Full-length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma.

In hepatitis B virus (HBV)-endemic countries, the majority of hepatocellular carcinoma (HCC) arises in HBV carriers. High frequency of mutations at nucleotides 1762(A-->T) and 1764(G-->A) in the core promoter region have been described in HCC. Due to the differences in genetic backgrounds, environmental risk factors and random cellular insertion sites, it is difficult to analyze the possible roles of HBV variants detected in different HCC patients. In a follow-up cohort study, an HBsAg-positive asymptomatic carrier was diagnosed HCC within 4 years. Eleven full-length HBV isolates, three from the first serum sample obtained 4 years pre-HCC, and eight from serum sample, peri-tumor and tumor tissue post-HCC of this individual were sequenced and used to transfect HepG2 cells. When sequences were compared between pre- and post-HCC isolates, no single mutation common to all post-HCC isolates that differed from pre-HCC isolates was found. Among all 11 isolates, there were 20 predicted amino acid substitutions shared by two or more post-HCC isolates. These were located in the S(5), X(4), core(4), polymerase(4), pre-S1(2) and pre-S2(1) proteins. Possible roles of amino acid substitutions and enhanced replication efficiency in cells transfected by post-HCC isolates are discussed.     

肝癌高发区广西隆安县乙型肝炎病毒株全基因序列分析

目的 探讨肝癌高发区广西隆安县乙型肝炎病毒(HBV)株的全基因序列是否存在地域特殊性。方法 用套式PCR(nPCR)扩增该县HBV无症状携带者血清HBV全基因，用克隆测序法测序并做同源性对比。结果 本病毒株共3215个碱基，基因型为C，血清型为adw。其P基因区有40处发生碱基点突变，导致11个氨基酸改变；S基因区的前S1、前S2和S基因分别有11、2和3个碱基点突变，分别导致3、1、1氨基酸改变，未发现“a”区突变；X基因区共有6个碱基点突变，导致4个氨基酸改变，其中出现能影响e抗原表达的双突变(nt1762A→T、1764G→A)；未发现前C区基因突变，C基因有13个碱基发生突变，导致2个氨基酸改变。本病毒株与越南北部病毒株高于同为C基因型的上海株、北京株、西藏株。结论 未发现肝癌高发区的本例HBV基因C型的隆安株，基因虽有肝癌高发性，但无地域特殊性。     

Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates

Summary.  We determined full-length nucleotide sequence of hepatitis B virus (HBV) genome in sera from 40 Japanese patients with HBsAg-positive hepatocellular carcinoma (HCC), in order to obtain information on HCC-specific characteristics, if any, of the HBV genome. Direct sequencing of the long distance PCR products starting from 50 μl of serum samples revealed that 95% of our isolates were of genotype C, and that mutations and deletions/insertions were very common. With respect to envelope protein genes, deletions and missense mutations were frequent in preS2, and the determinant a domain of HBsAg was rich in “antibody-escape” mutations. Within the precore/core region, the most remarkable mutation was the replacement of proline of wild type by other amino acids at codon 130 of the core gene, which was found in 58% of our isolates, while precore-stop mutation was found in 45%. Most interestingly, however, about 90% of our isolates had mutations at nt positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter, which had been implicated in “e-suppressive” phenotype of HBV genome. G-to-A at nt 1613 and C-to-T at nt 1653 within enhancer II and T-to-C/A at nt 1753 within core promoter were also evident: 38%, 53%, and 40%, respectively. It was interesting that some of the characteristics observed in our isolates form HCC patients had been previously implicated in fulminant hepatitis and/or acute exacerbation of chronic hepatitis. Received May 18, 1998 Accepted July 18, 1998     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates from Iran

Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma

DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti-HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma.     

Epidemiology of chronic hepatitis B virus infection,hepatocellular carcinoma,and hepatitis B virus-induced hepatocellular carcinoma

Approximately 360 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at high risk of developing hepatocellular carcinoma (HCC). Chronic HBV infection is the most prevalent cause of this tumour, accounting for 55% of global cases, and 89% of those in endemic regions for HBV infection. Relative risks for developing HCC in the presence of chronic HBV infection may be as high as 49 in case-control studies, and 98 in cohort studies. HCC is the sixth most common cancer in the world today, with approximately 630,000 new cases occurring each year. It ranks third in annual cancer mortality rates. Approximately 80% of HCCs occur in developing countries where HBV infection is endemic, with the highest incidences being in the Asia-Pacific region, and sub-Saharan Africa. In the chronic carriers of the virus who are at greatest risk of developing HCC, the infection is acquired at birth or in the early months or years of life, either perinatally or horizontally, and frequently becomes chronic. The risks are greater in males, and older individuals, and are increased by co-exposure to aflatoxin B₁, the presence of cirrhosis, obesity, or diabetes mellitus, and possibly co-infection with hepatitis C virus. Viral factors that influence the risk of HCC are high viral load, the presence of certain mutations, and genotypes. Although the incidence of chronic HBV infection is beginning to decrease as a result of the universal infant immunization programme, HBV-induced HCC incidence is projected to increase for at least another two decades.     

Biological features of hepatitis B virus isolates from patients based on full‐length genomic analysis

The mechanisms for HBV persistence and the pathogenesis of chronic HB have been shown mainly due to defects in host immune responses. However, HBV isolates with different biological features may also contribute to different clinical outcomes and epidemiological implications in viral hepatitis B (HB). This review presents interesting biological features of HBV isolates based on the structural and functional analysis of full‐length HBV isolates from various patients. Among isolates from children after failure of HB vaccination, 129L mutant at the ‘a’ determinant was found with normal binding efficiency to anti‐HBs, but with reduced immunogenicity, which could initiate persistent HBV infections. Isolates from fulminant hepatitis (FH) B patients were not all highly replicative, but differences in capacities of anti‐HBs induction could be involved in the pathogenesis of FH. The high replicative competency of isolates from hepatocellular carcinoma (HCC) patients could result in enhanced immune‐mediated cytopathic effects against HBV viral proteins, and increased transactivating activity by the X protein. The mechanism of a double‐spliced variant in enhancing replication of the wild‐type virus is presented. The importance of integrating structural and functional analysis to reveal biological features of HBV isolates in viral pathogenesis is discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma.

DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti-HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma.     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates in Cambodia

Although it is known that Cambodia is one of the high endemic area of hepatitis B virus (HBV) infection, molecular characterization of HBV circulating in this country has not been reported. In this study, pre-S gene of HBV from 12 Cambodian patients was sequenced. Phylogenetic analysis based on the pre-S gene sequence revealed that 8 out of 12 isolates (66.7%) belonged to HBV/C1 and remaining four (33.3%) were HBV/B4. Furthermore, complete genomic sequences were also determined for three Cambodian HBV isolates. They all comprised of 3,215 bp long and two of them belonged to subgenotype B4, which had recombination event with genotype C in the precore/core gene confirmed by SimPlot and BootScanning analyses. Our results showed that both HBV strains belonged to subgenotypes B4 and C1, which are circulating in this country. This is the first report on molecular characterization of the HBV prevalent in Cambodia.     

Primary hepatocellular carcinoma and hepatitis B virus infection in korea

Serological evidence of hepatitis B virus (HBV) infection and serum alphafetoprotein (AFP) were assayed in sera from 112 Korean patients with primary hepatocellular carcinoma (PHC) and from 63 age- and sex-matched controls. Serological evidence of HBV infection was found in 100% of PHC patients and in 97% of controls. The majority of PHC patients (87%) were positive for hepatitis B surface antigen (HBsAg). In contrast, only 14% of control individuals were positive for HBsAg, but 82% were positive for antibody to HBsAg (anti-HBs). Hepatitis B e antigen (HBeAg) was detected in a high percentage (38%) of HBsAg-positive PHC patients, but in none of the nine HBsAg-positive control individuals. Serum AFP was detectable in 83% of PHC patients but in only one of 63 controls (1.5%). These results document that HBV infection may be the mjor factor in the development of PHC in this country.     

Hepatitis B virus (HBV)-DNA-positive hepatocellular carcinoma following hepatitis B virus infection in a child

Integrated hepatitis B virus (HBV) DNA sequences were found in neoplastic liver tissue of a hepatitis B surface antigen (HBsAg)-negative child who had previously suffered from HBsAg-positive chronic active hepatitis and was anti-HBs and anti-hepatitis B core (HBc) positive at the time of tumor development. Reintegration pattern was consistent with the presence of a single integration site of the HBV genome into cellular DNA, and clonal proliferation of such infected cells. A normal liver, tested in the same experiment with the same amount of total DNA, was negative for viral DNA sequences. These findings support the possible oncogenic role of HBV in the development of liver cancer, not only in adults, but also in children, even in patients who are negative for HBsAg at the time of tumor diagnosis.     

Full-length sequence analysis of hepatitis E virus isolates: showing potential determinants of virus genotype and identity

The complete genome sequence of a genotype 4 strain of hepatitis E virus (CH-YT-HEV02) from a patient (in Yantai, China) has been determined. Phylogenetic analysis showed that CH-YT-HEV02 belongs to genotype 4, subtype 4a. However, the phylogenetic analysis indicated that it was most closely related to JKO-CHiSai98C (AB197673) strain, sharing only 91.6 % sequence identity with it. Judging from the phylogenetic tree based on the full-length nucleotide sequences of all 70 genotype 4 HEV isolates retrieved from GenBank up to May, 2013, the CH-YT-HEV02 isolates could serve as a Yantai-indigenous strain. A broader comparison with other genotype isolates revealed that there are a few conserved amino acids in the HVR region of different HEV genotypes, and two amino acid motifs in ORF2 and ORF3 might serve as signatures of genotype diversity of HEV.     

Comparison of full length sequences of hepatitis B virus isolates in hepatocellular carcinoma patients and asymptomatic carriers of Korea

Relatively few genomic sequences of Korean hepatitis B virus (HBV) isolates are available. Moreover, no comparative study has been made between the full-length genomes of Korean HBV isolates and clinical status. To evaluate mutations in HBV isolates obtained from chronically infected HBV patients in terms of clinical significance, we determined the genomic sequences of HBV isolates obtained from three hepatocellular carcinoma (HCC) patients (He52, He53, and He82) and from three asymptomatic carriers (He74, He100, and He127). A comparison of sequence variations showed that the HBV isolates from the three HCC patients showed higher frequencies of mutation than the isolates from the three asymptomatic carriers. Three characteristic mutation patterns were identified in the HBV isolates from the HCC patients, which distinguished the HBV isolates from the asymptomatic carriers. First, HBV isolates from the three HCC patients both had double mutations in a core promoter (T1762/A1764) and a precore mutation (A1896). Second, although these isolates belonged to genotype C, 11 amino acids deletions in the preS1 region, specific for HBV genotype D, were detected in the isolates of two HCC patients (He52 and He82). Third, mutations (I127T/N, K130M, and V131I) at three codons in the carboxy functional region of X protein were observed in isolates from all three HCC patients. Additionally, phylogenetic analysis based on the entire HBV sequences showed that all six isolates belonged to genotype C2, as do other Korean strains.     

Microvessel density and clinicopathological characteristics in hepatitis C virus and hepatitis B virus related hepatocellular carcinoma

AIMS: To compare intratumorous microvessel density (MVD) and clinicopathological features in two different groups of hepatocellular carcinoma (HCC), namely: hepatitis B virus (HBV) related HCC (B-HCC) and HCV related HCC (C-HCC). METHODS: Fifty consecutive cases each of B-HCC and of C-HCC were studied. Microvessel numbers were assessed by staining for the antigen CD34; in each case, three areas with the highest numbers of microvessels were counted in both the intratumorous and the surrounding non-tumorous tissue; the mean value represented the final MVD. RESULTS: Patients with B-HCC were significantly younger than those with C-HCC (mean age, 60.1 (SD, 4.1) v 66.4 (4.3) years); no significant differences were seen for sex or Child's class distribution. The tumour diameter was larger in B-HCCs than in C-HCCs (mean, 5.6 (SD, 1.8) v 3.8 (1.8) cm). Tumour microsatellite formation was significantly higher in C-HCCs (12 v 4 cases). No differences were found for histological subtype, degree of differentiation, tumour encapsulation, and vascular invasion. The mean MVD value was significantly higher in tumorous (mean, 54 (SD, 13.8) v 38 (8.9)) and in the surrounding non-tumorous liver tissue (mean, 15 (SD, 4.3) v 7 (3.1)) of C-HCCs. CONCLUSIONS: C-HCCs present as smaller tumours in older patients, with a higher incidence of tumour microsatellite formation and higher MVD values both in the tumorous and the non-tumorous areas, suggesting a link between HCV infection, angiogenesis, and hepatocarcinogenesis.     

Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection

Antibody to hepatitis B surface antigen (HBsAg) (anti‐HBs) can exist in patients with chronic hepatitis B virus (HBV) infection. To date, little is known about the association of concurrent HBsAg and anti‐HBs (concurrent HBsAg/ anti‐HBs) with hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinical relevance of concurrent HBsAg/anti‐HBs with preS deletion mutations and HCC in chronic HBV infection. A total of 755 patients with chronic HBV infection were included consecutively at a tertiary center. Logistic regression analysis was used to identify risk factors for HCC, and serum HBV DNA was amplified, followed by direct sequencing to detect preS deletions. The prevalence of concurrent HBsAg/anti‐HBs was 6.4% (48/755) and all HBVs tested were genotype C. HCC occurred more frequently in the concurrent HBsAg/anti‐HBs group than in the HBsAg only group [22.9% (11/48) vs. 7.9% (56/707), P = 0.002]. In multivariate analyses, age >40 years [odds ratio (OR), 14.712; 95% confidence interval (CI), 4.365–49.579; P < 0.001], male gender (OR 2.431; 95% CI, 1.226–4.820; P = 0.011), decompensated cirrhosis (OR, 3.642; 95% CI, 1.788–7.421; P < 0.001) and concurrent HBsAg/anti‐HBs (OR, 4.336; 95% CI, 1.956–9.613; P < 0.001) were associated independently with HCC. In molecular analysis, preS deletion mutations were more frequent in the concurrent HBsAg/anti‐HBs and HCC groups than in the HBsAg without HCC group (42.3% and 32.5% vs. 11.3%; P = 0.002 and 0.012, respectively). In conclusion, concurrent HBsAg/anti‐HBs is associated with preS deletion mutations and may be one of the risk factors for HCC in chronic HBV infection with genotype C. J. Med. Virol. 81:1531–1538, 2009. © 2009 Wiley‐Liss, Inc.     

Genotype characterization and phylogenetic analysis of hepatitis B virus isolates from Iranian patients

Hepatitis B virus (HBV) is one of the major causative agents of acute and chronic liver disease worldwide and is believed to be responsible for a million deaths annually. Eight genotypes of HBV, A to H, have been described on the basis of similarity of the complete genomes sequence. Although, it is reported that the predominant HBV genotype in the Mediterranean area and the middle east is genotype D, there are no reports on HBV genotypes prevalent in Iran. In this study, the C and S regions of HBV from 26 chronic hepatitis B Iranian patients were amplified and sequenced. Phylogenetic analysis revealed that all Iranian HBV isolates sequences were classified into genotype D with bootstrap values of 100%, 73%, and 100% (1,000 replicates each) for S, C, and preS2 regions, respectively. The mean percent intra-distance of S and C regions were 0.8% and 2.3%, respectively. The mean percent inter-distance of S and C regions between Iranians and genotype D isolates were 1.7% and 3.0%, respectively, and the range of mean percent nucleotide distance of S and C regions between Iranians and the other reference isolates were 7.9%-17.5% and 4.8%-14.7%, respectively. Thirteen out of 23 HBV C region sequences showed nucleotide "A" at position 1896 (precore mutant) in C region. Nucleotide 1858 showed presence of "T" in all isolates. No insertion or deletion was found in both regions. SimPlot and BootScanning analyses did not show any recombination between Iranian isolates and other genotypes in both regions.     

Frequent integration of hepatitis B virus DNA in noncancerous liver tissue from hepatocellular carcinoma patients

To investigate the relationship between hepatocarcinogenesis and integration of hepatitis B virus (HBV) DNA in the cellular DNA of the liver, we studied the integration of HBV DNA in various noncancerous regions of the liver from 31 patients using Southern blot analysis. Of 13 patients without hepatocellular carcinoma (HCC), 4 had heterogeneously integrated HBV DNA. Of the latter four patients, two had chronic liver disease, and two had nonspecific histological changes. In contrast, integration of HBV DNA was found in noncancerous tissue from 11 of 18 patients with HCC. In eight patients, homogeneous integration was found in noncancerous tissue, and restriction fragments of integrated HBV DNA were different from those found in cancerous tissue. Moreover, integration of HBV DNA was found in all portions examined from the same liver, and homogeneously integrated HBV DNA showed different restriction patterns in different areas. These results suggest that integration of HBV DNA may occur in heterogeneous sites of cellular DNA before hepatocarcinogenesis. Subsequently, multi-focal clonal populations develop from these hepatocytes, especially frequently in the case of HCC. Integrated HBV DNA may play an important role in the clonal growth of hepatocytes, although the development of HCC requires additional factors.     

Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma

Chronic hepatitis B infection is a major cause of hepatocellular cancer (HCC). The pathogenesis of the carcinogenesis is not fully understood. Viral proteins such as the X protein and the truncated middle S protein have been implicated to be transactivators. In order to investigate whether any mutations within relevant parts of the hepatitis B virus (HBV) genome could be associated with the development of HCC, the genomes of 16 HBV strains from chronic HBV carriers with HCC were studied. Serum samples were subjected to PCR and the HBV DNA sequenced subsequently. Genotypes A-D were represented. The sequence analysis showed that an especially high proportion, 50% (CI 95%, 25-75%), of the patients with HCC carried HBV mutants with deletions or insertions in the N-terminal half of the pre-S2 region or had a point mutation in the start codon of pre-S2 compared with controls with chronic HBV infection, 21% (CI 95%, 3-39%). A high proportion (69%) also had mutations at position 1762 (A --> T) and/or 1764 (G --> A) in the core promoter region, but the proportion of core promoter mutations was no different from what was found in a control group of HBV carriers without HCC (68%). The pre-S2 variants, which involve deletions of immunogenic regions, may have a survival advantage as they are mostly found in long-standing HBV infection. There were no other mutations found frequently within the region coding for the X protein.     

Establishment of a human hepatocellular carcinoma cell line releasing hepatitis B virus surface antigen

A new hepatocellular carcinoma cell line, DELSH-5, derived from operative wedge biospy from a HBsAg sero- and tissue-positive patient, has been continuously propagated in vitro for nearly 22 months. The cells not only resemble hepatocytes on light and electron microscopic examination but also possess biosynthetic markers of the latter such as albumin and alpha-foetoprotein which were demonstrated in the supernatant medium as well as in the tumour cell cytoplasm. Karyology of cloned cells shows moderate aneuploidy, the major model chromosome number being 61. Though in the initial few passages HBsAg could not be detected, from the 13th passage onwards this viral component could be consistently demonstrated in small amounts in the concentrated supernatant medium by the macro- and micro-ELISA techniques. The immunohistochemical techniques as well as electron microscopy have failed to demonstrate any virus component inside the cell. The cell line reported here is the third of its kind which will act as a useful laboratory model to obtain pure HBsAg and to study the hepatitis-B-virus--liver-cell interaction with particular reference to the oncogenic potential of the virus.