Yan Chen 1, Kojiro Michitaka 1,2, *, Hiroshi Matsubara 1, Kazuhisa Yamamoto 3, Norio Horiike 1, Morikazu Onji 1

Transcription

1 Journl of Heptology 38 (2003) Complete genome sequence of heptitis B virus (HBV) from ptient with fulminnt heptitis without precore nd core promoter muttions: comprison with HBV from ptient with cute heptitis infected from the sme infectious source Yn Chen 1, Kojiro Michitk 1,2, *, Hiroshi Mtsubr 1, Kzuhis Ymmoto 3, Norio Horiike 1, Morikzu Onji 1 1 Third Deprtment of Internl Medicine, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime , Jpn 2 Endoscopy Center, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime , Jpn 3 Deprtment of Gstroenterology, Mtsuym Shimin Hospitl, Mtsuym, Ehime, Jpn Bckground/Aims: There is pucity of informtion regrding heptitis B virus (HBV) from ptients with fulminnt heptitis (FH) without precore (pre-c, nt 1896) nd core promoter (CP, nt 1762, 1764) muttions. Methods: Pre-C nd CP muttions were studied in eight ptients with FH nd 26 ptients with cute heptitis (AH) due to HBV. One ptient with FH (FH1) ws infected with HBV without these muttions. Interestingly, the ser of the infectious source (IS1) nd of ptient with AH (AH1) infected from IS1 were vilble. Complete HBV genomes from these three ptients were nlyzed. Results: These muttions were found in seven of eight FH nd five of 26 AH (P, 0.01). HBV from FH1, IS1 nd AH1 belonged to genotype D. Nucleotide difference between FH1 nd AH1 ws six of 3182 bses (nt 493, 998, 1173, 2928, 3067, nd 3078). Two nd five substitutions of deduced mino cid sequences were found in the pre-s1 nd polymerse regions, respectively. The sme nucleotide substitutions t nt 493, 1173, 2928 nd 3067 were found in severl ptients with FH in our lbortory or GenBnk. Conclusions: These six nucleotide substitutions of HBV DNA could be cndidtes of muttions relting to FH. q 2002 Europen Assocition for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Heptitis B virus; Fulminnt heptitis; Precore muttion; Core promoter muttion; Heptitis B virus genotype; Genotype D; Infectious source 1. Introduction Heptitis B virus (HBV) cuses vriety of diseses, including self-limiting cute heptitis (AH), fulminnt heptitis (FH), chronic heptitis, liver cirrhosis nd heptocellulr crcinom. Approximtely 1% of ptients with cute HBV infection develop FH. Mny studies hve been reported concerning the reltion between virl muttions nd the severity of disese. Precore muttion t nt 1896 (G to A) nd core promoter muttions t nt 1762 (A to T) Received 17 April 2002; received in revised form 10 September 2002; ccepted 17 September 2002 * Corresponding uthor. Tel.: ; fx: E-mil ddress: mich@m.ehime-u.c.jp (K. Michitk). nd nt 1764 (G to A) hve been reported to relte with FH [1 8]. Both muttions re known to result in the reduction of the production of HBe ntigen (HBeAg) by trnsltionl or trnscriptionl level. The mechnism of this reltion hs been studied, nd it hs been shown tht these muttions induce not only the reduction of HBeAg but lso enhnced repliction of HBV [9 14]. Besides the supposition tht HBeAg directly suppresses virl repliction, severl mechnisms for enhnced virl repliction in these mutnt strins hve been reported, such s the efficient encpsidtion of pregenomic RNA by structurl rerrngement, the chnge of trnscriptionl trnsctivtion ctivity of X protein, nd the emergence of the heptocyte nucler fctor binding site by core promoter muttion [9,11,13,15]. However, severl reports did not find tht n ssocition /02/$20.00 q 2002 Europen Assocition for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S (02)

2 Y. Chen et l. / Journl of Heptology 38 (2003) Tble 1 Primers used in this study Primer Sequence nt position Primers for PCR of frgment A SP3 sense 5 0 -CTCTCTTTTTTGCCTTCTGAC ( ) AP5 nti-sense 5 0 -CCTCCTAGTACAAAGACCTT ( ) SP4 sense 5 0 -GACACCGCCTCTGCTCTGTAT ( ) AP1 nti-sense 5 0 -TCCCCCAACTCCTCCCAGTCCTT ( ) Primers for PCR of frgment B B1 sense 5 0 -CTCTCTCGGAAATACACCTC ( ) B3 sense 5 0 -TGGATCCTTCGCGGGACGTCCTT ( ) P6 nti-sense 5 0 -TTCCCACCTTATGAGTCCAA ( ) Primers for sequencing SS1 sense 5 0 -CTCCACCACATTCCACCAAG (1 20) SS4 nti-sense 5 0 -CCGTACAATATGTTCCTGTGG ( ) S5 sense 5 0 -GGCTTTCGCAAGATTCCTATGG ( ) SS2 nti-sense 5 0 -GAGGAGCCACAAAGGTTCCGC ( ) SS5 sense 5 0 -CCACAGGAACATATTGTACGG ( ) AP1 nti-sense 5 0 -TCCCCCAACTCCTCCCAGTCCTT ( ) B3 sense 5 0 -TGGATCCTTCGCGGGACGTCCTT ( ) B2 nti-sense 5 0 -GTAACTCCACAGAAGCTCCA ( ) P5 sense 5 0 -AAGGTCTTTGTACTAGGAGG ( ) P6 nti-sense 5 0 -TTCCCACCTTATGAGTCCAA ( ) SP4 sense 5 0 -GACACCGCCTCTGCTCTGTAT ( ) SP6 sense 5 0 -TTGGACTCATAAGGTGGGAA ( ) AS3 nti-sense 5 0 -GACTCTGTGGTATTGTGAGGA ( ) nt ws numbered from the unique EcoRI site of the HBV genome. exists between these muttions nd severity of disese [16 22]. Although host-derived fctors my be importnt in the severity of heptitis, severl investigtors hve shown tht muttion in the pre-s2 strt codon, muttion in the core region, nd insertion of nucleotides my lso be relevnt [23 25]. In this study, we nlyzed the complete genome of HBV without precore (nt 1896) nd core promoter (nt 1762, 1764) muttions from ptient with FH nd from ptient with AH infected from the sme infectious source. The purpose of this study is to find the novel cndidte muttions relting to FH. 2. Ptients nd methods 2.1. Ptients Eight ptients with FH (ges rnging from 18 to 61 yers, medin 44 yers; five mles nd three femles) nd 26 ptients with AH (ges rnging from 20 to 74 yers, medin 34 yers; 14 mles nd 12 femles) were enrolled in this study. All of them were Jpnese ptients with spordic heptitis with cute HBV infection. Among them, one ptient with FH (ptient FH1) nd one ptient with AH (ptient AH1) were infected from the sme infectious source (ptient IS1). HBV from IS1 ws lso studied. Fig. 1. Positions of primers nd mplicons for sequencing.

3 86 Y. Chen et l. / Journl of Heptology 38 (2003) Tble 2 Differences of HBV DNA sequence from ptients with fulminnt heptitis (FH1), cute heptitis (AH1) nd the infectious source (IS1) by direct sequencing nt M32138 T T G T C G FH1 T A A C A G IS1 T T G C A G AH1 G T G A C C Sequence of M32138 ws written s prototype sequence becuse this strin hs stndrd sequence of HBV genotype D mong 41 strins HBV genotype D submitted in GenBnk [28]. The dignostic criteri for cute HBV infection ws positivity for IgM type ntibody to heptitis B core (IgM-nti-HBc) nd positivity for nti- HBc with low titer ð,90% in 200-fold diluted serum). FH is defined s severe liver dysfunction with prolonged prothrombin time (,40%) nd the onset of heptic encephlopthy within 8 weeks of the first symptoms in n individul without underlying liver disese. All of AH nd FH ptients were positive for nti-hbc with low titer nd IgM-HBc, nd were negtive for IgM type ntibody to heptitis A virus (IgM-nti-HA), ntibody to heptitis C virus (nti-hcv) nd HCV-RNA. Acute phse serum smples were obtined nd stored t 280 8C until used. The clinicl bckgrounds of the three ptients (FH1, AH1, IS1) were s follows. Ptient FH1 is 18-yer-old femle. Her physicl signs (heptic encephlopthy, grde IV) nd clinicl dt (totl bilirubin, T.bil 5.0 mg/dl, lnine minotrnsferse, ALT 7620 IU/l, prothrombin time, PT 36%, heptitis B surfce ntigen (HBsAg) 1ve, HBeAg 1ve, IgM-nti-HBc 1ve, nti-hbc 97% in undiluted serum nd 88% in 200-fold diluted serum on dmission) were typicl for ptient with FH-B. Ptient IS1 is 19-yer-old mn. His trnsminse level ws slightly elevted (ALT 49 IU/l) nd he ws positive for HBsAg, HBeAg, nd nti- HBc (99% in 200-fold diluted serum). His level of DNA polymerse (DNA- P) ws high (6665 cpm). Trnsmission of HBV from IS1 to FH1 ws suspected, becuse they hd sexul contct 2 months before the onset of FH. Fifteen months following the onset of FH1, ptient AH1 (20 yers old, femle) felt mild generl mlise. Generl mlise continued for 3 months before she consulted hospitl. Liver function tests showed bnormlity, however the degree of her heptitis ws not severe. Her lbortory dt were s follows: T.Bil 1.4 mg/dl, ALT 189 IU/l, HBsAg 1ve, HBeAg 1ve, IgM-nti-HBc 1ve, DNA-P 585 cpm. It hs been proven tht she ws negtive for HBsAg nd nti-hbc 1 yer before when she donted blood t the Red Cross, nd tht she hd history of sexul contct with IS1 few months before she felt generl mlise. She ws positive for HBeAg nd DNA-P for more thn 1 month, nd her liver histology indicted tht her heptitis ws in trnsition from AH to chronic heptitis. She ws treted with interferon, nd HBeAg nd HBsAg becme negtive. Therefore, it ws suspected tht FH1 nd AH1 were infected from the sme infectious source, though the severity of heptitis differed gretly. The serum smple from IS1 ws obtined 1 week fter the onset of FH1. The purpose of this study ws explined, nd written informed consent ws obtined from ll ptients or from their fmilies Serologicl virl mrkers HBsAg (AxSYM HBsAg, Dinbot, Tokyo, Jpn), nti-hbc (AxSYM HBc, Dinbot), IgM-nti-HBc (AxSYM HBc-M, Dinbot), HBeAg (AxSYM HBeAg, Dinbot), nti-hbe (AxSYM HBeAb, Dinbot), IgM-nti-HA (AxSYM HA-M, Dinbot), nti-hcv (Imcheck-F-HCV, Kokusi-Shiyku, Kobe, Jpn) nd HCV RNA (Amplicor HCV, Roche Dignostics, Mnnheim, Germny) were ssyed using commercil kits. Serum DNA-P ws mesured by Kpln s method with some modifictions [26] Isoltion, mplifiction nd sequencing of HBV DNA DNA ws extrcted from ser. Briefly, 50 ml of serum smple ws incubted with lysis buffer contining proteinse K. DNA ws extrcted using phenol chloroform solution nd precipitted with ethnol. HBV DNA ws mplified by polymerse chin rection (PCR). Primers used in this study re shown in Tble 1. For mplifiction of the core promoter nd precore regions, semi-nested PCR ws performed using primers B1, B3, nd B2 (B1 nd B2 for firstround PCR, nd B3 nd B2 for second-round PCR). Sequencing ws done by direct sequencing using commercilly vilble kit (BigDye Termintor Cycle Sequencing FS Redy Rection Kit, Applied Biosystems, Almed, CA) Complete genome sequence HBV DNA from FH1, AH1, nd IS1 were studied for complete genome sequence, becuse they were proven to be infected with HBV without precore nd core promoter muttions. To obtin the full-length HBV DNA sequence, two mplicons were obtined by PCR (Fig. 1). One ws frgment A, 2936 bses in length (nt 1994 to 1747), which ws mplified using primers SP3 nd AP5 for the first PCR nd SP4 nd AP1 for the second PCR. The other frgment B, 1080 bses in length (nt 1399 to 2478), ws mplified using primers B1 nd P6 for the first PCR nd B3 nd P6 for the second PCR. Sequencing ws done by direct sequencing, using the method described bove. Positions of sequencing primers re shown in Fig. 1 nd Tble 1. The ccurcy of the sequence ws ensured by the identifiction of the sequence dt of the complete genome obtined by sense sequencing primers nd tht obtined by nti-sense sequencing primers Subcloning nd sequencing Subcloning ws done in three prts of HBV DNA from ptients FH1 nd AH1. One ws frgment C (nt , mplified using primers SS1 nd SS2), the second ws frgment D (nt , mplified using primers SP6 nd AS3) nd the lst ws frgment E (nt , mplified using primers B3 nd B2). They were inserted into the plsmid vector (pt7bluevector) nd trnsfected into competent cells using Perfectly Blunt Cloning Tble 3 Vritions of HBV DNA sequence from the ptients with fulminnt nd cute heptitis infected from the sme infectious source studied by subcloning nt FH1 T10/10 A15/20 A13/20 C10/10 A10/10 G10/10 A20/20 G20/20 G20/20 T5/20 G7/20 AH1 G10/10 T10/10 G10/10 A10/10 C10/10 C10/10 A20/20 G20/20 G20/20

4 Y. Chen et l. / Journl of Heptology 38 (2003) Tble 4 Differences of deduced mino cid sequence (ORF) of HBV protein between FH1 nd AH1 kits (Novgen, Drmstdt, Germny). Ten or 20 clones for ech frgment were nlyzed for sequencing. Primers SS1 nd SS2 for frgment C, SP6 nd AS3 for frgment D, nd B3 nd B2 for frgment E were used for sequencing HBV genotyping HBV genotype ws determined bsed on the restriction frgment length polymorphism ptterns of the S gene sequence [27] Sequencing of other ptients In severl prts of HBV DNA, where the nucleotide sequence ws different between the HBV strins from ptient FH1 nd AH1, direct sequencing ws done in HBV from five ptients with FH nd 11 ptients with AH using suitble PCR nd sequencing primers. HBV strins from ptients with FH or AH whose complete HBV sequences were vilble from GenBnk nd previous literture were lso compred nd nlyzed Sttisticl nlysis Sttisticl nlysis ws performed using the chi-squred test (or Fisher s exct test when pplicble). P vlues of less thn 0.05 were considered sttisticlly significnt. 3. Results Amino cid sequence (ORF) PreS1-S2-S (389 ) Polymerse (832 ) FH1 I L R N A F N AH1 L F S T P V I ORF, open reding frme;, mino cid Comprison of HBV nucleotide sequence from FH1 nd AH1 After initil screening of HBV DNA sequence t precore (pre-c, nt 1896) nd core promoter (CP, nt 1762, 1764) region in eight ptients with FH nd 26 ptients with AH, we found tht ptient with FH (FH1) ws infected with HBV without these muttions. As described in Section 2, ser from AH1 nd IS1 were lso vilble. The complete Tble 5 HLA types of the ptient with fulminnt nd cute heptitis infected from the sme infectious source HLA FH1 AH1 A A24 A24 B B61 B70 B51 B35 C Cw3 Cw1 DRB DQB DPB HBV nucleotide sequences of three ptients (FH1, AH1 nd IS1) were nlyzed nd found to hve nucleotide length of 3182 bses (ccession number AB for ptient FH1, AB for ptient AH1, nd AB for ptient IS1). These three strins did not hve muttions in the core promoter (nt 1762, 1764) nd precore (nt 1896) regions. Tble 2 shows the differences of nucleotides mong these three strins. The nucleotide difference between strins FH1 nd IS1, between strins IS1 nd AH1, nd strins FH1 nd AH1 were 2/3182, 4/3182, nd 6/3182, respectively. The three HBV strins showed very high homology (.99.8%); therefore, we concluded tht both FH1 nd AH1 were infected from IS1. The sequencing dt of cloned HBV DNA t the six positions, where the nucleotide sequences were different between strins FH1 nd AH1, re shown in Tble 3. The sequences of the mjor clones were sme s tht of direct sequencing, nd the sequence of minor clones of FH1 were sme s tht from AH1. At nucleotide positions 1762, 1764, nd 1896, no clones from FH1 nd AH1 showed mutnt sequences HBV nucleotide in promoter nd enhncer region No muttions were found in the core promoter (nt ) [29], pre-s promoter (SP1) (nt ) [30], nd X promoter (nt ) [31] in FH1 nd AH1. In the enhncer region, no muttions were found in Enhncer II (nt ) [32], but one difference nt 1173A in FH1 nd nt 1173G in AH1 ws found in Enhncer I (nt ) [32]. On the other hnd, muttion t nt 3067 from C to A in the FH1 strin resulted in the emergence of n AT-rich sequence in the pre-s1 region (ATACTA in prototype nd AH1, nd ATAATA in FH1) (nt ) Amino cid sequence in four open reding frmes Comprison of deduced mino cid sequence of four open reding frmes between FH1 nd AH1 strins were exmined. Amino cid sequences were completely identicl in the pre-s2, S, X nd precore to core regions. Two differences were found in the pre-s1 region, nd five were found in the polymerse (P) region (Tble 4). Among five differences in the P region, three were locted in the Spcer region (208th, 254th, nd 258th mino cid) nd two were locted in DNA polymerse/reverse trnscriptse domin (Pol/RT,457th nd 625th mino cid) HLA type HLA types of ptient FH1 nd ptient AH1 re shown in Tble 5. HLA locus A ws A24 nd identicl between the two ptients. Other HLA types were different Genotypes nd sequence in other ptients Beside FH1 nd AH1, five ptients with FH nd ten ptients with AH were studied for genotypes nd sequences of six positions where nucleotide sequences were different between the strins of FH1 nd AH1. HBV forms FH1, AH1

5 88 Y. Chen et l. / Journl of Heptology 38 (2003) Tble 6 Nucleotide sequences of HBV from ptients with fulminnt nd cute heptitis in our lbortory, in GenBnk nd in previous literture No. of strins nt (%) FH 40 T 3 (7.5) A 1 (2.5) A 5 (12.5) C 12 (30.0) A 4 (10.0) G 38 (95.0) A 23 (57.5) G 21 (52.5) G 18 (45.0) A 22 (55.0) T 39 (97.5) C 7 (17.5) A 26 (65.0) T 13 (32.5) A 1 (2.5) T 17 (42.5) A 18 (45.0) A 22 (55.0) C 14 (35.0) G 27 (67.5) T 2 (5.0) C 22 (55.0) del 1 (2.5) T 1 (2.5) G 1 (2.5) T 1 (2.5) del 1 (2.5) AH b 14 G 2 (14.3) T 14 (100) G 3 (21.4) A 14/ (100) C 1 (7.1) C 1 (7.1) A 10 (71.4) G 10 (71.4) G 11 (78.6) A 12 (85.7) C 10 (71.4) T 13 (92.9) G 12 (85.7) T 4 (28.6) A 4 (28.6) A 3 (21.4) T1 (7.1) A 1 (7.1) Six strins from our lbortory including FH1, 24 strins from GenBnk (AF297621, AF297622, AF297623, AF297624, AF [22]. AB [34]. AB031266, AB031267, AB [35]. AF X97850, X97849, X97848 [36]. X98072 [25]. D50521, D50522 [37]. AY AF AF L08805 [38]. X80925, X80924 [20]. AJ L27106 [39]). Ten strins from previous literture [21,33]. Genotype A: 5 strins, Genotype B: 8 strins, Genotype C: 10 strins, Genotype D: 17 strins. del, deletion. Bold letters re the sequence of FH1. b Eleven strins from our lbortory including AH1, 3 strins from GenBnk (D16665 [40]. D1666 [41]. AB [35]); Genotype A: 2 strins, Genotype C: 11 strins, Genotype D: 1 strin. Bold letters re the sequence of AH1. nd IS1 belonged to genotype D. All other HBV strins from our lbortory belonged to genotype C except two strins from AH which belonged to genotype A. From previous literture [21,33] nd from GenBnk, full genome sequence of 34 HBV strins from FH nd three from AH were vilble. Tble 6 shows the nucleotide sequence t six positions nd core promoter (nt 1762T, 1764A), precore (nt 1896A) of HBV strins from our lbortory, from previous literture nd from GenBnk. Though the sttisticl difference ws not found, nt 493T, 998A, 1173A, 2928C nd 3067A were seen only in HBV strins from ptients with FH Muttion in precore or core promoter Among eight ptients with FH nd 26 with AH in this study, the frequency of muttions t nt 1762 (A to T) or 1764 (G to A) in the core promoter region or t nt 1896 (G to A) in the precore region ws significntly higher in ptients with FH thn tht in ptients with AH (7/8 vs. 5/26, P, 0:01) (Tble 7). Only HBV from FH1 did not hve these muttions mong eight ptients with FH. 4. Discussion In this report, we nlyzed serum smples from ptient with FH nd from ptient with AH who were infected from the sme infectious source. It is known tht some or mny subpopultions of different HBV strins re present in chronic HBV crrier, so it could be cusble tht different HBV strins in IS1 preferentilly trnsferred to FH1 nd AH1 [42]. It could not be clrified whether the difference of FH1 nd AH1 ws cused by the selection of preferentil strins for hosts or the chnge of dominnt strin in IS1 during the period between the onset of FH1 nd AH1. However, nlysis of HBV genomes from ptients FH1 nd tht from AH1 reveled tht they hd very close homology. It could be suspected tht few differences of nucleotide sequence between the two HBV strins might be the cuse of the difference of severity of heptitis. The six nucleotide substitutions found in this study my be the cndidtes of muttions which ffect the severity of the disese. Possible mechnisms for chnging severity of heptitis by virl muttions re (1) chnge of immunogenicity, function, or locliztion of HBV protein by mino cid substitution, (2) chnge of the mount of virl protein by nucleotide substitution t the promoter or enhncer, nd (3) chnge of ctivity of virl repliction by nucleotide substitution t the promoter or enhncer. Substitutions t nt 3067, 3078, nd t nt 493, 1173 which cuse the mino cid substitution in the pre-s1 nd pol/rt domin of polymerse protein, respectively, my be cndidtes for importnt substitutions by chnging mino cid sequence. Furthermore, nucleotide substitution t 3067 mkes TA-rich sequence in the upstrem of pre-s2. The six positions shown in this study were not described in reltion to severity of heptitis in previous literture. However, it hs been Tble 7 The frequency of core promoter (nt 1762 A to T, nt 1764 G to A) nd precore (nt 1896 G to A) muttion in ptients with fulminnt nd cute heptitis n 1762T (%) 1764A (%) 1896A (%) Any of the 3 muttions Fulminnt heptitis 8 7 (87.5) 7 (87.5) 4 (50.0) 7 (87.5) Acute heptitis 26 5 (19.2) 5 (19.2) 5 (19.2) 5 (19.2) P vlue Fisher s exct test.

6 Y. Chen et l. / Journl of Heptology 38 (2003) reported tht chnges of nucleotides or mino cids in the pre-s1 region ffect the secretion of virl protein or virl repliction [43,44], nd the positions found in this study (nt 2928, 3067, 3078) locte in pre-s1. On the other hnd, nucleotide substitution t 1173 is locted in the enhncer I region. Muttion in the enhncer I region my ffect the repliction of HBV [45]. We screened the full genome sequence of HBV from FH nd AH in GenBnk nd from previous publictions, becuse it is more importnt to compre HBV genome between ptients with FH nd AH thn between FH nd chronic HBV infection. Nt 493T, 1173A, 2928C nd 3067A were found in other ptients with FH nd not in ny ptient with AH. We could not find ny full genome sequence dt of HBV genotype D from ptients with AH from GenBnk or from previous publictions. AH cused by genotype D is very rre in Jpn, therefore it ws impossible for us to compre the HBV genotype D genome from ptients with FH nd tht with AH. More dt re needed regrding the nucleotide sequence t these positions of genotype D HBV from ptients with AH; however, these substitutions re thought to be cndidtes for muttions relting to FH by chnging the mino cid sequence or trnscriptionl ctivity. These substitutions re worth further study. It is suspected tht both virl fctors nd host fctors ffect the severity of the disese. Anlysis of HLA types in two ptients in this study reveled tht they shred the sme HLA A24, though their other HLA types differed. It hs been reported tht mjority of HLA A24-positive Jpnese hve HLA-A2402 [46]. The motif of the mino cid sequence for the epitope of HLA-A2402 hs been reported (Thr or Phe t position 2 nd Phe, Leu, Ile, or Trp t C terminus) [47,48]. In this study, mino cid substitutions did not result in the emergence or disppernce of the HLA A2402 motif in HBc protein or HBs protein which were thought to be the trget protein of cytotoxic T lymphocytes. However, the epitope of other HLA in HBV proteins should be further nlyzed. In this study, we determined tht precore (nt 1896) nd/or core promoter muttions (nt 1762, 1764) were not found in ptient with FH infected with genotype D, but were found in ll ptients with FH infected with genotype C, which is mjor HBV genotype in Jpn [49]. It is suspected tht these muttions hve close reltion with FH in genotype C, nd tht virl fctors which influence the severity of heptitis my be different mong different genotypes. The exct frequency of these muttions needs to be clrified in ech HBV genotype in ptients with FH nd with AH. In the present study, six nucleotide substitutions were found between ptient with FH nd ptient with AH infected from the sme infectious source. These substitutions could be cndidtes for novel muttions which ffect the severity of heptitis. However, it is still uncertin which fctors, host fctors or virl fctors, ffect the severity of the disese. Host fctors need to be studied, s well s the effect of these muttions on virl repliction nd on the production of virl proteins. Acknowledgements This work ws supported by the non-a, non-b heptitis study group from the Ministry of Helth nd Welfre, Jpn. References [1] Omt M, Eht T, Yokosuk O, Hosod K, Ohto M. Muttions in the precore region of heptitis B virus DNA in ptients with fulminnt nd severe heptitis. N Engl J Med 1991;324: [2] Crmn WF, Fgn EA, Hdziynnis S, Kryinnis P, Tssopoulos NC, Willims R, et l. Assocition of precore genomic vrint of heptitis B virus with fulminnt heptitis. Heptology 1991;14: [3] Ling TJ, Hsegw K, Rimon N, Wnds J, Ben-Porth E. A heptitis B virus mutnt ssocited with n epidemic of fulminnt heptitis. N Engl J Med 1991;324: [4] Kosk Y, Tkse K, Kojim M, Shimizu M, Inoue K, Yoshib M, et l. Fulminnt heptitis B: induction by heptitis B virus mutnts defective in the precore region nd incpble of encoding e ntigen. Gstroenterology 1991;100: [5] Sto S, Suzuki K, Akhne Y, Akmtsu K, Akiym K, Yunomur K, et l. Heptitis B virus strins with muttions in the core promoter in ptients with fulminnt heptitis. Ann Intern Med 1995;122: [6] Tnk S, Yosib M, Iino S, Fukud M, Nko H, Tsud F, et l. A common source outbrek of fulminnt heptitis B in hemodilysis ptients induced by precore mutnt. Kidney Int 1995;48: [7] Friedt M, Gerner P, Lusch E, Trubel H, Zbel B, Wirth S. Muttions in the bsic core promoter nd the precore region of heptitis B virus nd their selection in children with fulminnt nd chronic heptitis B. Heptology 1999;29: [8] Teo EK, Ostpowicz G, Hussin M, Lee WM, Fontn R, Lok ASF, The US ALF Study Group. Heptitis B infection in ptients with cute liver filure in the United Sttes. Heptology 2001;33: [9] Lmberts C, Nssl M, Velhgen I, Zentgrf H, Schroder CH. Precore-medited inhibition of heptitis B virus progeny DNA synthesis. J Virol 1993;67: [10] Moriym K, Okmoto H, Tsud F, Myumi M. Reduced precore trnscription nd enhnced core-pregenome trnscription of heptitis of heptitis B virus DNA fter replcement of the precore-core promoter with sequences ssocited with e ntigen-seronegtive persistent infections. Virology 1996;226: [11] Bumert TF, Rogers SA, Hsegw K, Ling TJ. Two core promoter muttions identified in heptitis B virus strin ssocited with fulminnt heptitis result in enhnced virl repliction. J Clin Invest 1996;98: [12] Buckwold V, Xu Z, Chen M, Yen T, Ou J. Effects of nturlly occurring muttion in the heptitis B virus bsl core promoter on precore gene expression nd virl repliction. J Virol 1996;70: [13] Kidd-Ljunggren K, Oberg M, Kidd AH. Heptitis B virus X gene 1751 to 1764 muttions: implictions for HBeAg sttus nd disese. J Gen Virol 1997;78: [14] Sterneck M, Klinin T, Gunther S, Fisher L, Sntntonio T, Greten H, et l. Functionl nlysis of HBV genomes from ptients with fulminnt heptitis. Heptology 1998;28: [15] Li J, Buckwold VE, Hon MW, Ou JH. Mechnism of suppression of heptitis B virus precore RNA trnscription by frequent double muttion. J Virol 1999;73: [16] Fery C, Gigou M, Smuel D, Bernuu J, Bismuth H, Brechot C. Low prevlence of precore muttions in heptitis B virus DNA in fulminnt heptitis type B in Frnce. J Heptol 1993;18: [17] Ling TJ, Hsegw K, Munoz SJ, Shpiro CN, Yoffe B, McMhon

7 90 Y. Chen et l. / Journl of Heptology 38 (2003) BJ, et l. Heptitis B virus precore muttion nd fulminnt heptitis in the United Sttes. A polymerse chin rection-bsed ssy for the detection of specific muttion. J Clin Invest 1994;93: [18] Lskus T, Rkel J, Nowicki M, Persing DH. Heptitis B virus core promoter sequence nlysis in fulminnt nd chronic heptitis B. Gstroenterology 1995;109: [19] Hsu SY, Chng MH, Lee CY, Hsieh KH, Ni YH, Chen PJ, et l. Precore mutnt of heptitis B virus in childhood fulminnt heptitis B: n infrequent ssocition. J Infect Dis 1995;171: [20] Kryinnis P, Alexopoulou A, Hdziynnis S, Thursz M, Seito S, Wtts R, et l. Fulminnt heptitis ssocited with heptitis B virus e ntigen-negtive infection: importnce of host fctors. Heptology 1995;22: [21] Sterneck M, Gunther S, Sntntonio T, Fischer L, Broelsch CE, Greten H, et l. Heptitis B virus genomes of ptients with fulminnt heptitis do not shre specific muttion. Heptology 1996;24: [22] Owiredu WKBA, Krmvis A, Kew MC. Moleculr nlysis of heptitis B virus genomes isolted from blck Africn ptients with fulminnt heptitis B. J Med Virol 2001;65: [23] Eht T, Omt M, Chung WL, Yokosuk O, Ito Y, Hosod K, et l. Muttions in core nucleotide sequence of heptitis B virus correlte with fulminnt nd severe heptitis. J Clin Invest 1993;91: [24] Pollicino T, Znetti AR, Ccciol I, Petit MA, Smedile A, Cpmo S, et l. Pre-S2 defective heptitis B virus infection in ptients with fulminnt heptitis. Heptology 1997;26: [25] Pult I, Chourd T, Wielnd S, Klemenz R, Yniv M, Blum HE. A heptitis B virus mutnt with new heptocyte nucler fctor 1 binding site emerging in trnsplnt-trnsmitted fulminnt heptitis B. Heptology 1997;25: [26] Kpln PM, Gerin JL. Heptitis B-specific DNA polymerse ctivity during post-trnsfusion heptitis. Nture 1974;249: [27] Mizokmi M, Nkno T, Orito E, Tnk Y, Skugw H, Mukide M, et l. Heptitis B virus genotype ssignment using restriction frgment length polymorphism ptterns. FEBS Lett 1999;450: [28] Tong S, Li J, Vitviski L, Trepo C. Active heptitis B virus repliction in the presence of nti-hbe is ssocited with virl vrints contining n inctive pre-c region. Virology 1990;176: [29] Yuh CH, Chng YL, Ting LP. Trnscriptionl regultion of precore nd pregenomic RNAs of heptitis B virus. J Virol 1992;66: [30] Chng HK, Wng BY, Yuh CH, Wei CL, Ting L. A liver specific nucler fctor intercts with the promoter region of the lrge surfce protein gene of humn heptitis B virus. Mol Cell Biol 1989;9: [31] Treinin M, Lub O. Identifiction of promoter element locted upstrem from the heptitis B virus X gene. Mol Cell Biol 1987;7: [32] Yuh CH, Ting LP. The genome of heptitis B virus contins second enhncer: coopertion of two elements within this enhncer is required for its function. J Virol 1990;64: [33] Petrosillo N, Ippolito G, Solforosi L, Vrldo P, Clementi M, Mnzin A, et l. Moleculr epidemiology of n outbrek of fulminnt heptitis B. J Clin Microbiol 2000;8: [34] Koseki T, Hongo S, Murki Y, Sugwr K, Mtsuzki Y, Nkmur K. Sequence nlysis of the entire genome of heptitis B virus from ptient with fulminnt heptitis. Ymgt Med J 1999;17: [35] Yus R, Tkhshi K, Dien BV, Binh NH, Morishit T, Sto K, et l. Properties of heptitis B virus genome recovered from Vietnmese ptients with fulminnt heptitis in comprison with those of cute heptitis. J Med Virol 2000;61: [36] Alexopoulou A, Kryinnis P, Hdziynnis SJ, Hou J, Pickering J, Luo K, et l. Whole genome nlysis of heptitis B virus from four cses of fulminnt heptitis: genetic vribility nd its potentil role in disese pthogenicity. J Virl Hept 1996;3: [37] Ashin Y, Enomoto N, Ogur Y, Skum I, Kuroski M, Izumi N, et l. Complete nucleotide sequences of heptitis B virus genomes ssocited with epidemic fulminnt heptitis. J Med Virol 1996;48: [38] Ogt N, Miller RH, Ishk KG, Purcell RH. The complete nucleotide sequence of pre-core mutnt heptitis B virus implicted in fulminnt heptitis nd its biologicl chrcteriztion in chimpnzees. Virology 1993;194: [39] Hsegw K, Hung J, Rogers SA, Blum HE, Ling TJ. Enhnced repliction of heptitis B virus mutnt ssocited with n epidemic of fulminnt heptitis. J Virol 1994;68: [40] Uchid T, Aye TT, Shimojim M, Gotoh K, Shikt T. Full-length nucleotide sequence of heptitis B virus (HBV) mutnt isolted from ptient with cute heptitis who did not exhibit serologicl mrkers for HBV infection. Int Heptol Commun 1994;2: [41] Uchid T, Gotoh K, Shikt T. Complete nucleotide sequences nd the chrcteristics of two heptitis B virus mutnts cusing serologiclly negtive cute or chronic heptitis B. J Med Virol 1995;45: [42] Sterneck M, Klinin T, Otto S, Gunther S, Fischer L, Burdelski M, et l. Neontl fulminnt heptitis B: structurl nd functionl nlysis of complete heptitis B virus genomes from mother nd infnt. J Infect Dis 1998;177: [43] Seyec JL, Chouteu P, Cnnie I, Guguen-Guillouzo C, Gripon P. Infection process of the heptitis B virus depends on the presence of defined sequence in the pre-s1 domin. J Virol 1999;73: [44] Bock CT, Kubick S, Mnns MP, Trutwein C. Two control elements in the heptitis B virus S-promoter re importnt for full promoter ctivity medited by CCAAT-binding fctor. Heptology 1999;29: [45] Bock CT, Mlek NP, Tillmnn HL, Mnns MP, Trutwein C. The enhncer I core region contributes to the repliction level of heptitis B virus in vivo nd in vitro. J Virol 2000;74: [46] Tokung K, Ishikw Y, Ogw A, Wng H, Mitsung S, Moriym S, et l. Sequence-bsed ssocition nlysis of HLA clss I nd II lleles in Jpnese supports conservtion of common hplotypes. Immunogenetics 1997;46: [47] Mier R, Flk K, Rotzchke O, Mier B, Gnu V, Stevnovic S, et l. Peptide motifs of HLA-A3, -24, nd -B7 molecules s determined by pool sequencing. Immunogenetics 1994;40: [48] Sobo Y, Sugi K, Tomiym H, Sito S, Fujiym S, Morimoto M, et l. Identifiction of heptitis B virus specific CTL epitopes presented by HLA-A*2402, the most common HLA clss I llele in Est Asi. J Heptol 2001;34: [49] Orito E, Ichid T, Skugw H, St M, Horiike N, Hino K, et l. Geogrphic distribution of heptitis B virus (HBV) genotype in ptients with chronic HBV infection in Jpn. Heptology 2001;34: