ABSTRACT
Acute hepatitis B virus (HBV) infection has been increasing through promiscuous sexual contacts, and HBV genotype A (HBV/A) is frequent in patients with acute hepatitis B (AHB) in Japan. To compare the geographic distribution of HBV genotypes in patients with chronic hepatitis B (CHB) in Japan between 2005 and 2006 and between 2000 and 2001, with special attention to changes in the proportion of HBV/A, a cohort study was performed to survey changes in genotypes of CHB patients at 16 hospitals throughout Japan. Furthermore, we investigated the clinical characteristics of each genotype and examined the genomic characteristics of HBV/A isolates by molecular evolutionary analyses. Of the 1,271 patients, 3.5%, 14.1%, and 82.3% were infected with HBV/A, -B, and -C, respectively. In comparison with our previous survey during 2000 and 2001, HBV/A was twice as frequent (3.5% versus 1.7%; P = 0.02). The mean age was lower in the patients with HBV/A than in those with HBV/B or -C. Based on phylogenetic analyses of 11 full-length genomes and 29 pre-S2/S region sequences from patients, HBV/A isolates were imported from Europe and the United States, as well as the Philippines and India. They clustered with HBV/A from AHB patients and have spread throughout Japan. HBV/A has been increasing in CHB patients in Japan as a consequence of AHB spreading in the younger generation through promiscuous sexual contacts, aided by a tendency of HBV/A to induce chronic hepatitis. The spread of HBV/A infection in Japan should be prevented by universal vaccination programs.
Hepatitis B virus (HBV), a member of the Hepadnaviridae, is a circular, partially double-stranded DNA virus and is one of the major causes of chronic liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC).
The HBV genome is composed of approximately 3,200 nucleotides. HBV is classified into eight genotypes, designated A to H, based on an intergroup divergence of 8% or more in the complete nucleotide sequence (3, 23, 26, 37). They have distinct geographical distributions and are associated with differences in clinical and virological characteristics, such as severity of liver disease and response to antiviral therapies (7, 8, 12, 13, 22, 28). Furthermore, subgenotypes have been reported for HBV/A, -B, and -C and named A1 to -3 (17, 38), B1 to -6 (31, 32, 40), and C1 to -6 (20, 31, 45). Equally, other genotypes are classified into subgenotypes. There have been increasing lines of evidence to indicate influences of HBV subgenotypes on the outcome of liver disease and the response to antiviral therapies (1, 39, 44).
In 2001, we reported the geographic distribution of HBV genotypes in Japan (27). Of the 720 Japanese patients with chronic HBV infection (CHB), 12 (1.7%) harbored HBV/A, 88 (12.2%) HBV/B, 610 (84.7%) HBV/C, 3 (0.4%) HBV/D, and 7 (1.0%) mixed genotypes. HBV/C was detected in over 94% of patients on the Japanese mainland, while HBV/B was found in 64% of those in Okinawa, the southernmost islands, and 44% of those in the Tohoku area in the northern part of the mainland.
Recently, acute HBV infection (AHB) has been increasing in Japan, predominantly through promiscuous sexual contacts. In addition, it was reported that HBV/A was more frequent in patients with acute hepatitis than in those with chronic hepatitis (29, 41, 49). Recent studies suggest that the chances for progression to chronic disease may differ among patients acutely infected with HBV of distinct genotypes (21, 25); patients infected with HBV/A run an increased risk of becoming HBV carriers. Hence, it is of utmost concern whether chronic HBV/A infection is increasing in Japan.
In the present study, we compared the geographic distribution of HBV genotypes in Japan during 2005 and 2006 with 2000 and 2001, with special attention to changes in the proportion of HBV/A. Furthermore, we investigated the clinical characteristics of each genotype and examined the genomic characteristics of HBV/A isolates by molecular evolutionary analyses.
MATERIALS AND METHODS
Patients.From September 2005 to October 2006, sera were collected from 1,370 consecutive patients with CHB at 16 representative hospitals that were liver centers in their respective regions throughout Japan for the purpose of investigating the geographic distribution of HBV genotypes in Japan. All of the patients were diagnosed after they had been followed for at least 12 months. Patients diagnosed with AHB were excluded from the study; they had a sudden onset of clinical symptoms of hepatitis, along with high-titer antibody to HBV core antigen of the immunoglobulin M class in serum. Their sera were tested for alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), and hepatitis B e antigen (HBeAg), as well as antibody to HBeAg (anti-HBe) (Dinabot, Tokyo, Japan). Four clinical diagnoses were established for them. The inactive carrier state was defined by the presence of HBV surface antigen (HBsAg) with normal ALT levels over 1 year (examined at least four times at 3-month intervals) and without evidence of portal hypertension. Chronic hepatitis was defined by elevated ALT levels (>1.5 times the upper limit of normal [35 IU/liter]) persisting over 6 months (with at least three bimonthly tests). Cirrhosis was diagnosed principally by ultrasonography (coarse liver architecture, nodular liver surface, blunt liver edges, and hypersplenism), platelet counts of <100,000/cm3, or a combination thereof. Histological confirmation by fine-needle biopsy of the liver was performed as required. HCC was diagnosed by ultrasonography, computerized tomography, magnetic resonance imaging, angiography, tumor biopsy, or a combination thereof.
The study protocol conformed to the 1975 declaration of Helsinki and was approved by the ethics committees of the respective institutions. Every patient or his/her next of kin gave informed consent to the purpose of the study.
Genotypes and subgenotypes of HBV.The six HBV genotypes (A to F) were determined serologically by enzyme immunoassay (EIA) using commercial kits (HBV Genotype EIA; Institutes of Immunology Co., Ltd., Tokyo, Japan). The method depends on the combination of epitopes on pre-S2 region products detected by monoclonal antibodies that were specific for each of them (46, 47). Subgenotypes of HBV/A, designated A1 and A2, were determined by direct sequencing of the pre-S2/S gene, followed by a phylogenetic analysis.
Quantification of HBV DNA and sequencing.HBV DNA levels in sera were quantitated with a commercial kit (Amplicor HBV Monitor; Roche Diagnostics, Basel, Switzerland) with a detection range from 2.6 to 7.6 log copies/ml. Nucleic acids were extracted from 100 μl of serum using the Qiaamp DNA Blood Minikit (Qiagen GmbH, Hilden, Germany). Eleven complete HBV/A genomes and 29 pre-S2/S region sequences were amplified by PCR with appropriate primer sets, as described previously (40). The amplified HBV DNA fragments were directly sequenced using the ABI Prism Big Dye kit version 3.0 (Applied Biosystems, Foster City, CA) in an ABI 3100 automated DNA sequencer (Applied Biosystems). All sequences were analyzed in both forward and reverse directions. Complete and partial HBV genome sequences were aligned using GENETYX version 11.0 (Software Development Co., Ltd., Tokyo, Japan).
Molecular evolutionary analysis of HBV.Reference sequences were retrieved from the DDBJ/EMBL/GenBank databases with their accession numbers for identification. To investigate the relationship between HBV isolates from patients with chronic and acute hepatitis B in Japan, HBV/A isolates (AH1 to -10) were randomly retrieved from them and sequenced in our previous study (29). Nucleotide sequences of HBV DNA were aligned by the program CLUSTAL X, and genetic distance was estimated by the six-parameter method (10) in the Hepatitis Virus Database (36). Based on these values, phylogenetic trees were constructed by the neighbor-joining method (30) with the midpoint rooting option. To confirm the reliability of the phylogenetic trees, bootstrap resampling tests were performed 1,000 times.
Statistical analysis.Categorical variables were compared between groups by the χ2 test or Fisher's exact test and noncategorical variables by the Mann-Whitney U test. A P value of less than 0.05 was considered significant.
Nucleotide sequence accession numbers.The DDBJ/EMBL/GenBank accession numbers of the complete genome sequences of HBV isolates JPN_CH1 to -11 are AB453979 to AB453989.
RESULTS
Distribution of HBV genotypes among patients with CHB.Of the 1,370 serum samples, the genotype could not be determined for 99 (7.2%) by EIA due to low HBsAg levels, leaving 1,271 for analysis in this study (Table 1). Of these, 206 (16.2%) were inactive carriers, 786 (61.8%) had chronic hepatitis, 175 (13.8%) cirrhosis, and 104 (8.2%) HCC. They had a mean age of 51.4 ± 14.0 years and included 766 (60.3%) men. They had a median HBV DNA level of 4.2 log copies/ml, and 399 (31.4%) of them were positive for HBeAg. Antiviral treatment had been given to 577 (45.4%) of them with interferon, lamivudine, adefovir pivoxil, or entecavir.
Characteristics of 1,271 CHB patients
The genotypes were HBV/A in 44 (3.5%), HBV/B in 179 (14.1%), HBV/C in 1,046 (82.2%), and HBV/D in 2 (0.2%) (Table 2). In comparison with our previous report on the distribution of genotypes in Japan in 2001 (27), HBV/A was more frequent in this study (3.5% versus 1.7%; P = 0.02). Of the 16 hospitals in this study, 10 overlapped with those in our previous report from 2001. In these 10 hospitals, HBV/A was more frequent in the present than in the previous survey (3.6% versus 1.7%; P = 0.04).
Distribution of HBV Genotypes
The distribution of HBV genotypes in Japan differed by geographic location (Fig. 1). HBV/C was the most prevalent in the majority of areas. In the Tohoku area, the northern part of the Japanese mainland (Honshu), HBV/B was more prevalent than in the other areas of the Japanese mainland. In Okinawa, the southernmost islands of Japan, HBV/B was predominant. Of note, HBV/A was more frequent in the Kanto area (9.5%), the metropolitan area, and Okinawa (9.1%) than in the other areas.
Geographic distribution of HBV genotypes in patients with chronic HBV infection in Japan during 2005 and 2006.
Clinical differences among HBV/A, -B, and -C.Clinical backgrounds were compared among the patients infected with HBV/A, -B, and -C (Table 3). HBeAg was significantly less prevalent in the patients infected with HBV/B than in those infected with HBV/A or -C (P < 0.01 for each). When the positivity of HBeAg was stratified by age, HBeAg was markedly less common in patients infected with HBV/B than in those infected with HBV/A or -C who were older than 40 years of age (7/157 [4.5%] versus 4/19 [21.1%] [P < 0.05] or 215/755 [28.5%] [P < 0.01]) (Fig. 2). There were no significant differences in HBV DNA levels among patients infected with the three genotypes. As antiviral treatments might have influenced the severity of liver disease, clinical states were compared among patients infected with HBV/A, -B, and -C who did and did not receive it; antiviral treatments did not affect the above-mentioned trends represented in Table 3 in age, diagnosis, and HBeAg, as well as ALT and HBV DNA levels (data not shown).
Prevalence of HBeAg among patients infected with HBV of different genotypes stratified by the age.
Clinical characteristics of individuals chronically infected with HBV of different genotypes
Additionally, we compared the distributions of age and liver diseases in patients infected with HBV/A, -B, and -C. In patients infected with HBV/C, the prevalence of cirrhosis and HCC increased in those older than 50 years of age compared to younger patients (Fig. 3), whereas in the patients infected with HBV/B, cirrhosis and HCC were rare in elderly patients. The proportion of patients younger than 40 years of age was higher in those infected with HBV/A than in those infected with HBV/B or -C (25/44 [56.8%] versus 22/179 [12.3%] or 288/1,046 [27.5%]; P < 0.01 for each), while cirrhosis and HCC were also found in those older than 50 years of age infected with HBV/A.
Distribution of HCC, cirrhosis, chronic hepatitis, and inactive carrier state among the 1,271 patients infected with HBV of different genotypes stratified by the age.
Coinfection with human immunodeficiency virus type 1 (HIV-1) was found in 6 of the 44 (13.6%) patients infected with HBV/A compared to only 3 of the 1,046 (0.3%) patients infected with HBV/C (P < 0.0001); it occurred in none of the 179 patients infected with HBV/B.
Phylogenetic analyses.Among the 44 HBV/A isolates, the complete genome was sequenced successfully in 11 (JPN_CH1 to -11). Seven of them were classified as HBV/A2 and four as HBV/A1. A phylogenetic tree was constructed based on the complete genome sequences of these 11 isolates, along with those from two patients with AHB and those from 40 HBV/A isolates retrieved from the database (Fig. 4). Of the seven HBV/A2 isolates, the four from patients with CHB in this study formed a cluster with the Japanese isolates retrieved from the database and two from patients with AHB. Of the other three isolates, JPN_CH5 clustered with French and U.S. isolates, JPN_CH6 with German isolates, and JPN_CH7 with Spanish and Italian isolates. All four HBV/A1 isolates in this study formed a cluster with Philippine and Indian isolates.
Phylogenetic tree constructed based on the complete genome sequences of HBV/A isolates. Those from 11 patients with chronic infection in this study are shown in boldface italic (JPN_CH1 to -11), along with two isolates (JPN_AH1 and -2) from patients with acute hepatitis in Japan reported in our previous study (17). Representative isolates were retrieved from the DDBJ/EMBL/GenBank databases, including 21 HBV/Ae, 10 HBV/Aa, and 2 HBV/Ac isolates, along with 7 HBV isolates representative of the other seven genotypes. Isolates from the databases are identified by accession numbers, followed by the country of origin. The bar at the bottom spans 0.01 nucleotide substitutions per site.
In addition, the pre-S2/S region sequences of a total of 29 isolates were determined, including the 11 isolates whose complete genomes were sequenced. Of these, 21 (72%) were classified as HBV/A2 and the remaining 8 as HBV/A1. A phylogenetic tree was constructed based on the pre-S2/S region sequences from the 29 isolates, along with those from 10 patients with AHB infected with HBV/A and 47 HBV/A isolates retrieved from the database (Fig. 5). The 21 HBV/A2 isolates in the present study formed a cluster with Japanese, American, and European isolates retrieved from the database and those from patients with acute hepatitis. In addition, some of them were highly homologous with each other. Likewise, HBV/A1 isolates from eight patients with chronic hepatitis in this study were highly homologous with those from two patients with acute hepatitis and isolates from the Philippines and India. Based on the phylogenetic analyses, HBV/A isolates were imported from Europe and the United States, as well as the Philippines and India, and had infiltrated throughout Japan.
Phylogenetic tree constructed based on pre-S2/S region sequences of HBV/A isolates. Those from 29 patients with chronic infection in this study are shown in boldface italic (JPN_CH1 to -29), along with 10 isolates (JPN_AH1 to -10) from patients with acute hepatitis in Japan reported in our previous study (17). Representative isolates were retrieved from the DDBJ/EMBL/GenBank databases, including 28 HBV/Ae, 10 HBV/Aa, and 2 HBV/Ac isolates and 7 HBV isolates representative of the other seven genotypes. Isolates from the databases are identified by accession numbers, followed by the country of origin. The bar at the bottom spans 0.01 nucleotide substitutions per site.
DISCUSSION
Perinatal transmission from carrier mothers to their babies has been the principal route for establishing persistent HBV infection in Asian countries (19). In Japan, passive and active immunoprophylaxis with HBV immune globulin and vaccine has been mandated for babies born to HBeAg-positive carrier mothers since 1986; this was extended to HBeAg-negative carrier mothers in 1995. As a result, HBsAg has become rare in Japanese born after 1986; it was detected in only 0.2% of first-time blood donors younger than 19 years of age in 2000 (24). However, AHB has been increasing in Japan, predominantly through promiscuous sexual contacts.
In Japan, HBV/A is detected rarely among patients with CHB but is frequent in those with acute hepatitis (14, 25, 29, 41, 43). Yotsuyanagi et al. reported the distribution of genotypes in 145 Japanese patients with AHB and found HBV/A in 27 (19%), HBV/B in 8 (5%), and HBV/C in 109 (75%) (49). HBV/A is more frequent in metropolitan areas than other areas. The majority of patients with HBV/A infection in metropolitan areas have had extramarital sexual contacts with multiple irregular partners, through which they could have contracted infection. In support of this view, among men who have sex with men (MSM) who are coinfected with HBV and HIV-1 in Tokyo, most were infected with HBV/A (15, 35).
In Japan, AHB in adulthood becomes chronic in only ∼1% of cases. This is much less than the progression to chronic disease (close to 10%) in Europe and the United States, where HBV/A prevails (34). Recent studies have suggested that the chances for persistence may differ among patients acutely infected with HBV of distinct genotypes (21, 25). In particular, acute infection with HBV/A may bring about an increased risk of progression to chronic disease. Therefore, an increase of acute infection with HBV/A would result in a surge of HBV/A among patients with CHB in Japan. In actuality, in comparison with our previous results during 2000 and 2001 (27), HBV/A was twice as frequent in this study (3.5% versus 1.7%; P = 0.02). HBV/A has been increasing in patients with CHB in the Kanto area, where HBV/A in patients with acute hepatitis is more frequent than in the other areas. In the islands of Okinawa, also, HBV/A was found to be prevalent in this study. Of the four patients infected with HBV/A there, two were coinfected with HIV-1. They were both MSM, and they were suspected to have been infected with HIV through sexual contacts on the Japanese mainland. It has been reported that HIV infection increases the probability that AHBs will become chronic (2, 11, 33, 48). Because they share routes of transmission and the risk for HIV-1 and HBV infections, approximately 90% of patients with AIDS have markers of past or ongoing HBV infection (18). Thus, HBV carriers are more frequent in the HIV-1-positive than in the HIV-1-negative population (4, 9). Among patients with HIV infection in Japan, 6.3% are HBsAg positive, in particular, 8.3% of HIV-infected MSM (16). In this study, coinfection with HIV was found in 6 of the 44 (13.6%) patients infected with HBV/A. All of them were men. Their median age was 27.7 ± 4.1 years, and five patients were positive for HBeAg. Thus, there is a possibility that HIV-1 and HBV/A coinfections are increasing among young people in Japan, and the high rate of HBeAg positivity may be influenced by immune suppression due to HIV infection.
In the phylogenetic analysis, the HBV/A2 isolates recovered in this study were homologous to those from Europe and the United States, and some of them clustered with the Japanese isolates. On the other hand, there were HBV/A1 isolates that formed a cluster with those from the Philippines and India. Furthermore, some isolates from patients with acute hepatitis who were infected with HBV/A in Japan were highly homologous to HBV/A isolates from patients with chronic hepatitis. This invites speculation that some HBV/A isolates were introduced into Japan from foreign countries, while others have already settled down there and spread from patients with chronic infection to their contacts. HBV/A would have been infiltrating throughout Japan by these two different routes.
Clinical differences among patients infected with HBV/A, -B, and -C were observed. The mean age was lower in the patients infected with HBV/A than in those infected with HBV/B or -C. As mentioned above, AHB patients infected with HBV/A have been increasing in the younger generation in Japan, and around 10% of them would have progressed to chronic infection. This is one of the reasons why the patients infected with HBV/A are younger than those infected with HBV/B or -C. Most patients infected with HBV/B were negative for HBeAg, while a high proportion of the patients infected with HBV/A and -C had it. In particular, this difference was remarkable in the patients who were older than 40 years of age. Thus, the seroconversion rate for the loss of HBeAg among younger people may be higher in infection with HBV/B than in that with HBV/A or -C. Inactive carriers were commoner in HBV/A than in HBV/C infection, as well.
These lines of evidence indicate that the activity of hepatitis is lower in HBV/B than HBV/C infection, and patients with HBV/B seroconvert from HBeAg to anti-HBe at young ages. In addition, cirrhosis and HCC were less frequent in the patients infected with HBV/B than in those infected with HBV/C. Therefore, the prognosis would be better in the patients infected with HBV/B than in those infected with HBV/C. These results are in accord with previous reports (5, 13, 28, 42). There have been few reports on the clinical features of patients with chronic hepatitis infected with HBV/A in Japan. Chu et al. have reported the distribution of HBV genotypes with reference to clinical characteristics in the United States (6). They have shown that HBV/A and HBV/C infections are accompanied by a higher frequency of HBeAg than HBV/B infection, while HBV/B is associated with a lower rate of hepatic decompensation than HBV/A and -C. In our study, inactive carriers were commoner, while cirrhosis and HCC were found less often in HBV/A than in HBV/C infection. HBeAg was more prevalent in the patients infected with HBV/A than in those infected with HBV/B who were older than 40 years of age. Therefore, it can be said that the prognosis is better for patients infected with HBV/A than for those infected with HBV/C; it may be poorer than for those infected with HBV/B.
In conclusion, HBV/A has been increasing among CHB patients in Japan. On the basis of phylogenetic analyses, some HBV/A isolates appear to have been imported from foreign countries. They clustered with HBV/A from AHB patients and have infiltrated throughout Japan. It is very likely that acute and chronic infections with HBV/A have been increasing in Japan. Obviously, immunoprophylaxis of perinatal HBV infection, implemented since 1986 on a national basis, has been insufficient to prevent horizontal HBV/A infection diffusing among high-risk groups by transmission routes shared by HIV infection. The foreseeable spread of HBV/A infection in Japan should be prevented by universal vaccination programs extended to high-risk groups or the general population.
ACKNOWLEDGMENTS
The study was supported in part by a grant-in-aid from the Ministry of Health, Labor and Welfare of Japan and a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology.
We thank T. Kimura and K. Sato, Institutes of Immunology Co., Ltd. (Tokyo, Japan), for determining HBV genotypes in this study and Takashi Saito, Yamagata University Hospital; Akihiro Matsumoto, Shinshu University Hospital; Yasuhiro Asahina, Musashino Red Cross Hospital; Yoshito Ito, University Hospital, Kyoto Prefectural University of Medicine; Keiko Hosho, Tottori University Hospital; Morikazu Onji, Ehime University Hospital; Tatsuya Ide, Kurume University Hospital; and Hiroshi Sakugawa, Hospital, University of the Ryukyus, for their help throughout this work.
Kentaro Matsuura wrote the study protocol and the first draft of the manuscript and performed the experiments and statistical analysis. Yasuhito Tanaka contributed to the experimental work and the final version of the manuscript. Shuhei Hige, Gotaro Yamada, Yoshikazu Murawaki, Masafumi Komatsu, Tomoyuki Kuramitsu, Sumio Kawata, Eiji Tanaka, Namiki Izumi, Chiaki Okuse, Shinichi Kakumu, Takeshi Okanoue, Keisuke Hino, Yoichi Hiasa, Michio Sata, and Tatsuji Maeshiro contributed to the collection of the samples and clinical data from patients and to the final version of the manuscript. Fuminaka Sugauchi, Shunsuke Nojiri, Takashi Joh, and Yuzo Miyakawa contributed to the final version of the manuscript. Masashi Mizokami had the original idea and did the planning of the study and contributed to the final version of the manuscript. All of the authors have seen and approved the final draft of the manuscript.
FOOTNOTES
- Received 29 October 2008.
- Returned for modification 17 December 2008.
- Accepted 2 March 2009.
- Copyright © 2009 American Society for Microbiology
