Hepatitis B virus (HBV) infection is a worldwidehealth problem. The prevalence of HBV carriers variesfrom 0.1 to 2% in low prevalent areas to 10 to 20% inhigh prevalent areas. Indonesia is among the HBV highprevalent countries (16). The identification of the com-mon determinant a and two pairs of allelic variations, d/yand w/r, has lead to the definition of the four majorsubtypes of hepatitis B surface antigen (HBsAg), adw,adr, ayw, and ayr (3, 13, 18, 37). Another classificationsystem, which is based on the sequence diversity of the

HBV genome has been developed and, currently eightgenotypes designated A to H have been identified (1, 4,23, 25, 35). It has been reported that distribution ofHBV subtypes and genotypes varies with different geo-graphical areas (2, 15, 18, 22, 24, 26). Also, clinicalmanifestations and therapeutic responses to antiviraltherapy might differ with different HBV genotypes (11,12, 39).

Hepatitis D virus (HDV), a defective RNA virus thatrequires a helper function of HBV for packaging and

Genotype and Subtype Analyses of Hepatitis BVirus (HBV) and Possible Co-Infection of HBV andHepatitis C Virus (HCV) or Hepatitis D Virus (HDV)in Blood Donors, Patients with Chronic Liver Disease and Patients on Hemodialysis in Surabaya, Indonesia

Maria I. Lusida1, 4, Surayah5, Hiroshi Sakugawa6, Motoko Nagano-Fujii7, Soetjipto2, 4, Mulyanto5, Retno Handajani2, 4, Boediwarsono3, Poernomo B. Setiawan3, Chairul A. Nidom2, 4,Shinji Ohgimoto7, and Hak Hotta*, 7

Departments of 1Microbiology, 2Biochemistry, and 3Internal Medicine, Faculty of Medicine & Dr. Soetomo Hospital, 4Tropi-cal Disease Center, Airlangga University, Surabaya, Indonesia, 5West Nusa Tenggara Hepatitis Laboratory, Mataram,Indonesia, 6First Department of Internal Medicine, University of the Ryukyus School of Medicine, Okinawa 903–0215,Japan, and 7Department of Microbiology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650–0017, Japan

Received July 2, 2003. Accepted September 10, 2003

Abstract: Four subtypes (adw, adr, ayw, and ayr) and eight genotypes (A to H) of the hepatitis B virus (HBV)have been identified. They appear to be associated with particular geographic distribution, ethnicity, andpossibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotypingwere carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors;patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patientson hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested inSurabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Ourresults also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers inSurabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, andapproximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower inHBV carriers with HCV co-infection than in those without HCV co-infection. We also found that preva-lence of hepatitis D virus (HDV) co-infection was <0.5% among HBV carriers in Surabaya.

Key words: Hepatitis B virus, Genotype, Subtype, Indonesia

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Microbiol. Immunol., 47(12), 969–975, 2003

Abbreviations: aa, amino acid; AH, acute hepatitis; BD, blooddonors; CH, chronic hepatitis; HBV, hepatitis B virus; HCC,hepatocellular carcinoma; HCV, hepatitis C virus; HD, hemodial-ysis; HDV, hepatitis D virus; LC, liver cirrhosis.

*Address correspondence to Dr. Hak Hotta, Department ofMicrobiology, Kobe University Graduate School of Medicine,7–5–1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650–0017, Japan.Fax: �81–78–382–5519. E-mail: hotta@kobe-u.ac.jp

transmission, plays an important role in the progressionof chronic liver damage in patients chronically infectedwith HBV. Co-infection of HBV and HDV relatively isinfrequent in the Far East, including Japan, with someexceptions like Miyako Island, Okinawa and some partsof Taiwan (29, 30). As for Indonesia, a country inSoutheast Asia, it was reported some 15 years ago thatHDV infection was rather rare in Bandung, westernJava (41). As no data is available on current HDVprevalence in Surabaya, eastern Java, it is worthwhile per-forming such an epidemiological study there. Also,hepatitis C virus (HCV) is another important hepatitisvirus. We previously reported the prevalence of HCVinfection and HCV subtypes among various patient pop-ulations in Surabaya, Indonesia (6, 9, 10, 34). At thattime, we noticed that there was a considerable number ofpatients co-infected with HCV and HBV.

In the present study, we determined subtypes andgenotypes of HBV isolates obtained from various patientpopulations in Surabaya, Indonesia. We also analyzedthe prevalence of co-infection of HCV or HDV in HBV-infected patients.

Materials and Methods

Serum samples. Sera were obtained from healthyblood donors (BD) at the Red Cross Blood TransfusionCenter in Surabaya, from patients with acute hepatitis(AH) or chronic liver disease, such as chronic hepatitis(CH), liver cirrhosis (LC), and hepatocellular carcinoma(HCC), and from patients on maintenance hemodialysis(HD) at Dr. Soetomo Hospital, Faculty of Medicine,Airlangga University, Surabaya, Indonesia. The serumsamples were tested for HBsAg by the reverse passivehemagglutination (RPHA) test, and HBsAg titers werequantified. HBsAg-positive sera further were tested forHBV subtypes and HBV genotypes.

Subtype and genotype analyses of HBV. Subtypes ofHBsAg were determined by a commercial EIA kit(HBsAg Subtype EIA, Institute of Immunology Co.,Ltd., Tokyo).

HBV genotypes were determined, as previouslydescribed with some modifications. In brief, HBV DNAextracted from sera by a phenol-chloroform method wassubjected to PCR over 35 cycles, with each cycle con-sisting of 1 min at 94 C, 1 min at 55 C, and 2 min at 72C, to amplify part of the surface gene using primers P7(5'-GTGGTGGACTTCTCTCAATTTTC-3', positions256 to 278) and P8 (5'-CGGTAWAAAGGGACTCAM-GAT-3', positions 796 to 776) (14). If the first-roundPCR amplification was negative, the second-round PCRwas performed using primers HBS1 (5'-CAAGGTAT-GTTGCCCGTTTG-3', positions 455 to 474) and HBS2

(5'-AAAGCCCTGCGAACCACTGA-3', positions 713to 694) (19, 38, 42) under the same condition describedabove. Nucleotide sequences of the amplified frag-ments were determined with the Big Dye Deoxy Termi-nator Cycle Sequencing kit (Perkin Elmer) and ABI377or ABI310 DNA sequencer (Applied Biosystem, Inc.),and amino acid sequences were deduced. With thenucleotide sequences of the samples and those avail-able from the international DNA data bank (GenBank,EMBL, DDBJ), a phylogenetic tree was constructedusing a computer program TreeView (27).

HBV genotypes also serologically were determined byEIA with monoclonal antibodies to genotype-specificepitopes in the preS2-region products (HBV GenotypeEIA, Institute of Immunology Co., Ltd.). Briefly, themethod involved capturing HBsAg in sera by immobi-lized antibodies against the common determinant “a”of HBsAg, and evaluated for reactivity with enzyme-labeled monoclonal antibodies to genotype-specific epi-topes (m, k, s, or u). Genotypes of HBV were deter-mined by the combination of the common epitope: b, andepitopes: su for genotype A, m for genotype B, ks forgenotype C, ksu for genotypes D and E, and k for geno-type F (40).

Serological analyses of HCV and HDV. Anti-HCVand anti-HDV in the sera were tested by using com-mercially available kits (HCV EIA II and Anti-DeltaEIA, respectively, Abbott Laboratories) according tothe manufacturer’s instructions.

Statistical analysis. The data obtained statisticallywere analyzed by χ2 test. A P value of �0.05 was con-sidered significant.

Nucleotide sequence accession numbers. The nucleo-tide sequence data reported in this paper will appear inthe DDBJ/EMBL/GenBank nucleotide sequence data-bases with the accession numbers AB113218 throughAB113243, and AB113273 through AB113300.

Results

HBV Subtypes and Genotypes in IndonesiaA total of 110 HBsAg-positive sera, which was

obtained from 11 BD (male/female [M/F]�11/0; meanage, 39.4 years), 2 AH (M/F�2/0; mean age, 40.5 years),37 CH (M/F�22/15; mean age, 37.0 years), 30 LC(M/F�19/11; mean age, 49.3 years), 23 HCC (M/F�20/3; mean age, 44.6 years), and 7 HD (M/F�6/1;mean age, 43.4 years), was analyzed for HBV subtypesand genotypes. HBV subtypes could be assigned to 78samples. Due to their low HBsAg titers (�5 units/ml),the remaining 32 samples could not be assigned anyHBV subtype. Of the 78 HBV isolates, 73 (94%) weresubtype adw, 4 (5%) ayw, and 1 (1%) adr (Table 1).

970 M. I. LUSIDA ET AL

We then performed HBV genotype analysis on thebasis of partial nucleotide sequences of the S gene ofHBV. Phylogenetic analysis revealed that all 54 samplesthat could be assigned an HBV subtype were classifiedinto genotype B (Fig. 1 and Table 1). An easier geno-typing assay system has been developed that utilizes anEIA method instead of PCR and sequencing. We wereinterested to compare the genotyping results obtainedwith this EIA method and ordinary sequencing analysis.

Of 49 HBV isolates of genotype B that had been con-firmed by ordinary sequencing analysis, 39 (80%) sam-ples were shown to possess a genotype B-specific epitope‘m’ together with the common epitope ‘b’ using theEIA method and, therefore, they were classified intogenotype B (Table 2). One sample had a genotype A-specific epitope ‘su’ and a genotype B-specific epitope‘m’ and, therefore, was considered to be a mixed infec-tion with genotypes A and B. Three samples (6%) onlyhad the common epitope ‘b’ and 6 samples (12%) failedto react to any monoclonal antibody probably due totheir low HBsAg titers. Thus, this EIA method failed toassign HBV genotype B to 9 (18%) of 49 samples tested.

Deduced amino acid (aa) sequences of a part ofHBsAg (aa 113 to 183) of HBV isolates obtained fromvarious patient populations were compared with eachother along with reported sequences of HBV genotypesA to H (Fig. 2). The result revealed that 31 of 44 (70%)HBV isolates from chronic liver disease, including CH,LC, and HCC, possessed one or more aa substitutions inthe region. On the other hand, only 2 of 10 (20%) iso-lates from BD had a substitution in this region, withthe difference between chronic liver disease and BDbeing statistically significant (P�0.01). We did not findany particular mutation that was significantly associatedwith CH, LC, or HCC.

Co-infection of HBV and HCV or HDV in IndonesiaCo-infection of HBV with HCV or HDV was studied.

Anti-HCV antibodies were detected in one of 11 (9%)

971HBV GENOTYPE AND HBs SUBTYPE IN INDONESIA

Table 1. HBV subtypes and HBV genotypes among HBV-infected subjects of various clinical conditions

Subtypea)

Patient groupadw ayw adr

Genotype Bb)

Blood donor (BD) 9 (100) 0 0 10 (100)Chronic hepatitis (CH) 24 (92) 2 (8) 0 16 (100)Liver cirrhosis (LC) 22 (100) 0 0 15 (100)Hepatocellular carcinoma (HCC) 14 (82) 2 (12) 1 (6) 13 (100)Acute hepatitis (AH) 2 (100) 0 0 n.d.Hemodialysis (HD) 2 (100) 0 0 n.d.

Total 73 (94) 4 (5) 1 (1) 54 (100)a) A total of 78 serum samples were tested for HBV subtypes. Parentheses indicate percentage of the respective

subtypes in each patient group.b) A total of 54 serum samples were tested for HBV genotypes. Parentheses indicate percentage of genotype B

in each patient group.

Fig. 1. Phylogenetic analysis of HBV isolates obtained fromIndonesian patients on the basis of partial S gene sequences.Nucleotide sequences of the partial S gene (nt 491 to 703) of HBVisolates in Surabaya and representative strains of HBV geno-types A to H were phylogenetically analyzed. All IndonesianHBV isolates were clustered along with the representative strainsof genotype B (DDBJ accession numbers D23678, D00329, andD00330), some of which are indicated with the isolate num-bers.

Table 2. HBV genotypes determined by PreS2 epitope ELISA kit

Total no. of HBV genotyping by EIAsamples bm (B) bmsu (B/A) b (Unclassifiable) NDa)

49 39 (80)b) 1 (2) 3 (6) 6 (12)a) Not detected due to low HBsAg titers.b) Numerals in parentheses indicate percentage.

BD, 8 of 91 (9%) patients with chronic liver disease(CH, LC, and HCC), and 4 of 7 (57%) HD patients(Table 3). Interestingly, among 13 patients co-infectedwith HBV and HCV, 9 patients (69%) were shown to

have low HBsAg titers (�5 units/ml) (data not shown).On the other hand, only 22 of 95 (23%) of those infect-ed with HBV alone had low HBsAg titers. The differ-ence between the two patient populations statistically was

972 M. I. LUSIDA ET AL

Fig. 2. Alignment of aa sequences of HBV isolates from various patient populations in Surabaya, Indonesia, and reported sequences ofgenotypes A to H. The sequences correspond to aa 113 to 183 of HBsAg. All tested Indonesian isolates belong to genotype B.Asterisks indicate the subtype ayw. The remaining Indonesian isolates shown in this figure belong to the subtype adw.

significant (P�0.01).Anti-HDV antibodies were detected in none of 209

HBsAg-positive individuals tested, which included 105BD, 90 patients with chronic liver disease (CH, LC,HCC), 7 with AH, and 7 on HD (Table 3). The resultssuggest that the prevalence of HDV was �0.5% inHBsAg-positive individuals in Surabaya.

Discussion

Our present results have revealed that 94% of HBVisolates in Surabaya, eastern Java, Indonesia belong tosubtype adw, followed by ayw (5%) and adr (1%) (Table1). This is comparable with a previous observation(21), as far as Surabaya is concerned. It also was report-ed that in Yogyakarta, central Java, adw (74%) wasmost prevalent, followed by adr (11%) and other com-pound subtypes (8). It should be noted, however, that theprevalence of each HBV subtype appears to markedlyvary with different geographical regions in Indonesia; adrwas most prevalent in Irian Jaya and some parts ofSumatra while ayw was most prevalent in Flores, Sumbaand Timor (21). Indonesia is a multiethnic country withan extensive territory. It is expected, therefore, that theprevalence of each HBV subtype vary with differentregions.

HBV genotypes also have a characteristic geograph-ic distribution (2, 15, 18, 22, 24, 26). For example,genotypes A and D are predominant in western countrieswhile genotypes B and C are prevalent in Asia. Sas-trosoewignjo et al. (31) isolated HBV isolates of geno-types B, C, and D in Java, Indonesia. On the otherhand, our present results have revealed that all the HBVisolates in Surabaya belong to genotype B (Table 1).Again, it is expected that the prevalence of each HBVgenotype varies with different regions surveyed. In thisconnection, it is worthwhile to state that HBV geno-type B in Surabaya might have a virological character-istic(s) different from that of the same genotype found inJapan, where HBV genotype B is associated with milderliver disease and barely is associated with HCC (26).

Sugauchi et al. (36) recently reported that HBV genotypeB strains in Asia other than Japan often had undergonegenetic recombination with genotype C over the pre-core region and the core gene of the viral genome, whilethose found in Japan had not. HBV genotype C hasbeen associated with HCC (26) and the genetic recom-bination with this genotype might render genotype Bstrains more pathogenic and more carcinogenic. Thismay explain why HBV genotype B in Surabaya often isassociated with HCC (Table 1), as is genotype C inJapan. As for the correlation between HBV genotypesand subtypes, the latter of which relies on antigenicity ofHBsAg encoded in the S gene of the viral genome, allHBV strains of subtype adw in this study were shown tobelong to genotype B, which has been reported in othercountries.

An EIA kit for HBV genotyping is now availablewhich makes genotype determination easier and morepractical, especially in large-scale studies. In this study,we used a commercial kit that utilizes monoclonal anti-bodies against genotype-specific epitopes in the preS2region. Our result showed that 80% of the tested sampleswere correctly genotyped by the EIA kit while genotypesof the remaining samples could not be determined prob-ably due to their low HBsAg titers (Table 2), with theresults being comparable with a previous study in whichgenotypes of 76.6% of the tested samples could be deter-mined by the EIA kit (20). Since all the HBV isolates inour study belonged to genotype B, we could not evaluatethe sensitivity and specificity of the EIA kit for the othergenotypes. One (2%) sample was positive for epitopes‘bmsu,’ which might suggest a possible mixed infec-tion of genotypes A and B, as previously has been report-ed (12). However, possible cross-reactivity of a particularisolate of genotype B also should be taken into consid-eration since the sequence results suggested single infec-tion of genotype B.

The prevalence of anti-HDV antibodies amongHBsAg-positive carriers was rather low (�0.5%), ifany, in Surabaya, Indonesia (Table 3). It previouslywas reported that there was no anti-HDV-positive subjects

973HBV GENOTYPE AND HBs SUBTYPE IN INDONESIA

Table 3. Possible co-infection of HCV or HDV with HBV

No. positive/no. tested (%)Patienta)

Anti-HCV Anti-HDV

Blood donor (BD) 1/11 (9) 0/105 (0)Chronic liver disease (CH, LC, HCC) 8/90 (9) 0/90 (0)Acute hepatitis (AH) n.t.b) 0/7 (0)Hemodialysis (HD) 4/7 (57) 0/7 (0)

Total 13/108 (12) 0/209 (�0.5)a) All the patients were tested positive for HBsAg.b) Not tested.

among HBsAg-positive pregnant women in Bandung,Indonesia (41). These results collectively suggest thatHDV infection is rare in Indonesia. In this connection, itshould be noted that in Malaysia anti-HDV antibodieswere not found in HCC patients (17), but were found in34% of intravenous drug users (IVDU) who were posi-tive for HBsAg (7) and in 0.9% of patients with acuteviral hepatitis (28).

Risk factors for acquisition of HBV infection include,as is the case with HCV infection, blood transfusionand intravenous drug use. Increased prevalence of HBVinfection also is found in persons with multiple sex part-ners. Mother-to-baby transmission is another commonroute of viral transmission. Our present results show thatHCV co-infection was found in as high as 60% ofHBsAg-positive HD patients (Table 3). This high preva-lence of HBV-HCV co-infection among HD patientsmost probably resulted from multiple transfusions ofcontaminated blood. Also, HBV-HCV co-infection wasseen in about 10% of HBV-infected BD and patientswith CH, LC, and HCC in Surabaya. It previously wasreported that HBV-HCV co-infection was found in only1 of 149 (0.7%) patients with CH, LC, and HCC inYogyakarta, Indonesia (8). Interestingly, most patientsco-infected with HBV and HCV had lower HBsAg titersthan those of patients only with HBV infection. Thisresult, together with previous clinical observations by oth-ers (16), suggests that HBV infection is suppressed byco-infecting HCV. In fact, experimental data using a cellculture system have demonstrated that the HCV coreprotein suppresses HBV replication and HBV geneexpression (5, 32, 33). Detailed molecular mechanismsof HCV-mediated interference of HBV replication ininfected individuals await further investigation.

The authors are grateful to Budi Arifah, Red Cross BloodTransfusion Center, Surabaya, Indonesia for providing them withserum samples obtained from blood donors. This work wassupported in part by a Grant-in-Aid from the Japan Society for thePromotion of Science, Japan.

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