HGV variants in China

Virus Research 73 (2001) 131– 144 www.elsevier.com/locate/virusres

Detection and genotyping of GBV-C/HGV variants in China Ling Lu a,b,e, Mun Hon Ng a, Bei-Ping Zhou c, Hong-Tao Luo d, Tatsunori Nakano e, Betty H. Robertson e, Stanley W.K. Im a,* a

Department of Microbiology, Uni6ersity Pathology Building, The Uni6ersity of Hong Kong, Queen Mary Hospital Compound, Pokfulam Road, Hong Kong b Department of Infectious Diseases, Third Affiliated Hospital, Research Center of Molecular Medicine, Sun Yat-sen Uni6ersity of Medical Sciences, Guangzhou City, Guangdong Pro6ince, People’s Republic of China c The Eastlake Hospital, Shenzhen City, Guangdong Pro6ince, People’s Republic of China d Department of Infectious Diseases, The First People’s Hospital, Foshan City, Guangdong Pro6ince, People’s Republic of China e Hepatitis Branch, DVRD/NCID, Centers for Disease Control and Pre6ention, Atlanta, GA 30333, USA Received 27 October 1998; received in revised form 16 October 2000; accepted 16 October 2000

Abstract We detected GBV-C/HGV sequences in the sera from 64 out of a total of 324 subjects in the south of China. In agreement with findings of others, we noted an especially high rate of infection among intravenous drug addicts and patients with chronic hepatitis C virus infection. The detection was achieved by nested PCR to amplify the 5% noncoding region (5%NCR) of the viral genome. Sequence analysis of the resulting 234 bp product revealed a total of 26 different sequences of which 25 were found to belong to the genotype G3, which is the most prevalent genotypes among Asian isolates, and one belonged to genotype G1, common among African isolates. The sequence divergence between the genotypes was largely clustered in a short variable region (V2) within the 5%NCR, and we showed that genotyping may be achieved equally well by analysis of this variable region as by the more detail analysis of the entire 5%NCR or of the entire viral genome. © 2001 Elsevier Science B.V. All rights reserved. Keywords: GBV-C/HGV; Genotype; China; 5% Noncoding region

1. Introduction GBV-C/HGV refers to a virus group within the family Flaviviridae (Zuckerman, 1996). The pro-

* Corresponding author. Tel.: +852-28554891; fax: + 85228551241. E-mail address: [email protected] (S.W.K. Im).

totypes of these viruses are GBV-A and GBV-B, isolated after serial transmission of sera from a patient with non A to E hepatitis in tamarins (Karayiannis et al., 1989; Simons et al., 1995a). Subsequently, GBV-C (Simons et al., 1995b) and HGV (Linnen et al., 1996) were identified as the human counterpart of GBV-A and GBV-B. Since GBV-C and HGV share 95% amino acid homology, they are referred to as GBV-C/HGV. Their

0168-1702/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 0 ) 0 0 2 3 1 - 8

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Fig. 1. Genomic organization of HGV-GD and strategy for its cloning and sequencing. The diagram shows the length of the HGV-GD nucleotide sequence. The ten cDNA fragments amplified by PCR or RACE PCR were 5%, a, b, c, d, e, f, g, h and 3%. For each fragment, four clones were sequenced. (5%) The fragment covered 1 – 125 nt, (a) fragment covered 111– 1781 nt, (b) fragment covered 1758– 3100 nt, (c) fragment covered 3080–4554 nt, (d) fragment covered 4535– 5343 nt, (e) fragment covered 5324– 6000 nt, (f) fragment covered 5978–6927 nt, (g) fragment covered 6908– 8489 nt, and (h) fragment covered 8480– 9093 nt, and (3%) fragment covered 9052– 9382 nt.

genomic organization is similar to that of hepatitis C virus (HCV), except that unlike HCV, the 5% noncoding region (5’NCR) of GBV-C/HGV is variable, with nucleotide variation clustering in three segments referred to as variable regions V1, V2 and V3 (Hsieh et al., 1997). The sequence variation within 5%NCR (Muerhoff et al., 1996; An et al., 1997) mirrors that observed within the entire viral genomes (Okamoto et al., 1997; Wang et al., 1997), and can be used to classify natural isolates into three genotypes, G1, G2 and G3. Genotype G1 (also referred to as GB) contains predominently African isolates, genotype G2 (or HG) includes isolates from North America and Europe, while most Asian isolates are of the genotype G3. The distribution of GBV-C/HGV genotypes in China has not fully been understood. One study in a southwestern province indicated the presence of all three genotypes (Wu et al., 1996), while another found only genotype G3 variants in the northwest and in the east (An et al., 1997). In this study, the distribution of GBV-C/HGV genotypes, in a southern province Guangdong that is adjacent to Hong Kong, was further examined, and the possibility of using V2 to determine GBVC/HGV genotype was exploited.

2. Materials and methods

2.1. Collection of sera Serum specimens from subjects with parenteral

exposure risk were collected in three cities in Guangdong Province of China. The three cities are Guangzhou, Shenzhen, and Foshan, all are within 200 km from Hong Kong. The 342 specimens were from individuals in the following risk groups: 105 intravenous drug abusers (IVDA); 80 commercial blood donors; 63 patients with chronic non-A-E hepatitis, 33 patients with chronic hepatitis C; and 61 patients with chronic hepatitis B. All these sera were tested with EIA for HBsAg, anti-HAV, anti-HCV and anti-HEV. The EIA anti-HCV negative sera were also tested with RT-PCR for HCV-RNA. HBV infection was diagnosed as HBsAg positive and absence of antiHAV, anti-HCV and anti-HEV. HCV infection was determined as anti-HCV and/or HCV-RNA positive and absence of HBsAg, anti-HAV and anti-HEV. For non-A-E hepatitis, the sera were negative to all the virus markers mentioned above.

Table 1 Serum HGV RNA detection Patient group

n

HGV RNA+

%

Intravenous drug addict Commercial blood donor Non-A-E hepatitis Chronic hepatitis C Chronic hepatitis B

80 105 63 33 61

37 9 6 7 5

46.3 8.6 9.5 21.2 8.2

Total

342

64

18.7

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133

Fig. 2. Multiple alignment of the 190 nt segment (excluding the primer sequences) of the HGV 5%NCR from 26 Chinese strains and ten published GBV-C/HGV genomes. The capital letters represent the sequences determined and the lower case letters represent the sequences used as primers. Matched nucleotide residues are represented by dots. Nucleotide substitutions are indicated. Gaps are represented by horizontal bars. Nucleotide numbering corresponds to that described for U44402. The shaded rectangle marks the variable region 2 (V2). Isolates 1, 3–4, 8–13, 17, 20, 22 and 23 were isolated from intravenous drug abusers; 28 and 30 – 33 from commercial blood donors; 37 and 40 from patients with non-A-E hepatitis; 41 – 43 and 45 from patients with chronic hepatitis C; and 47 and 50 from patients with chronic hepatitis B.

2.2. Detection of HGV RNA by nested PCR and cDNA cloning The QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) was used for RNA extraction. The RNA was transcribed into cDNA using the SuperScript™ Preamplification System (Gibco BRL, Gaithersburg, USA) according to the manufacturer’s instruction. The cDNA from each sample was amplified by nested PCR with two sets of primers. The first round of PCR was performed with an outer primer set N1/N2 and the second round with an inner primer set N3/N4. The location of

the primer sequences with reference to U44402 (Linnen et al., 1996) in the 5%NCR were 112 –136 nt for N1, 452 –476 nt for N2, 161 –185 nt for N3, and 376 –398 nt for N4. The final expected product was 238 bp in length, including the 50 bp of the primers. One microlitre of the first round PCR product was used as template for the second round. Both rounds were run for 30 cycles of 94, 55 and 72°C, each for 40 s, using reagents from the PCR core kit (Boehringer Mannheim, GmbH, Germany). Products of the second-round PCR were analyzed by electrophoresis on 3% agarose gel. The PCR products of appropriate size were

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purified and cloned into the pT-ADv vector (Clontech, Palo Alto, USA) for sequencing.

2.4. Determination of the complete genome of HGV-GD

2.3. Analysis of cDNA sequences

The strategy for elucidating the complete HGVGD genome by PCR and RACE PCR is depicted in Fig. 1. The cDNA was synthesized from the serum of a child with acute icteric non-A-E hepatitis from whose serum GBV-C/HGV RNA was first detected by nested RT-PCR. The child was seronegative for HAV, HBV, HCV, and HEV diagnostic markers as determined with EIA or PCR. The PCR was performed using the Expand™ Long Template PCR System (Boehringer, Mannheim, GmbH, Germany) and the RACE PCR was carried out using the 5% RACE System and 3% RACE System (Gibco BRL, Gaithersburg, USA). All the products of appropriate sizes were subsequently purified by LMP agarose gel electrophoresis and cloned into the pT-ADv vector (Clontech) for sequencing.

cDNA clones were sequenced in both directions by using fluorescent dye terminator cycle sequencing method with an ABI 377 sequencer and the dRhodamine terminator kit (Applied Biosystem, Foster, USA). Nucleotide sequences were compiled and aligned using GCG Package (version 10.0, Genetics Computer Group, Madison, WI). Phylogenetic trees were plotted using the PHYLIP package (version 3.572, Phylogeny Inference Package, University of Washington, Seatle, WA) and the LASERGENE software (DNASTAR, Madison, WI). Maximum likelihoods were analyzed with PHYLIP package and mapped with PUZZLE software (version 4.0, University Munchen, Munich, Germany) (Strimmer and von Haeseler, 1997).

Fig. 3. Phylogentic tree plotted with 5%NCR sequences of HGV from our 26 isolates and ten published reference sequences listed in Fig. 2. Our isolates were marked by Arabic numerals and the ten reference sequences labeled with English abbreviations. Three circles encompass three groupings and the three percentages represent values of bootstrap analyses. The ruler beneath the tree measures the genetic distances between sequences, which was calculated with Kimura’s model of the DNADIST program from the PHYLIP package. One unit scale represents a distance of 0.01 substitution per nucleotide position. The maximum distance obtained was 0.1850 (between isolate 22 and GBV-C) and the minimum distance obtained was 0.0000 (between Gsi85 and Gsi93). Bootstrap analysis was carried out using the program of SEQBOOT, DNADIST, NEIGHBOR and CONSENSE from PHYLIP with 1000 random resampling times.

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Table 2 Designation, Genbank Accession number, and genotype of the GBV-C/HGV isolates used in the phylogenetic analysis of V2a in Fig. 4b Acc. No.c

Isolated

Geo. Reg.e

Typef

References

G1 Af003186 Af003181 C21596

Zai – 11 Zai – 6 Af-13

Zaire Zaire Africa

Group 1 Group 1 GB type

Af003180 U59540 C21605

Zai – 5 23 Af-96

Zaire West Africa Africa

Group 1 Subtype 1a GB type

Af003182 Af003184 U59541 U36380 U87882 C21595

Zai – 7 Zai – 9 24 GBV-C HGV09 Af-107

Zaire Zaire West Africa West Africa USA Africa

Group 1 Group 1 Subtype la G1 Genotype IIg GB type

Af003179 U59555 D84542

Zai – 1 39 CHNaLC14

Zaire West Africa China

Group 1 Subtype 1b Group 1

U59549 U59548 U59547 U59553 C21701 U87883 U59545 Af017536 Af003178 C21601

33 32 31 37 KORS131 GV10 28 11 Zai – 2 Af-53

West Africa West Africa West Africa West Africa Korea USA West Africa China Zaire Africa

Subtype lb Subtype lb Subtype lb Subtype 1b GB type Genotype IIg Subtype la G1 Group 1 GB type

U59544 C21598

27 Af-42

West Africa Africa

Subtype la GB type

C21604

Af-87

Africa

GB type

C21597

Af-26

Africa

GB type

Smith et al. (1997) (Af003152–86) Smith et al. (1997) (Af003152–86) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Smith et al. 1997) (Af003152–86) Muerhoff et al. (1996) (U59518–58) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Smith et al. (1997) (Af003152–86) Smith et al. (1997) (Af003152–86) Muerhoff et al. (1996) (U59518–58) Okamoto et al. (1997), An et al. (1997) (U86148–58) Ding et al. (1997) (U87874–91) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Smith et al. (1997) (Af003152–86) Muerhoff et al. (1996) (U59518–58) Kato et al. (1997) (D86481–90), Wu et al. (1996) (D84533–43) Muerhoff et al. (1996) (U59518–58) Muerhoff et al. (1996) (U59518–58) Muerhoff et al. (1996) (U59518–58) Muerhoff et al. (1996) (U59518–58) Park et al. (1997) (C21698, C21700–06) Ding et al. (1997) (U87874–91) Muerhoff et al. (1996) (U59518–58) This study (Af017534–59) Smith et al. (1997) (Af003152–86) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Muerhoff et al. (1996) (U59518–58) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685)

G2 Af003168 D90600 Af003175 C21676

Ed – 34 GT110 Ed – 74 Sa-29

Scotland Japan Scotland Saudi

Group 2a G2 Group 2a HG type

Smith et al. (1997) (Af003152–86) Okamoto et al. (1997) Smith et al. (1997) (Af003152–86) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685)

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136 Table 2 (Continued) Acc. No.c

Isolated

Geo. Reg.e

Typef

References

U59522 D84543 U44402 Af019320 Af019318 Af019323 Af003163 Af019321 U59524 U87875 Af019319 U59518 C21685

5 CHNaLC189 PNF2161 HD88 HD59 HD95 Pak – 554 HD89 7 HGV02 HD84 1 Sa-97

Europe China USA Spain Spain Spain Pakistan Spain USA USA Spain USA Saudi

Subtype 2a Group 2 G2 Close to HGV1 Close to HGVl Close to HGV1 Group 2a Close to HGV1 Subtype 2a Genotype I Close to HGV1 Subtype 2a HG type

D84539 Af003159 D86488 Af019315 D84536 Af019308 U59529 Af019317 Af019299 U59535 U63715 C21639

CHNaHCC59 Pak – 512 Ja-03 HD48 CHNaCH8 HD30 12 HD54 HD1 18 C-EA Ja-S4

China Pakistan Japan Spain China Spain Europe Spain Spain USA East Africa Japan

Group 2 Group 2a Group 2 Close to HGV1 Group 2 Close to HGV1 Subtype 2b Close to HGV1 Close to HGV1 Subtype 2b Group 2b HG type

Muerhoff et al. (1996) (U59518–58) Kato et al. (1997) (D86481–90), Wu et al. (1996) (D84533–43) Linnen et al. (1996), Okamoto et al. (1997) Forns et al. (1997) (Af019299–323) Forns et al. (1997) (Af019299–323) Forns et al. (1997) (Af019299–323) Smith et al. (1997) (Af003152–86) Forns et al. (1997) (Af019299–323) Muerhoff et al. (1996) (U59518–58) Ding et al. (1997) (U87874–91) Forns et al. (1997) (Af019299–323) Muerhoff et al. (1996) (U59518–58) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Kato et al. (1997) (D86481–90), Wu et al. (1996) (D84533–43) Smith et al. (1997) (Af003152–86) Kato et al. (1997) (D86481–90) Forns et al. (1997) (Af019299–323) Kato et al. (1997) (D86481–90), Wu et al. (1996) (D84533–43) Forns et al. (1997) (Af019299–323) Muerhoff et al. (1996) (U59518–58) Forns et al. (1997) (Af019299–323) Forns et al. (1997) (Af019299–323) Muerhoff et al. (1996) (U59518–58) Erker et al. (1996), Smith et al. (1997) (Af003152–86) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685)

G3 AF017558 D86485 Af017556 U76894 U86150 Af017541 C21624

50 Ja-02 45 T338 LZ3 22 Is-21

China Japan China Taiwan China China Isreal

G3 Group 3 G3 Taiwan typeh HGV type 3 G3 New type

C21625

Is-28

Israel

New type

D87262 U76893 Af017538 Af017557 Af017540 Af017537

HGV Gsi85 T-138 13 50 20 12

Japan Taiwan China China China China

G3 Taiwan type G3 G3 G3 G3

This study (Af017534–59) Kato et al. (1997) (D86481–90) This study (Af017534–59) Hsieh et al. (1997) (U76892–4) An et al. (1997) (U86148–58) This study (Af017534–59) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Nakao et al. (1997) Hsieh et al. (1997) (U76892–4) This study (Af017534–59) This study (Af017534–59) This study (Af017534–59) This study (Af017534–59)

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Table 2 (Continued) Acc. No.c

Isolated

Geo. Reg.e

Typef

References

D86484 D86486 Af017555 U86157 U86156 U86154 U86158 U86155 C21626

Ja-09 Ja-07 47 SH2 SH1 Nj1 SH3 Nj2 Ja-12

Japan Japan China China China China China China Japan

Group 3 Group 3 G3 HGV type HGV type HGV type HGV type HGV type New type

C21704

KORS201

Korea

New type

Kato et al. (1997) (D86481–90) Kato et al. (1997) (D86481–90) This study (Af017534–59) An et al. (1997) (U86148–58) An et al. (1997) (U86148–58) An et al. (1997) (U86148–58) An et al. (1997) (U86148–58) An et al. (1997) (U86148–58) Mukaide et al. (1997) (C21595–99, C21601, C21604–05, C21624–26, C21631, C21637, C21639–40, C21642, C21676, C21682, C21685) Park et al. (1997) (C21698, C21700–06)

3 3 3 3 3

a Of the 236 isolates analyzed, 45 were grouped in G1, 104 in G2, and 87 in G3. In this table, only 83 isolates with distinct V2s are listed. b Bold values refers to isolate with variable region V2 completely identical with those of other isolates. Af003180: Af003183, Af003185. U59541: U59542. U59555: U59556–58. U59549: U59550–52. U59553: U59554. U59545: C21682, U59546. Af017536: Af003176, U87874. Af003178: C21599, U59543. AF017558: C21651. D87262: Ab003673–75, Ab003678–79, C21631, C21642, D83498, D86481, D86483, D87250, D87263, D90601. Af017557: U86148–49. AF017540: C21706, D83500, D87253, U59538. Af017537: Af003177, Af017534–35, Af017539, Af017542–54, Af017559, C21647, C21652–55, C21700, C21703, D84533–35, D84540– 41, D86489–90, D87249, D87251–52, D87254, U59539, U76892, U86151–53. U86155: C21702, D86482. U59522: Af019312, U59523. U44402: Af003152–58, Af003160–62, Af003164–67, Af003169–74, Af019300, Af019303–04, Af019307, Af019310–11, Af019313–14, Af019316, C21637, C21649, C21698, C21705, D84537–38, D86487, D87255, U45966, U59519–21, U59525–28, U87874, U87876–78, U87881, U87885–91. Af019321: Af019322. Af019315: Af019305–06. U59529: U59530–34, U59536–37. Af019299: Af019309. U63715: Af019301–02, C21640, U87879–80. c Accession number in Genbank. d Isolate designation. e Geographic region of isolation. f Presumed or determined type/genotype/subtype in the related references. g In its original publication, genotype I resembles HGV (G2) and genotype II resembles GBV-C (G3). h In its original publication, Taiwan type is distinct from American (G2) and West African type (G1). It is consistent to the Asian type (G3).

3. Results

3.1. Detection and genotyping of GBV-C/HGV isolates We tested 324 sera for the presence of 5%NCR sequence of GBV-C/HGV by nested PCR and 64 of which were found positive as determined by the presence of the expected 238 bp product (Table 1). The high rate of detection of GBV-C/HGV among the intravenous drug addicts and chronic hepatitis C patients confirm earlier findings by other investigators (Dawson et al., 1996; Linnen

et al., 1996; Schleicher et al., 1996; Schlueter et al., 1996; Schreier et al., 1996; Stark et al., 1996; Alter et al., 1997; Tacke et al., 1997). Sequence analysis of the 64 amplicons revealed 26 distinct sequences. These sequences, which are available in Genbank (Accession no. AF017534 – AF017559), were compared with the corresponding segments of 10 published GBV-C/HGV isolates. They include GBV-C (U30382), the prototype strain in genotype G1 found in west Africa; GBV-C-EA (u63715), the east African isolate of genotype G2; Pnf2l61 (u44402) and R10291 (u45966), the American isolates of geno-

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type G2; HGV-Iw (d87255) and GT110 (d90600), the Japanese isolates of genotype G2; HGVC964 (u75356), the first Chinese isolate of uncertain genotype; and GT230 (d90601), Gsi85 (d87262) and Gsi93 (d87263), the Japanese isolates of genotype G3 (Simons et al., 1995b; Erker et al., 1996; Linnen et al., 1996; Shao et al., 1996; Nakao et al., 1997; Okamoto et al., 1997). As the multiple alignment shown in Fig. 2, when the primer regions are excluded the nucleotide variations cluster between 186 and 213 nt (shaded region in Fig. 2), which is known as variable region 2 (V2) (Hsieh et al., 1997). The V2s of our isolates are distinct, but closely resemble the Japanese isolates. The genotype of our isolates was established from a phylogenetic tree (Fig. 3) and verified through a bootstrap analysis. Twenty-five of our isolates were grouped into genotype G3, together with the Japanese isolates GT230, Gsi85 and Gsi93. One isolate, 11 was categorized into genotype G1, but none into genotype G2.

3.2. Genotyping using V2

Fig. 4. Phylogenetic tree of V2 region from 82 distinct HGV isolates presented in Table 2. The tree was constructed using the Megalign program from the Lasergene (DNASTAR) package. The accession numbers appeared after the horizontal lines from top to bottom are in an exact order to those listed in Table 2. The grayed numbers mark the sequences isolated in this study.

As shown in Fig. 2, the bulk of nucleotide variations are located in the V2 region. This suggested to us that this 38 nt segment may be exploited for direct determination of GBV-C/ HGV genotype. To test this notion, we repeated genotyping of our 26 sequences with 209 published GBV-C/HGV strains from different geographic areas, which included Africa, North America, East Asia, the Middle East and Europe. The genotype of these 209 isolates has been previously proposed or established based upon analyses of long sections of nucleotide sequences, varying in size from several hundred nucleotides to the entire genome (Simons et al., 1995b; Erker et al., 1996; Muerhoff et al., 1996; An et al., 1997; Ding et al., 1997; Forns et al., 1997; Fukushi et al., 1996; Hsieh et al., 1997; Kato et al., 1997; Kondo et al., 1997; Linnen et al., 1996; Mukaide et al., 1997; Nakao et al., 1997; Okamoto et al., 1997; Park et al., 1997; Shao et al., 1996; Smith et al., 1997; Wu et al., 1996). Their basic informa-

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Fig. 5. Maximum likelihood analysis of V2 region from 82 distinct GBV-C/HGV isolates. (A) Phylogenetic tree of equal branch length plotted with PHYLIP package. DNAML program was used to generate the treefile and DRAWTREE used to construct the tree. Double stars (**) represent a positive branch (PB0.01) statistically significant to clustering. Seven branches were named representing seven isolates with their clustering not statistically proven in this analysis and therefore, subjected to further evaluation by maximum-likelihood-mapping. (B) Maximum-likelihood-mapping statistics analyzed with PUZZLE software, for the clustering of seven unproven isolates. Totally, seven mappings were analyzed with each mapping for each one of the seven unproven sequences and the rest of the six unproven sequences excluded. Thus, in each mapping, an unproven sequence was assigned as a group to be determined and all the other well-resolved sequences of three genotypes (G1, G2, G3) as three groups to receive its mapping. Under each isolate there are two triangles each with its three angles corresponding to three GBV-C/HGV grenotypes, namely the left angle corresponding to G3, the up angle to G2 and the right angle to G1. Inside the upper seven triangles, the three likelihoods are represented as points. And within the lower seven triangles, the number of quartets are given in seven areas. In the result, all seven sequences are grouped into genotype G2 since each of their clustering percentages is over 94% within the G2 area.

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tion and V2 analysis are summarized in Table 2. It tells that all the previously proposed genotypic identities, including our 26 isolates, can be distinguished by analysis of V2 region. Fig. 4 presents phylogenetic tree constructed using 82 distinct V2 sequences from the 235 isolates, showing their phylogenetic relations. All isolates are accounted for in the three genotypes G1, G2 and G3. The maximum likelihoods of clustering of these 82 distinct V2 sequences were analyzed, which resulted in a more phylogenetic tree with equal branch lengths (Fig. 5(A)) showing that the clustering of all 82 isolates is consistent with the grouping in Fig. 4. However, excluding seven sequences (branches named in Fig. 5(A)) from the genotype G2, the clustering of all the rest of the isolates is statistical significant (P B0.01). The seven statistically unproven sequences were further analyzed by maximum likelihood-mapping. The method is based on an analysis of the maximum likelihoods for three fully resolved tree topologies that can be computed for four or four groups of sequences. The three likelihoods are represented as points inside an equilateral triangle. The triangle is partitioned into seven regions. The center of the triangle represents a starlike evolution whereas the three corners represent three clades that have been well-resolved and the three intermediate regions between corners reflect the difficulty in distinguishing between two of the three clades. The more points distributed in a certain region of a particular corner, the bigger support for the mapped sequence to join in the

well-resolved clade. Seven mappings were analyzed with each mapping for each one of the seven unproven sequences and the rest of the six unproven sequences excluded. The resulting seven pairs of triangles are presented in Fig. 5(B). It shows that each of the mapping over 94% of the quartets, as represented by points, were distributed into the top corner of the corresponding triangle supporting the classification of the seven unproven sequences into the clade of genotype G2.

3.3. Comparison of genotyping by analysis of V2 and complete genome The complete sequence of one isolate from our collection was delineated. It was designated HGVGD and its Genbank accession number is AF006500. To further substantiate the determination of GBV-C/HGV genotype by V2 region, we took HGV-GD and 28 other complete GBV-C/HGV sequences which we retrieved from Genbank, and subjected them to phylogenetic analysis (Simons et al., 1995b; Erker et al., 1996; Linnen et al., 1996; Shao et al., 1996; Nakao et al., 1997; Okamoto et al., 1997; Takahashi et al., 1997; Wang et al., 1997; Bukh et al., 1998; Katayama et al., 1998). Three phylogenetic trees verified through bootstrap analyses are shown in Fig. 6. Fig. 6(A) was generated from comparing complete genomes, Fig. 6(B) from V2 region comparison, and Fig. 6(C) derived from comparison of de-

Fig. 6. Phylogenetic tree of 29 GBV-C/HGV sequences. (A) Comparing their complete nucleotide sequences. (B) Comparing their V2 regions (C) Comparing their complete amino acid sequences predicted from the nucleotide sequences. References to isolates: Ab003288– 91, 93 to Takahashi et al. (1997). Af031827-29 to Bukh et al. (1998). Ab008335 and D87708– 15 to Katayama et al. (1998). D87262 and D87263 to Nakao et al. (1997). D90600 and D90601 to Okamoto et al. (1997). U36380 to Simons et al. (1995b). U44402 and U45966 to Linnen et al. (1996). U63715 to Erker et al. (1996). U75356 to Zhou et al. (1996). U94695 to Wang et al. (1997). Ab008342 was only published in Genbank DataBase. Af006500 refers the isolate of HGV-GD in this study. G1, G2 and G3 label the HGV genotypes. The percentages represent the values of bootstrap analyses each resampled 1000 times. The horizontal bar beneath each tree indicates a unit of nucleotide or amino acid substitutions per site. From PHYLIP package, the genetic distances of trees A and B were calculated using Kimura two-parameter method and of tree C calculated using Dayhoff PAM matrix. The Neighbor-Joining program was used to generate treefile for trees A and C while the Fitch program used for tree B. Bootstrap analyses were carried out using the program of SEQBOOT, DNADIST/PROTDIST, NEIGHBOR/FITCH, and CONSENSE with 1000 random resampling times.

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Fig. 6. (Continued)

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duced amino acids. With no discrepancy, all three approaches produced comparable genotype results, although the genetic distances between the isolates showed some variations.

4. Discussion In the course of examining the 5%NCR of GBVC/HGV isolated from 64 individuals in Guangdong province of China, we have discerned 26 distinct sequences. In line with current understanding, we observed that nucleotide variations in these sequences are found mainly in the variable region V2, a small span of 38 nt within the 5%NCR. We further observed that genotyping of our 26 sequences using V2 instead of the longer 5%NCR segment produced the consistent results. This has led us to explore the usage of V2 region for determination of GBV-C/ HGV genotype. For this purpose, we repeated genotyping by using V2 analysis on an additional 209 published GBV-C/HGV isolates. The genotypes of all 209 isolates have been previously proposed from analysis of long sections of the genome, varying in size from several hundred nucleotides to the entire genome. Their genotypes were confirmed by V2 analysis. Essentially the same conclusion could be drawn from our further study using 29 complete GBV-C/ HGV sequences. One of these was an isolate of our collection, the rest were retrieved from Genbank. These conclusions were valid by analysis of the complete genomes, deduced amino acids, and V2 regions itself, the 29 isolates being consistently typable within three genotypes, the G1, G2 or G3. While the genomic organization of the GBV-C/ HGV is similar to that of other Flaviviruses, it appears to have distinct characteristics (Leary et al., 1996). Between 70% and 75% of variations concentrate in three identifiable regions called variable regions V1, V2 and V3, and they are located in the 5%NCR (Hsieh et al., 1997). Other parts of the genome (viz. E2, NS3, NS5b) are relatively conserved and deemed not suitable for genotyping (Kao et al., 1996; Tsuda et al., 1996; Muerhoff et al., 1997; Pickering et al., 1997; Smith et al., 1997;

Viazov et al., 1997). On the other hand, the small variable region V2, where genomic variations concentrate, is proven in this study to be useful for genotyping, especially when large numbers of isolates are involved. Our 26 isolates from Guangdong province of China are more closely related to the Japanese variants than to the African, North American and European variants. All but one has been shown to belong to G3 together with the Japanese isolates. However, our understanding of the geographic distribution of GBV-C/HGV genotypes in China is incomplete. Nearly half of the isolates in Guangxi, a southwestern province that is adjacent to Vietnam, belong to G2 (Wu et al., 1996). This differs from our study and from results obtained from the northeast and along the east coast (An et al., 1997). All of the isolates tested by An and colleagues belonged to genotype G3. Available data therefore indicate that genotype G3 is more prevalent in China. Intravenous drug abusers accounted for 46% of the GBV-C/HGV RNA positive sera in our study, and this falls in the reported range of 40–50% (Schlueter et al., 1996; Schreier et al., 1996; Stark et al., 1996; Tacke et al., 1997). The extent of drug abuse in China is largely underestimated and represents an important source of GBV-C/HGV transmission. The prevalence of GBV-C/HGV RNA positive sera in hepatitis C patients was also in agreement with published data (Dawson et al., 1996; Linnen et al., 1996; Schleicher et al., 1996; Alter et al., 1997), and significantly higher than that in non-A-E hepatitis and hepatitis B. The high rate of ‘coinfection’ with HCV was not surprising since both HCV and GBV-C/HGV are blood borne agents (Choo et al., 1989; Wang et al., 1996).

Acknowledgements This work was supported by the Committee on Research and Conference Grants (CRCG) of The University of Hong Kong, the Industrial Support Fund from the industrial department of the government of Hong Kong Special Administrative Region, and a special grant for high-technology development in the year 1996 –1997 from the Com-

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