Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India

Journal of Medical Virology 78:1164–1174 (2006)

Phylogenetic Relatedness and Genetic Diversityof Hepatitis B Virus Isolates in Eastern India

Arup Banerjee,1 Fuat Kurbanov,2 Sibnarayan Datta,1 Partha Kumar Chandra,1

Yasuhito Tanaka,2 Masashi Mizokami,2 and Runu Chakravarty1*1ICMR Virus Unit, ID & BG Hospital Campus, Kolkata, West Bengal, India2Department of Clinical Molecular Informative Medicine and Department of Internal Medicineand Molecular Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Hepatitis B virus (HBV) has been classified intoeight genotypes, and several subgenotypes,distinctly distributed geographically. The geno-types A and D were previously reported to bepredominant in India. Recent studies indicatedevidence of circulation of genotype C in Easternpart of India. With the aim to confirm thephylogenetic relation and molecular geneticcharacteristics of the HBV circulating in Kolkata,themost populous city in Eastern India, 11 strainswere isolated and the complete genomesequences were analyzed. Phylogenetic analysisdetermined; three genotype C (adr-serotype)isolates closely related with C1 (Cs) subgenotypereferences from South East Asia, and threegenotype A (adw2-serotype) isolates, related toAsia-variant references of subgenotype A1 (Aa).Whereas, five genotype D (ayw2, ayw3 serotype)isolateswerehighly divergent; onewas related tosubgenotypeD1, two to subgenotypeD3, and theremaining twoclusteredwith a singlegenotypeDisolate from Japan belonging to an unclassifiedsubgenotype. Together, these two isolates dif-fered from HBV D1–D4 subgenotypes by nucleo-tide differences ranging from 5.0 to 5.49%,probably indicating a new subgenotype, whichwe designate as D5. All serotype ayw3 ofgenotype D isolates had specific amino acidsubstitution Threonine at codon 118 andMethio-nine at codon 125 in antigenic determinant ofsurface gene that has not been reported pre-viously in isolates from other parts of India. Inconclusion; using the completegenomeanalysesthis study has confirmed circulation of thegenotype C in Eastern part of India and demon-strated considerable genotypic heterogeneity ofthe IndiangenotypeD. J.Med.Virol. 78:1164–1174, 2006. � 2006 Wiley-Liss, Inc.

KEY WORDS: HBV Eastern India; HBV geno-type; HBV subgenotype; geno-type C; subgenotype D5

INTRODUCTION

Hepatitis B virus (HBV) has been classified into nineserological types, adw2, adw4, adr, adrq-, ayw1, ayw2,ayw3, ayw4, and ayr that were distinguished by a singleamino acid change at positions 122 (d or y) and 160 (r orw) in the small surface protein [Courouce-Pauty et al.,1983]. Later HBV has been classified into genotypesbased on HBV genome nucleotide sequence divergenceexceeding 8% [Okamoto et al., 1988]. So far eightgenotypes (A to H) of HBV with distinct geographicaldistribution have been reported [Okamoto et al., 1988;Norder et al., 1994; Stuyver et al., 2000; Arauz-Ruizet al., 2002]. A number of studies have confirmed thatthe genotypic heterogeneity of HBV is associated withdiverse clinical outcomes of HBV infection [Kao et al.,2000; Orito et al., 2001; Akuta et al., 2003; Sugauchiet al., 2003; Tanaka et al., 2004].

Furthermore, recent studies demonstrated the pre-sence of several sub-genotypes within widely spreadgenotypes. Genotype A is further sub-classified intoAfro-Asian A1 (Aa) predominant in Asia and Africancountries, A2 (Ae) prevalent mainly in European-NorthAmerican countries [Bowyer et al., 1997; Kramvis et al.,2002; Tanaka et al., 2004; Sugauchi et al., 2004a; Kewet al., 2005] and more recently, A3 (Ac) [Mulders et al.,2004; Kurbanov et al., 2005] described in Cameroon,Central Africa. Genotype B is sub-classified into sub-genotypes differing in their geographical distribution,with B1 (Bj) dominating in Japan, B2 (Ba) in China andVietnam, B3 found in Indonesia, and B4 confined to

Grant sponsor: Indian Council of Medical Research (ICMR),NewDelhi; Grant sponsor: University Grants Commission (UGC).

*Correspondence to: Dr. Runu Chakravarty, ICMR Virus Unit,Kolkata, ID & BG Hospital Campus, GB-4, 1st Floor (East Wing),57, Dr. Suresh Chandra Banerjee Road, P.O. Beliaghata, Pin—700 010, Kolkata, West Bengal, India.E-mail: [email protected].

Accepted 26 April 2006

DOI 10.1002/jmv.20677

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2006 WILEY-LISS, INC.

Vietnam [Sugauchi et al., 2002b, 2003, 2004b; Norderet al., 2004]. Sub-genotype of genotype C, C1 (Cs)is common in South-East Asia, and Bangladesh, C2(Ce) in Japan, Korea, and China [Huy et al., 2004; Chanet al., 2005]; and C3 in Oceania comprising strainsspecifying adrq-, and C4 specifying ayw3 is encounteredin Aborigines from Australia [Sugauchi et al., 2001;Norder et al., 2004]. This pattern of definedgeographicaldistribution was less evident for genotype D subgeno-types, recently designated as D1–D4 [Norder et al.,2004].

Many studies have shown a relation between HBVgenotypes and mutations in the precore and basal corepromoter (BCP) region that affect production of HBeAg,which is associated with viral replication. Among them,the A1896 mutation in the precore region is restrictedto HBV isolates that have T at nt 1858, common ingenotypesB,C, andD.Whereas, the double substitutionat positions 1762–1764 has been described to occurpreferentially in isolates having C instead of T at nt1858 frequently found in genotype A and F and C also[Alestig et al., 2001].

India is a vast country with ethnically and linguisti-cally diverse population. Several waves of migrationshave influenced thegenetic diversity of thepopulation indifferent periods of time [Majumder, 1998]. HepatitisB is endemic in Indiawith an estimated nationalHBsAgcarrier rate of 4%, it represents a major etiology ofchronic liver diseases as reported from different parts ofthe country. Serological studies showed that subtypeadw and ayw are circulating in Southern, Northern,and Western India [Thyagarajan et al., 1979, 1996;Courouce-Pauty et al., 1983; Shanmugham, 1990].However, genetic analyses of the indigenous IndianHBV strains circulating in the subcontinent, the secondlargest pool of chronic HBV infection in the world, arelimited [Sarin, 1999].

The present study is the first report of the entiregenome sequence analysis for the genotype C strainsfound in Eastern India. The strains were isolated fromchronic hepatitis B carriers residing in Kolkata, themost populous city in Eastern India. In addition, weanalyzed complete genome of genotypes A andD strainspreviously typed by RFLP.

MATERIALS AND METHODS

Source of HBV DNA

A total of 11 HBsAg positive serum samples used inthis study, were obtained from chronic carriers hospi-talized in different units of Kolkata, coming to our Unitfor HBV DNA detection before receiving the antiviraltreatment. All of them were ethnic Bengali, residents ofKolkata.

Serological Markers and GenotypeDetermination

The HBsAg, HBeAg, and anti-HBe, were examinedusing commercial diagnostic kits (Biomerieux, Boxtel,

The Netherlands). Hepatitis B virus genotypes weredetermined by RFLP [Banerjee et al., 2005].

HBV DNA Extraction

Serum samples were stored at �808C until assay.DNA was extracted from 100 ml of serum by usingQIAamp DNA Blood Mini Kit (Qiagen GmbH,Hilden, Germany) in accordance with manufacture’sinstructions.

HBV DNA Amplification and Quantitation

Eleven complete genomes of HBV were sequencedbyusing theprimers reportedpreviously [Sugauchi etal.,2001]. Briefly, the complete genome of HBV for eachsample was amplified as two overlapping fragments AandB.For fragmentA (nt 18–1803) andB (nt 1611–256),nested PCR was performed to amplify eight overlappingfragments by using the internal primers.

HBV DNA was quantified using ABI 7000 sequencedetection system (Applied Biosystems, Foster City, CA)allowing detection up to 100 viral DNA copies/reaction[Abe et al., 1999].

Complete Genome Sequencing

Two overlapping HBV DNA fragments covering theentire genome sequence were amplified using specificprimers and PCR conditions described previously[Sugauchi et al., 2001]. Amplified HBV DNA fragmentswere sequenced directly using a Prism Big Dye v3.0 kit(Applied Biosystems) on the ABI 3100 DNA automatedsequencer (Applied Biosystems). All sequences wereanalyzed in both, forward and reverse directions.The nucleotide sequence data reported in this articlesubmitted to the DDBJ/EMBL/GenBank, accessionnumbers DQ315776–DQ315786.

Sequence Analyses

HBV genomeswere assembled usingGENETYX v12.0(SoftwareDevelopmentCo.,Tokyo, Japan),alignedusingCLUSTALXv1.83 [Thompsonetal., 1997], sequencepairwise distances (percent nucleotide divergence overcomplete genome sequences) were estimated usingClustal method implemented in MEGALIGN software[Clewley andArnold, 1997]. Complete genome sequenceswere examined for presence of intergenotypic recombina-tion as described previously [Robertson et al., 1995] withusing bootscan analysis implemented in SimPlot soft-wareprogram[Lole et al., 1999] andconfirmedmanually.The conventionalEcoRI positionwas taken as nucleotideposition 1.

Phylogenetic Analysis

Reference sequences were retrieved from DDBJ/GeneBank database. Alignments were done usingthe CLUSTAL W method [Thompson et al., 1997], andphylogenetic Neighbor Joining tree (NJ)was constructedwith genetic distances corrected by 6-parameter

J. Med. Virol. DOI 10.1002/jmv

HBV Full Genome Analysis in Eastern India 1165

method [Gojobori et al., 1982], bootstrap resampling wasperformed 1,000 times.Subgenotypes were assigned as described previously;

A1 (Aa), A2 (Ae), A3 (Ac) [Bowyer et al., 1997; Kramviset al., 2002; Tanaka et al., 2004; Sugauchi et al., 2004aKewet al., 2005],C1 (Cs),C2 (Ce) [Huyet al., 2004;Chanet al., 2005], C3, C4 and D1–D4 [Norder et al., 2004].Recombination was investigated using SimPlot [Loleet al., 1999].

RESULTS

HBsAg Serotypes and Genotypes of HBV

A total of 11 HBsAg positive serum samples used inthis studywere obtained from chronic carriers (10malesand 1 female, mean age 34.0 years, range 8–64 years) inEastern India (EI). Table I summarized the details ofserological, clinical status, subtypes, and genotypes.The subtypes were deduced from the sequences. Threeadr subtype genotype C strains were isolated from thespecimens of the patients one of whom was HBeAg-negative (EI0423) and the other two were HBeAg-positive (EI0398 & EI03188). In addition, three-adw2genotypes A (EI0386, EI03101, EI057), and five geno-type D, of these three (EI00615, EI0399, EI02456) withayw3 and other two (EI03194, EI0388) with ayw2strains, were analyzed.

Genetic Distances and Phylogenetic Analysis

Eastern IndianHBV/C. PreliminaryphylogeneticNJ tree was calculated using complete genomesequences isolated in this study along with 600 reportedsequences (with exclusion of the most related ones)retrieved from DDBJ/EMBL/GenBank. A total of 46HBV references, including all known human HBVgenotypes; 18 subgenotype C1 (Cs), 12 subgenotype C2(Ce), and 2 each from subgenotypes C3 and C4 wereselected for the final tree (Fig. 1). Three Indian HBV/Cstrains obtained in this study were grouped togetherwith previously reported genotype C strains, within C1(Cs) cluster, along with Thailand andMyanmar isolateswith 100%bootstrap index distinctly fromC2 (Ce). All ofthe Indian strains were closely related among eachother, with 98.7–100% sequence homology, indicatingcirculation of homologous viral strain in the populationstudied.The nucleotide sequence divergence throughout the

complete genome among Eastern Indian strains andthose corresponding to C1 (Cs) and C2 (Ce) from othergeographic destinations consisted of 2.01� 0.89% and4.42�0.38%, respectively (Table II). The intra-geno-type divergence was 1.8%. Furthermore, the threestrains were shown to be most closely related in termsof evolutionary distance (0.0105, 0.0129, and 0.0119substitutions per position, respectively) to a strainisolated in Thailand (accession no. AF068756). Phylo-genetic relation among the strains was also confirmedusing four different ORFs and showed the resultssimilar to that of the whole genome. Simplot analysis


TABLE

I.Dem

ographic,Virolog

icalCharacteristics

ofPatien

tsIn

fected

WithHBV

Gen

otypeA,C,andD

inEasternIn

dia

Sample

no.

Age

Sex

(M/F)

HbeA

g/

anti-H

Be

Clinical

status

ALT

(IU/L)

Log

10

copies/ml

Gen

ome

length

(bp)

Subtype/

subgen

otype

Nucleo

tide(nt)

substitution

inbasa

lcore

promoter

(BCP)andprecore

region

1,762/

1,764

1,809/

1,812

1,858

1,862

1,896

1,899

EI0386

28

M�/þ

HCC

34

3.722

3197

adw2/A1(A

a)

T/A

T/T

CT

GA

EI03101

30

M�/þ

HCC

130

3.58

3218

adw2/A1(A

a)

T/A

T/T

CT

GG

EI057

55

Mþ/�

LC

53

3.255

3218

adw2/A1(A

a)

T/A

T/T

CG

GG

EI0423

22

F�/þ

CLD

78

5.563

3203

adr/C1(C

s)T/A

G/C

TG

GG

EI0398

50

Mþ/�

CLD

87

6.352

3212

adr/C1(C

s)T/A

G/C

TG

GG

EI03188

50

Mþ/�

CLD

50

5.639

3212

adr/C1(C

s)T/A

G/C

TG

GG

EI00615

64

M�/þ

ASC

47

7.15

3182

ayw3/D

3A/G

G/C

TG

GG

EI03194

23

Mþ/�

CLD

36

4.188

3095

ayw2/D

3A/G

G/C

TG

GA

EI0388

21

M�/þ

CLD

119

4.2

3212

ayw2/D

1A/G

G/C

TG

GA

EI02456

23

Mþ/�

ASC

52

6.48

3119

ayw3/D

5T/A

G/C

TG

GG

EI0399

8M

�/þ

CLD

122

6.646

3182

ayw3/D

5A/G

G/C

TG

GG

EIrepresentEasternIn

dia

follow

edbysa

mple

no.

ASC,asymptomaticcarrier;CLD,ch

ronic

liver

disea

se;LC,liver

cirrhosis;HCC,hep

atocelluarcarcinom

a.

1166 Banerjee et al.

revealed no signs of recombination in any sequence(data not shown).

Eastern Indian HBV/D and HBV/A. The phylo-genetic relation of three Indian HBV/A and five HBV/Disolates from this study with those previously reported50 selected reference strains of different genotype fromDDBJ/EMBL/Genbank (including 29 of HBV/D and 11ofHBV/A isolates) was demonstrated in the separateNJ

tree (Fig. 2). One of our HBV/D strains (EI0388)was grouped together with D1 references and two(EI00615, EI03194) were clustered in D3 clade (Fig. 2).The remaining two strains (EI02456 and EI0399)distinctly clustered together with a published isolatefrom Japanese blood donor (accession no. AB033558),whichwas outlying fromall designatedD subgenotypes.The bootstrap index was 100%, and the evolutionary


Fig. 1. Neighbor joining (NJ) phylogenetic tree was constructedbased on the complete genome sequences of 46 HBV reference-strainsfrom DDBJ/EMBL/GenBank; including 18 and 12 sequences from thetwo major groups of the subgenotype C1 (Cs) and C2 (Ce), respectivelyand two each from HBV subgenotype C3 and C4. The bootstrap valuesobtained from 1,000 replicas are given at the internal nodes. HBV

strain from Eastern India (EI0423, EI0398, EI03188) marked by *wassequenced in the present study. The capital letters A to H designateHBV genotypes. The sequences of four subgenotypes of genotype Cwere denoted by C1 (Cs), C2 (Ce), C3, C4. Reference sequences aredenoted by their accessionnumber and the strain isolated fromEasternIndia are indicated by EI followed by isolates number.


distance among these strains was 0.02147–0.02315substitutions per site. The nucleotide differences of theoutlying cluster and those of subgenotypes D1, D2, D3,and D4 was ranging from 5.0 to 5.49% (Table II). Thephylogenetic relation among the strains was alsoconfirmed using four different ORFs and showed theresults similar to that of the whole genome. Simplotanalysis revealed no signs of recombination in anysequence (data not shown).Three of the genotype A strains reported here

clustered with Asian isolates referring to A1 (Aa)subgenotype distinctly from African strains as shownin Figure 2.

Sequence Analysis of 4 DifferentORFs of 11 Complete Genome

The genomes of two of three Indian HBV/C strains(EI0398 and EI03188) were 3,212 nucleotides (nt) long,and the third one (EI0423) was 3,203 nt, due to the 9 ntdeletion in the pre-S2 region (49–57 nt) (Table I). Allthe genotype C samples studied had T1762/A1764double mutation in the BCP region. None of them hadA1896 precore region mutation, although they hadT1858. Amino acid analysis of genotypeC samples alongthe differentORF showed that Core andPre-S/S regionswere highly conserved, however, a short deletion ofthree amino acids Tyr- Phe-, Pro (YFP) at codon 20–22was observed inPre-S2 region of the strain isolated fromHBeAg-negative carrier (EI0423).When our genotypeCstrains were compared to the subgenotypes’ referencestrains, we found amino acid homology with C1 (Cs)sequences as shown in Table III, which is consistentwith phylogenetic relatedness of Eastern Indian strainswith C1 (Cs).Genotype D isolates were found to be variable in the

genome sequence length (Table I) having variousinsertions and deletion in the S gene; 30 nt-insertion(EI0388) within nt 2,878–2,907 resulting addition of 10aminoacids betweencodon15and codon16of thePre-S1region, deletion of 87nt from2,996 to 3,082nt (EI03194),deletion of 17 codon at the start of Pre-S2 including thestart codon of the Pre-S2 (EI02456), both insertion anddeletion in Pre-S region (EI0399). However, in all thiscases, open reading frames were not affected. Presenceof insertion anddeletions amonggenotypeDstrainswas

not associated with HBeAg status. In the BCP region,the double mutation A to T at 1762 and G to A at 1764(T1762/A1764) was found in only one (EI02456) of thegenotype D strains. All of these samples had Gat position 1896 (wild-type) in the precore regionirrespective of the HBeAg status. However, 1899 G toA (Asp29) mutation in precore region was found intwo strains (EI03194, EI0388). All Eastern Indiangenotype D strains had the T1858 and G1862 corre-sponding to the HBV pregenomic encapsidation signalsequence (Table I). Both the EI02456 and EI0399isolates showed some unique amino acid substitution(in Pre-S1 A/N27R, S85A, in Pre-S2 T11A, R48K, L54P,in P gene S78T, K90N/Y, and in X region F30V, T36S,P40S, V92I) in the entire genome, which was similar tothe sequence from the only phylogeneticaly relatedreference sequence (Japanese blood donor isolateAB033558) (Table IV). These amino acid substitutionswere not found among other genotype D isolates in thepresent study.Mutations at codon S43L in Pre-S region,at codon V119F, R208Q, GI235-236RV, and CY591-592SW in P gene were also present among genotype D4sequences. T125Mmutation in S gene region was foundin three Eastern Indian strains (EI02456, EI0399, andEI00615) which were also common among subgenotypeD3 and in sequences from Japan (AB033558) (Table IV).

In contrast to the genotype C and D, the genotypeA (adw2) strains in this study had conserved sequencesthroughout the genome.One of the strains fromHBeAg-negative carrier (EI0386, 3197 nt) had 21-nucleotidedeletion at the start of the Pre-S1 region similar to thatof the Nepalese isolate (retrieved from DDBJ/EMBL/GenBank AB116090), while the remaining two strains(EI03101, EI057) had genomic length 3,218 nt. All hadT1762/A1764 mutation in the BCP region. In addition,T1809, T1812 was found in all of the samples, whichwere the characteristics of Asian genotype A strains.Except EI057, other two had T at 1862 position inprecore region (Table I). The amino acid motifs wereconserved in all the three genotype A sequences.

DISCUSSION

Hepatitis B virus genotypesA andDwere known to bepredominant in India [Thakur et al., 2002; Arankalleet al., 2003; Gandhe et al., 2003; Sugauchi et al., 2004a].


TABLE II. Genetic Distance Between Sub-Genotype C and D With Eastern Indian Samples

Eastern Indian sample

Genotype C Genotype D

Sub-genotype EI0398, EI03188, EI0423 Sub-genotype EI0388 EI00615, EI03194 EI02456, EI0399

C1 (Cs) 2.01� 0.89 D1 2.63� 0.35 3.22� 0.31 5.19� 0.25C2 (Ce) 4.42� 0.38 D2 3.20� 0.14 3.38� 0.52 5.14� 0.24C3 6.1� 0.86 D3 3.49� 0.33 1.63� 0.71 5.09� 0.25C4 8.36� 1.1 D4 4.38� 0.48 3.86� 0.50 5.45� 0.58

D5 4.971 4.82� 0.05 2.38� 0.04


However, a recent report published fromSouthern Indiaindicated that theHBVgenotypeCwas foundamong thepatients from Eastern India [Vivekanandan et al.,2004]. However, no sequence information of the HBV/C strains was available. In our previous study [Banerjee

et al., 2005], using the restriction fragment lengthpolymorphism method (RFLP) and limited partial S,PreCore/Core sequencing, we also found the genotype Cstrains in Eastern India in addition to the predominantgenotypes D and A. Thus, the presence of genotype C


Fig. 2. A phylogenetic tree constructed using NJ method, based oncomplete genome sequences of 58 HBV isolates (8 from the presentstudymarked by *, 29 of different subgenotypes of HBV/D as describedby Norder et al. [2004] along with 21 HBV isolates of other differentHBV genotypes) retrieved from the DDBJ/EMBL/GenBank. Thenumber represents the percentages of bootstrap replicates (of 1,000

total) for the node. The capital letters A toH designateHBV genotypes.The sequences of subgenotypes of genotypeDwere denoted byD1 toD4and a new subgenotype D5 found in the present study. Referencesequences are denoted by their accession number and the strainisolated from Eastern India are indicated by EI followed by isolatesnumber.



TABLE

III.

Com

parisonof

AminoAcidsin

thePre-S1/S2/S

andPolymerase

(P)Gen

e,Betwee

nSub-G

enotypes

CWithEasternIn

dianSamples

Sub-gen

otypeC/cou

ntry

Sgen

e(cod

onnumber)

Pgen

e(cod

onnumber)

Pre-S1

Pre-S2

Primingregion

Spacer

RT

51

54

60

62

73

125

227

136

143

232

234

237

249

252

304

354

358

C1

(Cs)

(con

sensu

s)/Sou

thEast

Asia

QA

VS

S/N

SL

T/I

QM

SP

SQ

QY

H

EI0398/EasternIn

dia

——

——

——

—I

——

——

——

——

—EI03188/EasternIn

dia

——

——

——

——

——

——

——

——

—EI0423/EasternIn

dia

——

——

N—

—I

——

——

——

——

—C2(C

e)(con

sensu

s)/

East

Asia

HE

AA

GT

SA

KL

RS

PR

HH

N

C3(con

sensu

s)/Polynesia

—E

AA

GT

SA

K—

RS

PR

HH

NC4

(con

sensu

s)/Australian

Aborigines

HE

AA

GT

SA

KL

RS

P—

H—

—

TABLE

IV.Com

parisonof

AminoAcidsin

thePre-S1/S2/S

Gen

e,Polymerase

(P)Gen

e,andX

Gen

eBetwee

nSub-G

enotypes

DWithEasternIn

dianSamples

Sub-gen

otypes

D

Envelop

eprotein

(cod

onnumber)

Polymerase

gen

e(cod

onnumber)

Xgen

ePre-S1

Pre-S2

SPrimingregion

Spacer

RT

27

85

103

11

43

48

54

118

122

125

127

160

78

90

119

178

208

235–36

458

591–92

30

36

40

91

D1(con

sensu

s)A

SN

TS

RL

TR

TP

KS

KV

ER

GI

NCY

FT

PV

EI0388EasternIn

dia

——

——

——

——

——

——

T—

——

——

—S-

——

——

D2(con

sensu

s)—

—N/D

——

——

V—

—T

——

—V/D

KS

——

——

——

—D3(con

sensu

s)—

——

——

——

——

T/M

P/T

——

——

D—

-F—

-HL

——

—EI03194EasternIn

dia

——

D—

——

——

——

——

T—

——

—G-

——

——

——

EI00615EasternIn

dia

——

——

——

——

—M

T—

——

—D

—-F

——

——

——

D4(con

sensu

s)N

—T

—L

——

——

——

——

—F

—Q

RV

—SW

—A

——

AB033558(D

5)

RA

DA

LK

P—

—M

T—

TN

FK

QRV

DSW

VS

SI

EI0399(D

5)EasternIn

dia

RA

DA

LK

P—

—M

T—

TY

VK

QGV

DSW

VS

SI

EI02456(D

5)Eastern

India

RA

D////

LK

P—

—M

T—

TN

FK

QRV

DSW

VS

SI

////represents

deletion.


among infected population from Eastern India requiredthe confirmation by complete genome sequence analy-sis. The present study is the first report of the entiregenome analyses of the isolates to confirm the circula-tion of hepatitis B virus genotype C strain in localpopulation of Eastern India. Additionally, we describedwhole genome analysis of indigenous genotype A and Dstrains, two of which might represent a new subgeno-type.

The genotype C has recently been classified into twomajor subgroups C1 (Cs)—prevalent in Southeast Asiaand C2 (Ce)—prevalent in East & Far East Asia,including China, Korea, and Japan [Huy et al., 2004;Chan et al., 2005]. All of the HBV genotype C strainsfrom Eastern India isolated in this study were phylo-genetically classified to the C1 (Cs) subgenotype. East-ern India is close geographically to the border of China,Myanmar, Bangladesh, and Thailand (Fig. 3), andthe high homology of the Indian isolates with thosepreviously reported in these countries indicates thepossible origin of the infection. This supports our earlierconclusion based on the partial S gene sequenceanalysis, which has also demonstrated that genotype Cin Eastern India had possibly been spread from Thai-land [Vivekanandan et al., 2004; Banerjee et al., 2005].The subjects of this study were ethnic Bengali, all werenatives ofKolkata. There is no documented evidence of acommon ancestor to the population of Eastern India andThailandwhowould share the sameHBV sub-genotype.However, presence of genotype C is reported fromBangladesh, which is another geographically close

country with population ethnically and linguistically,related to the one we studied.

Moreover, the C1858 variant, which was previouslyfrequently found in the South East Asian genotype Cstrains [Alestig et al., 2001] was not observed either inThailand strains [Sugauchi et al., 2002a], or amongthe genotype C strains in this study. Further large-scale population studies are required to investigate theepidemic routes of the virus spread.

Recombination between HBV genotypes is commonevent in countries where different genotypes areprevalent [Morozov et al., 2000], however, the presentstudy found no evidence of recombination in the strainsstudied as was confirmed by phylogenetic analysesusing different ORFs and Simplot analysis.

Aswaspreviously reported, thegenotypeD is themostpredominant genotype in India, and the strains circu-lating in this country were classified into subgenotypesD1 andD2 [Norder et al., 2004]. In addition, the presentstudy extended the idea about HBV/D diversity in thiscountry. Out of three subtype ayw3 HBV/D strains, onewas classified as subgenotype D3 and two others formeda distinctive sub cluster with a previously unclassifiedstrain AB033558 fromJapan [Norder et al., 2004]. Sincethese sequences differed distinctly from HBV isolatesD1, D2, D3, and D4 with nucleotide differences rangingfrom 5.0 to 5.49%, it can be defined as another distinctsubgenotype, that is, D5. Genotype D is uncommon inJapan and it has recently been reported from only aparticular area of Japan. However from the presentstudy, the possible link between the subgenotype


Fig. 3. Geographical location and HBV genotype distributions in India and its neighboring countries.


D5 from Eastern India and Japan could not beascertained.The T118V/A in the antigenic determinant of Surface

gene was commonly found among genotype D isolatesfromotherparts of India. In our study, the specificaminoacid substitution T at codon 118 and M at 125 in theantigenic determinant was found among genotype Disolates (Table IV). This mutation was not found in anyof the surface gene sequences submitted so far fromother parts of Indian mainland [Gandhe et al., 2003;Sugauchi et al., 2004a]. It was reported from only onesample from Nicobar islands [Arankalle et al., 2003].Therefore, the spread of this mutation and D3 sub-genotype among Eastern Indian population should bemonitored among different risk groups.Hepatitis B virus isolates with C-1858 hinders the

development of A1896 mutation in the precore regionabolishing HBeAg expression. These nucleotide var-iants are frequently found among genotypes A [Li et al.,1993] and F strains [Arauz-Ruiz et al., 1997] and havealso been reported in some genotype C isolates [Chanetal., 2005].GenotypeCandDstrains in our studyhadTat 1858; however, none of the HBeAg negative samplesin both D and C strain had A1896 mutation. BCPmutation (T1762/A1764) was more common amonggenotypes A and C than among genotype D in thepresent study.We found in frame deletion in the Pre-S1 and Pre-S2

among five isolates, three from genotype D (EI03194,EI0388 and EI02456) and one each with genotype A(EI0386) and C (EI0423). The regions exposed at thesurface particle, Pre-S1 and Pre-S2 together with theregion from amino acid 100 to 160 in the S gene, arehighly immunogenic and are potentially under selectivepressure by the host immune system and thus deletionmutations are favored at this site. Several in vitro studypreviously documented that pre-S1 mutants involvingthe binding sites for transcription factors of the Spromoter, such as nuclear binding factor (NF1) andsurface promoter (SP1), might lead to a decrease inHBsAg secretion, accumulation of HBsAg and a largeprotein in the cytoplasm which could give rise tosevere, prolonged hepatocellular injury [Melegariet al., 1994]. Pre-S1/Pre-S2 deletion was reported inAsia including Vietnam, Nepal, China, and India [Huyet al., 2003, Chaudhuri et al., 2004]. The deletionobserved in one hepatocellular carcinoma (HCC)patients (genotype A, EI0386) was also observed in asample reported from Nepal (AB116090). The deletionobserved in genotype C in sample EI0423 at nt 49–57was observed in a sequence reported from Myanmar.Over all, the viral load of the chronic cases with Pre-Smutation was lower (4.83�1.15) than that of wild type(5.44� 1.64), it will be very early to comment on itsrole in chronicity and development of hepatocellularcarcinoma in the course of HBV infection in ourstudy population. Large number of study is needed toconfirm it.As all our sequence data were gained by direct

sequencing of PCR products, we cannot exclude the

possibility that viral subpopulationswith other genomicsequences were also present in the sera studied. Yet, aseach sequence was determined from at least twoindependent amplifications, the resulting data certainlyrepresent the highly predominant species of HBV in therespective samples.

In our study, genotype D samples were found to be ofvariable length,whichwasalso reported in other studies[Hasegawa et al., 1994; Bozdayi et al., 2004; Chaudhuriet al., 2004; Doung et al., 2004; Sallam and Tong, 2004].All the genotype A isolates sequenced belonged to thesubgroup A1 (Aa) as previously described from Indianstrains [Sugauchi et al., 2004a].

In conclusion, thepresent study reflects the importantresults indicating the genetic diversity of HBV genomein this population. With full genome analysis, we havecharacterized the HBV genotype A, C, and D isolatedfrom Eastern India. Genotype C strains belonged to thesubgroup C1 (Cs) from Southeast Asia and were relatedto Thailand strains; genotype A to the subgroup A1 (Aa)and the most predominant genotype D is highlydivergent. Genotype D was distributed among sub-genotype D1, D3 and probably with a new subgenotypeD5. Pre-S deletion is common in all the genotypes;especially D. Subtype ayw3 had specific amino acidsubstitution T at codon 118 and M at codon 125 in anti-genic determinant of surface gene, which has not beenreported previously in isolates from other parts of India.

ACKNOWLEDGMENTS

Arup Banerjee and Sibnarayan Datta thank IndianCouncil of Medical Research (ICMR), New Delhi andUniversity Grants Commission (UGC), New Delhirespectively for providing research fellowship. Authorsalso thank all the technicians of Department of ClinicalMolecular Informative Medicine and Department ofInternal Medicine and Molecular Science, Nagoya CityUniversity Graduate School of Medical Sciences,Nagoya, Japan and ICMR Virus Unit, Kolkata for theirexcellent technical assistance.

REFERENCES

Abe A, Inoue K, Tanaka T, Kato J, Kajiyama N, Kawaguchi R, TanakaS, Yoshiba M, Kohara M. 1999. Quantitation of Hepatitis B VirusGenomic DNA by Real-Time Detection PCR. J Clin Microbiol37:2899–2903.

Akuta N, Suzuki F, Kobayashi M, Tsubota A, Suzuki Y, Hosaka T,Someya T, Saitoh S, Arase Y, Ikeda K, Kumada H. 2003. Theinfluence of hepatitis B virus genotype on the development oflamivudine resistance during long-term treatment. J Hepatol38:315–321.

Alestig E, Hannoun C, Horal P, Lindh M. 2001. Phylogenetic origin ofhepatitis B virus strains with precore C-1858 variant. J ClinMicrobiol 39:3200–3203.

Arankalle VA, Murhekar KM, Gandhe SS, Murhekar MV, RamdasiAY,Padbidri VS, Sehgal SC. 2003.HepatitisB virus: Predominanceof genotype D in primitive tribes of the Andaman and Nicobarislands, India (1989–1999). J Gen Virol 84:1915–1920.

Arauz-Ruiz P, Norder H, Visona KA, Magnius LO. 1997. Molecularepidemiology of hepatitisBvirus inCentralAmerica reflected in thegenetic variability of the small S gene. J Infect Dis 176:851–858.

Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO. 2002. GenotypeH: A new Amerindian genotype of hepatitis B virus revealed inCentral America. J Gen Virol 83:2059–2073.



Banerjee A, Banerjee S, Chowdhury A, Santra A, Chowdhury S,Roychowdhury S, Panda CK, Bhattacharya SK, Chakravarty R.2005. Nucleic acid sequence analysis of basal core promoter/precore/core region of hepatitis B virus isolated from chroniccarriers of the virus from Kolkata, Eastern India: Low frequencyof mutation in the precore region. Intervirology 48:389–399.

Bowyer SM, vanStadenL,KewMC,SimJG. 1997.Aunique segment ofthe hepatitis B virus group A genotype identified in isolates fromSouth Africa. J Gen Virol 78:1719–1729.

Bozdayi AM,AslanN,BozdayiG, TurkyilmazAR,Sengezer T,WendU,ErkanO,Aydemir F, Zakirhodjaev S,Orucov S,BozkayaH,GerlichW, Karayalcin S, Yurdaydin C, Uzunalimoglu O. 2004. Molecularepidemiology of hepatitis B, C and D viruses in Turkish patients.Arch Virol 149:2115–2129.

Chan HL, Tsui SK, Tse CH, Ng EY, Au TC, Yuen L, Bartholomeusz A,Leung KS, Lee KH, Locarnini S, Sung JJ. 2005. Epidemiologicaland virological characteristics of 2 subgroups of hepatitis B virusgenotype C. J Infect Dis 191:2022–2032.

Chaudhuri V, Tayal R, Nayak B, Acharya SK, Panda SK. 2004. Occulthepatitis B virus infection in chronic liver disease: Full-lengthgenomeand analysis ofmutant surface promoter.Gastroenterology127:1356–1371.

Clewley JP, Arnold C. 1997. MEGALIGN. The multiple alignmentmodule of LASERGENE. Methods Mol Biol 70:119–129.

Courouce-Pauty AM, Plancon A, Soulier JP. 1983. Distribution ofHBsAg subtypes in the world. Vox Sang 44:197–211.

Doung TN, Horiike N, Michitaka K, Yan C, Mizokami M, Tanaka Y,Jyoko K, Yamamoto K, Miyaoka H, Yamashita Y, Ohno N, Onji M.2004. Comparison of genotypes C and D of the hepatitis B virus inJapan: A clinical and molecular biological study. J Med Virol72:551–557.

Gandhe SS, Chadha MS, Arankalle VA. 2003. Hepatitis B virusgenotypes and serotypes in western India: Lack of clinicalsignificance. J Med Virol 69:324–330.

Gojobori T, Ishii K, Nei M. 1982. Estimation of average number ofnucleotide substitutions when the rate of substitution varies withnucleotide. J Mol Evol 18:414–423.

HasegawaK,HuangJ,RogersSA,BlumHE,LiangTJ. 1994.Enhancedreplication of a hepatitis B virus mutation associated with anepidemic of fulminant hepatitis. J Virol 68:1651–1659.

Huy TT, UshijimaH,WinKM, Luengrojanakul P, Shrestha PK, ZhongZH, Smirnov AV, Taltavull TC, Sata T, Abe K. 2003. Highprevalence of hepatitis B virus pre-s mutant in countries where itis endemic and its relationship with genotype and chronicity. J ClinMicrobiol 41:5449–5455.

Huy TT, Ushijima H, Quang VX, Win KM, Luengrojanakul P, KikuchiK, Sata T, Abe K. 2004. Genotype C of hepatitis B virus can beclassified into at least two subgroups. J Gen Virol 85:283–292.

Kao JH, Chen PJ, Lai MY, Chen DS. 2000. Hepatitis B genotypescorrelate with clinical outcomes in patients with chronic hepatitisB. Gastroenterology 118:554–559.

Kew MC, Kramvis A, Yu MC, Arakawa K, Hodkinson J. 2005.Increased hepatocarcinogenic potential of hepatitis B virus geno-type A in Bantu-speaking sub-saharan Africans. J Med Virol75:513–521.

KramvisA,WeitzmannL,OwireduWK,KewMC. 2002.Analysis of thecomplete genome of subgroup A’ hepatitis B virus isolates fromSouth Africa. J Gen Virol 83:835–839.

Kurbanov F, Tanaka Y, Fujiwara K, Sugauchi F, Mbanya D, Zekeng L,Ndembi N, Ngansop C, Kaptue L, Miura T, Ido E, Hayami M,Ichimura H, Mizokami M. 2005. A new subtype (subgenotype) Ac(A3) of hepatitis B virus and recombination between genotypes Aand E in Cameroon. J Gen Virol 86:2047–2056.

Li JS, TongSP,WenYM,Vitvitski L, ZhangQ,TrepoC. 1993.HepatitisB virus genotype A rarely circulates as an HBe-minus mutant:Possible contribution of a single nucleotide in the precore region.J Virol 67:5402–5410.

Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, NovakNG, Ingersoll R, Sheppard HW, Ray SC. 1999. Full-length humanimmunodeficiency virus type 1 genomes from subtype C-infectedseroconverters in India, with evidence of intersubtype recombina-tion. J Virol 73:152–160.

Majumder PP. 1998. People of India: Biological diversity and affinities.Evol Anthropol: Issues, News, Rev 6:100–110.

Melegari M, Bruno S, Wands JR. 1994. Properties of hepatitis B viruspre-S1 deletion mutants. Virology 199:292–300.

Morozov V, Pisareva M, Groudinin M. 2000. Homologous recombina-tion between different genotypes of hepatitis B virus. Gene 260:55–65.

Mulders MN, Venard V, Njayou M, Edorh AP, Bola Oyefolu AO,Kehinde MO, Muyembe Tamfum JJ, Nebie YK, Maiga I, Ammer-laan W, Fack F, Omilabu SA, Le Faou A, Muller CP. 2004. Lowgenetic diversity despite hyperendemicity of hepatitis B virusgenotype E throughout West Africa. J Infect Dis 190:400–408.

Norder H, Courouce AM, Magnius LO. 1994. Complete genomes,phylogenetic relatedness, and structural proteins of six strains ofthe hepatitis B virus, four of which represent two new genotypes.Virology 198:489–503.

Norder H, Courouce AM, Coursaget P, Echevarria JM, Lee SD,Mushahwar IK, Robertson BH, Locarnini S, Magnius LO. 2004.Genetic diversity of hepatitis B virus strains derived worldwide:Genotypes, subgenotypes, and HBsAg subtypes. Intervirology47:289–309.

Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M,Miyakawa Y, Mayumi M. 1988. Typing hepatitis B virus byhomology in nucleotide sequence: Comparison of surface antigensubtypes. J Gen Virol 69:2575–2583.

OritoE,MizokamiM,SakugawaH,MichitakaK, IshikawaK, IchidaT,Okanoue T, Yotsuyanagi H, Iino S. 2001. A case-control study forclinical and molecular biological differences between hepatitis Bviruses of genotypes B and C. Japan HBV Genotype ResearchGroup. Hepatology 33:218–223.

Robertson DL, Hahn BH, Sharp PM. 1995. Recombination in AIDSviruses. J Mol Evol 40:249–259.

Sallam TA, Tong CYW. 2004. African links and hepatitis B virusgenotypes in the Republic of Yemen. J Med Virol 73:23–28.

Sarin SK. 1999. Changing terminology of hepatitis B and C carrier:Consensus statement. Ind J Gastroenterol 18:S15–S20.

Shanmugham J. 1990. Hepatitis B virus: prevalence of HBsAgsubtypes: The Indian picture. In: Thyagarajan SP, SubramanianS, edtors. Proceedings of the Indo-UK Symposium on ‘Updates inViral Hepatitis.’ p 78–83.

Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF,Rossau R. 2000. A new genotype of hepatitis B virus: Completegenome and phylogenetic relatedness. J Gen Virol 81:67–74.

Sugauchi F, Mizokami M, Orito E, Ohno T, Kato H, Suzuki S, KimuraY, Ueda R, Butterworth LA, Cooksley WG. 2001. A novel variantgenotypeCof hepatitisBvirus identified in isolates fromAustralianAborigines: Complete genome sequence and phylogenetic related-ness. J Gen Virol 82:883–892.

Sugauchi F, Chutaputti A, Orito E, Kato H, Suzuki S, Ueda R,Mizokami M. 2002a. Hepatitis B virus genotypes and clinicalmanifestation among hepatitis B carriers in Thailand. J Gastro-enterol Hepatol 17:671–676.

Sugauchi F, Orito E, Ichida T, KatoH, SakugawaH,KakumuS, IshidaT, Chutaputti A, Lai CL,UedaR,MiyakawaY,MizokamiM. 2002b.Hepatitis B virus of genotype Bwith or without recombination withgenotype C over the precore region plus the core gene. J Virol 76:5985–5992.

Sugauchi F, Orito E, Ichida T, KatoH, SakugawaH,KakumuS, IshidaT, Chutaputti A, Lai CL, Gish RG, Ueda R, Miyakawa Y,MizokamiM. 2003. Epidemiologic and virologic characteristics of hepatitis Bvirus genotype B having the recombination with genotype C.Gastroenterology 124:925–932.

Sugauchi F, Kumada H, Acharya SA, Shrestha SM, Gamutan MT,KhanM,GishRG, TanakaY,Kato T,Orito E,UedaR,MiyakawaY,Mizokami M. 2004a. Epidemiological and sequence differencesbetween two subtypes (Ae andAa) of hepatitis B virus genotypeA. JGen Virol 85:811–820.

Sugauchi F, Kumada H, Sakugawa H, Komatsu M, Niitsuma H,Watanabe H, Akahane Y, Tokita H, Kato T, Tanaka Y, Orito E,Ueda R, Miyakawa Y, Mizokami M. 2004b. Two subtypes ofgenotype B (Ba and Bj) of hepatitis B virus in Japan. Clin InfectDis 38:1222–1228.

Tanaka Y, Hasegawa I, Kato T, Orito E, Hirashima N, Acharya SK,GishRG,KramvisA,KewMC,YoshiharaN,ShresthaSM,KhanM,Miyakawa Y, Mizokami M. 2004. A case-control study fordifferences among hepatitis B virus infections of genotypes A(subtypes Aa and Ae) and D. Hepatology 40:747–755.

Thakur V, Guptan RC, Kazim SN,Malhotra V, Sarin SK. 2002. Profile,spectrumand significance ofHBVgenotypes in chronic liver diseasepatients in the Indian subcontinent. J Gastroenterol Hepatol 17:165–170.



Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG.1997. The CLUSTAL_X windows interface: Flexible strategies formultiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 25:4876–4882.

Thyagarajan SP, Subramanian S, Solomon S, Panchanadam M,Madangopalan N. 1979. Antigenic subtypes of HBsAg: Theirdistribution and pattern of occurrence among blood donors andpatientswith liver diseases and leprosy inTamilnadu, India. JTropMed Hyg 82:62–66.

Thyagarajan SP, Jayaram S, Mohanavalli B. 1996. Prevalence of HBVin the general population of India. In: Sarin SK, Singal AK, editors.Hepatitis B in India. Problems and prevention. New Delhi, India:CBS. p 5–16.

Vivekanandan P, Abraham P, Sridharan G, Chandy G, Daniel D,Raghuraman S, Daniel HD, Subramaniam T. 2004. Distribution ofhepatitis B virus genotypes in blood donors and chronically infectedpatients in a tertiary care hospital in southern India. Clin InfectDis38:e81–e86.



Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India

Documents

Transcript of Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India