Zoonotic risk of hepatitis E virus (HEV): A study ... - Wiley Online Library

Hepatology Research 2009; 39: 1153–1158

Original Article

hepr_558

doi: 10.1111/j.1872-034X.2009.00558.x

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Zoonotic risk of hepatitis E virus (HEV): A study of HEV infection in animals and humans in suburbs of Beijing Yibin Chang,1 Ling Wang,1* Jiabao Geng,1 Yonghong Zhu,1 Hongwei Fu,1 Furong Ren,2 Lingjun Li,1 Xiaojuan Wang1 and Hui Zhuang1* 1

Department of Microbiology, Peking University Health Science Center, and 2Beijing Red Cross Blood Center, Beijing, China

Aim: To investigate hepatitis E virus (HEV) infection among different animals and workers in pig farms and slaughterhouses, and analyze the genotype of HEV isolated in this study. Methods: Serum samples were collected from adult swine, cows, sheep, younger swine (< 3 months), and workers in pig farms and slaughterhouses (professional group). Fecal samples were collected from younger swine in the south suburbs of Beijing. Anti-HEV antibody was evaluated by direct sandwich enzyme immunoassay. HEV RNA was extracted from fecal samples and ampliﬁed by nested reverse transcription polymerase chain reaction (RT-nPCR). The PCR products were sequenced, and the sequence homology and phylogenetics of the HEV strains isolated from swine were analyzed.

60.73% (99/164), 42.51% (105/247) and 20.29% (522/2572), respectively. The HEV RNA positivity rate of fecal samples was 22.89% (19/83) and 16/19 samples were positive for HEV RNA ampliﬁed with both primers, HEV open reading frame (ORF)1 and HEV ORF2. Sequence analysis of these 16 samples showed that there were two groups, designated BJ-1 and BJ-2. The nucleotide homology of BJ-1 and BJ-2 was 99%. Phylogenetic analysis indicated that both of these groups belonged to genotype 4d.

Conclusion: Workers in pig farms and slaughterhouses were more likely to contract HEV infection than the general population because of close contact with swine with a high prevalence of anti-HEV.

Results: The anti-HEV positivity rates in adult swine, cows, sheep, younger swine, professional group and general population were 98.23% (222/226), 29.35% (54/184), 9.80% (20/207),

Key words: genotype, hepatitis E virus, subgenotype, zoonosis

INTRODUCTION

infection has been identified after ingestion of meat from HEV-infected deer or swine have been reported, and the HEV sequence isolated from the meat was the same as that isolated from the patients, definitively demonstrating zoonotic transmission of HEV.2 Various species of animals, such as non-human primates, rodents, swine, cows and horses, have been found with a high prevalence of anti-HEV antibody3–10 since Meng11 isolated HEV from swine in 1997. Some laboratory studies have shown that human HEV could infect monkeys, swine and rats,3,12,13 and swine HEV could infect rhesus monkeys,3,14 and these results strongly suggested the zoonotic risks of HEV. Based on the generally accepted sequence analysis, HEV isolates identified worldwide have been classified into four genotypes (genotypes 1–4).15,16 All these genotypes except genotype 2 which has been reported to only infect humans have been isolated from both humans and animals.2,5,6 Genotype 5, which is unusual in that its genome lacks

H

EPATITIS E VIRUS (HEV), which was previously referred to as an enterically transmitted non-A, non-B hepatitis virus, is the causative agent of acute self-limited or fulminant hepatitis E, and is considered to be endemic in many developing countries where sanitation conditions are suboptimal. However, recent studies have documented hepatitis E in individuals from several industrialized nations without any history of travel to endemic countries.1 HEV is transmitted primarily by the fecal–oral route. Cases in which HEV Correspondence: Assoc. Professor Ling Wang, Department of Microbiology, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing 100191, China. Email: [email protected] *These authors contributed equally to the study. Received 21 December 2008; revision 4 May 2009; accepted 7 May 2009.

© 2009 The Japan Society of Hepatology

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open reading frame (ORF)3 was recently found in avian species,17 and subsequent studies showed that this avian HEV could infect turkeys but not rhesus monkeys in laboratory conditions.18,19 Genotypes 1 and 2 have been considered responsible for large hepatitis E epidemics in the world, such as the hepatitis E that occurred in the Xingjiang Uighur Autonomous Region in China in 1986–1988,20 whereas HEV genotypes 3 and 4 usually have induced sporadic infection. In China, it was found previously that HEV genotypes 1 and 4 were dominant in humans and genotype 4 was the prevailing type in swine. However, genotype 3 HEV has recently been found to be widespread in pig farms of Shanghai suburbs.21,22 Many studies have been carried out in China to compare human HEV and animal HEV in order to better understand HEV zoonosis. Our research group has engaged in a series of studies on the zoonotic risks of HEV in China, including recent successful establishment of a rhesus monkey HEV infection model using swine HEV of subgenotypes 4a and 4b, which further demonstrated the possibility of cross-species infection of HEV.14 The aim of the current study was to investigate HEV infection among various animals and people working in the pig farms and slaughterhouses in the south suburbs of Beijing, and to analyze the genotype and subgenotype of HEV isolated in this study in order to obtain more information about zoonotic risks of HEV in China.


Enzyme-linked immunosorbent assay All the serum samples were evaluated for anti-HEV antibody by the method of direct sandwich enzyme immunoassay developed by Wantai Biopharmaceutical (Beijing, China). All assay procedures were carried out according to the manufacturer’s instructions. Briefly, amino acids 394–604 of the HEV genotype 1 ORF2 were expressed in Escherichia coli as a fusion protein with Glutathione-S-Transferase (GST). After purification, GST was removed from the recombinant polypeptide by cleavage with thrombin. Microtiter plates coated with this polypeptide were used to capture antibodies from 50 mL of serum. Bound antibodies were detected using this same polypeptide labeled with horseradish peroxidase (HRP). Information about these assays has been described in previous studies.5,14,23–25

Nested reverse transcription polymerase chain reaction (RT-nPCR)

METHODS

Total RNA was extracted from 100 mL of serum or a 10% fecal suspension solution (1 g fecal dissolved into 10 mL phosphate buffered saline [PBS] solution, pH 7.4) according to the instructions with the kit (Z5100; Promega, Madison, WI, USA). All samples were analyzed with RT-nPCR using primers (Table 1) which were designed based on HEV genotype 1–4 (GenBank accession numbers of the reference HEV strains: AB108537, AJ272108, AY594199, M74506, AF060668, 11093, 11092,L08816,M94177 and L25547). RT-nPCR was carried out as previously described.14,26 The final PCR product was analyzed by 1.5% (w/v) agarose gel electrophoresis.

Serum and fecal samples

Sequencing

S

The expected PCR products amplified from swine fecal samples were purified with a Gel Purify Recovery Kit

EVEN HUNDRED AND eighty-one serum samples were collected from adult swine (226), cattle (184), sheep (207) and younger swine (164) from several slaughterhouses and stock farms, and 83 fecal samples from younger swine were collected from two pig farms. A total of 247 human blood samples were collected, including 52 from individuals working in livestock farms and 195 from individuals working in slaughterhouses (professional group, aged 18–59 years; average age, 37 years; 162 male [65.59%] and 85 female [34.41%]). All the above samples were from the south suburbs of Beijing. 2572 serum samples (aged 18–52 years; average age, 32 years; 1621 male [63.02%] and 951 female [36.98%]) were selected from blood donors of Beijing Red Cross Blood Center. All samples were stored at -80°C pending use.


Table 1 Sequences of primers for nested reverse transcription polymerase chain reaction Primer Sequence P1 P2 P3 P4 S1 S2 S3

Site (nt)†

5′-AGGCT CCTTG CRTCA CTA-3′ 56–73 5′-GCCYT KGCGA ATGCT GTG-3′ 112–129 5′-CRCGG GTAGG RGCRG TAT-3′ 406–389 5′-CCMGT CTCRG CRGSA AAR-3′ 512–495 5′-ATGTY CGYAT YYTWG TCCA-3′ 5887–5905 5′-TGGCG YTCKG TTGAG ACCTC Y-3′ 5967–5987 5′-CDGCC GACGA AATCA ATTCT G-3′ 6369–6349

R = A/G; Y = C/T; S = C/G; W = A/T; K = G/T; D = G/A/T; M = A/C. †Site on the genome of Chinese swine strain (GenBank accession no.: AY594199).


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Table 2 Prevalence of anti-HEV antibodies in animals and humans Sample category

Case no.

Anti-HEV positive no.

Anti-HEV positivity rate %

Adult swine Young swine† Adult cow Adult sheep Pig farm workers General population Slaughterhouse workers

226 164 184 207 52 2572 195

222 99 54 20 35 522 70

98.23 60.73 29.35 9.80 67.31 20.29 35.90

†Two of the serum samples from young swine were HEV RNA positive.

(Axygen, Union City, CA, USA) and cloned into a pGEM-T vector (Promega). The target PCR fragments were sequenced bilaterally (Sangon, Shanghai, China).

Statistical analysis Data comparisons were performed using the c2-test with Fisher’s exact test. Differences were considered significant at P < 0.05.

Phylogenetic analysis The bilateral sequence was spliced by the Bioedit ver. 4.8.10 software and then BLASTed in the National Center for Biotechnology Information (NCBI). The homology between different isolated strains and different genotypes of HEV were analyzed with Vector NTI Suite ver. 9.0 software. The resemblance of nucleotide sequences was calculated with GeneDoc software, and phylogenetic analysis was carried out with TreeView ver. 1.6.5 software. The reference sequences15 for phylogenetic analysis of HEV ORF2 were retrieved from GenBank (genotype 1: M73218, 11092, 11093, M80581, 98292, 99441 and 10330; genotype 2: M74506; genotype 3: AF060668; AF060669 and AF082843; genotype 4: AJ272108, AY594199, AF296162, AF117275, AF134812 and AJ34417; genotype 4a: AF296162, AF117275, AJ344171 and AF134812; genotype 4b: AB124818, AF117280, AJ344186, AF103940 and AJ428856; genotype 4c: AB107367, AB105888, AB105902, AB094223, AB082545 and AB079762; genotype 4d: AY596308, AJ428853, AY594199 and AJ272108).

RESULTS Prevalence of anti-HEV antibodies

T

HE ANTI-HEV antibody positivity rates for adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. For general population and professional groups, the anti-HEV antibody positivity rates were 20.29% (522/2572) and 42.51% (105/ 247), respectively (Table 2).

Phylogenetic analysis Nineteen of 83 fecal samples from younger swine tested HEV RNA positive when amplified with HEV ORF1 primers, and 16 of them were also HEV RNA positive with HEV ORF2 primers. Sequence analysis of these 16 samples showed that there were two groups of HEV which we designated as BJ-1 (11 samples) and BJ-2 (5 samples). The nucleotide homology of BJ-1 and BJ-2 (GenBank accession no.: FJ 480213 and FJ 480212) was 99%. Phylogenetic analysis based on comparison of

Table 3 Homology comparison of partial sequences of isolated hepatitis E virus (HEV) strains with HEV genotype 1–4 (%)

BJ-2 BJ-1 4 3 2 1

1

2

3

4

BJ-1

BJ-2

75–77 76–77 72–80 74–77 75–78 89–98

73 73 70–78 71–73 100

77–78 76–78 69–79 90–91

81–91 81–91 78–92

99 100

100

BJ-2 BJ-1 4d 4c 4b 4a

4a

4b

4c

4d

BJ-1

BJ-2

85–87 84–86 83–87 83–87 82–87 91–98

84–88 84–86 82–88 82–87 84–92

84–88 84–89 82–87 88–99

88–91 88–91 90–99

99 100

100


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AY594199

100 52

AY596308

77

AJ428853 Subgenotype 4d

AJ272108

90

100

71

BJ-1

(FJ 480213)

BJ-2

(FJ 480212)

AB124818

54

AF117280 Subgenotype 4b

AF344186

85

61

AF103940

75 61

AJ428856

Genotype 4

AB079762 Ab082545

100 97

59

AB094223 Subgenotype 4c AB105902

33

AB105888

100 93

AB107367

AF134812 AJ344171

99

Subgenotype 4a AF117275

99 100

AF296162 AF060669

93

AF080843 M74506

44 100

86

Genotype 3

AF060668.

100

Genotype 2

D11092 D11093 M80581 X98292

100

Genotype 1

X99441

74

D10330

90 100

M73218

0.05

Figure 1 Phylogenetic analysis of partial nucleotide sequences of hepatitis E virus open-reading frame 2.



partial ORF2 (362 bp) sequences of all 16 swine HEV indicated that BJ-1 and BJ-2 belonged to genotype 4d. The homologies of HEV ORF2 nucleotide sequences between BJ-1, BJ-2 and genotypes 1–4 were 75–77%, 73%, 76–78% and 81–91%, respectively (Table 3). The identities of HEV ORF2 nucleotide sequences between BJ-1, BJ-2 and genotypes 4a, 4b, 4c and 4d were 84–87%, 84–88%, 84–89% and 88–91%, respectively. Phylogenetic analysis with Treeview software also showed that BJ-1 and BJ-2 were on the branch of the HEV 4 genotype and belonged to the 4d subgenotype (Fig. 1). Only two serum samples from younger swine were RNA positive when amplified with both HEV ORF1 and ORF2 primers, and the sequences of the positive HEV RNA were the same as BJ-1.

DISCUSSION

M

ANY STUDIES HAVE suggested that hepatitis E is a zoonosis, and more and more animal species have been found infected by HEV. Swine are suspected to be the principal animal reservoir, especially in geographic regions where swine and human HEV strains are genetically closely related.2,5 This study showed that the anti-HEV antibody positivity rate was the highest in adult swine (98.23%) among the animals enrolled in this study, and the HEV RNA positivity rate in fecal samples in younger swine was as high as 22.89%. Moreover, two serum samples from young swine were positive for HEV RNA amplified with both primers HEV ORF1 and HEV ORF2. However, we were unable to match them to the fecal samples with positive HEV RNA. This result suggested that younger swine may be an important source of HEV infection, and hygienic management of the feces of young swine may be helpful in controlling the transmission of HEV. Furthermore, comparing the prevalence of anti-HEV in the general Chinese population (20.29%), we found that HEV infection rate of people working in pig farms (67.31%) and slaughterhouse (35.90%) was significantly higher (P < 0.0001). This result was similar to that in previous studies where HEV infectivity rates were higher in professional workers than in the general population in other countries.27–29 This indicated that HEV infection in this professional group, and particularly in the individuals working in pig farms may be associated with close and frequent contact with younger pigs, which in turn strongly suggests the possibility of HEV transmission from swine to humans. However, there is no scientifically cogent evidence supporting this point of view as we

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could not detect HEV RNA in the professional group. Nonetheless, our results supported the hypothesis that swine are the dominant animal reservoir and that younger swine are most important in HEV transmission between swine and humans. Although HEV strains have been isolated from several animal species, most isolates have been found in pigs. Investigations conducted in commercial pig herds in various countries have shown that swine HEV infection is common and widespread.5,30,31 In China, most HEV isolated from swine were genotype 4 (4a in Xinjiang and Beijing; 4b in Guangxi and Beijing; 4c in Liaoning and Henan; and 4d in Guangdong, Beijing and Xinjiang15) except the genotype 3 isolated from swine in Shanghai recently.21,22 In the present study, the swine HEV isolates shared high sequence identity (91%) with HEV genotype 4 of human strains which was isolated from a patient in the Beijing area (GenBank accession no.: AJ272108),32 and phylogenetic analysis confirmed that the swine isolated strains belonged to the subgenotype 4d, which was the same as the strain from the patient as showed in Figure 1. Previous phylogenetic analysis showed that genotype 4 HEV in the Beijing area could be subdivided into three subgroups, 4a, 4b and 4d, which have been found circulating among humans and swine. Subgroup 4a predominated in prevalence over subgroups 4b and 4d in both human and swine isolates.15 All previous studies have suggested that the distributions of HEV genotypes and subgenotypes differed in age, and geography.33,34 The close genetic relationship between human and swine HEV found in the present study suggests the possibility of zoonotic infection with genotype 4 HEV in the south suburbs of Beijing.

ACKNOWLEDGMENTS

W

E ARE GRATEFUL to Dr Michael A. McNutt for proofreading the manuscript. This work was funded by the 863 National High Technology Research and Development Program of China (grant 2006A 02Z453) and the National Natural Science Foundation of China (grant 30570063).

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