Toshiki Ehata

Mutations in the Precore Region of Hepatitis B Virus DNA in Patients with Fulminant and Severe Hepatitis

BACKGROUND The presence of the hepatitis B e antigen (HBeAg) in serum is known to be a marker of a high degree of viral infectivity. However, fulminant hepatitis may occur in persons who are negative for HBeAg. A single point mutation has been reported to produce a stop codon in the precore region of hepatitis B virus DNA and prevent the formation of the precore protein required to make HBeAg. To determine whether a precore-mutant virus is causally related to severe liver injury, we analyzed the entire precore region in viral strains isolated from patients with fatal cases and uncomplicated cases of hepatitis B. METHODS Serum was obtained from 9 patients with fatal hepatitis B (5 with fulminant and 4 with severe exacerbations of chronic hepatitis) and 10 patients with acute, self-limited hepatitis B. Serum samples from a sex partner implicated as the source of the virus in one case of fulminant hepatitis were also studied. The 87 nucleotides in the precore region of the hepatitis B virus were amplified by the polymerase chain reaction and then directly sequenced. RESULTS Of the nine patients with fatal hepatitis, seven had retrievable hepatitis B DNA: In all seven there was a point mutation from G to A at nucleotide 1896 of the precore region, converting tryptophan (TGG) to a stop codon (TAG). In contrast, this mutation was not found in the 10 patients with acute, self-limited hepatitis B. The hepatitis B DNA from the implicated source contained a sequence with the stop-codon mutation that was identical to the sequence in her partner, who had fulminant hepatitis. CONCLUSIONS The presence of a mutant viral strain is associated with and may be involved in the pathogenesis of fulminant hepatitis B and severe exacerbations of chronic hepatitis B.

Variations in codons 84-101 in the core nucleotide sequence correlate with hepatocellular injury in chronic hepatitis B virus infection.

Individuals with chronic hepatitis B virus (HBV) infection are generally divided into asymptomatic healthy carriers and patients with chronic liver disease. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes (CTL). To investigate the possible pressure site from CTL, the entire core region of HBV DNA was sequenced in 30 subjects (10 asymptomatic healthy carriers and 20 patients with chronic liver disease). No significant changes in the nucleotide sequence and deduced amino acid residue were noted in the 10 healthy carriers. In contrast, a cluster of changes in a small segment of 18 amino acids (codons 84-101 from the start of the core gene) was found in 15 of the 20 chronic liver disease patients. All these 15 patients had advanced liver diseases (chronic active hepatitis and cirrhosis), whereas only mild liver disease (chronic persistent hepatitis) was found in the five patients without mutations. These data suggest that the region with mutation clustering is the major target of CTL, and that the mutations evolve under the pressure of immune selection.

Mutations in core nucleotide sequence of hepatitis B virus correlate with fulminant and severe hepatitis.

Infection with hepatitis B virus leads to a wide spectrum of liver injury, including self-limited acute hepatitis, fulminant hepatitis, and chronic hepatitis with progression to cirrhosis or acute exacerbation to liver failure, as well as an asymptomatic chronic carrier state. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes. To investigate the reason why the extreme immunological attack occurred in fulminant hepatitis and severe exacerbation patients, the entire precore and core region of hepatitis B virus DNA was sequenced in 24 subjects (5 fulminant, 10 severe fatal exacerbation, and 9 self-limited acute hepatitis patients). No significant change in the nucleotide sequence and deduced amino acid residue was noted in the nine self-limited acute hepatitis patients. In contrast, clustering changes in a small segment of 16 amino acids (codon 84-99 from the start of the core gene) in all seven adr subtype infected fulminant and severe exacerbation patients was found. A different segment with clustering substitutions (codon 48-60) was also found in seven of eight adw subtype infected fulminant and severe exacerbation patients. Of the 15 patients, 2 lacked precore stop mutation which was previously reported to be associated with fulminant hepatitis. These data suggest that these core regions with mutations may play an important role in the pathogenesis of hepatitis B viral disease, and such mutations are related to severe liver damage.

Precore mutations and core clustering mutations in chronic hepatitis B virus infection

BACKGROUND Mutant hepatitis B virus is often associated with severe liver damage. The purpose of this study is to elucidate the relationship between mutations in hepatitis B precore/core gene and the severity of liver damage. METHODS The hepatitis B precore/core gene from 20 patients with chronic hepatitis B virus infection was studied by polymerase chain reaction and direct sequencing. RESULTS Missense mutations in the core gene were only found in patients with chronic active hepatitis. Three mutation clustering regions of core gene, codons 48-60, 84-101, and 147-155, had higher substitution rates than other regions. All patients with chronic active hepatitis had missense mutation(s) either in codons 84-101 or in codons 48-60. There was a trend of increasing substitutions in the precore/core gene from e antigen-positive asymptomatic carriers to e antibody-positive patients with chronic active hepatitis. CONCLUSIONS These data suggest that (1) severe liver damage in chronic hepatitis B virus infection is related to the clustering missense mutations in codons 48-60 and 84-101 of core gene and that (2) the emergence of precore stop codon mutation and missense mutations around the carboxy-terminal processing site of precore/core protein (codons 147-155) may be the adaptive mechanisms of hepatitis B virus to decrease production and secretion of viral protein and retain the viral persistence.

The Two Different States of Hepatitis B Virus DNA in Asymptomatic Carriers (HBe-Antigen-Positive versus Anti-HBe-Positive Asymptomatic Carriers)

During the course of hepatitis B virus (HBV)infection, there exists a long period of normal liverfunction tests with different states of HBeAg/Ab. As thestate of HBV in asymptomatic carriers was not well characterized, we quantitatively andqualitatively examined HBV in both HBeAg-positive andanti-HBe-positive asymptomatic carriers. Sera from 10HBeAg-positive and 27 anti-HBe-positive asymptomatic carriers were analyzed. The amount of HBV DNAwas determined by dot-blot hybridization and polymerasechain reaction. The mutations in precore and coreregions, spanning 636 nucleotides, of hepatitis B virus were examined by directly sequencing theamplified HBV DNA. HBV DNA was detected in all 10 HBeAg-positive cases, whereas it was found in only 7 of 27(26%) anti-HBe-positive cases by the nested PCR method. The mean amount of HBV DNA inHBeAg-positive cases was 109.1±0.7copies/ml, while that in anti-HBe-positive cases was101.0±1.5 copies/ml. There were no 0.7missense mutations in the entire precore and core genes of HBV DNA taken fromHBeAg-positive asymptomatic carriers. In contrast, manymutations (mean 9.0 ± 3.3, range 6-14) weredetected in the core gene of seven anti-HBe-positiveasymptomatic carriers including two cases with increments of themutations. Analysis of the precore region revealed threewild-type and four mutant-type (including one coexistingwith wild-type) cases. These data suggest that HBV exists in quite different ways in“asymptomatic” carriers; in the HBeAg-positive phase HBV probably coexists with the host andremains as the wild type, whereas in theanti-HBe-positive phase a drastically reduced amount of HBV with many mutationsremains, probably as a consequence of the long-lastinginteraction with the host. Nevertheless, such smallamount of virus could cause fulminant hepatic failure. It is important to make further clinical andvirological investigations in order to understand thestate of asymptomatic carrier.

Detection and direct sequencing of hepatitis B virus genome by DNA amplification method

Hepatitis B virus (HBV) DNA was detected with amplification by the polymerase chain reaction method. Cloned HBV DNA equivalent to one virus genome (3 x 10(-6) pg) was detectable by ethidium bromide staining after 50 cycles of polymerase chain reaction. By applying this method, presence of HBV DNA was studied in 23 hepatitis B surface antigen (HBsAg)-positive and 11 HBsAg-negative sera from patients with chronic liver disease. Hepatitis B virus DNA was positive in 8 of 8 hepatitis B e antigen (HBeAg)-positive, in 2 of 2 HBeAg- and anti-HBe-negative, and in 12 of 13 anti-HBe-positive sera. Hepatitis B virus DNA was undetectable in all HBsAg-negative sera even with amplification. To confirm specificity, the amplified product was directly sequenced. Sequences around 122nd and 160th codon of HBs gene, which determines subtypes d/y and w/r, respectively, were analyzed. The results were compatible with recent reports regarding the relation between HBV subtypes and HBV DNA sequence at those codons. Hepatitis B virus DNA could be detected at the level of one virion by gene amplification method, and its sequence could be determined by direct sequencing in a few days.

Detection of mutations in the enhancer 2/core promoter region of hepatitis B virus in patients with chronic hepatitis B virus infection: Comparison with mutations in precore and core regions in relation to clinical status

To investigate the meaning of the mutations in the enhancer 2/core promoter (Enh2/CP) region of hepatitis B virus (HBV) during the chronic HBV infection, mutations were examined in the Enh2/CP region (carboxyl half of X region) and their correlation with mutations in the precore and core regions in relation to the presence of chronic liver disease. The entire nucleotide sequences of the Enh2/CP region were determined by direct sequencing of the amplified products derived from 30 cases with chronic HBV infection. The results were compared to the mutations in the precore and core regions. In the Enh2/CP region, 91 generally scattered nucleotide substitutions were detected. There were 11 substitutions in the 10 asymptomatic healthy carriers (mean, 1.1/case) and 80 in the 20 chronic liver disease patients (4.0/case). The most frequent substitutions from A to T at nucleotide 1764 and from G to A at nucleotide 1766 were seen in none of the 10 asymptomatic carriers and in 14 (70%) of the 20 chronic liver disease patients. Comparisons of mutations in the precore and core regions revealed that 14 of 16 patients with mutations in the core region had the mutations in the Enh2/CP region and/or a precore stop codon mutation. These data suggest that mutations in the Enh2/CP and precore regions may affect the expression of the core and HBeAg peptides and might be involved in the pathogenesis of chronic liver disease. J. Med. Virol. 57:337–344, 1999.

GB virus-C RNA in Japanese patients with hepatocellular carcinoma and cirrhosis

BACKGROUND/AIMS The involvement of non-B, non-C virus in the incidence of hepatocellular carcinoma (HCC) is not yet known. We have therefore examined the occurrence of GBV-C RNA in such patients. METHODS One hundred and eleven patients diagnosed as having HCC and 67 patients with cirrhosis without HCC were examined for the prevalence of GBV-C RNA by nested reverse transcription polymerase chain reaction with primers located at the helicase region. Sera were obtained and kept at -20 degrees C until analysis. RESULTS GBV-C RNA was positive in 11/111 (9.9%) cases with HCC, in 10/74 (13.5%) anti-HCV positive cases, in 1/25 (4%) HBsAg positive cases, and in 0/8 (0%) anti-HCV and HBsAg negative cases. GBV-C RNA was also positive in 7/67 (10.4%) cases with cirrhosis, in only 1/18 (5.6%) anti-HCV and HBsAg negative cases, in 4/33 (12.1%) anti-HCV positive, and in 2/14 (14.3%) HBsAg positive cases. The clinical background of patients with anti-HCV positive HCC who were also positive for GBV-C RNA did not differ from the background of those negative for GBV-C RNA. CONCLUSIONS GBV-C is unlikely to be a major etiologic agent of non-B, non-C chronic liver diseases and HCC in Japan.

Concentrating missense mutations in core gene of hepatitis B virus : evidence for adaptive mutation in chronic hepatitis B virus infection

To elucidate the temporal relationship between liver damage and mutation(s) in hepatitis B virus core gene, serial sera from a progressive liver disease patient and an asymptomatic carrier were studied. By direct sequencing, missense mutations in the core gene were only found in serum from the progressive liver disease patient during the period with frequent exacerbation. Using methods of cloning and sequencing, missense mutations were also found in clones derived from the progressive liver disease patient at a relatively early phase, but strains with a missense mutation from earlier sera did not exist in sera of a later period. Furthermore, there was a tendency of concentrating missense mutations in clones derived from the progressive liver disease patient. These data suggested that missense mutations in the core gene that occurred at an earlier phase might evoke an immune response to eliminate mutated virus and that concentrating missense mutations during a phase of exacerbation might be a result of adaptive mutation.

Detection of hepatitis B virus precore stop codon mutants by selective amplification method: Frequent detection of precore mutants in hepatitis B e antigen positive healthy carriers

The precore region of hepatitis B virus (HBV) is indispensable for secretion of e antigen protein. Therefore, the precore stop codon mutants may play an important role in the process of e antigen seroconversion. However, the presence of the mutants in hepatitis B e antigen positive carriers has not been fully studied because of difficulties in detecting the mutants in the presence of large amounts of wild‐type viruses. To overcome this, a sensitive method has been developed to detect the presence of G to A stop codon mutants at codon 28 of precore region. Primers for polymerase chain reaction (PCR) were devised to introduce restriction enzyme site Sty I for wild‐type viruses and Dde I for the mutants. The amplification products with these primers were digested with Sty I to exclude the products from wild‐type viruses. The remaining amplicon from precore mutants were re‐amplified, and the presence of precore mutant was confirmed with Dde I digestion. The presence of precore mutants was examined in 61 HBV carriers by the method combining PCR and restriction enzyme digestion. Approximately 0.1% of precore mutant DNA among 106 copies of wild‐type virus DNA was detectable by this method. The presence of the precore mutants was detected in seven of 10 (70%) e antigen positive asymptomatic carriers, and in 29 of 36 (81%) e antigen positive patients with chronic liver diseases, and in all 15 (100%) anti‐e antibody positive patients with chronic liver diseases.