者血清中HBV-DNA检出阳性214例(55.01%),阴性175例(44.99%),HBV-DNA拷贝数在1×103-水平的例数最高,为102例(26.22%),其次为1×104-水平,41例(10.54%),两者比较有性差异(P<0.001);基因变异方面,HBV-DNA载量在1×105-水平发生前-C区变异比例为71.43%(15例),发生BCP区变异比例为52.38%(11例),两者比较有性差异(P<0.001),并且HBV-DNA载量越高基因变异发生比例越大;214例HBeAg阴性HBV-DNA阳性的慢性乙肝患者基因分型与基因变异结果中,基因分型A型6例(2.80%),B型84例(39.25%),C型106例(49.53),D型7例(3.27),混合型11例(5.14%),两者比较有性差异(P<0.001);而发生前-C区变异结果中A型发生比例为16.67%(1例),B型发生比例为36.90%(31例),C型发生比例为44.34%(47例);在BCP区变异结果中B型发生比例为19.05%(16例),C型发生比例为26.42%(28例),两者比较有性差异(P<0.001),A、D型与混合型均未检出变异。结论 HBeAg阴性HBV-DNA阳性的慢性乙肝患者病毒复制水平处于一个相对较低水平;HBV-DNA载量越高基因变异发生比例越大;本地区乙肝病毒基因分布以B、C型为主,其中B、C基因型HBeAg阴性的慢性乙肝患者基因变异发生率较高。

隐匿性HBV感染相关S基因突变对HBsAg检测的影响及其机制

目的探讨隐匿性HBV感染(OBI)相关S基因突变对多种抗-HBs及HBsAg临床检测试剂的反应性。方法9种代表性S基因突变株(M1–M9,其中M1–M5为本课题组首次报道)分离自1例OBI患者和3例OBI献血人员血清。分别采用野生型及突变型S基因重组质粒转染中国仓鼠卵巢细胞,分析9种突变株HBsAg表达及其突变蛋白对抗体和检测试剂反应的影响。结果采用HBsAg罗氏诊断试剂Elecsys定量检测9种突变株胞内HBsAg水平,均较野生株的水平明显下降;相同样品用抗-His抗体检测显示仅M1、M6和M7的HBsAg-His融合蛋白表达水平与野生株比较明显降低。6种抗-HBs与各突变HBsAg的反应性结果(S/CO值)显示,与野生株相比,多数突变株与6种抗-HBs的反应性明显降低,M1、M4、M7和M9产生的HBsAg与抗体4及M9产生的HBsAg与抗体6反应性最差(S/CO<1)。6种商品化HBsAg临床试剂检测结果显示,多数突变株与6种HBsAg试剂反应性较野生株明显降低(P<0.05),ELISA试剂D、E和F漏检率分别为11.1%、22.2%和55.6%。结论本实验涉及的突变HBsAg与抗-HBs的结合力降低是引起OBI表现的主要原因之一。当前临床常用HBsAg检测试剂对突变HBsAg的检测能力存在明显不足,亟待提高。