A stop codon in the precore genome of the hepatitis B virus (HBV) in anti-HBe positive HBV carriers may be associated with a more progressive form of HBV infection. Earlier studies, however, were mainly performed in patients from the Mediterranean area who had severe infection. The aim of this study was to evaluate the prevalence of precore mutants in an unselected population living in northern Europe. Twenty of 42 of these patients are infected predominantly with a virus strain, which has the typical stop codon in the precore genome, characterised by a mutation at base 83. In six patients there was an additional G to A mutation at base 86 of the precore genome. Statistical analysis showed no difference between the patients with or without a stop codon in the precore genome. When patients with a double mutation at base 83 and 86 of the precore genome were compared with the other anti-HBe positive HBV carriers, however, the corresponding clinical data were worse. Therefore we suggest, that it is not the stop codon in the precore gene itself, but the occurrence of a double mutation at bases 83 and 86, which is associated with a more severe course of disease in anti-HBe positive HBV carriers.
This study aimed to investigate the ratios of precore stop mutant (codon 28; TGG to TAG) to total viremia in 53 HBeAg-positive patients with chronic hepatitis B by amplification-created restriction site assays along the course of HBeAg-to-anti-HBe seroconversion. At baseline, 11% had exclusive wild-type hepatitis B virus (HBV), 15% had exclusively precore mutant, and 74% had mixed viral strains. Precore mutant ratios correlated little with age, sex, or HBV DNA levels (all P > 0.1), but correlated modestly with alanine aminotransferase (ALT) levels (P = 0.05). The intervals from presentation to anti-HBe seroconversion correlated significantly with ALT and precore mutant ratios in univariate analysis but with only precore mutant ratios in multivariate analysis (P = 0.003). Precore mutant ratios at baseline were significantly higher (P < 0.001) in six patients with persistent high viremia and ALT elevation after anti-HBe seroconversion (group 1) than in 47 with remission (group 2). All group 1 patients had exclusive precore mutant after anti-HBe seroconversion, as did only 14 (30%) of the group 2 patients (P = 0.003). Among group 2 patients, precore mutant ratios at baseline or after anti-HBe seroconversion showed no significant difference between 34 patients with sustained remission and 13 with relapse. Cirrhosis developed in 50% (5 of 10) of patients with precore mutant ratios >50% at baseline but only in 12% (5 of 43) of those with precore mutant ratios of <50% at baseline (P < 0.05). In conclusion, precore mutant of variable ratios was frequently detected in HBeAg-positive patients with chronic hepatitis B. Precore mutant ratios tended to correlate with ALT levels and anti-HBe seroconversion, but high precore mutant ratios were associated with persistent hepatitis after anti-HBe seroconversion and increased risk of cirrhosis.
After perinatal transmission of hepatitis B virus, infants of anti-HBe positive HBsAg carrier mothers may develop fulminant hepatitis B. Previously it has been suggested, that fulminant hepatitis B in adults was associated with specific mutations in the HBV-genome. The aim of this study was to investigate, whether specific viral variants are associated with fulminant hepatitis B in young infants.
The complete HBV-genomes of five mothers and their infants with fulminant hepatitis were isolated from the sera, amplified and directly sequenced.
Between 6 and 43 base pair exchanges between the HBV genomes of the infants and their mothers were identified. The mutations spread over the entire virus genome. Nucleotide exchanges in the basic core promotor and precore region were identified in all cases. A heterogeneous virus population was detected in four mothers.
Many new mutations were proved to emerge during fulminant hepatitis B in infants, who had been perinatally infected. HBeAg negative variants were the predominant population in all children, whereas these mutants could only be detected as subpopulations in four mothers. The data suggest that the selection of a specific HBeAg negative viral strain may be associated with the development of fulminant hepatitis B in children.
Hepatitis B virus (HBV) genotypes have distinct geographic distribution. Moreover, much genetic variability has been described in the precore (PC) and basal core promoter (BCP) regions of the HBV genome. The local prevalence of HBV genotypes and mutations has not been well studied. The aim of the present study is to determine the prevalence of HBV genotypes and mutations in the PC and BCP region in HBV strains in Karachi.
A total of 109 chronic hepatitis B patients with detectable HBV DNA by a PCR assay were enrolled in the study. Sera were tested for HBeAg, anti-HBe antibody and liver profile. HBV genotypes and mutations in the PC and BCP regions were detected by INNO-LiPA line-probe assays.
Of the 109 patients investigated, 38 (35%) were HBeAg positive while 71 (65%) were HBeAg negative. Genotype D was present in 100% of the patients. Two patients had co-infection with genotype A. There was no significant difference in the baseline characteristics, mean ALT levels, and presence of clinical cirrhosis in patients with HBeAg positive or negative strains with or without PC and BCP mutations. Of the 38 HBeAg positive patients, 9 (24%) had PC and BCP mutations. In the HBeAg negative patient group, mutations were detected in 44 (62%) of the strains investigated. More than one mutation was common, seen in 26 (37%) patients with HBeAg negative disease and 6 (16%) patients with HBeAg positive disease. Twelve (17%) HBeAg negative patients had dual T1762 and A1764 mutations. None of the HBeAg positive patients had T1762 mutation. Mutations were undetectable in 27 (38%) of patients with HBeAg negative disease.
Our study shows that type D is the main HBV genotype in Karachi, Pakistan. Significant numbers of patients infected with this genotype have PC and BCP variants. Mutations at more than one site are common. Patients harboring these mutants do not differ significantly in their clinical presentation from patients having wild type infection.
During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.
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.
HBV DNAs from 29 anti-HBe Brazilian samples were characterized by nucleotide sequencing of PCR products from precore region. Among them, 18 isolates presented C at nt 1858 (mostly genotype A strains). The 11 remaining isolates (genotypes D and F) had T1858. The stop codon mutation at nt 1896 was found in seven isolates (24% of the total and 63% of the isolates that had T1858). The frequency of the double substitution at positions 1762-1764 was surprisingly low (20%) among C1858 isolates. An association between A1896 and TGA 1762-1764 mutations was observed among genotype D isolates: these showed either none of the two mutations or both. Furthermore, strains mutated at positions 1896 and/or 1762-1764 also presented an elevated number of other, less common substitutions in the core promoter and precore regions.
The data reported here are not in accordance with some reports from other parts of the world. In half of the isolates, none of the mutations previously described could explain the anti-HBe phenotype.
Hepatitis B virus (HBV) genotypes may influence HBeAg seroconversion rates, mutational patterns in the precore (PC) and core promoter (CP) regions, severity of liver disease, and response to antiviral treatment. Development of rapid, simple, and standardized assays to detect viral genotypes and common mutations in the PC and CP regions can accelerate research on the clinical significance of these variants. We aim to assess the accuracy of a line probe assay in determining HBV genotypes and detecting HBV PC and CP variants. HBV genotypes in 701 patients and PC and CP variants in 600 patients with chronic HBV infection from China and the United States were studied using the INNO-LiPA assay. All but one (99.9%) sample were classified by the genotyping assay. All eight genotypes, i.e., A to H, were found. The INNO-LiPA genotyping assay results were completely concordant with those of sequencing. Using the INNO-LiPA PC assay, 99.8 and 94.7% samples were classifiable in the PC and CP regions, respectively. The PC assay results were completely concordant with those of sequencing in all samples that showed either wild-type or variant sequence. The line probe assay was more sensitive in detecting mixtures than was direct sequencing. By INNO-LiPA, only 50 and 27% of the samples, with mixed wild-type and variant sequence in the PC and CP region, respectively, showed mixed sequence by direct sequencing. INNO-LiPA is rapid, sensitive, and reliable—thus enabling accurate determination of HBV genotypes and detection of PC and CP variants in a large population of patients.
This study investigated the prevalence of the precore G1896A mutation in Chinese patients with hepatitis B e antigen (HBeAg) negative HBV infection and its relation to serum HBV pre-S1 antigen. The overall prevalence of the precore G1896A mutation was 72.6% in HBeAg-negative Chinese patients with detectable serum HBV DNA. The prevalence of the precore G1896A is significantly higher in Chinese HBeAg-negative patients with chronic hepatitis B than that in inactive HBV carriers with detectable serum HBV DNA. Serum pre-S1 and the precore G1896A mutation were simultaneously detected in most of Chinese HBeAg-negative patients.
Hepatitis B virus; precore mutation; pre-S1
There are cases of hepatitis involving occult hepatitis B virus (HBV) infection in which, even though the HB surface antigen (HBsAg) is negative, HBV-DNA is detected by a polymerase chain reaction (PCR). We conducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-year-old female. A diagnosis of non-B non-C chronic hepatitis was made. Although HBV markers, such as HBs antibody (anti-HBs), anti-HBc, HBeAg and anti-HBe, were negative, HBV-DNA was positive. Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR. Sequence analysis of the two obtained bands was conducted by direct sequencing. Compared with the control strains, the ATG (Methionine) start codon in the X region had mutated to GTG (Valine). It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.
Hepatitis B virus; X region; Mutation; Non-B non-C chronic hepatitis; Occult infection
Hepatitis B virus (HBV) causes a chronic infection in 350 million people worldwide and greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. The majority of chronic HBV carriers live in Asia. HBV can be divided into eight genotypes with unique geographic distributions. Mutations accumulate during chronic infection or in response to external pressure. Because HBV is an RNA-DNA virus the emergence of drug resistance and vaccine escape mutants has become an important clinical and public health concern. Here, we provide an overview of the molecular biology of the HBV life cycle and an evaluation of the changing role of hepatitis B e antigen (HBeAg) at different stages of infection. The impact of viral genotypes and mutations/deletions in the precore, core promoter, preS, and S gene on the establishment of chronic infection, development of fulminant hepatitis and liver cancer is discussed. Because HBV is prone to mutations, the biological properties of drug-resistant and vaccine escape mutants are also explored.
hepatitis B virus; persistent infection; genotype; mutant
Hepatitis B virus (HBV) has eight genotypes which have distinct geographical distributions. Studies comparing differences in the clinical outcomes of infections caused by strains with genotype-related variations in the HBV genome have largely compared genotypes B and C and genotypes A and D but not all four genotypes. The present study included 196 HBV-infected patients attending an infectious diseases outpatient clinic in Sweden. The age and geographic origin, liver function, HBeAg and anti-HBe status, and the presence or absence of HBV DNA were analyzed for each patient. HBV DNA was detected in 144 patients, and the HBV genotype and the core promoter and precore sequences were determined for the isolates from 101 of these patients. Among the patients who might be considered most likely to be nonviremic, namely, anti-HBe-positive HBV carriers with normal alanine aminotransferase (ALT) levels, 65% had detectable HBV DNA and were thus viremic. Among the viremic patients, HBeAg-positive patients were more likely to have elevated ALT levels than anti-HBe-positive patients. HBV genotypes A to F were represented in the study, and their distributions coincided accurately with the origin of the patient. A significantly higher number of genotype D-infected patients were anti-HBe positive and had elevated ALT levels (42% of genotype D-infected patients but 0% of patients infected with genotypes B and C). Genotype D strains with mutations in the core promoter and precore regions were significantly correlated with elevated ALT levels in the patients. The differences were not age related. Therefore, in this large-scale cross-sectional study, genotype D appears to be associated with more active disease.
Hepatitis B virus (HBV) mutants unable to synthesize HBV e antigen have been described in association with fulminant hepatitis. We have cloned and sequenced the entire viral genome of an HBV strain associated with an epidemic of fulminant hepatitis. This strain contained, in addition to two G-to-A mutations in the precore region (nucleotides 1898 and 1901), numerous other mutations in conserved nucleotide positions resulting in significant amino acid substitutions in HBV gene products. We introduced either or both of the two G-to-A mutations into wild-type HBV by oligonucleotide-directed mutagenesis and generated replication-competent constructs of these mutants as well as the fulminant strain. Viral antigen synthesis, transcription, and replication were analyzed after transfection into human hepatoma cells. All viral constructs produced and secreted similar levels of envelope proteins (HBV surface antigen). Analysis of cellular lysate for core-specific immunoreactivity demonstrated a much higher level of core-associated antigens in cells transfected with the fulminant strain. While cells transfected with mutant and wild-type HBV DNAs synthesized similar levels of viral RNAs, the fulminant strain directed the synthesis of a much higher level of core-associated replicative intermediates (as well as virion particles) than the wild type and mutants with either or both of the precore mutations. Increase in the encapsidation of pregenomic RNA into core particles likely the basis for the enhanced replication associated with the fulminant strain. Our study suggests that an HBV mutant with enhanced viral replication may be important in the pathogenesis of fulminant hepatic failure, and mutations other than the precore mutations may be responsible for this variant behavior.
The −1G mutant HBV is more prevalent in individuals co-infected with HIV/HBV than in individuals infected with HBV alone and in some cases is the dominant virus in circulation. This mutant is created by the deletion of a dGMP (−1G) from the guanine rich homopolymer sequence located at nts 2,085–2,090 (numbering from EcoRI site as position 1) in the HBV core gene. This deletion causes a frameshift generating a premature stop codon at 64Asn in the HBV core gene (codon 93 in the precore gene), that truncates the precore protein, precursor of the secreted hepatitis B “e” antigen (HBeAg), and the core protein which forms the viral nucleocapsid. However, the replication phenotype of the −1G mutant HBV is unknown. An in vitro cell culture model in which hepatoma cells were transiently transfected with infectious cDNAs was used to show that the −1G mutant HBV is incapable of autonomous replication and, as expected, replication was restored to wild-type (wt) levels by supplying HBV core protein in trans. Although the −1G mutation had no deleterious effect on intracellular HBV-DNA levels, high levels of −1G mutant HBV relative to wt HBV reduced virus secretion and HBeAg secretion relative to empty vector controls. Importantly, the −1G mutant HBV also caused intracellular retention of truncated precore protein in the endoplasmic reticulum (ER) and Golgi apparatus. Together, these effects may be contributing to the increased pathology observed in the setting of HIV/HBV co-infection.
HBV; HIV; co-infection; precore; HBeAg; virus replication; ER stress
Previous studies have proved the presence of several distinct types of mutations in hepatitis B virus (HBV) infections, which are related to the progression of liver disease. However, few reports have detailed the mutation frequencies and mutation patterns in the precore/core (preC/C) region, which are based on the clinical status and HBeAg serostatus. Our aim in this study is to investigate the relationships between the preC/C mutations and clinical severity or HBeAg serostatus from patients chronically infected with HBV genotype C. A total of 70 Korean chronic patients, including 35 with hepatocellular carcinoma (HCC), participated in this study. HBV genotyping and precore/core mutations were analyzed by direct sequencing. All patients were confirmed to have genotype C infections. Mutations in the C region were distributed in a non-random manner. In particular, mutations in the MHC class II restricted region were found to be significantly related to HCC. Six (preC-W28*, C-P5H/L/T, C-E83D, C-I97F/L, C-L100I and C-Q182K/*) and seven types (preC-W28*, preC-G29D, C-D32N/H, C-E43K, C-P50A/H/Y, C-A131G/N/P and C-S181H/P) of mutations in the preC/C region were found to be related to HCC and to affect the HBeAg serostatus, respectively. In conclusion, our data indicated that HBV variants in the C region, particularly in the MHC class II restricted region, may contribute to the progress of HCC in chronic patients infected with genotype C. In addition, we found several distinct preC/C mutations in the Korean chronic cohort, which affect the clinical status of HCC and HBeAg serostatus of patients infected with genotype C.
A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (P < 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles—T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764—were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (P < 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.
This study was carried out to determine the effects of hepatitis B virus genotypes, core promoter mutations (A1762G1764→T1762A1764) as well as precore stop codon mutations (TGG→TAG) on HBeAg expression and HBeAg/ anti-HBe status. Study was also performed on the effects of codon 15 variants (C1858/ T1858) on the predisposition of precore stop codon mutations (TGG→TAG). A total of 77 sera samples were analyzed. Fifty one samples were successfully genotyped of which the predominant genotype was genotype B (29/ 51, 56.9 %), followed by genotype C (16/ 51, 31.4 %). Co-infections by genotypes B and C were observed in four samples (7.8 %). To a lesser degree, genotypes D and E (2.0 % each) were also observed. For core promoter mutations, the prevalence was 68.8 % (53/ 77) for A1762G1764 wild-type and 14.3 % (11/ 77) for T1762A1764 mutant while 9.1 % (7/ 77) was co-infected by both strains. The prevalence of codon 15 variants was found to be 42.9 % (33/ 77) for T1858 variant and 16.9 % (13/ 77) for C1858 variant. No TAG mutation was found. In our study, no associations were found between genotypes (B and C) and core promoter mutations as well as codon 15 variants. Also no correlation was observed between HBeAg/ anti-HBe status with genotypes (B and C) and core promoter mutations.
HBV; Genotypes; HBeAg; Core Promoter Mutation; 1858 Variants; Precore Stop Codon Mutation
We have determined the structure of the core capsid of an unusual variant of hepatitis B virus, genotype G (HBV/G) at 14 Å resolution, using cryo-electron microscopy. The structure reveals surface features not present in the prototype HBV/A genotype. HBV/G is novel in that it has a unique 36- bp insertion downstream of the core gene start codon. This results in a twelve amino acid insertion at the N-terminal end of the core protein, and two stop codons in the precore region that prevent the expression of HBeAg. HBV/G replication in patients is associated with co-infection with another genotype of HBV, suggesting that HBV/G may have reduced replication efficiency in vivo. We localized the N-terminal insertion in HBV/G and show that it forms two additional masses on the core surface adjacent to each of the dimer-spikes and have modelled the structure of the additional residues within this density. We show that the position of the insertion would not interfere with translocation of nucleic acids through the pores to the core interior compartment. However, the insertion may partially obscure several residues on the core surface that are known to play a role in envelopment and secretion of virions, or that could affect structural rearrangements that may trigger envelopment after DNA second-strand synthesis.
core-like particles; electron microscopy; Hepatitis B virus
Hepatitis B virus (HBV) precore G1896A mutation is associated with Hepatitis B e antigen (HBeAg) seroconversion. This mutation and the adjacent G1899A mutation also appear to associate with increased risk of hepatocellular carcinoma. Quantitative mutant dynamics may help determine the potential of these mutants as clinical biomarkers. However, a reliable method to quantify either mutant is not available, partly because the viral genome has polymorphisms in general and the precore mutations are complex.
(1) To develop a reliable and ultrasensitive assay for the quantification of HBV G1896A and/or G1899A mutants. (2) To obtain preliminary data on the quantities of the precore mutants in patients.
A SimpleProbe real time PCR assay was developed to quantify the HBV precore mutants. Dual melting analysis and a primer-probe partial overlap approach were used to increase detection accuracy. A wild-type selective PCR blocker was also developed to increase mutant detection sensitivity.
The assay correctly identified the precore sequence from all 62 patient samples analyzed. More than 97% of precore sequences in the GenBank can be recognized. Mutant detection sensitivity reached 0.001% using a wild type-selective PCR blocker. At least one precore mutant can be detected from all 20 HBeAg-positive individuals who were negative for precore mutations by DNA sequencing.
The reliability of this ultrasensitive mutation quantification assay was demonstrated. The same approaches may be useful for the detection of other clinically significant mutations. Evolution of the precore mutants warrants further studies.
mutation; HBV; precore; quantification; SimpleProbe; qPCR
To assess the prevalence and clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations in Taiwan, a cohort of 200 Taiwanese chronic hepatitis B patients was analyzed. The HBV genotypes and sequences of the precore and the core promoter regions were determined in 66 asymptomatic carriers and 134 patients who had liver biopsy-verified chronic hepatitis and liver cirrhosis. The HBV e-antigen (HBeAg)-negative patients had a higher frequency of mutations at core promoter nucleotides 1753 and 1773 and precore nucleotides 1846, 1896, and 1899 than HBeAg-positive patients. Among the 200 patients, the frequencies of genotype C, T1762 and A1764, C1753, T1766 and A1768, and A1896 mutations increased and the frequencies of T or G1752, T1773, G1799, and C1858 mutations decreased with advancing liver diseases. These factors were different between those with HBeAg-positive status and those with HBeAg-negative status. Based on multiple logistic regression analysis, the risk factors of liver cirrhosis for 200 patients were the presence of T1762 and A1764 mutations (odds ratio [OR] = 11.11; 95% confidence interval [CI] = 3.91 to 31.25; P < 0.001), age ≥35 years (OR = 3.42; 95% CI = 1.33 to 8.77; P = 0.011), and genotype C (OR = 2.87; 95% CI = 1.21 to 6.81; P = 0.017). Further categorical analysis found that 62.1% of patients with genotype C, T1762 and A1764 mutations and age ≥35 years had liver cirrhosis. None of the 55 patients infected with the genotype B, A1762 and G1764 wild type and age <35 years showed liver cirrhosis. In conclusion, our data suggest that pathogenic differences between HBeAg-positive and -negative patients may exist. In Taiwan, HBV genotype C and the T1762 and A1764 mutations may play a role in HBV-related liver cirrhosis, and these could serve as molecular markers for prediction of the clinical outcomes of chronic HBV patients.
By using an amplification-created restriction site method, the precore TAG mutant of hepatitis B virus was detected in 6 (75%) of 8 acute fulminant hepatitis B patients, 7 (58%) of 12 acute self-limiting hepatitis B patients, 35 (81%) of 43 hepatitis B virus surface antigen carriers with fulminant hepatitis, and 42 (70%) of 60 hepatitis B virus surface antigen carriers with chronic hepatitis. The precore TAG mutant prevails in acute and chronic hepatitis B of various severity in this area where hepatitis B is endemic.
The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >106 copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies.
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 × 109 to 5.7 × 109 copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 × 107 to 4.6 × 107 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.
AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.
The level of hepatitis B virus (HBV) DNA in serum reflects the replicative activity of HBV. To compare serum HBV DNA levels in different states of hepatitis B, 47 sera of patients with HBeAg-positive chronic hepatitis B, 4 sera of patients with HBeAg-negative chronic hepatitis B, 40 samples of patients after HBeAg seroconversion during alpha interferon treatment, 57 sera of inactive HBsAg carriers, and 42 sera of patients who had recovered from chronic hepatitis B more than 12 months prior to blood collection were checked for the presence of HBV DNA with the Amplicor HBV Monitor Test. In patients with HBeAg-positive chronic hepatitis B, the median of serum HBV DNA levels (8.3 × 108 copies/ml) was significantly higher than that for patients after HBeAg seroconversion (6.2 × 103 copies/ml) and than that for inactive HBsAg carriers (5.6 × 103 copies/ml). None of the patients who had recovered from hepatitis B had detectable HBV DNA in serum. Quantitative PCR proved to be a valuable tool for identification of different states of HBV infection. This technique was found to be a good method for determination of serum HBV DNA levels both for patients with HBeAg seroconversion and for inactive carriers who showed low viremia not detectable by conventional hybridization assays.
More than 300 million people worldwide are chronically infected with hepatitis B virus (HBV). Considering the very short generation time for a virus, and the high error rate associated with the reverse transcription step of HBV replication, decades of HBV infection are probably equivalent to million years of human evolution. The most important selective force during the natural course of HBV infection appears to be the immune response. The development of anti-HBe antibody in hepatitis B patients usually correlates with reduction of HBV viremia. As a consequence, escape mutants of anti-HBe are selected. The core promoter mutants express less HBe antigen (HBeAg) through transcriptional down regulation, while precore mutants express truncated products. We recently identified additional mutations that modulate HBeAg translation initiation, proteolytic cleavage, and secondary structure maintenance through a disulfide bond. The core promoter mutants have been associated with the development of fulminant hepatitis during acute infection and liver cancer during chronic infection. Consistent with their enhanced pathogenicity, core promoter mutants were found to replicate at up to 10-fold higher levels in transfected human hepatoma cells than the wild-type virus. Moreover, some core promoter mutants are impaired in virion secretion due to missense mutations in the envelope gene. These virological properties may help explain enhanced pathogenicity of core promoter mutants in vivo.
Hepatitis B virus; HBeAg; naturally occurring mutations; immune escape; replication; secretion