Hajime Tokita

Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan.

ABSTRACT Among 87 patients who were previously treated for acute hepatitis of unknown etiology between 1992 and 2001 at five hospitals in Japan, 11 (13%) patients were positive for immunoglobulin M-class antibodies to hepatitis E virus (HEV) by enzyme immunoassay and had detectable HEV RNA by reverse transcription-PCR with two independent sets of primers derived from well-conserved genomic areas in open reading frames 1 and 2. Clinical HEV infection was significantly associated with male sex (9 of 11 versus 29 of 76 patients [P < 0.01]) and older age (52 ± 11 [mean ± standard deviation] versus 41 ± 17 years [P < 0.05]), and its prevalence differed by geographic region (6 to 25%), with a higher rate in the northern part of Japan. At admission, the 11 patients with HEV-associated hepatitis had elevated alanine aminotransferase levels of 914 to 4,850 IU/liter, and all but 1 had elevated bilirubin levels of 1.5 to 24.0 mg/dl. The 11 HEV isolates were of genotype III or IV and were segregated into three groups with intergroup nucleotide differences of 9.5 to 22.0%. Phylogenetic analysis revealed that four isolates of genotype III were closely related to a Japanese isolate, while the other four isolates of the same genotype were nearest those from the United States. The remaining three isolates were close to known isolates of genotype IV in China and Taiwan but shared less than 88% identity with them. These results indicate that multiple genotypes of HEV cocirculate in Japan and contribute to the development of sporadic acute hepatitis, with the prevalence differing by age, sex, and geographic region.

Influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis B virus infection.

The outcome of acute hepatitis B virus (HBV) infection is variable, influenced by host and viral factors. From 1982 through 2004, 301 patients with acute HBV infection entered a multi‐center cross‐sectional study in Japan. Patients with fulminant hepatitis (n = 40) were older (44.7 ± 16.3 vs. 36.0 ± 14.3 years, P < .0017), less predominantly male (43% vs. 71%, P = .0005), less positive for hepatitis B e antigen (HBeAg) (23% vs. 60%, P < .0001), less infected with subgenotype Ae (0% vs. 13%, P < .05), and more frequently with Bj (30% vs. 4%, P < .0001) than those with acute self‐limited hepatitis (n = 261). Precore (G1896A) and core‐promoter (A1762T/G1764A) mutations were more frequent in patients with fulminant than acute self‐limited hepatitis (53% vs. 9% and 50% vs. 17%, P < .0001 for both). HBV infection persisted in only three (1%) patients, and they represented 2 of the 23 infected with Ae and 1 of the 187 with the other subgenotypes (9% vs. 0.5%, P = .032); none of them received antiviral therapy. In multivariate analysis, age 34 years or older, Bj, HBeAg‐negative, total bilirubin 10.0 mg/dL or greater, and G1896A mutation were independently associated with the fulminant outcome. In in vitro transfection experiments, the replication of Bj clone was markedly enhanced by introducing either G1896A or A1762T/G1764A mutation. In conclusion, persistence of HBV was rare (1%) and associated with Ae, whereas fulminant hepatitis was frequent (13%) and associated with Bj and lack of HBeAg as well as high replication due to precore mutation in patients with acute HBV infection. (HEPATOLOGY 2006;44:326–334.)

Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11a) genetic groups.

Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279-9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups.

A second-generation method of genotyping hepatitis C virus by the polymerase chain reaction with sense and antisense primers deduced from the core gene

A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmonds classification.

THE ENTIRE NUCLEOTIDE SEQUENCES OF THREE HEPATITIS C VIRUS ISOLATES IN GENETIC GROUPS 7-9 AND COMPARISON WITH THOSE IN THE OTHER EIGHT GENETIC GROUPS

We have proposed that hepatitis C virus should be classified into eleven genetic groups (types) which further divide into more than 80 genotypes (subtypes). However, only eight genetic groups (1-6, 10 and 11) have been defined on the basis of the full-length sequence. Hence, the entire nucleotide sequences of three HCV isolates in genetic groups 7-9 have now been determined. Phylogenetic analysis over the full-length sequences of these three isolates, along with 30 more in the other eight genetic groups, indicated that genetic groups 6-9 and 11 have bifurcated from a common branch and groups 3 and 10 from another. In the former branch groups 7 and 11, and groups 8 and 9, are closely related. Consequently, HCV can be classified into either eleven (1-11) or six groups (1; 2; 3 and 10; 4; 5; 6-9 and 11), allowing a clear separation of group and genotype similarity within the NS5b region or a subregion of 1093 nt. When pairwise comparison of 1093 nt in the NS5b sequence was performed on 106 HCV isolates of 36 genotypes in eleven genetic groups, they were classified into either eleven (1-11) or six (1; 2; 3 and 10; 4; 5; 6-9 and 11) genetic groups. However, group and genotype similarities were not clearly separable in either classification. The overlapping range was smaller using the classification into eleven genetic groups as compared to six genetic groups (2.7 vs 4-7%). These results indicate that HCV might not have evolved in the two-tiered fashion, at least in a strict sense.

Risk factors for the development of hepatocellular carcinoma among patients with chronic hepatitis C who achieved a sustained virological response to interferon therapy

Background and Aim:  Hepatitis C virus (HCV)‐infected patients who responded to interferon (IFN) treatment with clearance of serum HCV RNA may rarely develop hepatocellular carcinoma (HCC). The aim of the present study was to elucidate the risk factors for liver carcinogenesis among such patients.

Two Subtypes of Genotype B (Ba and Bj) of Hepatitis B Virus in Japan

We have previously reported 2 subtypes of hepatitis B virus (HBV) genotype B, one of which has the recombination with genotype C over the precore region plus core gene (Ba) and the other of which does not (Bj). A restriction fragment-length polymorphism method with 2 endonucleases was newly developed for distinguishing between subtypes Ba and Bj and was applied to 313 carriers of HBV genotype B in Japan. Subtype Ba was detected in 38 (12%) and subtype Bj in 275 (88%) of the carriers of HBV genotype B. Hepatitis B e antigen in serum was found more frequently in patients with chronic infection with subtype Ba than in those with chronic infection with subtype Bj (8 [32%] of 25 vs. 25 [9%] of 273; P<.01). The new method for distinguishing between Ba and Bj by restriction fragment-length polymorphism would be useful in examining the distribution of these 2 subtypes in situations in which HBV genotype B is prevalent.

T1653 Mutation in the Box a Increases the Riskof Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Virus Genotype C Infection

BACKGROUND Most patients with chronic hepatitis B virus infection become carriers of inactive virus after hepatitis B e antigen seroconversion; however, a subgroup of patients have persistent abnormal transaminase levels and develop hepatocellular carcinoma after seroconversion. METHODS In an age-matched case-control study, 40 carriers of inactive virus (mean age+/-standard deviation [SD], 50.9 +/- 11.1 years), 40 patients with chronic hepatitis (mean age+/-SD, 50.2 +/- 8.9 years), and 40 patients with hepatocellular carcinoma (mean age+/-SD, 50.7 +/- 9.4 years) who were infected with hepatitis B virus genotype C and had test results positive for antibody to hepatitis B e antigen were analyzed. RESULTS The prevalence of T1653 in the box alpha was significantly higher among patients with hepatocellular carcinoma than among carriers of inactive virus who did not have hepatocellular carcinoma (70% vs. 25%; P < .0001) or chronic hepatitis (70% vs. 35%; P = .003). Mutations in the basic core promoter region (T1762/A1764) were frequently found in all groups, regardless of clinical status (in 77.5% of carriers of inactive virus, 77.5% of patients with chronic hepatitis, and 90% of patients with hepatocellular carcinoma). In the multivariate analysis, the presence of T1653, an alanine aminotransferase level of > or = 37 U/L, and a platelet count of < 18 x 10(4) platelets/mm3 were independent predictive values for hepatocellular carcinoma (odds ratio [95% confidence interval], 5.05 [1.56-16.35], 12.56 [3.05-51.77], and 11.5 [3.47-38.21], respectively). High alpha -fetoprotein level was the only independent predictive value for T1653 in patients with hepatocellular carcinoma (odds ratio, 12.67; 95% confidence interval, 1.19-134.17]). Among patients with test results positive for antibody to hepatitis B e antigen who had hepatocellular carcinoma and were infected with different genotypes of hepatitis B virus, the prevalence of T1653 was 40%, 15%, 25%, 25%, 67%, and 23% in patients infected with hepatitis B virus genotypes Aa, Ae, Ba, Bj, C, and D, respectively (P<.05 for genotype C vs. genotypes Ae, Ba, Bj, or D). CONCLUSIONS Our data indicate that the addition of T1653 mutation in the box alpha to the basic core promoter mutation increases the risk of hepatocellular carcinoma in patients with hepatitis B virus genotype C.

Full-length genomic sequence of a hepatitis C virus genotype 2c isolate (BEBE1) and the 2c-specific PCR primers

SummaryWe sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.

Hepatitis C virus RNA and antibodies among blood donors in Beijing.

Blood units from voluntary as well as commercial donors in Beijing, China, were tested for hepatitis C virus RNA and antibodies, and for serological markers of hepatitis B virus infection. HCV RNA was detected less frequently in 1909 voluntary donors (5 (0.3%)), than in 1017 commercial donors (58 (5.7%)) (p < 0.001). Antibody to hepatitis C virus was detected by the second-generation enzyme immunoassay in 55 (87%) of 63 blood units with viremia. Evidence of present or past infection with hepatitis B virus was common both in voluntary (43.9%) and commercial (46.4%) donors. There were eight (13%) sera with HCV-RNA in which hepatitis C virus antibodies were not detectable by second-generation enzyme immunoassay. Of 63 HCV-RNA samples from donors, 33 (52%) were of genotype II, 18 (29%) of III and one (2%) of II + III. HCV-RNA in the remaining 11 (17%) were not classifiable into any of the genotypes I, II, III, IV and V. Genotype II was more frequent in viremic donors with elevated alanine aminotransferase levels (13/18 or 72%) than in those with normal levels (20/45 or 44%). These results indicate a low prevalence of hepatitis C virus infection in the general population in Beijing, and the limitations of identifying sera with viremia by second-generation enzyme immunoassay.