ADONIS 08.1593199100079D
Journal of Gastroenterologyand Hepatology (1991) 6 , 491-498
LIVER AND BILIARY Molecular epidemiology of hepatitis B virus in Indonesia RETNO I. SASTROSOEWIGNJO,*+ BERNARDUS SANDJAJA' AND HIROAKI OKAMOTO* *Immunology Division, Jichi Medical School, M inamikawachi-Machi, Tochigi-Ken, Japan, +Department of Microbiology, Medical Faculty, University of Indonesia, Jakarta, Indonesia and *Jayapura Public Health Laboratory, Zrian Jaya, Indonesia Abstract The S-gene sequences of hepatitis B virus (HBV) from 22 carriers in several islands of Indonesia were amplified by polymerase chain reaction, and XbaI-SpeI fragments corresponding to nucleotides 93-529 (437 base pairs) in the S gene were sequenced. The 22 sequences, along with the 5 reported sequences from Indonesia, were compared with each other, and with the corresponding sequences of 20 clones from other countries including China, France, Great Britain, Japan, Kenya, Papua New Guinea, Philippines, USA and USSR. When the 27 HBV DNA clones of various subtypes from Indonesia were classified by the homology in the nucleotide sequence into the five genotypes, twelve belonged to genotype B (subtype adw 7 and ayw 5 ) , 13 to genotype C (adw 1, adr 10, ayr 1 and ar I), and 2 to genotype D (ayw); none belonged to genotype A or E. Different subtypes of clones in the same genotype indicated that point mutations inducing d-to-y or w-to-r phenotypic changes would be common among Indonesian carriers. Comparison of the translation products of XbaI-SpeI fragments, now available for 47 HBV DNA clones of different genotypes (A 4; B 14; C 21; D 7; E I), identified several amino acids characteristic to or influenced by the five genotypes as well as those highly conserved by clones of different genotypes.
Key words: genotypes, hepatitis B surface antigen, hepatitis B virus, molecular epidemiology, subtypes, viral evolution.
INTRODUCTION Hepatitis B surface antigen (HBsAg) has the group-specific determinant named a and, in addition, one or the other member from each of two sets of subtypic determinants called d andy as well as w and r.',' As a result, the four major subtypes are known as adw, adr, ayw and ayr. Subtypes of HBsAg breed true through passages and have been proposed as the phenotypic expression of the four major genotypes of hepatitis B virus (HBV).3 HBsAg subtypes have distinct geographical distribution, and are useful in a number of clinical, epidemiological and anthropological HBsAg is the translation product of the S gene of the HBV genome, capable of encoding 226 amino acid residues. A single amino acid substitution determines the expression of d/y as well as wIr allele.9-'2 The determinant d is specified by the amino acid 122 of lysine, and y by that of arginine. Similarly, the determinant w is specified by the amino acid 160 of lysine, and r by that of arginine. Such amino acid substitutions, in turn, are ascribable to an A-to-G point mutation at nucleotide 365 or 479, which converts codon 122 or 160 for lysine (AAGIABA) to arginine (AGG/AGA).lo-''
HBV can be classified, by the homology of the entire nucleotide sequence, into at least five genotypes with intragroup differences of < 5.6% and inter-group differences of > 8.0%.13Nucleotide substitutions characteristic of the five genotypes are distributed on the HBV DNA so evenly that genotyping can be achieved by sequencing only a few hundred nucleotides.13 Because d-to-y or w-to-r change are the results of single point mutations, the five genotypes do not always correlate with HBsAg subtypes. In an attempt to study the molecular epidemiology of the HBV genome in Indonesia, XbaI-SpeI fragments were sequenced on HBV DNA clones from plasma samples of 22 asymptomatic carriers living in various islands of the country. They were compared with each other and with the corresponding sequences of 23 r e p ~ r t e d ' ~ -and ' ~ 2 unpublished HBV DNA clones of various genotypes and subtypes.
METHODS Plasma samples Plasma samples were obtained at random from blood donors who were positive for HBsAg and lived in several cities in
Correspondence: Hiroaki Okamoto MD, Immunology Division, Jichi Medical School, Minamikawachi-Machi, Tochigi-Ken 329-04, Japan. Accepted for publication 4 February 1991.
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islands of Indonesia, including Jakarta (Java Island), Padang (Sumatra Island), Balikpapan (Kalimantan Island), Manado (Sulawesi Island), Bajawa (Flores Island) and Jayapura (Irian Jaya).
Serological tests HBsAg was determined by reversed passive haemagglutinatjon with a commercial assay kit (Mycell HBsAg, Institute of Immunology Co. Ltd, Tokyo, Japan). Subtypes of HBsAg were determined by enzyme immunoassay with monoclonal antibodies with a commercial kit (HBsAG SUBTYPE EIA, Institute of Immunology).
Molecular cloning and DNA sequencing HBV DNA in each plasma sample was liberated by digestion with proteinase K, and then extracted with phenol/ chloroform by the procedure described in detail elsewhere. ” An S-gene sequence of HBV DNA was amplified by polymerase chain reaction2*(PCR) by the method described previously with primer S 1 sequenced 5 ’-TCGTGTTACAGGCGGGGTTT-3’ (nt 38-57 in the S gene) and primer S2 with a sequence of S’-CGAACCACTGAACAAATGGC-3’ (nt 531-550 of the complementary strand).’* HBV DNA amplification products, after 25 cycles of PCR, were digested with XbaI and SpeI (Takara Biochemicals, Kyoto, Japan). Obtained XbaI-SpeI fragments of 437 base pairs (bp) were cloned into the XbaI site of MI3 mpl I phage vector, and the sequence spanning nt 93-529 in the S gene (437 bp) was determined by dideoxy chain termination method29using an M13 universal primer or a synthetic oligonucleotide primer with a sequence of 5’-CTGCGGCGTTTTATCAT-3’ (nt 229-245 in the S gene). Two clones were propagated from each of the 22 plasma samples and the sequences of XbuI-SpeI fragments were determined. The representative clone, without deletion or insertion within the sequence at issue, was adopted for comparison with each other and with the reported 23 HBV DNA clone^'^-^' and 2 unpublished ones.
RESULTS Subtypes of HBsAg in carriers from various islands of Indonesia The results of subtyping HBsAg in plasma samples from 144 carriers in Indonesia are summarized in Table 1. Of the four major subtypes of HBsAg, adw, adr and uyw accounted for the majority (139 (97%)); only 1 sample was of subtype ayr. There was an apparent geographic variation of HBsAg subtypes. Subtype udw was predominant in Java Island (35/ 45; 78%) and Kalimantan Island (8/8; 100%); adr was commonly seen in Sumatra Island (11/14; 79%), Sulawesi Island (16/26; 62%) and Irian Jaya (7/14; 50%); u p was prevalent in Flores Island (34/37; 92%). Subtypes of HBsAg samples from Irian Jaya were different from the other
Table 1 Subtypes of HBsAg in asymptomatic carriers in various islands of Indonesia Regular subtypes Atypical ayr subtypes
Island (city)
No. adw adr a p
Jawa (Jakarta) Sumatra (Pandang) Sulawesi (Manado) Irian Jaya (Jayapura) Flores (Bajawa) Kalimantan(Balikpapan) Total (YO)
4 5 3 5 8 2 0 0 1 4 2 1 1 1 0 0 2 6 6 1 6 4 0 0 1 4 1 7 1 1 4“ 3 7 3 0 3 4 0 0 8 8 0 0 0 0 144 55 42 42 1 4 (38%) (29%) (29%)(0.7O/0) (2.8%)
*Two samples were subtyped as ar and the remaining two as adwr.
islands in that they included a single uyr sample and four samples of atypical subtypes - that is, two each of ar and udwr.
Genotypes of HBV DNA clones from carriers in Indonesia Two clones were propagated from each of the 22 plasma samples from Indonesian carriers. They were divergent by 1-6 nucleotides within 437 bp covering a part of the S gene as the results of point mutations. A deletion of nt 336 of A was observed in one of the two clones from a Manado carrier, and nt 506 of G was missed in one of the two from an Irian Jaya carrier; the other clone from each carrier; without frameshift mutation, was selected to represent the HBV DNA clone in them. For the remaining 20 samples, one clone was selected randomly. XbaI-SpeI fragments of HBV DNA clones, representing nt 247-683 in the S gene (437 bp), from 27 carriers in Indonesia were compared with each other for the sequence homology (Table 2). Among them, five had been reported p r e v i ~ u s l y ’ ~ and ~ ’ ”the ~ ~remaining ~ ~ ~ ~ 22 were determined de novo. There were two different categories of divergences in the sequence, one ranging form 0.5 to 4.1% and the other from 5.5 to 8.9%; no differences in a range in between (4.25.5%) were observed between any two clones. Based on an intra-group difference of < 4.1% and inter-group difference of 2 5.4’/0, the 27 HBV DNA clones were classified into three genotypes. With the sequence homology to the four genotypes reported by Okamoto et ~ 1 . and ’ ~ another described by Vaudin et al.,” they were categorized into genotype B, C or D. The 12 clones of genotype B were of subtype either adw or uyw, and both clones of genotype D were of subtype uyw. Ten of the 13 clones of genotype C were of subtype udr; there was one each clone of subtype udw, ayr or ar. Some regional influences were seen on the genotypes of HBV DNA clones. All 4 clones from Flores Island were of genotype B, and all 5 clones from Irian Jaya Island were of genotype C. Clones from Jawa Island were heterogeneous in
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Table 2 Two-by-two analysis of an S-gene sequence of HBV DNA clones from carriers in various districts of Indonesia* Clone number
Clone no. geno/ subtype and origint 1
1* Jaw B adw 2* Jaw B adw 3* Sum B adw 4*Sul Badw 5 Sul Badw 6 Flo B adw 7 Kal Badw 8 SumB ayw 9 Sul Bayw 10 Flo Bayw 11 Flo Bayw 12 Flo B ayw 13* Jaw Cadw 14 Jaw C a d r 15 Jaw C a d r 16 SumC adr 17 SumCadr 18 Sul C a d r 19 Sul C a d r 20 Sul C a d r 21 Iri C adr 22 Iri C adr 23 Iri C adr 24 Iri C ayr 25 Iri C a r 26 Jaw Dayw 27 Jaw Dayw
2
3
4
5
0.5 0.9 0.9 0.5 0.5 0.5 1.4 1.4 1.4 0.9 0.5 0.5 0.7 0.2 1.6 1.4 1.1 1.1 1.1 1.1 1.1 0.7 0.9 0.9 0.9 0.5 1.4 1.4 1.4 0.9 0.9 0.9 0.9 0.5 1.1 1.1 1.1 0.7 6.6 6.6 6.9 6.4 6.9 6.9 6.6 6.6 6.6 6.4 6.4
6.6 6.4 6.4 6.2
6.4 6.2 6.2 6.4 6.2 6.2 6.2 5.9 5.9
5.9 5.9 5.9 5.9
6.4 6.4 6.2 6.2 6.2 5.9 5.9 5.5 6.2 7.2 7.6 7.8 7.6 8.0 8.2 7.8 7.8 8.0 6.4 7.6 6.4 6.4 7.4 6.2
6.2 5.9
5.7 7.2 7.8 7.6 6.4 6.2
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.1 2.1 1.4 1.60.9 1.8 1.4 0.7 1.6 0:7 1.8 1.1 2.1 1.1 0.9 1.4 0.7 1.6 0.7 0.5 1.6 0.9 1.8 0.9 0.7 6.2 6.4 7.6 7.1 6.9 6.4 6.6 6.9 7.3 7.1 6.2 6.4 7.1 7.1 6.9 5.9 6.2 6.4 6.9 6.6 5.9 6.2 6.9 6.9 6.6 5.7 5.9 6.6 6.6 6.4 5.7 5.9 6.6 6.6 6.4 5.9 6.2 6.9 6.9 6.6 5.7 5.9 6.6 6.6 6.4 5.7 5.7 6.6 6.4 6.2 7.1 7.2 8.5 8.0 7.8 7.6 7.8 8.9 8.0 7.8 7.3 7.6 8.7 8.2 8.0 6.4 6.4 6.9 6.6 6.4 6.2 6.2 6.6 6.4 6.2
0.9 1.1 0.7 7.3 6.9 7.6 7.1 7.3 6.9 7.1 6.6 7.1 6.6 6.9 6.4 6.9 6.4 7.1 6.6 6.9 6.4 6.6 6.2
8.2 7.8 8.2 7.8 8.5 8.0 6.9 6.4 6.6 6.2
7.1 7.3 2.7 7.1 2.3 1.4 6.9 2.1 1.1 0.7 6.9 2.1 1.1 0.7 6.6 1.8 0.9 0.5 6.6 2.1 1.1 0.7 6.9 2.1 1.1 0.7 6.6 1.8 0.9 0.5 6.6 3.4 3.9 3.4 8.0 1.8 3.2 2.7 8.0 2.1 3.7 3.2 8.2 1.6 3.9 3.4 6.6 6.2 7.3 7.1 6.4 6.4 7.1 6.9
0.5 0.2 0.5 0.5 0.2 3.2 2.5 3.0 3.2
0.2 0.5 0.5 0.2 3.2 2.5 3.0 3.2
0.2 0.2 0.0 3.0 2.3 2.7 3.0 6.9 6.9 6.6 6.6 6.6 6.4
0.5 0.2 3.2 2.5 3.0 3.2
0.2 3.2 2.5 3.0 3.2
-
3.2 3.9 4.1 6.9 6.9 6.6 7.1 6.6 6.6 6.4 6.9 3.0 2.3 2.7 3.0
2.3 3.0 7.3 7.8
3.4 7.3 7.3 7.6 7.8 1.6 -
*XXbaI-SpeI fragments of the S gene (nucleotides 93-529) of HBV DNA clones from 27 carriers in various districts of Indonesia were compared for sequence homology, and classified into genotypes by intra-group difference of C 4.1% and inter-group difference of 2 5.5%. Inter-group differences 2 5.5% are shown in bold face. tAbbreviations for islands in Indonesia - Flo: Flores; Iri: Irian Jaya; Jaw: Jawa; Kal: Kalimantan; Sul: Sulawesi; Sum: Sumatra. *Sequences reported previously - 1: pRTB299;” 2: pIDW420;I3 3: pPAD744;” 4: P M N D ~ ~13: ~ ;~~I W ’ K146.l~
l r i a n Jaya
D Figure 1 Map of Indonesian archipelago. Cities where serum samples were obtained are identified with numbers. Genotypes and subtypes of cloned HBV genomes are shown. Key: 1 = Padang: B (adw:l, ayw: l), C (adr:2); 2 = Jakarta: B (adw:2), C (adw:l, adr:2), D (ayw:2); 3 = Balikpapan: B (adw:l); 4 = Bajawa: B (adw: 1, ayw:3); 5 = Manado: B (adw:2, ayw:l), C (adr:3); 6 = Jayapura: C (adr:3, ayr:l, ar: 1).
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genotypes, distributing in B, C and D. Likewise, clones from Sumatra and Sulawesi Islands included those of genotypes B and C. The single clone from Kalimantan Island was of genotype B. Fig. 1 shows topographical relationships of the six islands in Indonesia with the cities where serum samples were taken, along with various geno- and subtypes of HBV.
Amino acid substitutions specific to or characteristic of HBV genotypes Amino acids 32-176 (145 residues) of the S-gene product were deduced from XbaI-SpeI fragments of 47 HBV DNA
clones. Only 130 residues (amino acids 40-169), with at least 1 substitution among the 47 clones, are listed for comparison in Table 3. Within 145 residues in the S-gene product, 93 (64%) were conserved by 47 HBV DNA clones. Some diversions were sporadic; others appeared to be under controls. All the conversions were given rise to by point mutations. In Table 3, amino acid substitutions influencing the expression of d/y and w/r alleles are indicated by arrows; lysine or arginine at position 122 specifies d or y determinant, while that at position 160 does w or r determinant.’-” The correlation between genotypes and the w/r allele was obvious. The determinant r was observed in 18 (86%) of 21
Table 3 Amino acid sequences of a part of the translation product of the S gene of 47 HBV DNA clones of different genotypes Genolsubtypes a n d origin adw adw adw adw
USA USA PHI KEN
5t B adw €3 B adw 7f B adw 8t B adw 9t B adw 107 B adw 1 1 B adw 12 B adw 13 B adw 14 B ayw 15 B ayw 16 B ayw 17 B ayw 18 B ayw
JPN JPN INA INA INA INA INA INA INA INA INA INA INA INA
C adw C adw C adw C adr C adr 247 C adr 25t C adr 26f C adr
JPN JPN INA JPN JPN JPN JPN CHN
27 C adr
INA
58 C idr
INA INA INA INA INA INA INA INA INA JPN INA INA
1’ 2t 3t 4’
A A A A
l9f 20t 217 22T 237
29 30 31 32 33 34 35 36 37t 38 39
C adr C adr C adr C adr C adr C adr C adr C adr C avr C ayr C ar
40t 417 42t 437 44t 45 46
D ayw FRA D ayw URS D ayw JPN D ayw PNG D ayw KEN D ayw INA D ayw INA
47+ E adw GBR
XbaI-SpeI fragments corresponding to nucleotides 93-529 in the S gene of different HBV DNA clones were decoded and presented in single letter abbreviations of amino acid residues. Of 145 amino acids at positions from 32 to 176, only 130 (amino acids 40-169) are shown; sequences outside them did not diverge among 47 clones. The origins of clones are indicated by three-letter codes for nations adopted by International Olympic Committee: CHN, China; FRA, France; GBR, Great Britain; INA, Indonesia; JPN, Japan; KEN, Kenya; PHI, Philippines; PNG, Papua New Guinea; USA, United States of America; URS, Union of Soviet Socialist Republics. ; ~pKNDW60 ~ (unpubl.); 5 : pJDW233;” +Cloneshave been reported previously - 1: pHBV-3200;” 2: pHBV933;17 3: ~ F D w 2 9 4 4: 9: ~pAD744;’~ 10; pMND122;” 19: ~ S K 6 1 920: ; ~ pAK66;” ~ 21: pIWK146;I322: pHBV16: pODW282;13 7: P R T B ~ ~8:~ p:IDW420;” ;~’ 1;” 23: pHBr330;” 24: pBRHBadr4;16 25: pNDR260;I3 26: P A D R - ~ ; *40: ~ Eco HBV DNA;14 41: pHB320;” 42: pYWB796;13 43: ~ P Y w 3 1 0 ; 44: ’ ~ pKNYW149 (unpubl.); 47: ~ ~ w - L S H . ‘ ~ Amino acid residues- A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine;G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine. Arrows indicate the positions of amino acids 122 and 160, lysine or arginine at which specifies d or y and w or r subtypic determinant respectively.
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HBV DNA clones of genotype C, whereas all 26 clones of genotype A, B, D or E expressed w. With a less close correlation, the determinant y was expressed by all 7 clones of genotype D, while d was found in all clones of genotype A or E, as well as in 9 of 16 (56%) clones of genotype B, and predominant in genotype C (18/21; 86%). The clone no. 39 of genotype C did not express d despite iysine at position 122, because it possessed Ala145 in place of G1~145.~' Aside from those specifying subtypic determinants, there were at least 18 amino acids, within 145 residues spanning positions 32-176, the expression of which was apparently allelic, and characteristic to certain genotypes. In addition, there were 3 amino acids which were under influence of a certain genotype, but were not necessarily restricted to it. Correlation of amino acid conversions with HBV genotypes are summarized in Table 4.
Table 4 Amino acid changes in the S-gene product characteristic to or influenced by HBV genotypes No. clones with amino acids in allele Position and members of allele Characteristic Genotype A 114:ThdSer 131:AsdThr Genotype B 56:GldPro 57: Ile/Thr 59: Ser/Asn 64 CydSer 85:CyslPhe Genotype C 47 :ThrNal 49: Pro/Leu 110:Leu/Ile 113:Thr/Ser 126:IlelThr Genotype D 46 :ThdPro 68:ThdIle 134:Tyr/Phe 159:Gly/Ala 168:AldVal Genotype E 117 :Thr/Ser Influenced 53:SerlLeu 122:LydArg 16O:LydArg
A
B
C
D
E
4/0 4/0
0/14 0/14
1/20 0121
0/7 0/7
0/1 011
0/4 0/4 0/4 014 0/4
14/0 13/0* 14/0 14/0 14/0
0/2l 0/2l 0121 0/21 0121
0/7 0/7 0/7 0/7 0/7
0/1 0/1 0/1 0/1 0/1
0/4 0/4 0/4 0/4 0/4
0114 19/lt 0/14 20/1 0/14 21/0 0/14 21/0 0114 20/0*
0/7 0/7 0/7 0/7 0/7
0/1 011 011 011 0/1
0/4 0/4 0/4 0/4 0/4
0/14 0/14 0/12$ 0/14 0/14
0/21 0/21 0/21 0/21 0121
7/0 7/0 6/1 7/0 7/0
0/1 0/0' 0/1 011
014
0/14
0/21
017
110
4/0 4/0 410
14/0 9/5 14/0
13/8 19/2 3/18
7/0 0/7 710
110 1/0 110
*Vals7in clone no. 16. tAla47in clone no. 39. *Amlz6in clone no. 27. ISer,,, in clone no. 13 and Ile13, in clone no. 14. *Ile,34in clone no. 47.
011
DISCUSSION Indonesia, an archipelago spanning some 2 million square kilometres in Southeast Asia, has a high endemicity for HBV. Carrier rates among voluntary blood donors range from 2.1 to 9.5% in 11 big cities; it is even higher at 17.5% in Jayapura City in Irian Jaya Island. The anthropological origin of Indonesians, who would have arrived at the islands by sea, is not established yet, nor is it known how early the ancestors migrated there. Based on cultural backgrounds, strong influences of people from South China, India, Mediterranean and European countries including Netherlands and Portugal, are conceived. There is evidence to indicate that South Chinese migrated to Indonesia for trading, Indians and Mediterraneans through religious spreading, and Portuguese and Netherlanders with political or religious aims. Subtypes and genotypes of HBV may be helpful in research along these lines, as has been useful in investigating the migration of Japanese ancestors. Because the Indonesian islands are separated by sea, hindering free surface migration of inhabitants among them, HBV strains of distinct subtypes andor genotypes would likely have been maintained in each individual island. Surrounding Indonesia, the subtype adr is prevalent in South China as well as in mainland China, adw and ayw in India, and ayw in Mediterranean countries, while adw is common in the Netherlands, and ayw in PortugaL8 The fact that adw is predominant in Jawa, adr in Sulawesi, and ayw in Flores (Fig. 1, Table 4) possibly reflects the influences of these countries. In view of d/y and w/r subtypic changes that are ascribable to single point mutations in HBV DNA,"-'* however, the subtype distribution is not readily taken as a clue in pursuing the migration of ancestors to the Indonesian Islands. Although the predominance of adw in Java and that of adr in the other islands imply the influence of surrounding countries, the analysis of nucleotide sequences favours the view that the Indonesian HBV strains of these two subtypes would be indigenous and that the different subtypes would have arisen by point mutations. On the basis of sequence analyses, the HBV genomes of subtype ayw in Flores Island would most likely be the result of mutation from those of subtype udw. HBsAg/up in this situation, therefore, would not reflect a foreign influence from Portugal. Similarly, in North Lombok, near Flores Island, the predominant ayw subtype might have been given .~' analysis rise to by a point mutation in H B V J ~ ~ WSequence of the uyw strain in North Lombok, for comparison with udw and ayw strains in Flores Island, would be required to substantiate this view, however. HBV DNA clones can be classified into five genotypes by differences in the entire nucleotide sequence of 8% or greater, including genotypes A-D proposed by Okamoto et ~ 1 . and ' ~ genotype E represented by a single HBV genome isolated by Vaudin et al.*' from a naturally infected chimpanzee. Nucleotide divergence distributes so evenly on the whole genome that the genotyping is feasible by sequencing only a few hundred nucleotides of HBV DNA.13 HBV replicates by the reverse transcription of an RNA
R . Sastrosoewignjo et al.
496
intermediate.32 This process involves errors, because it is not controlled by proofreading enzymes.33y34Some mutations are associated with phenotypic changes. For example, subtypic changes occur as the result of a single point mutation which converts a single amino acid residue.'-12 Assuming the rate of nucleotide substitution at 1.4-3.2 x lO-'/site per year,23evolution of HBV genomes into different types would have started at least 3000 years ago. Recently, Orito et al. attempted genetic classification by a similar approach, and classified the reported 12 HBV genomes into four c a t e g o r i e ~They . ~ ~ found that sequence divergences among HBV genomes far exceeds those between HBV and animal or avian hepadnaviruses, and estimated the evolution of viruses of different host ranges at 5000 years ago or earlier. By means of PCR, S-gene sequences of 22 HBV DNA clones were amplified to determine the sequence of XbaISpeI fragment. Amplification of DNA sequences by PCR with the Taq polymerase reaction involves potential misincorporation of nucleotides. Saiki et al. estimated the frequency of overall error, during amplification for 30 cycles, at o.25°/~.2sThis ratio, applied to an S-gene sequence of 437 nucleotides (XbaI-SpeI fragment) multiplied for 25 cycles, would account for < 1 error, far less than the > 24 (6.4%) divergent nucleotides which are required to create intergroup differences in the present study. Misincorporation in amplification by PCR, therefore, would not seriously affect our genotyping criteria. The 27 HBV DNA clones from Indonesian carriers, including 22 determined in the present study and 5 reported previ~usly,'~~ were ' ~ ~classified ~ ' ~ ~ ~ into genotype B, C or D (Table 2). All 4 clones from Flores Island were of genotype B, and all 5 clones from Irian Jaya Island were of genotype C; HBV genomes of a particular genotype appear to have been maintained in these islands. In contrast, 7 HBV clones from Java Island were heterogeneous, including genotypes B, C and D; 11 clones from the other three islands were of genotype either B or C (Fig. 1). With the limitation of the small number of clones dealt with in the present study, these results appear to indicate that Java Island would have been the centre of transportation, attracting people from the other islands. We then compared amino acid sequences, within the range of XbaI-SpeI fragment carrying 145 codons, among 47 clones including 23 previously r e ~ o r t e d ' ~and - ~ ~2 unpublished ones. A number of amino acid substitutions characteristic of HBV genotypes emerged, all of which were ascribable to point mutations. Obviously enough, the great majority of amino acids (93 of 145; 64%) were preserved by HBV DNA clones of different genotypes. Although mutations may occur evenly in various sites of HBV DNA sequence, some selective force would have to be operating for the survival of mutants with point mutations at restricted nucleotides in the S gene. Amino acid substitutions responsible for subtypic changes are partly associated with HBV genotypes. For instance, Arglz2for the determinant y was observed in all clones of genotype D and also in some clones of genotypes B and C, while LysIZ2for d was seen in all clones of genotype A or E,
and also in the majority of clones of genotype B or C. Arg,,, for the determinant r was restricted to genotype C, but not a hallmark for it because it was not possessed by 3 of 21 clones. The influence of an amino acid at a position away from Lysl22 on the expression of d determinant was seen in one of the clones subtyped as ar by the immunological method. This was clone no.39 of genotype C, which had LyslZ2 characteristic of the determinant d. HBsAg particles in the plasma, from which it was propagated, did not express the determinant d , however. The clone had AlaI45,unlike all the other 46 clones (with either determinant d or y), which possessed Glyl4~.These results confirmed our previous proposal that the expression of d would be under the influence of amino acid 144 or 145 in the S-gene product.30 Ohnuma et al. reported that the microconformation maintained by the -Cys12l-Cys124- bond would constitute a microconformation for a common antigenic determinant of HBsAg detectable by monoclonal antibody (no. 5 124).36 This disulfide bond was expressed by 46 of 47 HBV DNA clones. A single clone, no. 18, possessed Argi2, and, therefore, the conformation would not be built in the HBsAg it encodes. They reported Ile126to be involved in an HBsAg determinant specific for genotype C, which is detectable by another monoclonal antibody (no. 4403). Their view was extended to 20 of 21 clones of genotype C, with the single exception of clone no. 27, which possessed ASp126, unlike the other 46 clones of any genotypes. HBV genotypes, with characteristic amino acid substitutions in the S-gene product, would find applications in virological and epidemiological research. Cloning and sequencing HBV DNA, however, are not feasible for genotyping on a large scale. Recently, Norder et al. reported genotypeassociated differences in the efficiency of oligonucleotide primers for amplifying HBV DNA by PCR.-17Their observations suggest that genotyping might be performed by evaluating the compatibility of HBV DNA templates with a few sets of primers in amplification by PCR. It is possible, also, that nucleotide substitutions intrinsic to genotypes may create specific restriction cleavage sites or distinct antigenic epitopes on the S-gene product. Should such become the reality, genotyping would be achieved by comparing restriction patterns with a few endonucleases or by immunological tests.
ACKNOWLEDGEMENTS We thank the following doctors for valuable materials, enlightenment, discussion and constructive criticism toward the completion of this work: M. Rustam of the Indonesian Red Cross Blood Center, Sujudi of University of Indonesia, S. Gunawan of the Ministry of Health, Indonesia; D. L. Peterson of Virginia Commonwealth University, USA; H. Suzuki of Yamanashi Medical College, M. Homma of Kobe University, Y. Miyakawa of Mita Institute, T. Yamanaka, S. Yotsumoto, and M. Mayumi of Jichi Medical School, Japan. This work was supported in part by the Japan Society for the Promotion of Science.
HBV genotypes in Indonesia
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tion of additional antigenic determinants of hepatitis B antigen. 3. Immunol. 1972; 109: 842-8. 3. LE BOUVIER G. L., MCCOLLUM R. W., HIERHOLZER W. J., IRWING. R., KRUGMAN S. & GILES J. P. Subtypes of Australia antigen and hepatitis-B virus. J A M A 1972; 222: 928-30. 4. MAZZURS., BURGERT S. & BLUMBERG B. S. Geographical distribution of Australia antigen determinants d, y and w. Nature ( L a d . ) 1974; 247: 38-40. 5 . YAMASHITAY., KURASHINA S., MIYAKAWA Y. & MAYUMI M. South-to-north gradient in distribution of the r determinant of hepatitis B surface antigen in Japan. 3. Infect. Dis. 1975; 131: 567-9. 6. NIELSENJ. O., LE BOUVIERG. L. & THECOPENHAGEN HEPATITIS ACUTE PROGRAM. Subtypes of Australia antigen among patients and healthy carriers in Copenhagen. A relation between the subtypes and the degree of liver damage in acute viral hepatitis. N . Engl. 3. Med. 1973; 288: 1257-61. 7. OKADAK., KAMIYAMA I., INOMATA M., I ~ M.,I MIYAKAWA Y. & MAYUMI M. e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N.Engl. 3. Med. 1976; 294: 746-9. 8 . COUROUCE-PAUTY A. M., PLANCON A. & SOULIER J. P. Distribution of HBsAg subtypes in the world. Vox Sang. 1983; 44: 197-211. 9. PETERSON D. L., PAULD. A., LAMJ., TRIBBY I. I. E. & ACHORDD. T. Antigenic structure of hepatitis B surface antigen: identification of the ‘d’ subtype determinant by chemical modification and use of monoclonal antibodies. 3. Immunol. 1984; 132: 920-7. 10. OKAMOTO H . , IMAIM., TSUDA F., TANAKA T., MIYAKAWA Y. & MAYUMI M. Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr. J . Virol. 1987; 61: 3030-4. 1 1 . OKAMOTO H., IMAIM., MIYAKAWA Y. & MAYUMI M. Sitedirected mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w. Biochem. Biophys. Res. Commun. 1987; 148: 500-4. H., TSUDA F., MIYAKAWA Y. & 12. YOTSUMOTOS., OKAMOTO MAYUMI M. Subtyping hepatitis B virus DNA in free or integrated forms by amplification of the S-gene sequences by the polymerase chain reaction and single-track sequencing for adenine. J. Vzrol. Methods 1990; 28: 107-16. 13. OKAMOTOH., TSUDAF., SAKUCAWA H. et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. 3. Gen. Virol. 1988; 69: 2575-83. 14. GALIBERT F., MANDARTE., FITOUSSIF., TIOLLAIS P. & CHARNAY P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature (Lond.) 1979; 281: 646-50. 15. VALENZUELA P., QUIROGA M., ZALDIVAR J., GRAYP. & RUTTERW. J. The nucleotide sequence of hepatitis B viral genome and the identification of the maior viral genes. In: Fields B. N., Jaenish R. & Fox C. F., eds. Animal Virus Genetics. Academic Press, London, 1980: 57-70.
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16. FUJIYAMA A., MIYANOHARA A., NOZAKIC., YONEYAMA T., OHTOMON. & MATSUBARA K. (1983) Cloning and structural analyses of hepatitis B virus DNAs, subtype adr. Nucl. A d s Res. 1983; 11: 4601-10. R., ICARASHI K.,SUGINOY. & 17. ONOY., ONDAH., SASADA NISHIOKAK. The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucl. Acids Res. 1983; 11: 1747-57. 18. KOBAYASHI M. & KOIKEK. Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene 1984; 30: 227-32. P., DREILINA D., PUMPEN P. & GRENE. 19. BICHKOV., PUSHKO Subtype ayw variant of hepatitis B virus. DNA primary structure analysis. FEBS Let. 1985; 185: 208-12. 20. SASTROSOEWIGNJO R. I., OKAMOTO H., MAYUMI M., WARSA U. c . & SUJUDI. The complete nucleotide sequence of an HBV DNA clone of subtype adw (pRTB 299) from Indonesia. I C M R Ann. 1985; S: 39-50. M. et al. Nucleotide 21. OKAMOTOH., I M I M., SHIMOZAKI sequence of a cloned hepatitis B virus genome, subtype ayr: comparison with genomes of the other three subtypes. 3. Gen. Virol. 1986; 67: 2305-14. 22. SASTROSOEWIGNJO R. I., OKAMOTO H., MAYUMI M., RusTAM M., WARSAu. C. & SUJUDI.The complete nucleotide sequence of an HBV DNA clone of subtype adw (pPAD 744) from Sumatra, Indonesia. I C M R Ann. 1986; 6: 99-106. 23. OKAMOTO H . , IMAIM., KAMETANI M., NAKAMURA T. & MAYUMI M. Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission. Japan. 3. Exp. Med. 1987; 57: 231-6. 24. GANR., CHUM., SHENL., QIANS. & LI Z. The complete nucleotide sequence of the cloned DNA of hepatitis B virus subtype adr in pADR-1. Sci. Sinica 1987; 30: 507-21. 25. SASTROSOEWIGNJO R. I., OMIS., OKAMOTO H., MAYUMI M., RUSTAM M. & SUJUDI.The complete nucleotide sequence of HBV DNA clone of subtype adw (pMND 122) from Manado in Sulawesi island, Indonesia. I C M R Ann. 1987; 7: 51-60. R . C., CHAVEZ C. C., OKAMOTO H. et al. Nucleotide 26. ESTACIO sequence of a hepatitis B virus genome subtype adw isolated from a Phdippino: comparison with the reported three genomes of the same subtype. 3. Casrroenterol. Hepatol. 1988; 3: 215-22. 27. VAUDINM., WOLSTENHOLME A. J., TSIQUAYE K. N., ZUCKERMAN A. J. & HARRISON T. J. The complete nucleotide sequence of the genome of a hepatitis B virus isolated from a naturally infected chimpanzee.3. Gen. Virol. 1988; 69: 1383-9. 28. SAIKIR. K., GELFANDD. H., STOFFELS. et al. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-91. 29. SANGER F., NICKLEN S. & COULSON A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. U S A 1977; 74: 5463-7. 30. OKAMOTO H . , OMIS., WANGY. er al. The loss of subtypic determinants in alleles, d/y or w/r, on hepatitis B surface antigen. Mol. Immunol. 1989; 26: 197-205. 31. CUNAWAN S., MULJANTO D. H., SUMARSIDI D., SULASTINI Hyperendemic HBV H. D., KARYONO W. & SOEWIGNYO. infection with rare HBsAg subtype in northern part of Lobok island. Proceeding of the Third Scientific Meeting of the Indonesian Association for the Study of the Liver. 1985. J. & MASONW. S. Replication of the genome of a 32. SUMMERS hepatitis B-like virus by reverse transcription of an RNA intermediate. 1982; Cell 29: 403-15. 33. HOLLAND J., SPINDLERK., HORODYSKI F., GRABAU E.,
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NICHOLS. & VANDEPOLS. Rapid evolution of RNA genomes. Science 1982; 215: 1577-85. D. A. & HOLLANDJ. J. Direct method for 34. STEINHAUER quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. J. Virol. 1986; 57: 219-28. -35. ORITOE., MIZOKAMI M., INA Y. et al. Host-independent evolution and a genetic classification of the hepadnavirus
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family based on nucleotide sequences. Proc. Nut1 Acad. Sci. USA 1989; 86: 7059-62. 36. OHNUMAH . , TAKAIE., MACHIDAA. et al. Synthetic oligopeptides bearing a common or subtypic determinant of hepatitis B surface antigen. 3. Immunol. 1990; 145: 2265-71. 37. NORDERH., HAMMAS B. & MACNIUSL. 0. Typing of hepatitis B virus genomes by a simplified polymerase chain reacti0n.J. Med. Virol. 1990; 31: 215-21.
Journal of Gastroenterologyand Hepatology (1991) 6 , 491-498
LIVER AND BILIARY Molecular epidemiology of hepatitis B virus in Indonesia RETNO I. SASTROSOEWIGNJO,*+ BERNARDUS SANDJAJA' AND HIROAKI OKAMOTO* *Immunology Division, Jichi Medical School, M inamikawachi-Machi, Tochigi-Ken, Japan, +Department of Microbiology, Medical Faculty, University of Indonesia, Jakarta, Indonesia and *Jayapura Public Health Laboratory, Zrian Jaya, Indonesia Abstract The S-gene sequences of hepatitis B virus (HBV) from 22 carriers in several islands of Indonesia were amplified by polymerase chain reaction, and XbaI-SpeI fragments corresponding to nucleotides 93-529 (437 base pairs) in the S gene were sequenced. The 22 sequences, along with the 5 reported sequences from Indonesia, were compared with each other, and with the corresponding sequences of 20 clones from other countries including China, France, Great Britain, Japan, Kenya, Papua New Guinea, Philippines, USA and USSR. When the 27 HBV DNA clones of various subtypes from Indonesia were classified by the homology in the nucleotide sequence into the five genotypes, twelve belonged to genotype B (subtype adw 7 and ayw 5 ) , 13 to genotype C (adw 1, adr 10, ayr 1 and ar I), and 2 to genotype D (ayw); none belonged to genotype A or E. Different subtypes of clones in the same genotype indicated that point mutations inducing d-to-y or w-to-r phenotypic changes would be common among Indonesian carriers. Comparison of the translation products of XbaI-SpeI fragments, now available for 47 HBV DNA clones of different genotypes (A 4; B 14; C 21; D 7; E I), identified several amino acids characteristic to or influenced by the five genotypes as well as those highly conserved by clones of different genotypes.
Key words: genotypes, hepatitis B surface antigen, hepatitis B virus, molecular epidemiology, subtypes, viral evolution.
INTRODUCTION Hepatitis B surface antigen (HBsAg) has the group-specific determinant named a and, in addition, one or the other member from each of two sets of subtypic determinants called d andy as well as w and r.',' As a result, the four major subtypes are known as adw, adr, ayw and ayr. Subtypes of HBsAg breed true through passages and have been proposed as the phenotypic expression of the four major genotypes of hepatitis B virus (HBV).3 HBsAg subtypes have distinct geographical distribution, and are useful in a number of clinical, epidemiological and anthropological HBsAg is the translation product of the S gene of the HBV genome, capable of encoding 226 amino acid residues. A single amino acid substitution determines the expression of d/y as well as wIr allele.9-'2 The determinant d is specified by the amino acid 122 of lysine, and y by that of arginine. Similarly, the determinant w is specified by the amino acid 160 of lysine, and r by that of arginine. Such amino acid substitutions, in turn, are ascribable to an A-to-G point mutation at nucleotide 365 or 479, which converts codon 122 or 160 for lysine (AAGIABA) to arginine (AGG/AGA).lo-''
HBV can be classified, by the homology of the entire nucleotide sequence, into at least five genotypes with intragroup differences of < 5.6% and inter-group differences of > 8.0%.13Nucleotide substitutions characteristic of the five genotypes are distributed on the HBV DNA so evenly that genotyping can be achieved by sequencing only a few hundred nucleotides.13 Because d-to-y or w-to-r change are the results of single point mutations, the five genotypes do not always correlate with HBsAg subtypes. In an attempt to study the molecular epidemiology of the HBV genome in Indonesia, XbaI-SpeI fragments were sequenced on HBV DNA clones from plasma samples of 22 asymptomatic carriers living in various islands of the country. They were compared with each other and with the corresponding sequences of 23 r e p ~ r t e d ' ~ -and ' ~ 2 unpublished HBV DNA clones of various genotypes and subtypes.
METHODS Plasma samples Plasma samples were obtained at random from blood donors who were positive for HBsAg and lived in several cities in
Correspondence: Hiroaki Okamoto MD, Immunology Division, Jichi Medical School, Minamikawachi-Machi, Tochigi-Ken 329-04, Japan. Accepted for publication 4 February 1991.
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islands of Indonesia, including Jakarta (Java Island), Padang (Sumatra Island), Balikpapan (Kalimantan Island), Manado (Sulawesi Island), Bajawa (Flores Island) and Jayapura (Irian Jaya).
Serological tests HBsAg was determined by reversed passive haemagglutinatjon with a commercial assay kit (Mycell HBsAg, Institute of Immunology Co. Ltd, Tokyo, Japan). Subtypes of HBsAg were determined by enzyme immunoassay with monoclonal antibodies with a commercial kit (HBsAG SUBTYPE EIA, Institute of Immunology).
Molecular cloning and DNA sequencing HBV DNA in each plasma sample was liberated by digestion with proteinase K, and then extracted with phenol/ chloroform by the procedure described in detail elsewhere. ” An S-gene sequence of HBV DNA was amplified by polymerase chain reaction2*(PCR) by the method described previously with primer S 1 sequenced 5 ’-TCGTGTTACAGGCGGGGTTT-3’ (nt 38-57 in the S gene) and primer S2 with a sequence of S’-CGAACCACTGAACAAATGGC-3’ (nt 531-550 of the complementary strand).’* HBV DNA amplification products, after 25 cycles of PCR, were digested with XbaI and SpeI (Takara Biochemicals, Kyoto, Japan). Obtained XbaI-SpeI fragments of 437 base pairs (bp) were cloned into the XbaI site of MI3 mpl I phage vector, and the sequence spanning nt 93-529 in the S gene (437 bp) was determined by dideoxy chain termination method29using an M13 universal primer or a synthetic oligonucleotide primer with a sequence of 5’-CTGCGGCGTTTTATCAT-3’ (nt 229-245 in the S gene). Two clones were propagated from each of the 22 plasma samples and the sequences of XbuI-SpeI fragments were determined. The representative clone, without deletion or insertion within the sequence at issue, was adopted for comparison with each other and with the reported 23 HBV DNA clone^'^-^' and 2 unpublished ones.
RESULTS Subtypes of HBsAg in carriers from various islands of Indonesia The results of subtyping HBsAg in plasma samples from 144 carriers in Indonesia are summarized in Table 1. Of the four major subtypes of HBsAg, adw, adr and uyw accounted for the majority (139 (97%)); only 1 sample was of subtype ayr. There was an apparent geographic variation of HBsAg subtypes. Subtype udw was predominant in Java Island (35/ 45; 78%) and Kalimantan Island (8/8; 100%); adr was commonly seen in Sumatra Island (11/14; 79%), Sulawesi Island (16/26; 62%) and Irian Jaya (7/14; 50%); u p was prevalent in Flores Island (34/37; 92%). Subtypes of HBsAg samples from Irian Jaya were different from the other
Table 1 Subtypes of HBsAg in asymptomatic carriers in various islands of Indonesia Regular subtypes Atypical ayr subtypes
Island (city)
No. adw adr a p
Jawa (Jakarta) Sumatra (Pandang) Sulawesi (Manado) Irian Jaya (Jayapura) Flores (Bajawa) Kalimantan(Balikpapan) Total (YO)
4 5 3 5 8 2 0 0 1 4 2 1 1 1 0 0 2 6 6 1 6 4 0 0 1 4 1 7 1 1 4“ 3 7 3 0 3 4 0 0 8 8 0 0 0 0 144 55 42 42 1 4 (38%) (29%) (29%)(0.7O/0) (2.8%)
*Two samples were subtyped as ar and the remaining two as adwr.
islands in that they included a single uyr sample and four samples of atypical subtypes - that is, two each of ar and udwr.
Genotypes of HBV DNA clones from carriers in Indonesia Two clones were propagated from each of the 22 plasma samples from Indonesian carriers. They were divergent by 1-6 nucleotides within 437 bp covering a part of the S gene as the results of point mutations. A deletion of nt 336 of A was observed in one of the two clones from a Manado carrier, and nt 506 of G was missed in one of the two from an Irian Jaya carrier; the other clone from each carrier; without frameshift mutation, was selected to represent the HBV DNA clone in them. For the remaining 20 samples, one clone was selected randomly. XbaI-SpeI fragments of HBV DNA clones, representing nt 247-683 in the S gene (437 bp), from 27 carriers in Indonesia were compared with each other for the sequence homology (Table 2). Among them, five had been reported p r e v i ~ u s l y ’ ~ and ~ ’ ”the ~ ~remaining ~ ~ ~ ~ 22 were determined de novo. There were two different categories of divergences in the sequence, one ranging form 0.5 to 4.1% and the other from 5.5 to 8.9%; no differences in a range in between (4.25.5%) were observed between any two clones. Based on an intra-group difference of < 4.1% and inter-group difference of 2 5.4’/0, the 27 HBV DNA clones were classified into three genotypes. With the sequence homology to the four genotypes reported by Okamoto et ~ 1 . and ’ ~ another described by Vaudin et al.,” they were categorized into genotype B, C or D. The 12 clones of genotype B were of subtype either adw or uyw, and both clones of genotype D were of subtype uyw. Ten of the 13 clones of genotype C were of subtype udr; there was one each clone of subtype udw, ayr or ar. Some regional influences were seen on the genotypes of HBV DNA clones. All 4 clones from Flores Island were of genotype B, and all 5 clones from Irian Jaya Island were of genotype C. Clones from Jawa Island were heterogeneous in
HBV genotypes ip Indonesia
493
Table 2 Two-by-two analysis of an S-gene sequence of HBV DNA clones from carriers in various districts of Indonesia* Clone number
Clone no. geno/ subtype and origint 1
1* Jaw B adw 2* Jaw B adw 3* Sum B adw 4*Sul Badw 5 Sul Badw 6 Flo B adw 7 Kal Badw 8 SumB ayw 9 Sul Bayw 10 Flo Bayw 11 Flo Bayw 12 Flo B ayw 13* Jaw Cadw 14 Jaw C a d r 15 Jaw C a d r 16 SumC adr 17 SumCadr 18 Sul C a d r 19 Sul C a d r 20 Sul C a d r 21 Iri C adr 22 Iri C adr 23 Iri C adr 24 Iri C ayr 25 Iri C a r 26 Jaw Dayw 27 Jaw Dayw
2
3
4
5
0.5 0.9 0.9 0.5 0.5 0.5 1.4 1.4 1.4 0.9 0.5 0.5 0.7 0.2 1.6 1.4 1.1 1.1 1.1 1.1 1.1 0.7 0.9 0.9 0.9 0.5 1.4 1.4 1.4 0.9 0.9 0.9 0.9 0.5 1.1 1.1 1.1 0.7 6.6 6.6 6.9 6.4 6.9 6.9 6.6 6.6 6.6 6.4 6.4
6.6 6.4 6.4 6.2
6.4 6.2 6.2 6.4 6.2 6.2 6.2 5.9 5.9
5.9 5.9 5.9 5.9
6.4 6.4 6.2 6.2 6.2 5.9 5.9 5.5 6.2 7.2 7.6 7.8 7.6 8.0 8.2 7.8 7.8 8.0 6.4 7.6 6.4 6.4 7.4 6.2
6.2 5.9
5.7 7.2 7.8 7.6 6.4 6.2
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.1 2.1 1.4 1.60.9 1.8 1.4 0.7 1.6 0:7 1.8 1.1 2.1 1.1 0.9 1.4 0.7 1.6 0.7 0.5 1.6 0.9 1.8 0.9 0.7 6.2 6.4 7.6 7.1 6.9 6.4 6.6 6.9 7.3 7.1 6.2 6.4 7.1 7.1 6.9 5.9 6.2 6.4 6.9 6.6 5.9 6.2 6.9 6.9 6.6 5.7 5.9 6.6 6.6 6.4 5.7 5.9 6.6 6.6 6.4 5.9 6.2 6.9 6.9 6.6 5.7 5.9 6.6 6.6 6.4 5.7 5.7 6.6 6.4 6.2 7.1 7.2 8.5 8.0 7.8 7.6 7.8 8.9 8.0 7.8 7.3 7.6 8.7 8.2 8.0 6.4 6.4 6.9 6.6 6.4 6.2 6.2 6.6 6.4 6.2
0.9 1.1 0.7 7.3 6.9 7.6 7.1 7.3 6.9 7.1 6.6 7.1 6.6 6.9 6.4 6.9 6.4 7.1 6.6 6.9 6.4 6.6 6.2
8.2 7.8 8.2 7.8 8.5 8.0 6.9 6.4 6.6 6.2
7.1 7.3 2.7 7.1 2.3 1.4 6.9 2.1 1.1 0.7 6.9 2.1 1.1 0.7 6.6 1.8 0.9 0.5 6.6 2.1 1.1 0.7 6.9 2.1 1.1 0.7 6.6 1.8 0.9 0.5 6.6 3.4 3.9 3.4 8.0 1.8 3.2 2.7 8.0 2.1 3.7 3.2 8.2 1.6 3.9 3.4 6.6 6.2 7.3 7.1 6.4 6.4 7.1 6.9
0.5 0.2 0.5 0.5 0.2 3.2 2.5 3.0 3.2
0.2 0.5 0.5 0.2 3.2 2.5 3.0 3.2
0.2 0.2 0.0 3.0 2.3 2.7 3.0 6.9 6.9 6.6 6.6 6.6 6.4
0.5 0.2 3.2 2.5 3.0 3.2
0.2 3.2 2.5 3.0 3.2
-
3.2 3.9 4.1 6.9 6.9 6.6 7.1 6.6 6.6 6.4 6.9 3.0 2.3 2.7 3.0
2.3 3.0 7.3 7.8
3.4 7.3 7.3 7.6 7.8 1.6 -
*XXbaI-SpeI fragments of the S gene (nucleotides 93-529) of HBV DNA clones from 27 carriers in various districts of Indonesia were compared for sequence homology, and classified into genotypes by intra-group difference of C 4.1% and inter-group difference of 2 5.5%. Inter-group differences 2 5.5% are shown in bold face. tAbbreviations for islands in Indonesia - Flo: Flores; Iri: Irian Jaya; Jaw: Jawa; Kal: Kalimantan; Sul: Sulawesi; Sum: Sumatra. *Sequences reported previously - 1: pRTB299;” 2: pIDW420;I3 3: pPAD744;” 4: P M N D ~ ~13: ~ ;~~I W ’ K146.l~
l r i a n Jaya
D Figure 1 Map of Indonesian archipelago. Cities where serum samples were obtained are identified with numbers. Genotypes and subtypes of cloned HBV genomes are shown. Key: 1 = Padang: B (adw:l, ayw: l), C (adr:2); 2 = Jakarta: B (adw:2), C (adw:l, adr:2), D (ayw:2); 3 = Balikpapan: B (adw:l); 4 = Bajawa: B (adw: 1, ayw:3); 5 = Manado: B (adw:2, ayw:l), C (adr:3); 6 = Jayapura: C (adr:3, ayr:l, ar: 1).
R. Sastrosomignjo et al.
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genotypes, distributing in B, C and D. Likewise, clones from Sumatra and Sulawesi Islands included those of genotypes B and C. The single clone from Kalimantan Island was of genotype B. Fig. 1 shows topographical relationships of the six islands in Indonesia with the cities where serum samples were taken, along with various geno- and subtypes of HBV.
Amino acid substitutions specific to or characteristic of HBV genotypes Amino acids 32-176 (145 residues) of the S-gene product were deduced from XbaI-SpeI fragments of 47 HBV DNA
clones. Only 130 residues (amino acids 40-169), with at least 1 substitution among the 47 clones, are listed for comparison in Table 3. Within 145 residues in the S-gene product, 93 (64%) were conserved by 47 HBV DNA clones. Some diversions were sporadic; others appeared to be under controls. All the conversions were given rise to by point mutations. In Table 3, amino acid substitutions influencing the expression of d/y and w/r alleles are indicated by arrows; lysine or arginine at position 122 specifies d or y determinant, while that at position 160 does w or r determinant.’-” The correlation between genotypes and the w/r allele was obvious. The determinant r was observed in 18 (86%) of 21
Table 3 Amino acid sequences of a part of the translation product of the S gene of 47 HBV DNA clones of different genotypes Genolsubtypes a n d origin adw adw adw adw
USA USA PHI KEN
5t B adw €3 B adw 7f B adw 8t B adw 9t B adw 107 B adw 1 1 B adw 12 B adw 13 B adw 14 B ayw 15 B ayw 16 B ayw 17 B ayw 18 B ayw
JPN JPN INA INA INA INA INA INA INA INA INA INA INA INA
C adw C adw C adw C adr C adr 247 C adr 25t C adr 26f C adr
JPN JPN INA JPN JPN JPN JPN CHN
27 C adr
INA
58 C idr
INA INA INA INA INA INA INA INA INA JPN INA INA
1’ 2t 3t 4’
A A A A
l9f 20t 217 22T 237
29 30 31 32 33 34 35 36 37t 38 39
C adr C adr C adr C adr C adr C adr C adr C adr C avr C ayr C ar
40t 417 42t 437 44t 45 46
D ayw FRA D ayw URS D ayw JPN D ayw PNG D ayw KEN D ayw INA D ayw INA
47+ E adw GBR
XbaI-SpeI fragments corresponding to nucleotides 93-529 in the S gene of different HBV DNA clones were decoded and presented in single letter abbreviations of amino acid residues. Of 145 amino acids at positions from 32 to 176, only 130 (amino acids 40-169) are shown; sequences outside them did not diverge among 47 clones. The origins of clones are indicated by three-letter codes for nations adopted by International Olympic Committee: CHN, China; FRA, France; GBR, Great Britain; INA, Indonesia; JPN, Japan; KEN, Kenya; PHI, Philippines; PNG, Papua New Guinea; USA, United States of America; URS, Union of Soviet Socialist Republics. ; ~pKNDW60 ~ (unpubl.); 5 : pJDW233;” +Cloneshave been reported previously - 1: pHBV-3200;” 2: pHBV933;17 3: ~ F D w 2 9 4 4: 9: ~pAD744;’~ 10; pMND122;” 19: ~ S K 6 1 920: ; ~ pAK66;” ~ 21: pIWK146;I322: pHBV16: pODW282;13 7: P R T B ~ ~8:~ p:IDW420;” ;~’ 1;” 23: pHBr330;” 24: pBRHBadr4;16 25: pNDR260;I3 26: P A D R - ~ ; *40: ~ Eco HBV DNA;14 41: pHB320;” 42: pYWB796;13 43: ~ P Y w 3 1 0 ; 44: ’ ~ pKNYW149 (unpubl.); 47: ~ ~ w - L S H . ‘ ~ Amino acid residues- A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine;G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine. Arrows indicate the positions of amino acids 122 and 160, lysine or arginine at which specifies d or y and w or r subtypic determinant respectively.
HBVgenotypes in Indonesia
495
HBV DNA clones of genotype C, whereas all 26 clones of genotype A, B, D or E expressed w. With a less close correlation, the determinant y was expressed by all 7 clones of genotype D, while d was found in all clones of genotype A or E, as well as in 9 of 16 (56%) clones of genotype B, and predominant in genotype C (18/21; 86%). The clone no. 39 of genotype C did not express d despite iysine at position 122, because it possessed Ala145 in place of G1~145.~' Aside from those specifying subtypic determinants, there were at least 18 amino acids, within 145 residues spanning positions 32-176, the expression of which was apparently allelic, and characteristic to certain genotypes. In addition, there were 3 amino acids which were under influence of a certain genotype, but were not necessarily restricted to it. Correlation of amino acid conversions with HBV genotypes are summarized in Table 4.
Table 4 Amino acid changes in the S-gene product characteristic to or influenced by HBV genotypes No. clones with amino acids in allele Position and members of allele Characteristic Genotype A 114:ThdSer 131:AsdThr Genotype B 56:GldPro 57: Ile/Thr 59: Ser/Asn 64 CydSer 85:CyslPhe Genotype C 47 :ThrNal 49: Pro/Leu 110:Leu/Ile 113:Thr/Ser 126:IlelThr Genotype D 46 :ThdPro 68:ThdIle 134:Tyr/Phe 159:Gly/Ala 168:AldVal Genotype E 117 :Thr/Ser Influenced 53:SerlLeu 122:LydArg 16O:LydArg
A
B
C
D
E
4/0 4/0
0/14 0/14
1/20 0121
0/7 0/7
0/1 011
0/4 0/4 0/4 014 0/4
14/0 13/0* 14/0 14/0 14/0
0/2l 0/2l 0121 0/21 0121
0/7 0/7 0/7 0/7 0/7
0/1 0/1 0/1 0/1 0/1
0/4 0/4 0/4 0/4 0/4
0114 19/lt 0/14 20/1 0/14 21/0 0/14 21/0 0114 20/0*
0/7 0/7 0/7 0/7 0/7
0/1 011 011 011 0/1
0/4 0/4 0/4 0/4 0/4
0/14 0/14 0/12$ 0/14 0/14
0/21 0/21 0/21 0/21 0121
7/0 7/0 6/1 7/0 7/0
0/1 0/0' 0/1 011
014
0/14
0/21
017
110
4/0 4/0 410
14/0 9/5 14/0
13/8 19/2 3/18
7/0 0/7 710
110 1/0 110
*Vals7in clone no. 16. tAla47in clone no. 39. *Amlz6in clone no. 27. ISer,,, in clone no. 13 and Ile13, in clone no. 14. *Ile,34in clone no. 47.
011
DISCUSSION Indonesia, an archipelago spanning some 2 million square kilometres in Southeast Asia, has a high endemicity for HBV. Carrier rates among voluntary blood donors range from 2.1 to 9.5% in 11 big cities; it is even higher at 17.5% in Jayapura City in Irian Jaya Island. The anthropological origin of Indonesians, who would have arrived at the islands by sea, is not established yet, nor is it known how early the ancestors migrated there. Based on cultural backgrounds, strong influences of people from South China, India, Mediterranean and European countries including Netherlands and Portugal, are conceived. There is evidence to indicate that South Chinese migrated to Indonesia for trading, Indians and Mediterraneans through religious spreading, and Portuguese and Netherlanders with political or religious aims. Subtypes and genotypes of HBV may be helpful in research along these lines, as has been useful in investigating the migration of Japanese ancestors. Because the Indonesian islands are separated by sea, hindering free surface migration of inhabitants among them, HBV strains of distinct subtypes andor genotypes would likely have been maintained in each individual island. Surrounding Indonesia, the subtype adr is prevalent in South China as well as in mainland China, adw and ayw in India, and ayw in Mediterranean countries, while adw is common in the Netherlands, and ayw in PortugaL8 The fact that adw is predominant in Jawa, adr in Sulawesi, and ayw in Flores (Fig. 1, Table 4) possibly reflects the influences of these countries. In view of d/y and w/r subtypic changes that are ascribable to single point mutations in HBV DNA,"-'* however, the subtype distribution is not readily taken as a clue in pursuing the migration of ancestors to the Indonesian Islands. Although the predominance of adw in Java and that of adr in the other islands imply the influence of surrounding countries, the analysis of nucleotide sequences favours the view that the Indonesian HBV strains of these two subtypes would be indigenous and that the different subtypes would have arisen by point mutations. On the basis of sequence analyses, the HBV genomes of subtype ayw in Flores Island would most likely be the result of mutation from those of subtype udw. HBsAg/up in this situation, therefore, would not reflect a foreign influence from Portugal. Similarly, in North Lombok, near Flores Island, the predominant ayw subtype might have been given .~' analysis rise to by a point mutation in H B V J ~ ~ WSequence of the uyw strain in North Lombok, for comparison with udw and ayw strains in Flores Island, would be required to substantiate this view, however. HBV DNA clones can be classified into five genotypes by differences in the entire nucleotide sequence of 8% or greater, including genotypes A-D proposed by Okamoto et ~ 1 . and ' ~ genotype E represented by a single HBV genome isolated by Vaudin et al.*' from a naturally infected chimpanzee. Nucleotide divergence distributes so evenly on the whole genome that the genotyping is feasible by sequencing only a few hundred nucleotides of HBV DNA.13 HBV replicates by the reverse transcription of an RNA
R . Sastrosoewignjo et al.
496
intermediate.32 This process involves errors, because it is not controlled by proofreading enzymes.33y34Some mutations are associated with phenotypic changes. For example, subtypic changes occur as the result of a single point mutation which converts a single amino acid residue.'-12 Assuming the rate of nucleotide substitution at 1.4-3.2 x lO-'/site per year,23evolution of HBV genomes into different types would have started at least 3000 years ago. Recently, Orito et al. attempted genetic classification by a similar approach, and classified the reported 12 HBV genomes into four c a t e g o r i e ~They . ~ ~ found that sequence divergences among HBV genomes far exceeds those between HBV and animal or avian hepadnaviruses, and estimated the evolution of viruses of different host ranges at 5000 years ago or earlier. By means of PCR, S-gene sequences of 22 HBV DNA clones were amplified to determine the sequence of XbaISpeI fragment. Amplification of DNA sequences by PCR with the Taq polymerase reaction involves potential misincorporation of nucleotides. Saiki et al. estimated the frequency of overall error, during amplification for 30 cycles, at o.25°/~.2sThis ratio, applied to an S-gene sequence of 437 nucleotides (XbaI-SpeI fragment) multiplied for 25 cycles, would account for < 1 error, far less than the > 24 (6.4%) divergent nucleotides which are required to create intergroup differences in the present study. Misincorporation in amplification by PCR, therefore, would not seriously affect our genotyping criteria. The 27 HBV DNA clones from Indonesian carriers, including 22 determined in the present study and 5 reported previ~usly,'~~ were ' ~ ~classified ~ ' ~ ~ ~ into genotype B, C or D (Table 2). All 4 clones from Flores Island were of genotype B, and all 5 clones from Irian Jaya Island were of genotype C; HBV genomes of a particular genotype appear to have been maintained in these islands. In contrast, 7 HBV clones from Java Island were heterogeneous, including genotypes B, C and D; 11 clones from the other three islands were of genotype either B or C (Fig. 1). With the limitation of the small number of clones dealt with in the present study, these results appear to indicate that Java Island would have been the centre of transportation, attracting people from the other islands. We then compared amino acid sequences, within the range of XbaI-SpeI fragment carrying 145 codons, among 47 clones including 23 previously r e ~ o r t e d ' ~and - ~ ~2 unpublished ones. A number of amino acid substitutions characteristic of HBV genotypes emerged, all of which were ascribable to point mutations. Obviously enough, the great majority of amino acids (93 of 145; 64%) were preserved by HBV DNA clones of different genotypes. Although mutations may occur evenly in various sites of HBV DNA sequence, some selective force would have to be operating for the survival of mutants with point mutations at restricted nucleotides in the S gene. Amino acid substitutions responsible for subtypic changes are partly associated with HBV genotypes. For instance, Arglz2for the determinant y was observed in all clones of genotype D and also in some clones of genotypes B and C, while LysIZ2for d was seen in all clones of genotype A or E,
and also in the majority of clones of genotype B or C. Arg,,, for the determinant r was restricted to genotype C, but not a hallmark for it because it was not possessed by 3 of 21 clones. The influence of an amino acid at a position away from Lysl22 on the expression of d determinant was seen in one of the clones subtyped as ar by the immunological method. This was clone no.39 of genotype C, which had LyslZ2 characteristic of the determinant d. HBsAg particles in the plasma, from which it was propagated, did not express the determinant d , however. The clone had AlaI45,unlike all the other 46 clones (with either determinant d or y), which possessed Glyl4~.These results confirmed our previous proposal that the expression of d would be under the influence of amino acid 144 or 145 in the S-gene product.30 Ohnuma et al. reported that the microconformation maintained by the -Cys12l-Cys124- bond would constitute a microconformation for a common antigenic determinant of HBsAg detectable by monoclonal antibody (no. 5 124).36 This disulfide bond was expressed by 46 of 47 HBV DNA clones. A single clone, no. 18, possessed Argi2, and, therefore, the conformation would not be built in the HBsAg it encodes. They reported Ile126to be involved in an HBsAg determinant specific for genotype C, which is detectable by another monoclonal antibody (no. 4403). Their view was extended to 20 of 21 clones of genotype C, with the single exception of clone no. 27, which possessed ASp126, unlike the other 46 clones of any genotypes. HBV genotypes, with characteristic amino acid substitutions in the S-gene product, would find applications in virological and epidemiological research. Cloning and sequencing HBV DNA, however, are not feasible for genotyping on a large scale. Recently, Norder et al. reported genotypeassociated differences in the efficiency of oligonucleotide primers for amplifying HBV DNA by PCR.-17Their observations suggest that genotyping might be performed by evaluating the compatibility of HBV DNA templates with a few sets of primers in amplification by PCR. It is possible, also, that nucleotide substitutions intrinsic to genotypes may create specific restriction cleavage sites or distinct antigenic epitopes on the S-gene product. Should such become the reality, genotyping would be achieved by comparing restriction patterns with a few endonucleases or by immunological tests.
ACKNOWLEDGEMENTS We thank the following doctors for valuable materials, enlightenment, discussion and constructive criticism toward the completion of this work: M. Rustam of the Indonesian Red Cross Blood Center, Sujudi of University of Indonesia, S. Gunawan of the Ministry of Health, Indonesia; D. L. Peterson of Virginia Commonwealth University, USA; H. Suzuki of Yamanashi Medical College, M. Homma of Kobe University, Y. Miyakawa of Mita Institute, T. Yamanaka, S. Yotsumoto, and M. Mayumi of Jichi Medical School, Japan. This work was supported in part by the Japan Society for the Promotion of Science.
HBV genotypes in Indonesia
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