New trends of HCV infection in China revealed by genetic analysis of viral sequences determined from first-time volunteer blood donors

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1 Journal of Viral Hepatitis, 2011, 18, doi: /j x New trends of HCV infection in China revealed by genetic analysis of viral sequences determined from first-time volunteer blood donors Y. Fu, 1 Y. Wang, 2 W. Xia, 1 O. G. Pybus, 3 W. Qin, 4 L. Lu 4,5 and K. Nelson 6 1 Guangzhou Blood Center, Guangzhou; 2 The Laboratory of Integrated Biosciences, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China; 3 Department of Zoology, University of Oxford, Oxford, UK; 4 Clinical Pathogen Research Center, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; 5 Division of Gastroenterology-Hepatology, and Nutrition, Department of Medicine, University of Utah, Salt Lake City, UT; and 6 Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA Received September 2009; accepted for publication November 2009 SUMMARY. Recently, we studied hepatitis C virus (HCV) sera-prevalence among first-time volunteer blood donors in China. From randomly selected 450 anti-hcv positive donors, we detected HCV RNA in 270 donors. In this study, we amplified HCV E1 and/or NS5B sequences from 236 of these donors followed by DNA sequencing and phylogenetic analysis. The results indicate new trends of HCV infection in China. The HCV genotype distribution differed according to the donorsõ region of origin. Among donors from Guangdong province, we detected subtypes 6a, 1b, 3a, 3b, 2a, and 1a at frequencies of 49.7%, 31.0%, 7.6%, 5.5%, 4.1%, and 2.1%, respectively. Among donors from outside Guangdong, we detected 1b, 2a, 6a, 3b, 3a, 6e, and 6n at frequencies 57.1%, 13.2%, 11.0%, 9.9%, 4.4%, 2.2%, and 2.2%, respectively. Although we found no significant differences among regions in age or gender, subtype 6a was more common (P < 0.001) in donors from Guangdong than those from elsewhere, whilst subtypes 1b (P < 0.02) and 2a (P < 0.001) were more frequent outside Guangdong. Disregarding origins, the male/female ratio was higher for subtype 6a-infected donors (P < 0.05) than for subtype 1b donors, whilst the mean age of subtype 2a donors was 8 10 years older (P < 0.05) than that for all other subtypes. Detailed phylogenetic analysis of our sequence data provides further insight into the transmission of HCV within China, and between China and other countries. The predominance of HCV 6a among blood donors in Guangdong is striking and mandates studies into risk factors for its acquisition. Keywords: blood donor, China, genotyping, HCV, sequence. INTRODUCTION Hepatitis C virus (HCV) is a blood-borne pathogen that presents a major threat to global public health. Worldwide, about 170 million people are infected with HCV [1], and the prevalence varies among countries [1 6]. HCV can cause chronic liver disease in 75 85% of the infected individuals. The outcomes include liver cirrhosis and hepatocellular carcinoma [7,8]. The rapid, global spread of HCV resulted mainly from transmission through blood transfusion [9]. Recently, in countries where donor screening is performed, new cases are Abbreviation: HCV, hepatitis C virus Correspondence: Yongshui Fu, Guangzhou Blood Center, 31 Lu Yuan Road, Guangzhou, Guangdong , China. fuyongshui1969@yahoo.com; and Ling Lu, Division of Gastroenterology-Hepatology, and Nutrition, Department of Medicine, University of Utah, 30N 1900E, SOM 4R118, Salt Lake City, UT 84132, USA. ling.lu@hsc.utah.edu often associated with injection drug use (IDU) and unsafe medical procedures. Other routes are also indicated [10]. Analysis of viral sequences has resulted in classifying HCV into six genotypes and >80 subtypes [11]. HCV genotypes vary in patterns of geographical distribution and therapeutic response. Subtypes 1a, 1b, 2a, 2b, and 3a are Ôglobal epidemicõ. Other genotypes are restricted to particular regions [12 14]. However, the geographical and genetic diversity of HCV is constantly evolving as result of modern transmission and increasing global travel. Hepatitis C virus classification is usually consistent throughout the genome. Recombination appears rare. Hence, the provisional assignment of HCV genotypes/ subtypes can be based on partial genomic regions. Sequences from 5 UTR are optimal for sensitive diagnosis but not sufficient to differentiate subtypes [15,16]. Sequences from the E1 and NS5B regions vary among strains and are suitable for genotyping [12]. Using sequence data from the two regions, we have determined HCV genotype distribution in different

2 New trends of HCV infection in China 43 areas of China. Overall, 1b is the most predominant followed by 2a. However, in Guangdong Province, 6a has replaced 2a as the second most common subtype [17]. The emergence of 6a is probably because of its close association with IDU. This transmission may have been aggravated in recent years. In our previous report, however, samples were only obtained from patients who may not adequately reflect the general population [17]. In this study, specimens were collected from first-time volunteer blood donors [18]. We performed detailed phylogenetic analysis of E1 and NS5B sequences to provide insights into HCV transmission within China, and between China and other countries. MATERIALS AND METHODS From January 2004 to December 2007, a total of first-time volunteer blood donors were recruited. Routine screening detected anti-hcv among 1877 donors. Among the 1877 donors, 450 were selected for RT-PCR, and 270 were found to be HCV RNA+ [18]. cdna from the latter 270 donors were retained and used in this study. Sequencing methods were as previously described [17]. Briefly, HCV fragments were amplified using the Primer STAR kit (Takara, Dalian, China). Amplicons were purified with the QIAquick PCR purification kit (QIAgen, Valencia, CA, USA). DNA was sequenced in both directions on an ABI Prism 3100 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). Sequences were aligned using the CLUS- TAL_X program ( Phylogenies were estimated using the maximum-likelihood method under the HKY+I+G 6 substitution model, implemented in the PHYML program ( Bootstrap resampling was performed using 500 replicates. A variety of referenced sequences were retrieved from Genbank and included for analyses (see Figs 1 4). Guidelines set by the Institution Review Board of the Guangzhou Blood Center and the University of Utah were strictly followed. Written informed consent was obtained from all participants when they donated blood. RESULTS Characteristics of the study group Of the 270 donors, 204 (75.6%) were men, and 66 (24.4%) were women; 178 (65.9%) were from Guangdong, and the remainder from elsewhere. Ages ranged from 21 to 54 years with a mean age of 34.4 years (SD = 6.79 years). Two hundred and sixty-eight donors are of Han ethnicity; one each originates from the Zhuang and Tujia ethnic groups [18]. Hepatitis C virus sequence amplification and classification Of the 270 donors, E1 and NS5B sequences were amplified from 212 and 229 donors, respectively. E1 and NS5B were both obtained for 205 donors, E1 only for 7 and NS5B only for 24. Collectively, amplification of either or both regions was successful for 236 donors (female/male = 57/179, age = 34.5 ± 6.67 years) but failed in 34 (female/male = 9/25, age = ± 6.95). HCV genotypes were determined with the following results: 1b in 97, 6a in 82, 2a in 18, 3a in 15, 3b in 17, 1a in 3, 6e in 2, and 6n in 2 (Table 1). Analysis of subtype 1b sequences In total, 97 subtype 1b isolates were identified: 84 were represented by both E1 and NS5B sequences, 1 represented by E1 only, and 12 by NS5B only. Almost all of the E1 sequences were grouped into five clusters, labelled A, B, C, D, and E containing 37, 29, 8, 2, and 2 sequences, respectively. The bootstrap supports for clusters A E were 83%, 44%, 99%, 88%, and 90%, respectively (Fig. 1). Although many reference sequences were included in the clusters, they were all of Chinese origin [14,17,19 22]. Figure 1 provides an expanded view of cluster A and B (each is collapsed into a branch in the main tree) that were investigated previously and found to coincide with the ÔCultural RevolutionÕ in China [23]. These two lineages appear no different to other branches but selectively spread in China. Clusters C, D, and E may have similar epidemiologic histories, although fewer isolates were identified. The NS5B sequences were also grouped into clusters A, B, C, D, and E, containing 38, 36, 10, 2, and 2 sequences, respectively. Bootstrap supports for clusters A E were 53%, 75%, 73%, 42%, and 24%. Figure 2 shows two further clusters, F and G, having bootstrap scores of 66% and 68%, respectively. All isolates in cluster F were from Yunnan [24], and all isolates in cluster G were from Beijing [25]. Figures 1 and 2 are compared, and the isolates from this study are located in similar positions, indicating reliable sequencing results and no recent viral recombination events or mixed HCV infection. In total, 87.6% (85/97) of the 1b isolates grouped into clusters A, B, or C (Table 2). Consistent with our previous report [17], cluster A is prevalent nationwide, and cluster B is more common in Guangdong. When the E1 and N5B sequences from identical isolates were concatenated, significant bootstrap supports were obtained for both clusters (not shown). Analysis of subtype 6a sequences Subtype 6a sequences were isolated from 82 donors; E1 and NS5B were sequenced from 75 donors, E1 from 4, and NS5B from 3. The E1 tree presents three clusters (denoted I, II, and III) containing 36, 9, and 19 isolates, respectively (Fig. 3). When 573 nt long sequences were analysed, the three clusters exhibited bootstrap scores of 75%, 91%, and 78%. When sequences were trimmed to 410 nt, bootstrap scores were correspondingly reduced to 70%, 83%, and 45%. The

3 GZ Y. Fu et al. 1b 1a 68% E sy2 bi169 bj168 AY (Shanghai) AF (Beijing) GZ0422 bj2 bj2445 sz % sz279 GZ0036 bj594 bj564 U14208 SINGAPORE M84754 (Taiwan) AB JAPAN D13729 INDONESIA D63857 JAPAN GZ % GZ0268 sz226 gz54104 D gz99323 sz894 AF (Jiangsu) sz865 sy9 zz1 V162 USA gz L16635 (Hong Kong) GZ0439 U14203 KOREA D00690 JAPAN GZ0227 S76540 (Taiwan) zz6 U14197 KOREA GZ % GZ0322 GZ0512 C GZ0028 GZ0011 GZ0558 GZ0006 GZ0255 GZ0445 M74888 PRC1 AF JAPAN GZ0218 AF JAPAN AF JAPAN V139 USA D10687 JAPAN D11168 JAPAN D10074 JAPAN V345 USA D28922 JAPAN D10934 (Hebei) km48 GZ0305 V463 USA L38318 BELGIUM U14199 KOREA X76413 FRANCE AF JAPAN sh767 AF (Zhejiang) AF TURKEY L16665 ITALY L16651 USA AF EGYPT L16663 PERU L16637 INDIA D13730 INDONESIA U14218 THAILAND L16654 GERMANY U14211 SINGAPORE L16648 (Taiwan) L16668 SOUTH AFRICA U80385 IRELAND Y09403 ITALY U14210 SINGAPORE D13734 INDONESIA AJ RUSSIA AJ RUSSIA AJ RUSSIA AJ RUSSIA U01214 KOREA V504 USA V296 USA V418 USA V419 USA V374 USA V377 USA V298 USA AF USA V381 USA V291 USA V309 USA X72976 HUNGARY X72978 CROTIA m2 m3 m1 V284 USA X72975 RUSSIA V288 USA V458 USA AF USA L16646 SWEDEN V368 USA GZ0539 L16636 (Hong Kong) gz51942 GZ0514 V350 USA V375 USA D28926 JAPAN D50480 JAPAN AB JAPAN AF JAPAN V152 USA V512 USA D50481 JAPAN V156 USA V142 USA V151 USA fs10 V304 USA V384 USA V503 USA U45476 GERMANY V279 USA V286 USA V351 USA L16667 ITALY V155 USA V352 USA V385 USA M74810 GERMANY V342 USA M74813 USA V164 USA V144 USA V348 USA V276 USA V379 USA D13731 INDONESIA gz53602 D14829 JAPAN AF JAPAN L20498 KOREA L12354 FRANCE V441 USA AF USA V136 USA L16629 DENMARK V149 USA V275 USA V285 USA M74809 ITALY D13406 JAPAN L16647 (Taiwan) gz3 V293 USA X76412 FRANCE B 49A 51B AF SINGAPORE V163 USA V141 USA V367 USA V310 USA V364 USA AY RUSSIA V415 USA AJ00009 AUSTRALIA V313 USA V247 USA 83% 44% GZ0538 gz53643 gz52540 zz3 GZ0130 GZ0469 gb13 AY (Hunan) GZ0113 AF (Anhui) sy4 GZ0210 GZ0570 GZ0437 GZ0438 GZ0471 GZ0326 GZ0431 GZ0018 EF (Hubei) GZ0029 GZ0258 AF (Jiangxi) bj162 GZ0048 sh12 sh321 GZ0436 GZ0201 gz5 sz874 fs5 GZ0124 sh664 GZ0328 AF (Zhejiang) sh666 GZ0544 AY (Shanghai) sh079 GZ0572 GZ0405 sh114 sh13 AF (Shanghai) sh002 AF (Shanghai) GZ0161 GZ0513 fs2 GZ0214 GZ0560 AF (Beijing) sz63 sy3 bj164 bj569 bj670 sy10 bj8386 EF (Hubei) sy6 bj516 gz51586 gb33 GZ0263 sy5 sh334 sy8 GZ0050 AF (Hubei) DW106 (Hubei) QC396 (Hubei) XS047 (Hubei) QC074 (Hubei) QC090 (Hubei) XS048 (Hubei) sh11 SZ213 gz53162 AF (Shandong) EF (Hubei) EF (Hubei) EF (Hubei) GZ0040 GZ0229 bj812 GZ0333 GZ0402 GZ0504 GZ0054 GZ0038 GZ0312 sz36 sz153 GZ0240 DW118 zz8 GZ0137 gz51969 gz99710 gz98739 GZ0016 GZ0154 GZ0108 GZ0004 gz53021 sz482 GZ0508 GZ0467 GZ0556 GZ0232 GZ0237 GZ0548 GZ0205 GZ0053 GZ0168 GZ0414 GZ0543 gb17 gz53998 GZ0315 zz11 zz5 sz125 GZ0162 sz4 GZ0562 zz10 GZ0158 GZ0030 GZ0452 GZ0412 GZ0039 GZ0332 fs7 gb5 GZ0456 gz7 fs4 gz4 GZ0242 fs9 sz747 gz52449 sz214 gz52999 GZ0424 zz bj9997 A B M Fig. 1 Subtype 1b phylogeny estimated from E1 region sequences (H77 positions: ). Subtype 1a sequence M62321 was used as an outgroup. Green circles label reference sequences from outside China, and red circles label sequences from this study. Sequences without a circle are Chinese isolates reported in other studies. Blue circles and dashed arrows represent the locations of clusters A and B, which are shown expanded in two boxes on the right. Three dashed circles indicate clusters C, D, and E and bootstrap support values are shown in italics. Scale bar represents 0.10 nucleotide substitutions per site.

4 SX02 New trends of HCV infection in China 45 1b 1a 92% XJB13 XJB27-47 XJXU2 XJB2 XJB16 XJB31 73% 24% HH068 QJ001 WS105 KM009 WS078 KM016 QJ005 QJ006 F QJ008 HH085 KM017 WS123 KM021 66% YN129 KM035 HH077 AF (Japan) shd-1 Shd-9 shd-4 sz865 GZ0218 shd-2 shd-7 shd-6 shd-8 sh767 D63857 (Japan) km48 P14 P15 P16 P25 68% G P26 P24 P21 P22 P23 HC-C2 P27 P28 P29 P30 P31 P32 P37 P38 P33 P34 P35 P36 gz % sz226 sz894 GZ0268 D GZ0527 gz54104 XJB27-39 U01214 (Korea) U14286 (Indonesia) sy9 S62220 (Japan) X91419 (India) zz6 L23442 (Germany) D50480 (Japan) M84754 (Taiwan) S71627 (Japan) U14291 (Korea) U14281 (Indonesia) U14308 (Singapore) U14305 (Singapore) U28021 (Honduras) fs10 AF (UK) AF (Thailand) AF (Sweden) AB (Japan) L38360 (Brazil) U14296 (Philippines) U14316 (Thailand) AB (Japan) AY (UK) AY D89815 (Japan) AF (Sweden) D10644 (Japan) AF (UK) U14311 (Singapore) U38289 (Madagascar) D10643 (Japan) AF (Sweden) gz53602 D50482 (Japan) D50481 (Japan) AJ (Germany) AJ (France) AY (UK) U14295 (Korea) AF (Argentina) L38356 (Belgium) U80346 (Ireland) X59934 (France) AF (Taiwan) L38372 (France) gz3 U14310 (Singapore) AJ (Australia) M58335 (Japan) L29575 (Belgium) P4 P3 P5 P2 P7 GZ0006 GZ0558 GZ0512 GZ0322 C GZ0028 GZ0011 GZ0255 GZ0075 GZ0009 GZ0056 U14290 (Korea) U14304 (Singapore) HN76 gz51942 GDM4 HK6573 U14284 (Indonesia) AJ (France) AY (UK) M87630 (Spain) U14307 (Singapore) GZ0227 GZ0051 GZ0539 GZ0445 GZ0514 sz279 sz2891 bj2 HeBei shd-5 bj169 bj168 sy2 bj594 bj2445 GZ0422 GZ0036 HN38M HN39 D00826 (Japan) D11168 (Japan) GZ0439 gz HK6552 GZ0305 zz1 L23443 (Spain) m2 m3 m1 U14285 (Indonesia) U14283 (Indonesia) HK6557 AF (Russia) E zz2 M % 23% (53%) B A GZ0121 HN11 GZ0014 GZ0467 GZ0137 GZ0205 GZ0162 GZ0240 GZ0424 GZ0237 GZ0168 GZ0242 GZ0556 GZ0452 GZ0562 GZ0536 GZ0039 GZ0332 GZ0053 GZ0232 GZ0543 GZ0030 GZ0414 GZ0303 GZ0154 GZ0548 GZ0108 GZ0220 GZ0358 GZ0306 GZ0412 GZ0004 GZ0016 GZ0315 GZ0508 HN2 sz153 sz36 zz7 gz53021 fs4 gz7 GZ0456 HK6553 gz99710 sz4 zz10 km10 bj564 HN25 sz63 zz11 zz8 gb5 sz747 gz53998 gz51969 zz5 sz125 gz98739 sz214 gz4 sz482 fs7 gz52449 fs9 gz52999 gb17 bj9997 P17 P1 P39 sh12 gb33 sh321 zz9 P20 sy4 gz51586 HCV-S sh002 sh114 sz874 sh13 gz5 sh079 bj162 P18 P19 shd-3 GZ0040 GZ0229 bj812 sh334 sy8 sy5 bj164 GZ0050 sh666 GZ0201 sy10 sy6 sy3 bj670 bj8386 GZ0328 GZ0544 sh11 bj516 HN78 HN30M HN50 bj569 SX1 HN55 sh664 HN37 GZ0214 GZ0572 GZ0048 GZ0124 GZ0431 GZ0129 GZ0263 GZ0258 GZ0504 GZ0054 GZ0038 GZ0333 GZ0312 GZ0049 GZ0437 GZ0438 GZ0570 GZ0471 GZ0113 GZ0405 zz3 fs5 fs2 gz53643 gb13 GZ0130 gz53162 GZ0326 GZ0436 GZ0161 GZ0538 GZ0513 GZ0029 GZ0560 gz52540 GZ0469 GZ0103 GZ0210 GZ0018 GZ0158 P Fig. 2 Subtype 1b phylogeny estimated from NS5B region sequences (H77 positions: ). Subtype 1a sequence M62321 was used as an outgroup. All labels are the same as those described in the Fig. 1 legend. In addition, yellow circles represent unpublished sequences obtained by us from Chinese patients coinfected with HIV-1 [40], and dashed circles indicate clusters F and G.

5 GZ0462 fs14 gb28 46 Y. Fu et al. GZ0465 GZ0131 GZ % I fs13 fs3 GZ0127 GZ0152 GZ0115 gz1 GZ0222 GZ0253 GZ0316 GZ0069 GZ0261 GZ0068 GZ0420 GZ0334 GZ0172 GZ0072 GZB0002 GZ0354 GZ0027 GZ0403 fs6 fs14 GZ0247 gz8 GZ0228 GZ0309 GZ0569 GZ0266 GZ0462 GZ0131 GZ0465 GZ0256 GZ0517 GZ0447 GZ0546 GZ0455 GZ0551 gb28 GZ0046 I gz1 fs13 fs3 GZ0266 GZ0115 fs6 GZ0546 GZ0152 GZ0256 GZ0334 GZB0002 GZ0569 gz GZ0309 GZ0247 PN118 PN135 PN311 PN094 PN128 BY438 BY448 BY674 BY522 BY449 BY580 PN123 GZ0403 GZ C BY720 BY725 GZ0253 GZ0068 GZ GZ0069 GZ0072 GZ0222 GZ0537 GZ0228 GZ0447 GZ0127 GZ0517 GZ % 91% III GZ0215 GZ0224 GZ0461 GZ0537 GZ0329 GZ0552 GZ0270 GZ0020 GZ0344 GZ0404 GZA0001 GZ0411 GZ0532 GZ0307 gb26 sz826 GZ0223 GZ0366 6a64 6a72 6a69 GZ0144 GZ0453 GZ0313 gb9 GZ0311 GZ0472 GZ0012 6a61 6a67 GZ0565 fs12 GZ0545 6a33 6a66 6a62 GZ0206 EUHK2 GZ0364 6a35 6a77 6a73 6a63 6a65 6a74 II GZ0367 gb14 GZ0216 GZ0074 GZ0343 GZ0111 GZ0167 GZ0044 GZ0047 GZ0510 GZ0045 GZ0221 GZ0466 GZ0225 GZ0351 GZC0003 fs1 GZ0534 GZ0207 GZE0003 sz593 GZ0458 GZ0034 GZ0104 VN571 Vietnam VN569 Vietnam VN11 Vietnam VN506 Vietnam VN538 Vietnam 70% 70% 87% III 86% IV II 6a35 GZ GZ0354 BY411 BY684 BY571 BY723 GZ0261 GZ0316 GZ QC158 Chinese Immigrant in Canada HK2 GZ0224 GZ0215 GZ0366 6a67 14A 6a fs12 6a33 6a66 6a63 BY604 PN155 PN017 gb9 GZ0565 6a64 GZ0329 VN11 Vietnam EUHK2 6a77 sz593 GZ0216 GZ0472 PN026 6a69 GZ0551 GZ0270 GZA0001 GZ0552 gb26 GZ0344 sz GZ0307 GZ0411 GZ0532 6a61 GZ0545 GZ A 8D 55D GZ0364 GZ0012 GZ0111 GZ0167 GZ0510 GZ0466 GZ0045 GZ0221 GZ0207 GZE0003 BY401 fs1 GZ0225 GZ0534 GZ0351 GZC0003 GZ0034 GZ0104 GZ0311 6a62 gb14 GZ0453 PN270 PN160 PN116 PN126 PN a72 6a74 GZ0367 GZ0313 GZ0144 PN326 PN028 PN005 PN100 PN312 PN269 PN061 PN264 6a65 PN080 13C GZ0404 GZ D GZ GZ0074 GZ0343 GZ0458 GZ0047 GZ0044 QC26 Vietnamese Immigrant in Canada % V VN506 Vietnam VN571 Vietnam PN090 PN HH075 HH064 HH093 HH081 2D D WS083 7A QC242 Vietnamese Immigrant in Canada QC296 Vietnamese Immigrant in Canada 6a 6b QC169 Vietnamese Immigrant in Canada VN569 Vietnam QC318 Vietnamese Immigrant in Canada QC168 Vietnamese Immigrant in Canada VN538 Vietnam D Fig. 3 Subtype 6a phylogeny estimated from E1 region sequences (H77 positions: ). Subtype 6b sequence D84263 was used as an outgroup. All labels are the same as those described in the Fig. 1 legend. In addition, five dashed circles indicate clusters I, II, III, IV, and V. The box on the left contains a smaller phylogeny estimated from longer 510 nt sequences, which shows stronger bootstrap support for clusters I, II, and III.

6 GZ0027 New trends of HCV infection in China 47 85% I 98% 98% II 99% III GZ0127 GZ0569 GZ0462 GZ0465 GZ0046 GZ0131 GZ0403 GZ0247 GZ0261 GZ0152 GZ0447 GZ0256 GZ0517 GZ0072 GZ0222 GZ0334 GZ0354 GZ0546 GZB0002 GZ0172 GZ0068 GZ0420 GZ0115 GZ0266 GZ0228 GZ0309 GZ0069 GZ0253 GZ0316 GZ0537 GZ0551 GZ0215 GZ0224 GZ0329 GZ0552 GZ0270 GZ0307 GZ0532 GZ0411 GZ0020 GZ0344 GZ0404 GZA0001 GZ0311 GZ0313 6a74 6a66 6a67 GZ0472 6a62 GZ0012 6a33 EUHK2 GZ0206 GZ0545 6a35 6a61 6a77 6a63 6a65 6a64 GZ0367 GZ0364 GZ0565 GZ0225 GZ0458 GZ0534 GZE0003 GZ0351 GZ0034 GZ0104 GZ0207 GZ0045 GZ0221 GZ0510 GZ0466 GZ0044 GZ0074 GZ0343 GZ0111 GZ0167 6a73 GZ0144 GZ0453 GZ0216 6a69 6a72 GZ0223 GZ0366 VN506 Vietnam VN11 Vietnam 80% II I GZ0344 GZ0552 6a64 GZ0532 GZ0270 GZ0307 GZ0411 GZ0565 fs12 6a61 HKGS24 HKG9P HKG19P HKGS31 GZ0404 TW12142 HKG22P HKGS21 HKGS1 6a33 HKGS25 6a35 LZ117 LZ165 LZ105 LZ099 LZ036 GZ0152 fs14 GZ0462 GZ0261 GZ0403 GZ0465 GZ0517 GZ0447 GZ0256 TW3759 GZB0002 gz8 GZ0420 GZ0253 GZ0068 GZ0247 GZ0266 GZ0115 HKG4P GZ0569 GZ0448 GZ0354 GZ0334 GZ0222 GZ0046 GZ0169 GZ0131 GZ0172 gz1 GZ0127 GZ0546 GZ0027 GZ0551 fs13 GZ0069 GZ0224 QC158 Chinese Immigrant in Canada GZ0316 GZ0228 GZ0309 GZ0537 GZ0072 GZ0215 gb28 VN693 Vietnam gb26 D52 HKG14P HKGS15 6a62 GZ0020 HKGS23 GZ0066 GZA0001 GZ0311 gb9 GZ0313 GZ0367 sz826 TW10520 TW346 TW12137 GZ0216 HKGS17 HKG3P Y12083 gb14 HKGS10 GZ0545 HKGS27 GZ0472 GZ0329 6a73 GZ0223 HKGS30 6a63 HKGS14 GZ0012 6a65 6a66 6a77 GZ0034 GZ0364 QC296 Vietnamese Immigrant in Canada D1 GZ0366 TW10350 GZ0144 D31 GZ0206 6a67 GZ0453 VN569 Vietnam VN571 Vietnam VN538 Vietnam III TW2442 HKGS29 GZ0534 GZ0104 GZ0044 GZ0207 GZ0074 GZ0343 GZ0111 GZ0167 GZ0045 GZ0221 GZ0466 GZ0510 GZ0351 GZ0225 GZ0458 sz593 fs1 D45 6a69 6a72 D2 VN11 Vietnam TW7458 TW9413 GZE0003 QC168 Canada VN826 Vietnam VN710 Vietnam VN824 Vietnam QC242 Vietnamese immigrant in Canada Vietnamese immigrant in the U.S. QC26 Vietnamese immigrant VN746 Vietnam D22 Vietnam QC318 Vietnamese immigrant in Canada VN853 Vietnam D16 D23 D75 VN865 Vietnam VN506 Vietnam VN753 Vietnam VN930 Vietnam D12 VN538 Vietnam D78 VN569 Vietnam VN689 Vietnam VN541 Vietnam VN555 Vietnam WS083 6a 6b D9 6a74 D17 VN571 Vietnam D Vietnamese Immigrant in the USS. QC169 Vietnamese Immigrant in Canada HH093 HH081 D Fig. 4 Subtype 6a phylogeny estimated from E1 region sequences (H77 positions: ) Subtype 6b sequence D84263 was used as an outgroup. All labels are the same as those described in the Fig. 1 legend. In addition, three dashed circles indicate clusters I, II, and III. The box on the left contains a smaller phylogeny estimated from a longer alignment of concatenated E1 + NS5B sequences (870 nt), which shows very high bootstrap support for clusters I, II, and III.

7 48 Y. Fu et al. Table 1 Hepatitis C virus genotypes and donor groupings Genotype 1a (n =3) 1b (n = 97) 2a (n = 18) 3a (n = 15) 3b (n = 17) 6a (n = 82) 6e (n =2) 6n (n =2) Total (n = 236) Men* Women Age (years) ± ± ± ± ± ± ± ± ± 6.77 E1 + NS5B E1 only NS5B only Guangdong (Group 1) Other areas (Group 2) Total (n = 236) 1b (n = 97) 6a (n = 82) Group Guangdong (n = 145) Non-Guangdong (n = 91) Guangdong (n = 45) Non-Guangdong (n = 52) Guangdong (n = 72) Non-Guangdong (n = 10) Men Women Ages (years) ± ± ± ± ± ± 4.64 *P < 0.05 was observed in 1b comparing with 6a. P < 0.05 was observed in 2a comparing with 1b, 3a, 3b, and 6a, respectively. Table 2 Cluster and origin of 1b isolates Province A (n = 39) B (n = 36) C (n = 10) D (n =2) Chongqing 1 Guangdong Guangxi 1 2 Hebei 1 Henan 3 1 Hubei 8 1 Hunan 8 8 Jiangsu 1 Jiangxi 2 Jilin 1 Liaoning 1 Shandong 1 Shanxi Shanghai 1 Sichuan 1 1 E (n =2) shorter alignment enabled the inclusion of more reference strains, which released two further clusters (denoted IV and V) having bootstrap scores of 90% and 78%, respectively. Clusters IV and V contained isolates from Yunnan, Hubei, and Guangxi [24,26,27]. Sixteen reference sequences were from Hong Kong, but none grouped within clusters I to V. Isolates from IDUs in Guangxi and Hubei appeared throughout the tree [26,27]. Generally, more derived sequences were from China [17], whereas more ancestral sequences were from Vietnam or immigrants from Vietnam. Among the NS5B sequences, 36, 10, and 16 grouped within clusters I, II, and III, respectively (Fig. 4). Only cluster I had a significant bootstrap score of 80%. In both trees, identical isolates were placed in the same clusters (Figs 3 & 4). Reference sequences of Vietnamese origin tended to locate nearer the tree base. Phylogenetic signal was increased by concatenating E1 and NS5B sequences from identical isolates: the rectangle in Fig. 4 shows the phylogeny. Clusters I, II, and III were again present and supported with bootstrap scores of 86%, 95%, and 88%, respectively. Only five Vietnamese reference sequences could be concatenated; they were placed near the tree root. Analysis of subtype 2a sequences Subtype 2a sequences were isolated from 18 donors; E1 and NS5B were sequenced from 16 donors, whilst E1 only and NS5B only from one each. In both E1 and NS5B trees, subtype 2a isolates do not exhibit a clear geographical pattern (Figure S1). In the E1 tree, we observed three clusters mainly composed of Chinese isolates [17]. The NS5B tree showed two notable clusters: one containing isolates from France and the other representing isolates from a single hospital in Beijing [25]. Analysis of subtype 3a sequences Subtype 3a sequences were isolated from 15 donors; E1 and NS5B were from nine donors, E1 only from one, and NS5B

8 New trends of HCV infection in China 49 only from five. In the both trees (Figures S2 & S3), our majority isolates formed a single cluster, supported by bootstrap scores of 81% and 60%. In the E1 tree, three wellsupported UK clusters were observed. In the NS5B tree, there was substantial geographical mixing: isolates from different continents tend to group together, whilst sequences from the same country were spread throughout the tree. Some geographical clusters were apparent but exhibited no strong bootstrap support. In contrast, sequences from China formed two distinct groups. Analysis of subtype 3b sequences Subtype 3b sequences were isolated from 17 donors; E1 and NS5B were from 15 donors and NS5B only from two. Some reference isolates from China were mingled with the strains from this study. They formed a single Chinese cluster with bootstrap support of 78% in the E1 tree and 44% in the NS5B tree. Other reference sequences were from South and Southeast Asia and placed nearer the root of the tree (Figure S4). Hence, subtype 3b likely originated from neighbouring countries and is now growing in China, particularly in Yunnan [24] and Guangxi [26] and among IDUs [27]. Analysis of subtype 1a sequences Subtype 1a isolates were sampled from three donors with both E1 and NS5B sequences amplified. We have previously sequenced a 1a strain in a patient from Shanghai [17]. Excluding this, no 1a sequences in Genbank were from China. Phylogenetically, the three 1a isolates appeared not to group with that from the Shanghai patient (Figure S5). In the E1 tree, the three isolates formed a cluster but only exhibited a bootstrap score of 44%. In the NS5B tree, the three isolates were dispersed among strains from other countries. Although six 1a strains from Taiwan formed a small cluster [28], they were not related to the isolates reported here. These results suggest that subtype 1a in China is resulted from sporadic importation events. Analysis of subtype 6e and 6n sequences Subtype 6e and 6n sequences were obtained from two donors each. Phylogenetic analysis grouped the two 6e isolates with GX004 and one 6n isolate close to KM42 (Figure S6). In the E1 tree, the Chinese 6e and 6n clusters were supported by bootstrap scores of 100% and 98%, respectively. In the NS5B tree, no strong support was obtained. GZ0355 was from a donor from Guangxi and was grouped with six subtype 6e isolates all from Guangxi. Similarly, GZ0203 was from a donor from Guizhou and closely grouped with six 6n isolates from Yunnan, which is adjacent to Guizhou [17,24]. Hepatitis C virus genotype distribution Hepatitis C virus genotypes were determined from 236 donors. We classified these donors into two groups. The Guangdong group contains 145 donors all from Guangdong Province. The non-guangdong group contains 91 donors all from other locations (Table 1). Within the Guangdong group, subtypes 6a, 1b, 3a, 3b, 2a, and 1a accounted for 49.7%, 31.0%, 7.6%, 5.5%, 4.1%, and 2.1%, respectively. Within the non-guangdong group, 1b, 2a, 6a, 3b, 3a, 6e, and 6n accounted for 57.1%, 13.2%, 11.0%, 9.9%, 4.4%, 2.2%, and 2.2%. Between groups, HCV genotype patterns differed. Subtype 6a was more frequent (P < 0.001) in Guangdong group; 1b (P < 0.02) and 2a (P < 0.001) more frequent in non-guangdong group (Table 1). The donorsõ age and gender are not statistically different between groups. HCV subtypes showed no significant relationship with the year of sampling (data not shown). Taking the 236 donors as a whole, 1b, 6a, 2a, 3b, 3a, 1a, 6e, and 6n accounted for 41.1%, 34.8%, 7.6%, 7.2%, 6.4%, 1.3%, 0.8%, and 0.8%, respectively. This subtype pattern resembles that previously reported [17]. Dividing the donors by HCV subtype, we observed two trends (Table 1). First, the male/female ratio among those infected with 6a was higher (P < 0.05) than among those infected with 1b. Second, the mean age of subtype 2a donors was older (P < 0.05) than that of donors infected with other strains. DISCUSSION In this study, we observed different patterns of HCV genotype distribution among two groups of blood donors. Subtype 6a was predominant in Guangdong group, whilst 1b and 2a predominant in non-guangdong group. In 2002, we completed a similar study of 139 patients from nine cities in China. We found that 1b, 2a, and 6a accounted for 66.2%, 13.7%, and 10.1% of infections, respectively. Importantly, among patients from Guangdong, subtype 6a has replaced 2a and become the second most common subtype, accounting for 21.2% (14/66). In contrast, no 6a has been detected among patients from other areas [17]. Findings from the current study indicate further spread of 6a infections in China. Among donors from Guangdong, 6a has become the dominant HCV genotype, accounting for 49.7%. Among donors from other areas, subtype 6a was also detected in 10.6%. Studies have identified 6a prevalence in Hong Kong, Macau, Thailand, and Vietnam. Other studies have found 6a in Singapore (U U908309) and Taiwan [28]. In Hong Kong, 6a has been detected in 27 30% of HCVinfected donors and 60% of HCV-infected IDUs. In Hong Kong, 6a appears to spread to the general population mainly through IDUs. We have postulated that subtype 6a in Guangdong was introduced from Hong Kong [17], because of the geographical proximity of the two locations and

9 50 Y. Fu et al. because the subtype was detected earlier in Hong Kong. Whilst this may be true for some cases, it is not sufficient to explain the recent 6a spread in mainland China. Phylogenetically, 6a sequences from Guangdong formed three clusters (denoted I, II, and III). Cluster I also contained sequences from IDUs from different regions of China, including cities in Guangxi province bordering Vietnam [26], Liuzhou in Guangxi [29], and Wuhan in Hubei province [27]. Clusters I and II may represent 6a strains originating in Guangdong and now starting to seed IDU networks elsewhere. Five subtype 6a isolates from IDUs in Taiwan [28] also grouped in cluster II, indicating the IDU networks extended from mainland China to Taiwan. Spreading of HIV in IDUs via this route has also been reported [30]. Cluster VI was among IDUs in Guangxi only. Cluster V could be an outcome of interchange between IDU networks in Yunnan and Guangxi [25 27,31]. Phylogenetically, subtype 6a sequences from Hong Kong are distinct from those isolated in mainland China [17], suggesting that the 6a circulation in mainland China may not be directly linked to that in Hong Kong. The causes of 6a emergence in China are unknown. Based on phylogenies, we hypothesize that subtype 6a originated in Vietnam, or perhaps pre-existed in southern China. A possible historical event for the importation of 6a to China was the emigration of Vietnamese to China from the late 1970s to early 1980s [32]. Hong Kong also received such emigrants, mostly from South Vietnam [33]. Many ethnic Chinese from North Vietnam fled to China and were resettled in Yunnan, Guangxi, and Guangdong provinces [34]. Some 6a strains may have entered the blood transfusion networks and undergone transmission among recipients, particularly before the discovery of HCV in 1989, or through unsafe medical procedures prior to the governmental ban on paid blood donors in 1998 [35]. Although no large-scale transfusion-linked infections were recorded, some individuals could have been infected by the use of contaminated plasma or unscreened medical infusions, such as foetal liver cells and therapeutic immunocytes that were injected commonly in China during 1990s [36,37]. Guangdong was the first region of mainland China to undergo economic reforms because of its proximity to Hong Kong. It was opened to outside investment and has undergone rapid economic growth. This has resulted in greater social exchange with other countries and has attracted millions of internal Ômigrant labourersõ [38]. The numbers of commercial sexual workers and IDUs have risen alongside this economic growth, providing more opportunity for 6a strains to spread to the general population [39]. In Guangdong, the relative prevalence of 6a has grown from undetectable levels to >20% of infections [17]. In this study, we demonstrate further 6a growth, such that it has become the dominant HCV strain, infecting 49.7% of donors in this study. The spread has occurred despite the outlawing of paid blood donors [35] and improved healthcare standards. Other transmission routes most likely explain the growth of 6a infection. Recent increases in the numbers of IDUs have been reported. IDUs can transmit 6a strains to distant areas, via known drug trafficking routes [31]. This is the first study of HCV genotype distribution among first-time volunteer blood donors in China. Subtype 1b was the most common, accounting for 41.1%. Most of these isolates were classified into two clusters, A and B. This is consistent with our previous study [17] and indicates that the geographical distribution of the two clusters in China remains largely unchanged. Overall, a marked decrease in the proportion of 1b infections was observed, down from 66.2% (92/139) in our previous report [17] to 41.1% (97/ 236) in the present study. Explanations are the relative increase in 6a infections and/or a larger portion of donors from Guangdong. However, if we consider only donors from outside Guangdong, a decreased 1b frequency is still observed (57.1%). Subtype 1b strains are likely more associated with transmission via blood transfusion and medical procedures. Effective measures to reduce these transmissions have lead to decreased 1b infections. In contrast, subtype 6a strains may be more linked to IDU and sexual transmission, which are both increasing risks in Guangdong. We found that subtype 2a infected donors were significantly older (8 10 years) than those infected by other HCV strains. Phylogenetically, 2a sequences from China did not form clear geographical clusters. The relative prevalence of 2a has declined in comparison to our previous report [17], which is apparent when the Guangdong group and non-guangdong group are considered separately or as a whole. Collectively, it is suggested that new cases of HCV 2a infection in China have been reduced. CONFLICTS OF INTEREST None reported. FINANCIAL SUPPORT The project described was supported by a grant from The National Institute of Allergy and Infectious Diseases (1R01AI A109, Dr Ling Lu), a grant from The Bureau of Health, Guangzhou Municipality, China (No.2008-ZDi-10, Dr Yongshui Fu), and a grant from The National Natural Science Foundation of China (No , Dr Yongshui Fu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sequences reported in this study have been submitted in Genbank. Their accession numbers are: GQ GQ REFERENCES 1 World Health Organization. Hepatitis C global prevalence. Wkly Epidemiol Rec 1999; 74:

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