Hepatitis B viral factors and clinical outcomes of chronic hepatitis B

Hepatitis B virus (HBV) infection is an important health problem and the major cause of chronic hepatitis, cirrhosis as well as hepatocellular carcinoma (HCC) worldwide. The natural history of chronic HBV infection can be divided into 4 dynamic phases in HBV carriers who acquire the virus early in life. In general, the frequency and severity of hepatitis flares in the immune clearance or reactivation phase predict disease progression in HBV carriers, and early HBeAg seroconversion typically confers a favorable outcome. In contrast, late or absent HBeAg seroconversion after multiple hepatitis flares accelerates the progression of chronic hepatitis to cirrhosis. Recently, several hepatitis B viral factors predictive of clinical outcomes have been identified. For example, serum HBV DNA level at enrollment is the best predictor of adverse outcomes (cirrhosis, HCC and death from liver disease) in adults with chronic HBV infection. In addition, HBV genotype C, basal core promoter (BCP) mutant and pre-S deletion mutant are associated with increased risk of HCC development. In conclusion, hepatitis B viral factors such as serum HBV DNA level, genotype and mutants have already been clarified to influence disease progression of chronic hepatitis B. Further studies are needed to investigate the pathogenic mechanism of each viral factor.     

乙型肝炎病毒变异与肝细胞癌发生关系

乙型肝炎病毒（hepatitisBvirus,HBV）基因组复制时,以病毒前基因组RNA作为模板合成子代病毒DNA,催化该过程的逆转录酶缺乏校对功能,所以HBV易出现变异。近年来,各国学者通过比较肝细胞癌（hepatocellular carcinoma,HCC）患者和非HCC患者的HBV基因序列,发现HBV基本核心启动子区的A1762T／G1764A变异或T1753V变异、增强子Ⅰ区的G1053A或G1229A变异、前S蛋白的F141L变异、前s2区基因缺失变异和x基因的截短变异,分别是HCC的易患因素,而前c区常见的G1896A变异,与HCC的发生无关。增强子Ⅱ区的C1653T变异在c基因HBV感染中可能与发生HCC有关,而在A基因型可能无关。     

Genotype-specific genomic markers associated with primary hepatomas, based on complete genomic sequencing of hepatitis B virus

We aimed to identify genomic markers in hepatitis B virus (HBV) that are associated with hepatocellular carcinoma (HCC) development by comparing the complete genomic sequences of HBVs among patients with HCC and those without. One hundred patients with HBV-related HCC and 100 age-matched HBV-infected non-HCC patients (controls) were studied. HBV DNA from serum was directly sequenced to study the whole viral genome. Data mining and rule learning were employed to develop diagnostic algorithms. An independent cohort of 132 cases (43 HCC and 89 non-HCC) was used to validate the accuracy of these algorithms. Among the 100 cases of HCC, 37 had genotype B (all subgenotype Ba) and 63 had genotype C (16 subgenotype Ce and 47 subgenotype Cs) HBV infection. In the control group, 51 had genotype B and 49 had genotype C (10 subgenotype Ce and 39 subgenotype Cs) HBV infection. Genomic algorithms associated with HCC were derived based on genotype/subgenotype-specific mutations. In genotype B HBV, mutations C1165T, A1762T and G1764A, T2712C/A/G, and A/T2525C were associated with HCC. HCC-related mutations T31C, T53C, and A1499G were associated with HBV subgenotype Ce, and mutations G1613A, G1899A, T2170C/G, and T2441C were associated with HBV subgenotype Cs. Amino acid changes caused by these mutations were found in the X, envelope, and precore/core regions in association with HBV genotype B, Ce, and Cs, respectively. In conclusion, infections with different genotypes of HBV (B, Ce, and Cs) carry different genomic markers for HCC at different parts of the HBV genome. Different HBV genotypes may have different virologic mechanisms of hepatocarcinogenesis.     

Transcriptional trans-activating function of hepatitis B virus

The ability of hepatitis B virus (HBV) to stimulate the expression of a cellular gene was investigated by using a transient-expression system. A plasmid in which the expression of the bacterial chloramphenicol acetyltransferase (cat) gene had been placed under the control of the DNA sequences that regulate the expression of the human beta-interferon gene was constructed. In Vero cells, cotransfection of the 2.7-kilobase BglII DNA fragment of HBV together with the test plasmid containing the cat gene resulted in stimulation of the expression of the cat gene. This HBV DNA fragment was specific in its trans-activation; no significant stimulation of CAT activity was observed in constructs when the promoter and enhancer elements were derived from the murine sarcoma viral long terminal repeat, Rous sarcoma virus, BK virus, or simian virus 40. Results of subcloning of the HBV DNA fragment indicate that the trans-activating function resides in a 944-base-pair EcoRV-BglII DNA fragment of the HBV genome that contains the X structural gene and its promoter element. Removal of the promoter from the X structural gene resulted in loss of the trans-activating function. A frameshift mutation within the X gene region also eliminated the trans-activating activity. These results suggest that the X antigen could play a role in HBV infections by activating the expression of cellular genes.     

The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.     

Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers

Background  

Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858.     

Specific mutations of basal core promoter are associated with chronic liver disease in hepatitis B virus subgenotype D1 prevalent in Turkey

The role of hepatitis B virus (HBV) genetics in the clinical manifestations of infection is being increasingly recognized. Genotype D is one of eight currently recognized major HBV genotypes. The virus is ubiquitous worldwide, but shows different features in different regions. One hundred and ninety‐eight patients with chronic HBV infection were enrolled in this study, 38 of whom had been diagnosed with cirrhosis of the liver and/or hepatocellular carcinoma. HBV DNA was isolated from the patients' blood samples and the entire genome and/or the basal core promoter/core promoter region sequenced. Phylogenetic analysis of the complete genomes revealed that subgenotype D1 is the most prevalent subgenotype in Turkey, but there was no definite phylogenetic grouping according to geography for isolates from different regions within Turkey, or for isolates in Turkey relative to other parts of the world. Turkish isolates tended to be genetically similar to European and central Asian isolates. Overall, HBV‐infection in Turkey appears to be characterized by early HBeAg seroconversion, a high incidence of the A1896 core promoter mutation and a small viral load. Genotype D characteristic mutations A1757 and T1764/G1766 were found in the BCP region. T1773 was associated with T1764/G1766 and a larger viral load. In conclusion, infection with HBV genotype D in Turkey has a similar clinical outcome to that of Europe and central Asia. Genotypic mutations in genotype D may be linked with disease prognosis in Turkey, but further studies with higher sample numbers and balanced clinical groups are needed to confirm this.     

A1762T/G1764A mutations of hepatitis B virus, associated with the increased risk of hepatocellular carcinoma, reduce basal core promoter activities

Hepatitis B virus (HBV) infection is a major global health problem that causes over one million deaths annually. A1762T and G1764A mutations in the basal core promoter are often present in HBV patients but seldom in asymptomatic carriers, and are highly correlated with the increased risk of HBV-associated hepatocellular carcinoma (HCC). In this study, for the first time, we show that the basal core promoter activity of HBV strains isolated from asymptomatic carriers is decreased when 1762A is mutated to 1762T or 1764G is mutated to 1764A by site directed mutagenesis. By contrast, the promoter activity of HBV strains isolated from HCC patients is increased when 1762T and 1764A are reversely mutated into 1762A and 1764G, respectively. 1764G contributes more promoter activity than 1762T. We also show that T1762A and G1764A double mutations synergize the reduction of the promoter activity. A mechanism of HBV evasion from host immunoresponse that may facilitate disease development is also discussed.     

Molecular aspects of hepatitis B viral infection and the viral carcinogenesis

Of many viral causes of human cancer, few are of greater global importance than the hepatitis B virus (HBV). Over 250 million people worldwide are persistently infected with HBV. A significant minority of these develop severe pathologic consequences, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Earlier epidemiological evidence suggested a link between chronic HBV infection and HCC. Further, the existence of related animal viruses that induce acute and chronic infections of the liver, and eventually HCC, confirms the concept that HBV belongs to one of the few human oncogenic viruses. Although it is clear that chronic HBV infections are major risk factors, relatively little is understood about how the viral factors contribute to hepatocarcinogenesis. This review will introduce molecular aspects of the viral infection, and highlight recent findings on the viral contribution to hepatocarcinogenesis.     

Variability in the precore and core promoter regions of HBV strains in morocco: characterization and impact on liver disease progression

Background

Hepatitis B virus (HBV) is one of the most common human pathogens that cause aggressive hepatitis and advanced liver disease (AdLD), including liver cirrhosis and Hepatocellular Carcinoma. The persistence of active HBV replication and liver damage after the loss of hepatitis B e antigen (HBeAg) has been frequently associated with mutations in the pre-core (pre-C) and core promoter (CP) regions of HBV genome that abolish or reduce HBeAg expression. The purpose of this study was to assess the prevalence of pre-C and CP mutations and their impact on the subsequent course of liver disease in Morocco.

Methods/Principal Findings

A cohort of 186 patients with HBeAg-negative chronic HBV infection was studied (81 inactive carriers, 69 with active chronic hepatitis, 36 with AdLD). Pre-C and CP mutations were analyzed by PCR-direct sequencing method. The pre-C stop codon G1896A mutation was the most frequent (83.9%) and was associated with a lower risk of AdLD development (OR, 0.4; 95% CI, 0.15–1.04; p = 0.04). HBV-DNA levels in patients with G1896A were not significantly different from the other patients carrying wild-type strains (p = 0.84). CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were associated with higher HBV-DNA level and increased liver disease severity. Multiple logistic regression analysis showed that older age (≥40 years), male sex, high viral load (>4.3 log₁₀ IU/mL) and CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were independent risk factors for AdLD development. Combination of these mutations was significantly associated with AdLD (OR, 7.52; 95% CI, 4.8–8; p<0.0001).

Conclusions

This study shows for the first time the association of HBV viral load and CP mutations with the severity of liver disease in Moroccan HBV chronic carriers. The examination of CP mutations alone or in combination could be helpful for prediction of the clinical outcome.     

Prediction of Clinical Outcomes in Hepatitis B E Antigen Negative Chronic Hepatitis B Patients with Elevated Hepatitis B Virus DNA Levels

Objectives

We investigated whether long-term clinical outcomes such as disease progression or inactive hepatitis B virus (HBV) carrier state can be predicted by baseline factors in hepatitis B e antigen (HBeAg)-negative HBV infected patients with an elevated viral load.

Methods

A retrospective cohort of 527 HBeAg-negative chronic HBV infected patients with an elevated viral load (HBV DNA ≥ 2,000 IU/ml) was assessed for disease progression defined by the development of hepatocellular carcinoma (HCC) or cirrhotic complication, as well as becoming an inactive carrier.

Results

During a median 3.6 years of follow-up, disease progression was detected in 46 patients (40 with HCC, 6 with cirrhotic complication), and 31 of 309 non-cirrhotic patients became inactive carriers. Older age, male gender, cirrhosis, high HBV DNA levels at baseline, and short antiviral therapy duration were independent risk factors for HCC. Low HBV DNA and quantitative hepatitis B surface antigen (qHBsAg) levels were independent predictors for becoming inactive carriers in patients without cirrhosis. In non-cirrhotic patients with both low qHBsAg and HBV DNA levels, the 5-year cumulative incidence of an inactive carrier was 39.8%, while that of disease progression was 1.6%.

Conclusion

HBeAg negative patients without cirrhosis can be closely monitored for becoming an inactive carrier when both HBV DNA and qHBsAg levels are low, as the risk of disease progression is low while incidence of an inactive carrier is high.     

HLA-DP Polymorphisms Affect the Outcomes of Chronic Hepatitis B Virus Infections,Possibly through Interacting with Viral Mutations

Genetic polymorphisms of HLA-DP have been associated with hepatitis B virus (HBV) persistence. We aimed to determine the effect of HLA-DP polymorphisms on the generation of HBV mutations and their interactions on the outcomes of HBV infection. rs3077, rs3135021, rs9277535, and rs2281388 were genotyped in 1,342 healthy controls, 327 HBV clearance subjects, and 2,736 HBV-positive subjects, including 1,108 hepatocellular carcinoma (HCC) patients, using quantitative PCR. HBV mutations were determined by sequencing. Multiplicative interactions of HLA-DP polymorphisms and viral mutations were assessed by multivariate logistic regression. rs3077 (from subjects with genotype CT combined with those from subjects with genotype TT [CT+TT] versus CC), rs3135021 (GA+AA versus GG), rs9277535 (GA+AA versus GG), and rs2281388 (CC versus CT+TT) significantly decreased HBV persistence. This effect was found only in genotype B HBV-infected subjects compared to HBV clearance subjects. HLA-DP polymorphisms promoting HBV clearance were associated with a lower prevalence of mutations increasing HCC risk (C1653T, T1674C/G, A1846T, G1896A and pre-S2 mutations and pre-S deletion in genotype C) and a higher prevalence of mutations decreasing HCC risk (G1652A, T1673C, T1674C, G1719T, G1730C, and G1799C in genotype B and A1727T in genotype C). Significant effects of viral mutations on cirrhosis and HCC were selectively evident in those with HLA-DP polymorphisms promoting HBV persistence. The interactions of C1653T, T1674C/G, and G1896A mutations with HLA-DP polymorphisms promoting HBV clearance significantly decreased cirrhosis risk. The interaction of rs9277535 AA with the T1674C/G or G1719T mutation in genotype C significantly decreased HCC risk. In conclusion, HLA-DP polymorphisms affect genotype B HBV clearance, regulate immune selection of viral mutations, and influence cirrhosis and HCC risks contributed by HBV mutations.     

Genome replication,virion secretion,and e antigen expression of naturally occurring hepatitis B virus core promoter mutants

The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.     

Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly

Protein-protein interactions can be regulated by protein modifications such as phosphorylation. Some of the phosphorylation sites (Ser155, Ser162 and Ser170) of HBV (hepatitis B virus) Cp have been discovered and these sites are implicated in the regulation of viral genome encapsidation, capsid localization and nucleocapsid maturation. In the present report, the dimeric form of HBV Cp was phosphorylated by PKA (protein kinase A), but not by protein kinase C in vitro, and the phosphorylation of dimeric Cp facilitated HBV core assembly. Matrix-assisted laser-desorption ionization-time-of-flight analysis revealed that the HBV Cp was phosphorylated at Ser87 by PKA. This was further confirmed using a mutant HBV Cp with S87G mutation. The S87G mutation inhibited the phosphorylation and, as a result, the in vitro HBV core assembly was not facilitated by PKA. In addition, when either pCMV/FLAG-Core(WT) or pCMV/FLAG-Core(S87G) was transfected into HepG2 cells, few mutant Cps (S87G) assembled into capsids compared with the wild-type (WT) Cps, although the same level of total Cps was expressed in both cases. In conclusion, PKA facilitates HBV core assembly through phosphorylation of the HBV Cp at Ser87.     

Pre-S Deletion and Complex Mutations of Hepatitis B Virus Related to Young Age Hepatocellular Carcinoma in Qidong,China

Background/Aim

To investigate the roles of biomedical factors, hepatitis B virus (HBV) DNA levels, genotypes, and specific viral mutation patterns on the progression of hepatocellular carcinoma (HCC) patients below 40 years of age in Qidong, China.

Methods

We conducted a case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence was determined in 49 HCC and 90 chronic hepatitis (CH) patients below 40 years of age. Mutation exchanges during follow-up in 32 cases were compared with 65 controls with paired serum samples. In addition, a consecutive series of samples from 14 HCC cases were employed to compare the sequences before and after the occurrence of HCC.

Results

After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positive, HBV DNA levels ≥4.00 log₁₀ copies/mL, pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations were associated with risk of young age HCC. Moreover, the presence of an increasing number of HCC-related mutations (pre-S deletion, T1762/A1764, and T1766 and/or A1768 mutations) was associated with an increased risk of young age HCC. Paired samples analysis indicated that the increased HCC risk for at-risk sequence mutations were attributable to the persistence of these mutations, but not a single time point mutation. The longitudinal observation demonstrated a gradual combination of pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations during the development of HCC.

Conclusion

High HBV DNA levels and pre-S deletion were independent risk factors of young age HCC. Combination of pre-S deletion and core promoter mutations increased the risk and persistence of at-risk sequence mutations is critical for HCC development.     

Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

Background

The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.

Methods

The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.

Results

Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.

Conclusions

Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.