Core promoter mutations (A(1762)T and G(1764)A) and viral genotype in chronic hepatitis B and hepatocellular carcinoma in Guangxi,China

Inhibition of virus-induced intracellular signaling pathways and viral infectivity are our ultimate goals in the development of effective antiviral agents to control human cytomegalovirus (HCMV) infections. The HCMV hyperimmune globulin may meet such criteria. In a human embryonic lung (HEL) fibroblast culture model, pretreatment of Towne strain HCMV with HCMV hyperimmune globulin was shown to inhibit viral infectivity successfully, as measured by a standard plaque assay. The extracellular viral titers and extracellular viral DNA, as measured by plaque assay and PCR, respectively, were also decreased. In addition, the HCMV hyperimmune globulin prevented HCMV from inducing the intracellular activation of NF-kappaB, Sp-1, and PI3-K signaling pathways. The PI3-K pathway was examined by following phosphorylation (activation) of two of its downstream kinases, Akt and p70S6K. HCMV hyperimmune globulin also prevented the production of immediate early, early, and late viral proteins. These studies show that HCMV hyperimmune globulin neutralization of HCMV prevents the earliest known events observed after viral envelope glycoproteins bind their cell membrane receptors, i.e., NF-kappaB, Sp-1 and PI3-K activation. This suggests that HCMV hyperimmune globulin not only can inhibit viral infectivity, but can also prevent the abnormal cellular signaling that may induce unwanted cellular proliferation or cytokine synthesis.     

Prevalence of HBV core promoter/precore/core mutations in Gambian chronic carriers.

One hundred forty-two precore/core sequences were obtained from Gambian chronic hepatitis B virus (HBV) carriers and the predominant variants defined. The two point mutations, from A to T and G to A at nt positions 1762 and 1764 in the basic core promoter region, were found in only 7/99 (7%) of the samples where this region was sequenced. These mutations were found in both HBeAg-positive and -negative patients. The precore stop-codon mutation at nt position 1896 was found in 14/51 (27%) of HBeAg-negative samples, which is a lower prevalence rate in comparison with other parts of the world with high carrier rates. In HBeAg-positive patients the core amino acid sequences were conserved, but after seroconversion to anti-HBe significantly more changes were apparent. Several of the amino acid substitutions found have been described previously been in wild-type viruses of other genotypes.     

Precore and core mutations in HBV from individuals in india with chronic infection

Ten hepatitis B surface antigen seropositive carriers (5 asymptomatic and 5 with chronic liver disease) were tested for HBeAg/anti-HBe and for HBV-DNA using the polymerase chain reaction. Five were DNA-positive, 2 with HBeAg and 3 with anti-HBe. Nucleotide sequences were determined for these 5 cases. Hepatitis B virus DNA from one cirrhotic carrier with anti-HBe had a mutation in the precore region (nucleotide position 1862) which may affect signal peptide cleavage and HBeAg synthesis. In the other 2 anti-HBe- and DNA-positive cases, a cirrhotic carrier and an asymptomatic case, there was a mutation at nucleotide position 1896 leading to a termination codon in the precore region. In all 5 patients, except for one or two missense mutations, there was no significant variation in the core region. © 1995 Wiley-Liss, Inc.     

C基因型乙型肝炎病毒前C区及基本核心启动子突变与疾病进展的相关性研究

目的探讨HBV前C区（PreC）及基本核心启动子（BCP）突变与慢性乙型肝炎病毒（HBV）感染者疾病进展的关系。方法收集88例慢性HBV感染者血清标本，包括36例无症状携带者（其中24例为HBV携带者，12例为HBsAg携带者）、36例慢性乙型肝炎（慢性乙肝）和16例肝硬化患者。所有标本均经型特异性引物PCR法鉴定为HBVC基因型，并用巢氏PCR法扩增HBVPreC和BCP基因片段，用PCR产物直接测序法测序，然后用ClustalW1．8软件进行序列分析。结果在50例HBeAg阳性患者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率和T1846突变率分别为12．5％、42．1％、100％和0％、5．3％、28．6％，差异均有统计学意义（P值分别为0．03和0．02）。在38例HBeAg阴性HBV感染者中，无症状携带者、慢性乙肝和肝硬化组的T1762／A1764双突变率分别为16．7％、58．8％和66．7％，差异无统计学意义（P=0．08）。肝硬化组的C／G1753突变率显著高于无症状携带者及慢性乙肝组（分别为55．6％、8．3％、11．8％，P=0．01），其A1896突变率也高于无症状携带者组（分别为55．6％、8．3％，P=0．01）。结论HBVT1762／A1764双突变与C基因型HBV慢性感染者的疾病进展有关。     

Hepatitis B e antigen‐negative mutations in the precore and core promoter regions in Korean patients

Most patients with hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B have variants of the hepatitis B virus (HBV) that include mutations in the precore or core promoter regions of the HBV genome. The aim of this study was to investigate the patterns of precore and core promoter mutations and their relationship to HBeAg expression in Korean patients. Four hundred seventy‐five Korean patients with chronic HBV infection between February 1995 and December 2003 were enrolled in this study. There were 236 HBeAg‐positive and 239 HBeAg‐negative patients. Blood samples were tested for HBsAg, anti‐HBs, HBeAg, hepatitis B e antibody (anti‐HBe), liver function tests, and serum HBV DNA. Mutations in the precore and core promoter regions were determined by direct sequencing. In the core promoter region, the C1740, C1753, T1762/A1764, and T1766 mutations were associated with HBeAg escape (all; P < 0.05). In the precore region, a higher frequency of the C1802, A1828, T1846, A1850, C1858, T1862, and A1896 mutations was found in HBeAg‐negative patients (all; P < 0.05). In particular, the A1896 mutation was associated with high serum levels of ALT and HBV DNA in HBeAg‐negative patients (P = 0.014 and 0.026, respectively). Mutations around the Kozak sequence (nucleotides 1809–1812) were found in 6.7% of patients and were not associated with undetectable HBeAg (P = 0.13). In Korean patients, various mutations in the precore and core promoter regions were associated with HBeAg escape and amelioration of hepatic inflammation in HBeAg‐ negative patients. Only the A1896 mutation contributed to HBeAg‐negative chronic hepatitis B. J. Med. Virol. 81:594–601, 2009 © 2009 Wiley‐Liss, Inc.     

Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.     

Hepatitis B virus BCP,precore/core,X gene mutations/genotypes and the risk of hepatocellular carcinoma in India

The study aims to characterize mutations of the HBV genome involving BCP, Precore/core and X regions and also defines HBV genotypes in patients of hepatocellular carcinoma (HCC). The study involved 150 HBV‐related HCC cases and 136 HBV‐related chronic liver disease patients without HCC as controls. HBV DNA was subjected to mutational analysis using SSCP technique, genotyping by RFLP, and direct nucleotide sequencing. HBV DNA was found in 58.7% (88/150) of the HCC cases and 74.3% (101/136) of controls. HBV mutants were observed in 44.3% of HCC cases and 43.2% of controls. HBV/D was prevalent amongst the patients and controls, followed by HBV/A. The prevalence of the TT1504 mutation in the X gene, the V1753 and T1762/A1764 mutations in the BCP region, and G1914 mutation in the core gene were significantly higher in the HCC group than in the non‐HCC group. Multivariate analyses showed that the TT1504, V1753, A1762T/G1764A, and the G1914 mutations and the patient's age, sex, and HBeAg status increased the risk of HCC development significantly. Also, patients with HCC had lower levels of serum albumin, viral load, and platelet counts but higher values of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, and Alpha feto‐protein than those of controls (P < 0.001 for all comparisons). HBV/D was the predominant genotype associated with HCC cases seen in India. The presence of different types of HBV mutations, age, sex, HBeAg status, and viral load was found to increase significantly the risk of HCC development in India. J. Med. Virol. 82: 1115–1125, 2010. © 2010 Wiley‐Liss, Inc.     

乙型肝炎病毒核心启动子及前C区突变检测基因芯片的制备及临床应用

目的 建立检测乙型肝炎病毒核心启动子及前C区突变基因芯片并探讨其临床应用的价值.方法 设计并合成针对核心启动子1762/1764、1814及前C区1896位点突变的特异性探针,制备寡核苷酸芯片.采用不对称PCR对该区域进行扩增,扩增产物与芯片杂交后分析结果,并评价该方法的特异性、灵敏度.采用该方法检测138例HBV DNA阳性血清标本.结果 该方法能够特异地检测乙型肝炎病毒核心启动子1762/1764、1814及前C区1896位点,灵敏度达1×101拷贝/μl.138例HBV DNA阳性血清标本中,T1762/A1764突变40例（28.99%）,C1814突变11例（7.97%）,A1896突变16例（11.59%）.前C区A1896突变在高拷贝组中明显高于低拷贝组（P＜0.01）.结论 本研究建立的基因芯片能够准确同时检测HBVV核心启动子区突变和前C区突变,前C区A1896突变可能与HBV复制状态有关.     

HBe antigen loss during lamivudine therapy is not caused by mutations in precore and core promoter genes in patients with chronic hepatitis B

HBe antigen (HBeAg) loss or seroconversion can occur during lamivudine therapy. The purpose of this study was to analyze nucleotide sequences in precore and core promoter regions, and examine the influence of mutations in these regions on the disappearance of HBeAg during lamivudine therapy. Serial serum samples were obtained from 51 patients (HBeAg loss in 26 patients) at commencement of therapy (baseline) and after 1 year of lamivudine therapy. Serum samples were amplified with PCR and nucleotide sequences of HBV were analyzed. At baseline, a precore stop codon mutation (A1896) was identified in 8 of 26 HBeAg loss patients and in 8 of 25 HBeAg non-loss patients. At 1 year, precore mutation was observed in 4 of 14 patients analyzed who showed HBeAg loss. At 1 year, however, a precore mutation was observed also in 3 of 9 analyzed patients who showed no HBeAg loss. Core promoter mutations were noted in 21 of 26 HBeAg loss patients and in 20 of 25 HBeAg non-loss patients. At 1 year, core promoter mutations were noted in 11 of 14 HBeAg loss patients and in 8 of 9 HBeAg non-loss patients. Our data suggested that during lamivudine therapy, core promoter and precore mutations do not influence HBeAg loss or seroconversion but may reduce the viral level upon HBeAg loss or seroconversion.     

Gradual accumulation of mutations in precore core region of HBV in patients with chronic active hepatitis: Implications of clustering changes in a small region of the HBV core region

The sequence in the precore and core region of the hepatitis B virus (HBV) genome in the serum of five chronic active hepatitis patients at four different stages in each individual were studied by polymerase chain reaction and DNA sequencing to determine the prevalence and type of precore and core mutants in each chronic active hepatitis (CAH) patient. Gradual changes of the virus genome in each CAH patient in precore and core regions were identified. Except for the virus from one patient, the mutant viruses showed gradual changes of genome sequences, which resulted in the generation of stop codons at the precore and core region, causing the association of active hepatitis in each patient even in the presence of anti-HBe. Mutational hot spots in the core region, which includes a clustering of changes in a small region of 14 amino acids (codons 84–97 from the start of the core gene) were found in all patients. This region of mutational hot spots in the core might be a major target of cytotoxic T lymphocytes (CTL), which has evolved under the pressure of immune selections, and these mutants might play a important role in the pathogenesis of viral hepatitis. © 1996 Wiley-Liss, Inc.     

Sequencing of human-viral DNA junctions in hepatocellular carcinoma from patients with HCV and occult HBV infection

DNA of free hepatitis B viruses (HBV) has been detected in the liver of patients infected with hepatitis C virus (HCV). It is unknown whether HBV DNA is integrated into such livers; if so, it may affect hepatocarcinogenesis. Hepatocellular carcinomas (HCCs) from 34 patients without HBV surface antigen (HBsAg) and with anti-HCV, and from 7 patients with HBsAg and without anti-HCV as controls, were examined, using the cassette-ligation-mediated polymerase chain reaction and primers based on HBV DNA sequence. In the controls, HBV DNA had been integrated into human DNA of all HCCs. On the basis of HBV DNA in tumor tissue, 23 of the 34 patients with anti-HCV had occult infection. Junctions between human DNA and HBV DNA were detected in 10 of the 34 patients without HBsAg and with anti-HCV. HBV DNA was integrated into chromosome 11q in 4 of the 10 HCCs with junctions. The DNA to either side of the human-viral junctions was sequenced. Clinically, the mean tumor size of these 10 HCCs was 39 mm; that of the 24 HCCs without integrated HBV was 25 mm. The surrounding tissue was cirrhotic in 2 of the 10 former HCCs and in 16 of the latter 24 HCCs. In conclusion, integrated HBV was detected in some patients with HCV infection; in these patients, the integrated DNA was associated with accelerated hepatocarcinogenesis.     

Basal core promoter, precore region mutations of HBV and their association with e antigen, genotype, and severity of liver disease in patients with chronic hepatitis B in India

Spontaneous mutations of hepatitis B virus (HBV) could influence the severity of liver disease. Since the basal core promoter (BCP) and the precore (Pc) regions are important for viral replication, these regions were examined for naturally occurring mutations and were correlated with the genotype, e antigen status, and severity of liver disease. In 82 patients with histologically confirmed chronic hepatitis B, the BCP and Pc regions were sequenced and aligned with known wild-type sequences. Sequence based HBV genotyping was done and HBV DNA was quantified. Thirty-three (40%) patients had decompensated chronic liver disease and the remaining patients had chronic hepatitis B. Forty-six (56%) patients were HBeAg positive. HBV genotype A was found in 28%, D in 65%, and B/C in 7.3%. The Pc G1896A mutation was more common in HBeAg-negative (33% vs. 2%, P < 0.01) patients and was genotype D specific. The Pc G1862T mutation was detected more often in HBeAg-positive than HBeAg-negative (37% vs. 11%, P < 0.01) patients and was genotype A specific (P < 0.01). BCP mutations at the 1,762/64 nucleotide positions were common in HBeAg negative than positive (36% vs. 13%, P < 0.05) and were equally common in different genotypes. TA 1-3 region mutations of the BCP were significantly higher in HBeAg-negative as compared to HBeAg-positive patients (78% vs. 26%, P < 0.01). BCP mutations had significantly higher HBV DNA levels. It is concluded that Pc G1862T mutant is Genotype A-specific but is not always associated with e antigen. The TA 1-3 rich mutations of BCP region are also associated with the absence of e antigen in Indian patients.     

Prevalence of basal core promoter and precore mutations in Chinese chronic hepatitis B patients and correlation with serum HBeAG titers

The A1762T and G1764A mutations in the basal core promoter (BCP) region and the G1896A mutation in the precore (PC) region of hepatitis B virus (HBV) genome are found commonly in HBeAg‐negative patients. Experiments in vitro suggest that BCP and PC mutation reduce and abolish HBeAg expression, respectively. In the present study, the prevalence of the BCP and PC mutations were determined in 207 patients with HBeAg positive chronic hepatitis B from China and correlated with the titers of serum HBeAg. None of the patients received antiviral therapy. The HBV genotype was determined by direct sequencing of the HBsAg gene. The BCP and PC mutations were detected by the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and confirmed by DNA sequencing. The HBeAg titer was measured by the microparticle enzyme immunoassay. Fifty‐one of the 207 patients (24.6%) were infected with genotype B and the remainder with genotype C. The BCP mutations were detected in 103 patients (50%) while the PC mutation was present in 43 (20.8%). Thirteen patients (6.3%) harbored both BCP and PC mutations. No significant difference in the titers of HBeAg was found between patients infected with the two HBV genotypes, but the presence of either the BCP or PC mutation was associated with reduced HBeAg titer (P < 0.05). The presence of both the BCP and PC mutations was accompanied by even lower HBeAg titer (P < 0.05). These findings confirm that in patients with HBeAg, the BCP and PC mutations reduced the expression of HBeAg. J. Med. Virol. 81:807–814, 2009. © 2009 Wiley‐Liss, Inc.     

A matched case-control study of hepatitis B virus mutations in the preS and core promoter regions associated independently with hepatocellular carcinoma

This study aimed to determine hepatitis B virus (HBV) mutations associated independently with the risk of hepatocellular carcinoma (HCC), as adjusted with other mutations in the preS and core promoter regions of HBV genotypes B and C. One hundred and forty HBV-infected patients with HCC and 280 HBV-infected patients without HCC who had intact data of HBV genotyping and DNA sequencing in both regions were involved in this age-, sex-matched case-control study. Univariate and two-step stepwise multivariate regression analyses were performed to determine factors associated with the risk of HCC. Of 39 mutations evaluated, 23 in genotype C and 6 in genotype B were associated with an increased risk of HCC in the univariate analysis. Multivariate analyses established that genotype C (adjusted odds ratio [AOR] = 3.3; 95% confidence interval [CI] = 1.1-9.8), viral load (≥10(4) copies/ml) (AOR = 2.4; 95% CI = 1.0-5.8), A2962G (AOR = 18.7; 95% CI = 7.5-46.7), preS2 start codon mutation (AOR = 12.5; 95% CI = 3.4-45.5), C105T (AOR = 0.1; 95% CI = 0.0-0.2), T1753V (AOR = 3.1; 95% CI = 1.1-9.2), and A1762T/G1764A (AOR = 2.9; 95% CI = 1.1-7.3) were associated independently with HCC, adjusted for factors including mutations in both regions. By using an estimating haplotype frequencies program, it was found that a haplotypic carriage with 105C and 2962G was significantly more frequent in the patients with HCC than in those without HCC and the frequency of haplotype 2962G-preS2 start codon wildtype-105C-1762T/1764A was 47.9% in the patients with HCC and 4.3% in those without HCC. Conclusively, A2962G and T105C are novel factors associated independently with HCC. Further prospective studies are needed to confirm the role of these mutations in the development of HCC.     

Evolution of precore/core promoter mutations in hepatitis B carriers with hepatitis B e antigen seroreversion

The evolution of precore stop codon mutation (A1896) and dinucleotide mutation (T1762/A1764) in the basic core promoter (BCP) of hepatitis B virus (HBV) genome during transient seroconversion and seroreversion of hepatitis B e antigen (HBeAg) remains unclarified. Five HBeAg-positive HBV carriers who experienced transient seroconversion followed by seroreversion of HBeAg (Group I, 3.3%) and 3 HBeAg-negative HBV carriers with documented reversion of HBeAg (Group II, 2.5%) in a prospective cohort of 272 patients with chronic hepatitis B were thus identified. The sequential changes at the precore nucleotide 1896 and BCP dinucleotide 1762/1764 were determined by polymerase chain reaction and direct sequencing. At enrollement, precore A1896 and BCP T1762/A1764 were noted in 4 (50%) and 1 (13%) of the eight patients. During a median follow-up period of 58 months (range: 31-76 months), 12 episodes of transient HBeAg seroconversion followed by seroreversion were encountered in Group I patients and 3 episodes of HBeAg seroreversion in Group II patients. Accompanying acute exacerbations were found in two-thirds of patients with either HBeAg seroconversion or seroreversion. Overall, precore nucleotide A1896 remained identical in 73% and 83% of the seroconversion and seroreversion events, respectively. BCP dinucleotide T1762/A1764 remained unchanged in 94% and 92% of the seroconversion and seroreversion events, respectively. At the end of follow-up, only one had both precore and BCP mutations. In conclusion, these data suggested that HBeAg seroreversion might be due to the lack of sustained precore and BCP mutations after HBeAg seroconversion. Although uncommon, HBeAg seroreversion can be associated with hepatitis exacerbation.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

Influence of hepatitis B virus X and core promoter mutations on hepatocellular carcinoma among patients infected with subgenotype C2

Hepatitis B virus (HBV) genotypes/subgenotypes and their related mutations in the HBV genome have been reported to be associated with hepatocellular carcinoma (HCC). To determine the HCC-associated mutations of the HBV genome in the entire X, core promoter, and precore/core regions, a cross-sectional control study was conducted comparing 80 Japanese patients infected with HBV C2 and suffering from HCC with 80 age-, sex-, and hepatitis B e antigen (HBeAg) status-matched patients without HCC (non-HCC group). Each HBeAg-positive group (31 with HCC; 29 without HCC) and HBeAg-negative group (49 with HCC; 51 without HCC) was also matched with respect to age and sex. The C1479, T1485, H1499, A1613, T1653, V1753, T1762/A1764, and A1896 mutations were frequent in this population. The prevalences of the T1653 mutation in the box alpha region and the V1753 and T1762/A1764 mutations in the basal core promoter region were significantly higher in the HCC group than in the non-HCC group (56% versus 30%, 50% versus 24%, and 91% versus 73% [P = 0.0013, P = 0.0010, and P = 0.0035, respectively]). The platelet count was significantly lower for the HCC group than for the non-HCC group (10.7 x 10(4) +/- 5.1 x 10(4) versus 17.3 x 10(4) +/- 5.1 x 10(4) platelets/mm(3) [P < 0.0001]). Regardless of HBeAg status, the prevalence of the T1653 mutation was higher in the HCC group (52% versus 24% [P = 0.036] for HBeAg-positive patients and 59% versus 33% [P = 0.029] for HBeAg-negative patients). In the multivariate analysis, the presence of T1653, the presence of V1753, and a platelet count of < or =10 x 10(4)/mm(3) were independent predictive factors for HCC (odds ratios [95% confidence intervals], 4.37 [1.53 to 12.48], 7.98 [2.54 to 25.10], and 24.39 [8.11 to 73.33], respectively). Regardless of HBeAg status, the T1653 mutation increases the risk of HCC in Japanese patients with HBV/C2.