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


  • The nucleotide sequences of HBV DNA isolates used in this study have been deposited in the international DNA database under accession numbers AB249373-AB249636.

  • Potential conflict of interest: Nothing to report.


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.)

Approximately 3 billion people, one half of the world population, have been exposed to hepatitis B virus (HBV), of whom approximately 350 million are persistently infected with it.1 Acute infection with HBV resolves in the great majority but can induce fulminant hepatitis or go on to become chronic. Host and viral factors may influence fulminant or chronic outcome of acute HBV infection, but they are not fully defined.

Eight genotypes have been detected by a sequence divergence greater than 8% in the entire HBV genome of approximately 3,200 nucleotides (nt), and designated by capital alphabet letters from A (HBV/A) to H in the order of documentation.2–5 They have distinct geographical distributions associated with severity of liver disease as well as response to antiviral therapies.6–8 Furthermore, subgenotypes have been reported for HBV/A, B, and C and named Aa/A1 (Asian/African type) and Ae/A2 (European type),9 Bj/B1 (Japanese type) and Ba/B2 (Asian type),10 as well as Cs/C1 (Southeast Asian type) and Ce/C2 (East Asian type).11–13 Increasing lines of evidence indicate that subgenotypes of HBV/A and B influence the replication of HBV and bear clinical relevance.14–16 Furthermore, genotypes affect mutations in precore region and core promoter, thereby influencing the expression of hepatitis B e antigen (HBeAg).8, 17

During the 23 years from 1982 to 2004, a multi-center cross-sectional study was conducted throughout Japan on 301 patients with acute hepatitis B. We examined the influence of genotypes/subgenotypes on their fulminant or chronic outcome. Furthermore, the influence of G1896A or A1762T/G1764A on replication of HBV was evaluated in an in vitro replication model.


HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBc, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; EIA, enzyme immunoassay; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; ALT, alanine aminotransferase.

Patients and Methods

Patients With Acute Hepatitis B.

During 1982 through 2004, 336 consecutive cases of acute hepatitis B were registered in 16 hospitals throughout Japan. These hospitals were from the following eight areas: Hokkaido (represented by J.-H. K. and S.H.), Tohoku (T.K. and K.S.), Kanto (H.T., Y.A. and K.I.), Koshin (E.T. and S.O), Tokai (A.O., Y.T., E.O., M.S., R.U., M.M., and S.K.), Kinki (T.O.), Honshu/Shikoku (Y.M., K.H., and M.O.), and Kyushu (H.Y. and H.S.). The diagnosis of acute hepatitis B was contingent on a sudden onset of clinical symptoms of hepatitis and detection of high-titered antibody to hepatitis B core antigen (anti-HBc) of IgM class in serum. Patients with initial high-titered anti-HBc (≥90% inhibition by a 1:200 diluted serum) were excluded; they were diagnosed as exacerbation of chronic hepatitis B. Patients with acute hepatitis A, hepatitis C, or human immunodeficiency virus co-infection, and drug- or alcohol-induced acute hepatitis also were excluded; hepatitis D virus infection was not examined because of its extreme rarity in Japan.18 Most of them were followed for clinical outcomes until the disappearance of hepatitis B surface antigen (HBsAg) during 24 weeks or longer after the presentation. The criteria of fulminant hepatitis are based on the report by Trey et al.,19 with a slight modification in 1981 (Inuyama symposium, Aichi, Japan): coma of grade II or higher and prothrombin time less than 40% developing within 8 weeks after the onset. Serum samples were collected at the presentation and had been stored at −80°C. HBV genotypes, HBV DNA, and HBeAg were determined, and clinical outcomes of acute hepatitis were analyzed. The study protocol conformed to the 1975 Declaration of Helsinki, and was approved by the Ethics Committees of the institutions. Every patient gave an informed consent for this study.

Serological Markers of HBV Infection.

HBsAg was determined by hemagglutination (MyCell; Institute of Immunology Co., Ltd., Tokyo, Japan) or enzyme immunoassay (EIA) (Axsym; Abbott Japan, Tokyo, Japan), and HBeAg by enzyme-linked immunosorbent assay (F-HBe; Kokusai Diagnostic, Kobe, Japan) or chemiluminescent EIA (Fujirebio Inc., Tokyo, Japan). Anti-HBc of IgM and IgG classes were determined by radioimmunoassay (Abbott Japan).

Genotypes and Subgenotypes of HBV.

The six major HBV genotypes (A-F) were determined serologically by EIA using commercial kits (HBV GENOTYPE EIA; Institute of Immunology). The method depends on the combination of epitopes on preS2-region products detected by monoclonal antibodies, which is specific for each of them.20 HBV/G was determined by a slight modification of the polymerase chain reaction (PCR) with specific primers.21

Subgenotypes of HBV/A designated Ae prevalent in Europe and Aa frequent in Africa as well as Asia,9 which corresponds to subgroup A′ originally reported by Bowyer et al.,22 were determined by PCR restriction fragment length polymorphism (RFLP) involving nucleotide conversions in an immediate upstream of the precore region that are specific for each of them.16, 23 HBV/Bj (Japanese type) lacking the recombination with C over the precore region and the core gene and Ba (Asian type) with the recombination were determined by its absence or presence on HBV DNA sequences, as well as RFLP based on specific nucleotide substitutions, after the methods described previously.15, 24

Subgenotypes of HBV/C, Cs (Southeast Asian type) found only in Southeast Asia, including Vietnam, Myanmar, Thailand, Laos, Bangladesh, Hong Kong, and Southern China, and Ce (East Asian type), found in Far East Asia, including Japan, Korea, and Northern China, were determined by the PCR-RFLP method described previously.12

Quantification of HBV DNA and Sequencing.

HBV DNA sequences spanning the S gene were determined by real-time detection PCR according to the method of Abe et al.,25 with the detection limit of 100 copies/mL. HBV DNA sequences bearing core promoter, precore region, and the core gene were amplified by PCR with hemi-nested primers by the method described previously.10 Negative samples were tested by another more sensitive second-round PCR with HB7F and HBV1917R (5′-CTC CAC AGT AGC TCC AAA TTC TTT A-3′). Thereafter, PCR products were directly sequenced with Prism Big Dye (Applied Biosystems, Foster City, CA) in the ABI 3100 DNA automated sequencer.

Construction of Plasmid and Site-Directed Mutagenesis of HBV DNA.

Serum samples were obtained from two patients infected with HBV/Bj and a patient with Ce. HBV DNA was extracted from 100 μL serum using QIAamp DNA blood kit (QIAGEN, GmbH, Hilden, Germany). Four primer sets were designed to amplify two fragments covering the entire HBV genome. Amplified fragments were inserted into pGEM-T Easy Vector (Promega, Madison, WI) and cloned in DH5a competent cells (TOYOBO, Osaka, Japan). At least five clones of each fragment were sequenced and the consensus sequence determined. Among them, those containing the consensus sequence were identified and adopted as templates for further construction. Finally, 1.24-fold the HBV genome (nt 1413-3215/1-2185), just enough to transcribe oversized pregenome and precore mRNA, was constructed into pUC19 vector (Invitrogen Corp., Carlsbad, CA). For site-directed mutagenesis, the wild-type HBV was digested by HindIII and EcoO65I and ligated with the fragment carrying T1762/A1764 to produce 1.24-fold the genome carrying the core-promoter double mutation. Similarly, 1.24-fold the HBV genome with the precore stop-codon mutation (1896A) was generated. Further details are available online at:

Cell Culture and DNA Transfection.

For the standard replication assay, 10-cm-diameter dishes were seeded with 1 × 106 Huh7 cells each. After 16 hours of culture, cells were transfected with 5 μg DNA construct using the FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) and harvested 3 days later. Transfection efficiency was measured by cotransfection with 1 μg reporter plasmid expressing secreted alkaline phosphatase and estimating its enzymatic activity in the culture supernatant.

Southern Blot Hybridization.

HBV DNA samples from cells at day 3 in culture were separated on 1.2% (wt/vol) agarose gel, transferred to a positive-charged nylon membrane (Roche Diagnostics), and hybridized with full-length HBV DNA labeled with alkaline phosphatase. Detection was performed with CDP-star (Amersham Biosciences, Piscataway, NJ), and signals were analyzed in the LAS-1000 image analyzer (Fuji Photo Film, Tokyo, Japan).

Statistical Analysis.

Categorical variables were compared between groups by the chi-squared test and non-categorical variables by the Mann-Whitney U-test. A P value less than .05 was considered significant. Multivariate analyses with logistic regression were used to determine independent factors for fulminant hepatitis. STATA Software (StataCorp LP, College Station, TX) version 8.0 was employed for analyses.


Demographic and Clinical Differences in Patients Infected With Various HBV Genotypes/Subgenotypes.

Genotypes of HBV were not classifiable in 28 (8%), and sufficient clinical data were not available in 7 (2%) of the 336 patients with acute hepatitis B. Exclusive of these 35 patients, 301 (90%) were left for evaluation of HBV genotypes in reference to clinical outcome.

HBV genotypes/subgenotypes were Aa in 10 (3%), Ae in 33 (11%), Ba in 22 (7%), Bj in 22 (7%), Cs in 11 (4%), Ce in 192 (64%), D in 5 (2%), and G in 6 (2%); none of them were infected with F or H (Table 1). All six patients with HBV/G were co-infected with another genotype; Ae in two, Ba in two, and Ce in the remaining two. The mean age was lower in the patients with HBV/Ae than Ba (P = .0001), Aa (P < .01), Bj or Cs (P < .05 for each) and Ce than Ba (P < .05). Men predominated in HBV infections with foreign (Ae and Ba) compared with domestic genotypes (Bj and Ce) (P < .05).

Table 1. Clinical Characteristics of Patients Acutely Infected With HBV of Distinct Genotypes/Subgenotypes
Aa (n = 10)Ae (n = 33)Ba (n = 22)Bj (n = 22)Cs (n = 11)Ce (n = 192)Da (n = 5)Gab (n = 6)
  • a

    Patients with HBV genotype D or G were not included in the analysis.

  • b

    All patients with HBV genotype G were co-infected with HBV of another genotype; Ae in two, Ba in two, and Ce in two.

  • c

    Exclusive of the 16 patients who died of fulminant hepatitis, 3 receiving liver transplantation and 10 without clinical data available.

  • d

    P = .0001, Ae vs. Ba. P < .01, Ae vs. Aa. P < .05, Ae vs. Bj or Cs.

  • e

    P < .05, Ba vs. Ce.

  • f

    P = .0001, Ae vs. Bj. P < .005, Ae vs. Ce.

  • g

    P < .005, Ba vs. Bj. P < .05, Ba vs. Ce.

  • h

    P < .005, Ae vs. Bj. P < .01, Ae vs. Ce. P < .05, Ae vs. Ba.

  • i

    P < .05, Ae vs. Bj.

  • j

    P < .01, Ba vs. Bj. P < .05, Ba vs. Ce.

  • k

    P < .005, Ae vs. Ce. P < .05, Ae vs. Bj.

Age (years)42.2 ± 13.131.2 ± 10.3d41.5 ± 10.7e43.5 ± 19.138.5 ± 11.136.3 ± 15.038.6 ± 20.842.7 ± 17.5
Men8 (80%)30 (91%)f19 (86%)g9 (41%)7 (64%)122 (64%)2 (40%)6 (100%)
HBeAg positive7 (70%)26 (79%)h11 (50%)8 (36%)8 (73%)101 (53%)1 (20%)4 (67%)
ALT (IU/L)1875 ± 7592070 ± 1113i2523 ± 11853472 ± 27202269 ± 9952610 ± 17192559 ± 16722142 ± 722
Duration of elevated ALT (weeks)c7.9 ± 5.89.5 ± 6.28.8 ± 3.7j6.0 ± 2.510.1 ± 7.57.7 ± 5.15.7 ± 2.19.8 ± 1.5
Total bilirubin (mg/dL)14.1 ± 10.39.0 ± 7.29.3 ± 5.910.9 ± 9.011.0 ± 13.89.8 ± 10.78.2 ± 2.213.0 ± 7.8
HBV DNA (log copies/mL)        
 Median (range)4.76 (2.90-8.08)6.08k (2.00-8.46)5.15 (2.00-8.19)4.93 (2.00-8.44)5.61 (2.00-8.50)4.94 (2.00-9.06)5.91 (2.00-8.37)5.97 (3.35-7.11)
 <2.00 (undetectable)0 (0%)1 (3%)2 (9%)3 (14%)2 (18%)28 (15%)1 (20%)0 (0%)
Medication with        
 Lamivudine1 (10%)9 (27%)2 (9%)5 (23%)2 (18%)28 (15%)4 (80%)2 (33%)
 Steroid03 (9%)05 (23%)1 (9%)16 (8%)00

HBeAg was detected in 79% of patients with HBV/Ae at a frequency much higher than that with Bj (P < .005), Ce (P < .001) or Ba (P < .05). HBeAg in four of the six (67%) patients with HBV/G was coded for by HBV of the other genotypes co-infecting them, because it has two stop codons and an insertion in the core gene that prohibit encoding HBeAg.21 HBV DNA levels as well as HBeAg-positive rates at the presentation were higher in HBV/Ae than Ce (P < .005) or Bj (P < .05) infection.

The peak alanine aminotransferase (ALT) level was higher in HBV/Bj than Ae infection (P < .05). Fulminant hepatitis was significantly more frequent in patients infected with HBV/Bj (55%) than the other genotypes (P < .05); it occurred in two of the five (40%) patients with HBV/D, also. In reflection of severe clinical course, the peak ALT level tended to be high in patients with HBV/Bj.

Presumed infection routes of 301 patients were sexual transmission in 172 (57%), blood transfusion in 4 (1%), medical accidents in 17 (6%), and unknown in the remaining 108 (36%).

Clinical Outcome of Patients With Acute Hepatitis B.

Fulminant hepatitis developed in 40 (13%) patients. To cope with severe acute liver disease, lamivudine and steroid were administered to 53 (18%) and 25 (8%) patients, respectively. Fulminant hepatitis led to death in 16 (5%) patients, and three (1%) received liver transplantation. Exclusive of the 40 patients with fulminant hepatitis who received various treatments and five without clinical data, 256 (85%) were followed for the chronic outcome (Fig. 1). Serum ALT levels stayed elevated for longer than 24 weeks for the diagnosis of chronic hepatitis in eight (3%) of them. Among them, five had cleared HBsAg from serum until then, and therefore, their liver function abnormality was not attributed to persistent HBV infection. Table 2 summarizes persistence of HBV infection in the 256 patients with acute hepatitis; 253 (99%) lost serum HBsAg by 6 months. Hence, HBV infection evolved into chronicity in only 3 of the 256 (1%) patients, representing 2 of the 32 (6%) infected with HBV/Ae and 1 of the 21 (5%) with Ba. All of the three with chronic outcome had low-titered IgG anti-HBc at the presentation, and two of them had been negative for HBsAg before the presentation. None of them had received lamivudine or steroid treatment during their acute phase of illness. Of the patients without antiviral therapy, chronic outcome was significantly more frequent in those infected with HBV/Ae than non-Ae genotypes (9% math image vs. 0.5% math image, P = .032).

Figure 1.

A flow diagram of 336 patients studied. Comparison was made between patients with fulminant and acute self-limited hepatitis (upper dotted area), and the chronicity was compared between patients with and without treatments (lower dotted area). *Of 301 patients, 37 were negative for HBV DNA, including 27 with acute and 10 with fulminant hepatitis.

Table 2. Persistence of HBV Infection in the Patients With Acute Hepatitis Who Did or Did Not Receive Lamivudine or Steroid
Aa (n = 8)aAe (n = 32)aBa (n = 21)aBj (n = 10)aCs (n = 10)aCe (n = 167)aD (n = 3)aG (n = 5)a
  • a

    Exclusive of 40 patients with fulminant hepatitis and 5 without clinical data available.

  • b

    Six patients received steroid along with lamivudine.

  • c

    P < .05, Ae vs. non-Ae.

Total (n = 256)3/256 (1.2%)02/32 (6%)c1/21 (5%)00000
Lamivudine (n = 36)b0/36 (0%)0/1 (0%)0/9 (0%)0/2 (0%)00/1 (0%)0/19 (0%)0/2 (0%)0/2 (0%)
Steroid (n = 16)b0/16 (0%)00/3 (0%)000/1 (0%)0/12 (0%)00
Neither3/210 (1.4%)0/7 (0%)2/23 (9%)c1/19 (5%)0/10 (0%)0/8 (0%)0/139 (0%)0/1 (0%)0/3 (0%)

Comparison Between Patients With Fulminant and Acute Self-Limited Hepatitis.

Table 3 compares demographic, clinical, and virological characteristics between the 40 patients with fulminant and the 261 with acute self-limited hepatitis for whom analysis was feasible. Patients with fulminant hepatitis were significantly older (44.7 ± 16.3 vs. 36.0 ± 14.3 years, P = .0017), less predominantly male (43% vs. 71%, P = .0005) and less often positive for HBeAg (23% vs. 60%, P < .0001) than those with acute hepatitis. Peak ALT and total bilirubin levels were higher for fulminant than acute hepatitis (P < .0001), reflecting severe hepatic lesions. Notably, the median HBV DNA level was lower in patients with fulminant than acute hepatitis (4.89 vs. 5.19 log copies/mL, P = .0178); the frequency of undetectable HBV DNA at the presentation was higher in fulminant hepatitis (25% vs. 10%, P = .0086). Lamivudine or steroid was given significantly more often to patients with fulminant hepatitis.

Table 3. Comparison Between Patients With Fulminant and Acute Self-Limited Hepatitis Who Were Infected With HBV
FeaturesFulminant (n = 40)Acute (n = 261)P Value
  • a

    Exclusive of 37 patients in whom precore region and core-promoter could not be amplified by PCR.

  • b

    T1753C/A/G and/or T1754C/A/G.

Age (years)44.7 ± 16.336.0 ± 14.3.0017
Men17 (43%)186 (71%).0005
HBeAg positive9 (23%)157 (60%)<.0001
ALT (IU/L)4315 ± 28892284 ± 1221<.0001
Total bilirubin (mg/dL)20.5 ± 16.48.3 ± 7.3<.0001
HBV DNA (log copies/mL)   
 <2.00 (undetectable)10 (25%)27 (10%).0086
 Lamivudine16 (40%)37 (14%).0003
 Steroid9 (23%)16 (6%).0022
 Aa1 (2.5%)9 (3%)NS
 Ae0 (0%)33 (13%).0121
 Ba1 (2.5%)21 (8%)NS
 Bj12 (30%)10 (4%)<.0001
 Cs1 (2.5%)10 (4%)NS
 Ce22 (55%)170 (65%)NS
 D2 (5%)3 (1%)NS
 G1 (2.5%)5 (2%)NS
 nt 1753 and/or nt1754b11/30 (37%)28/234 (12%).0003
 A1762T/G1764A15/30 (50%)39/234 (17%)<.0001
 G1896A16/30 (53%)21/234 (9%)<.0001
 G1899A7/30 (23%)8/234 (3%)<.0001

There were marked differences in the distribution of genotypes between patients with fulminant and acute hepatitis. HBV/Ae was less frequent (0% vs. 13%, P = .0121), whereas Bj was more often (30% vs. 4%, P < .0001) in patients with fulminant than acute hepatitis. Although HBV/Ce tended to be less frequent in patients with fulminant than acute hepatitis (55% vs. 65%), the difference fell short of being significant.

Precore stop-codon mutation (G1896A) and core-promoter double mutation (A1762T/G1764A) were more frequent in patients with fulminant than acute hepatitis (53% vs. 9% and 50% vs. 17%, respectively, P < .0001 for each). Likewise, mutations in core-promoter at nt 1753 or nt 1754, and G1899A mutation were more frequent in patients with fulminant than acute hepatitis (P = .0003 and P < .0001, respectively).

Factors Independently Associated With the Development of Fulminant Hepatitis.

Various factors found in association with fulminant hepatitis were evaluated for the independence in multivariate analysis (Table 4). Age 34 years or older (odds ratio 3.47 [95% confidence interval 1.09-11.02], P = .035), HBV/Bj (7.00 [1.74-28.23], P = .006), HBeAg-negative (3.34 [1.07-10.51], P = .039), total bilirubin ≥10.0 mg/dL (18.82 [4.32-81.98], P < .0001) and G1896A (4.16 [1.27-13.66], P = .019) were independent risk factors for the development of fulminant hepatitis.

Table 4. Multivariate Analysis for Factors Independently Associated With Fulminant Hepatitis
FactorsOdds Ratio95% Confidence IntervalP Value
  • a

    Median values.

  • b

    T1753C/A/G or T1754C/A/G.

Age (yr)   
ALT (IU/L)   
Total bilirubin (mg/dL)   
HBVDNA (log copies/mL)   
 Lamivudine (−)1  
 Lamivudine (+)2.6500.814-8.625.1056
 Steroid (−)1  
 Steroid (+)2.5150.668-9.472.1728
 nt 1753 and/or 1754b   

In view of the majority of Japanese patients who were infected with Bj or Ce, mutations in the precore region and core-promoter were compared between those with fulminant and acute self-limited hepatitis for each subgenotype (Fig. 2). G1896A and A1762T/G1764A were significantly more frequent in patients with fulminant than acute hepatitis infected with either HBV/Bj or Ce (56% vs. 0% and 67% vs. 0% for Bj or 44% vs. 11% and 56% vs. 22% for Ce, respectively, P ≤ .01 for all). For the patients infected with HBV/Bj, in particular, precore and core-promoter mutations were highly frequent in those with fulminant hepatitis (56% and 67%, respectively), whereas they occurred in none of those with acute hepatitis. G1899A was equally frequent in both patients with fulminant and acute hepatitis infected with HBV/Bj; it was rarely seen in those with Ce. Mutations involving nt 1753 or nt 1754 tended to be more frequent in patients with fulminant than acute hepatitis.

Figure 2.

Frequencies of precore and core-promoter mutations compared between patients with fulminant and acute self-limited hepatitis who were infected with HBV/Bj (A) or Ce (B).

Replication of the Wild-Type HBV as Well as Precore and Core-Promoter Mutants In Vitro.

Full-length HBV DNA of the wild-type HBV/Bj from a patient with chronic hepatitis B was incorporated with G1896A or A1762T/G1764A mutation in vitro. Another plasmid was constructed with HBV/Bj_58 carrying G1896A from a fulminant patient. Figure 3 compares densities of migration patterns of the wild-type, precore, and core-promoter mutants in Southern blotting analysis. The wild-type HBV/Bj displayed a band for single-stranded (ss) HBV DNA and an additional band for double-stranded (ds) HBV DNA. Of note, the densities of these bands were far greater for HBV/Bj mutants incorporated with precore or core-promoter mutation, as well as Bj_58 with the precore mutation, thereby indicating much enhanced replicative activity of precore or core-promoter mutant in vitro. Although the intracellular HBV DNA level for the wild-type HBV/Bj was comparable with that for the wild-type Ce (Fig. 3), the extracellular HBV DNA level in culture media was approximately threefold higher for Bj than Ce (P < .01) (Sugiyama M et al., manuscript in submission).

Figure 3.

Southern blot analysis for replicative activity of the wild-type HBV clones (HBV/Ce_wild and Bj_wild), as well as mutants with precore (Bj_PCm) or core-promoter (Bj_CPm) mutation, and Bj_58 with precore stop-codon mutation obtained from a patient with fulminant hepatitis.


A nationwide survey of genotypes/subgenotypes in patients with acute HBV infection from Japan during the past 2 decades has examined their influence on fulminant and chronic outcomes. The study was feasible in a country where mass vaccination has not been performed because of an extremely high efficacy of immunoprophylaxis on babies born to carrier mothers; it has decreased the persistent HBV carrier rate from 1.4% to 0.3%.26 Acute HBV infection keeps increasing, however, predominantly through promiscuous sexual contacts in Japan.

Fulminant hepatitis developed rather frequently in 40 of the 301 (13%) patients. This is likely due to selection bias because the study included only patients who were hospitalized for acute hepatitis B. Exclusion of subclinical cases of acute HBV infection would have overestimated the incidence of fulminant hepatitis. Regardless of such a selection bias, influence of HBV genotypes/subgenotypes was evident in comparison with the 40 patients with fulminant and the 261 with acute self-limited hepatitis. Remarkably, none of the 33 patients infected with HBV/Ae developed fulminant hepatitis. In sharp contrast, 12 of the 22 (55%) patients infected with HBV/Bj developed it. Furthermore, both precore (G1896A) and core-promoter (A1762T/G1764A) mutations were detected significantly more frequently in patients with fulminant than acute self-limited hepatitis. In infection with HBV/Bj, in particular, the frequency of core-promoter mutation was much higher in the patients with fulminant (67%) than that reported in those with chronic hepatitis (16%).27 Precore and core-promoter mutations are very frequent in patients with fulminant hepatitis from Asia28–30 and the Middle East.31 The failure in detecting these mutations in Western countries32–35 could be attributed to frequent HBV/Ae and rare Bj there. In multivariate analysis, HBeAg-negative, HBV/Bj, and the precore stop-codon mutation for G1896A were independent risk factors for the development of fulminant hepatitis (Table 4). Various mutations at nt 1753 for enhanced HBV replication,36 as well as those adjacent at nt 1754 prevailing in patients with fulminant hepatitis,37 occurred more frequently in patients with fulminant than acute self-limited hepatitis. Host factors, such as age and total bilirubin, contributed to the development of fulminant hepatitis as well (Table 4).

In vitro replication analysis demonstrated the intracellular HBV DNA level of the wild-type HBV/Bj comparable with that of the wild-type Ce (Fig. 3). The extracellular HBV DNA level of HBV/Bj-clone, however, was much higher than those of the other genotypes, indicating its strong inclination to be secreted from cells (Sugiyama et al., manuscript in submission). Such a high concentration of HBV/Bj in the circulation of patients would rapidly and extensively promote infection of hepatocytes.

Enhanced replication capacities of precore (G1896A) and core-promoter (A1762T/G1764A) mutants for HBeAg-minus and -reduced phenotypes, respectively, were demonstrated in a replication model in vitro (Fig. 3). These observations were concordant with those in previous reports38, 39; however no data are available on the replication of HBV/Bj in vitro, either of the wild-type or variants with these mutations. Extremely high intracellular and extracellular expressions of viral DNA were observed for the HBV/Bj clone with precore stop-codon mutation from a patient with fulminant hepatitis. These results might implicate high replication due to mutations of precore region and core-promoter in the induction of fulminant hepatitis. In support of this view, Bocharov et al.40 have proposed that enhanced HBV replication would efficiently stimulate immune reactions, represented by the cytotoxic T lymphocyte response, suggesting that enhanced replication by HBV/Bj or precore/core-promoter mutation might lead to fulminant hepatitis.

That HBV DNA levels were lower in patients with fulminant than acute hepatitis, despite a high replication capacity of HBV/Bj incriminated in the development of fulminant hepatic failure, may seem surprising. Because destruction of hepatocytes proceeds swiftly in patients with fulminant hepatitis, hepatic mass for HBV to thrive would have been extremely reduced in them at presentation. As a consequence, some patients with fulminant hepatitis B are without serum HBsAg; they are diagnosed by high-titered IgM anti-HBc.41 On the contrary, HBV DNA levels were higher in the patients with HBV/Ae than Bj (Table 1); those with Ae tend to delay reducing HBV DNA, some of whom have chronic outcome. Combined, correlating HBV DNA levels with the clinical outcome in acute HBV infection would be difficult.

A wide variation has been seen in the rate of persistence after acute HBV infection in adulthood. No chronic outcomes of acute hepatitis B were seen in female recipients of red blood cells contaminated with HBV (0/28)42 or patients in an acupuncture-associated outbreak (0/35).43 In marked contrast, they ranged from 0.2% (14/715) in Greece44 through 2.7% (1/37) in university students in Taiwan45 to 10.4% (5/8) in Alaskan Eskimos46 and 12.1% (7/58) in Germany.47 HBV genotypes are implicated in a high rate of persistence in European countries where HBV/A is predominant.48 In Japan, also, adulthood infection tends to persist longer with HBV/A than B or C (23% math image vs. 13% ⅛ or 12% math image).49 In the current series on 256 patients with acute hepatitis B in Japan who were followed rigorously, HBV infection persisted in only three (1%), representing 2 of the 32 (6%) with HBV/Ae and 1 of the 21 (5%) with Ba. Hence, 99% of patients lost their HBsAg by 6 months. Persistence of HBV observed in the patients with HBV/Ae (6%) is less frequent than that in 4 of the 31 (13%) patients with Ae from a hospital in metropolitan Tokyo.49 The difference would be ascribable, at least in part, to lamivudine given to some patients in this study (18%). All patients treated with lamivudine recovered from acute hepatitis, whereas none of the three patients with chronic outcome had received antiviral treatment during their acute phase of illness, indicating that lamivudine might be able to prevent the chronic outcome. Likewise, some patients from metropolitan Tokyo, in whom HBV persisted,49, 50 had received immunosuppressants in the acute phase of infection before referral to their hospital.

Using cell culture and chimeric mice models for the replication system of different genotype/subgenotype clones, we have observed that the replication of HBV is the highest for HBV/Bj or C and the lowest for Aa/Ae (Sugiyama M et al., manuscript in submission). It is probable that the propensity of HBV/A infection to chronicity would be due to less intensive immune response against its slow viral dynamics. Taken together, the infection with HBV/A appears to persist longer than those with the other genotypes; this needs to be confirmed by further investigation in patients from various countries.

In conclusion, persistence of HBV after acute infection is rare and occurs more often in patients infected with HBV/Ae than others. Fulminant outcome is frequent in hospitalized patients and associated with HBV/Bj accompanied by the lack of serum HBeAg as well as high replication due to precore stop-codon mutation (G1896A), a finding supported by an in vitro replication model.