Swine as a Principal R e servoir of Hepatitis E Virus That Infects Humans in Eastern China
Yingjie Zheng,1,a Shengxiang Ge ,2,a Jun Zhang, 2 Qingshun Guo, 2 Mun Hon Ng, 2 Fadi Wang, 3 Ningshao Xia, 2 and Qingwu Jiang 1
1 Department of Epidemiology, School of Public Health, The Key Laboratory on Public Health Safety, Ministry of Education, Fudan University,Shanghai, 2 Centre for Research on Medical Molecular Virology of Fujian Province, Xiamen University,Xiamen, and 3 Centre for Disease Prevention and Control, Deqing County, Huzhou City, Zhejiang, China
Background and methods.
Genotype IV hepatitis E virus (HEV) has been isolated from humans and swine.To study the relationship between the human and swine reservoirs, we estimated their respective viral burden, analyzed the genetic makeup of the virus populations, and assessed the risk of infection associated with swine farming.
Results.
In 2 swine-farming districts of eastern China , 9.6% of swine and 0.3% of healthy human subjects excreted HEV in stool, as did 68.8% of patients with confirmed HEV infection. The virus population circulating in humans consisted of genotype I and at least 4 phylogenetically distinct subgroups of genotype IV viruses, 2 of which concurrently circulated among swine. Persons engaged in occupations related to swine farming were found to have a 74% higher risk of infection than those engaged in other occupations, and persons living in communities downstream of the swine farms were found to have a 29% higher risk of infection than persons living in communities upstream.
Conclusions.
Genotype IV HEV is freely transmitted between humans and swine. Because the size of the swine population and its viral burden are much larger than those of humans, transmission of the virus most likely is directed from swine to humans. Infection can be acquired through contact with swine and their waste.
Hepatitis E virus (HEV), a member of the genus Hepevirus, is a positive-strand RNA virus that is morphologically similar to caliciviruses [1]. HEV isolates can be separated into at least 4 phylogenetically related groups (genotypes), which differ with respect to geographic distribution, host range, and pattern of infection, but different genotypes cannot be distinguished serologically and are cross-protective [2–4]. Genotype I is prevalent in Asia and Africa [5–10]; it has been isolated only from humans and has been associated with large waterborne outbreaks in these areas. Genotype II was isolated from a fecal sample collected during an outbreak of non-A, non-B hepatitis in Mexico in 1986 [2]. Genotype III is prevalent in swine herds in the United States [11, 12], Europe [13, 14], Canada [15], Mexico [16], New Zealand [17], South Korea [18], Japan [19], and Thailand [16]. HEV could cause rare sporadic cases and foodborne outbreaks of acute hepatitis in areas hitherto considered to be nonendemic for hepatitis E; this could account for the low but significant levels of anti-HEV seroprevalence in such areas [20], especially among persons with swine-related occupations [21, 22]. Genotype III isolates from swine and humans in the United States [12], the United Kingdom [14], and Japan [19] are genetically closely related, which suggests that swine could be a reservoir for the virus, although it is possible (albeit unlikely) that humans and swine might share a common reservoir for the virus. Genotype IV has been present in swine since at least 1985, and the virus was first detected in humans in 1993 in China [23, 24]. The virus is mainly distributed in China [25–27], Japan [28,29], India [23, 30], Indonesia , and Vietnam [31, 32],
NOTE. Fecal samples were obtained from swine herds in Shanghai in 2002 and Deqing in 2004, and bile samples were obtained from swine carcasses in abattoirs in Deqing in 2004. Serum samples were obtained from 1720 healthy subjects in Deqing in 2004, from 3047 healthy blood donors in Huzhou in 2002, and from 16 patients with serologically proven HEV infection in Shanghai and Deqing in 2004–2005.
a Serum samples from the healthy subjects and blood donors were screened for anti-HEV IgM antibodies, and 97 of 127 reactive samples were further tested for HEV RNA.The occurrence of HEV in these samples was calculated as the product of the prevalence of anti-HEV IgM and the detection rate of HEV RNA determined for the reactive samples.(127/4767 × 10/97 = 0.3%).
and it has a wide host range, being prevalent in humans, swine,and some other animals [25, 30, 33]. It is associated with sporadic cases and foodborne outbreaks [33, 34], but it is not known to cause large waterborne outbreaks, like genotype I viruses. As is the case with genotype III viruses, isolates of genotype IV viruses from swine and humans are closely related genetically, which suggests that swine could be a reservoir for the virus [13]. The present study, which was conducted in 2 principal swine-farming districts of eastern China , aimed to ascertain whether swine are a significant source of HEV for human hepatitis E disease by analyzing the occurrence of HEV in humans and swine, determining the phylogenetic relationship between concurrent isolates from swine and humans, and assessing risk of infection attributable to swine farming.
SUBJECTS, MATERIALS, AND METHODS
Samples and study subjects.
A total of 282 fecal samples and 160 bile samples were obtained from swine in 2002 and 2004 and tested for the presence of HEV RNA. The swine fecal samples were obtained from swine farms in 2 principal swinefarming districts of eastern China ; one is located in Deqing in Zhejiang Province , and the other is located near Shanghai . The bile samples were obtained from abattoirs in Deqing. Human serum samples were obtained from 16 patients with serologically confirmed diagnoses of HEV infection who were admitted to hospitals in Shanghai or Deqing, 1720 healthy subjects residing in Deqing, and 3047 blood donors residing in Huzhou City , 120 km from Deqing in Zhejiang Province . All study subjects provided informed consent. Samples from healthy subjects and blood donors were screened for anti-HEV IgM, and reactive samples were used for isolating the virus. Reversetranscription polymerase chain reaction (RT-PCR) was performed for the detection of the viral genome, and the amplified product was sequenced for genetic analysis.
A cross-sectional seroepidemiologic study was conducted in Deqing, which is criss-crossed by waterways that generally flow in a northeasterly direction. Serum samples were obtained from 3 groups of subjects for the determination of anti-HEV IgG: 340 subjects residing in the immediate vicinity of Deqing, who worked in swine farming–related occupations (e.g., swine farmers, slaughterhouse workers, veterinarians, butchers, and meat vendors), and 1295 subjects residing 1 6 km downstream of the farming district and 425 subjects residing 1 30 km upstream of the swine-farming district, none of whom worked in swine farming–related occupations.
Methods.
Anti-HEV IgG and IgM levels were determined using commercial ELISAs (Wan Tai Pharmaceutical). The ELISAs were performed with a recombinant peptide corresponding to aa 394–606 of the major structural protein specified by open reading frame (ORF) 2 of the HEV genome. Testing was performed in accordance with the manufacturer's instructions.Total RNA was extracted from 250 m L of sample, using Trizol (Gibco). RT-PCR was performed as described elsewhere [26]. Briefly, a 150-nt segment of ORF2, nt 6317–6466 relative to HEV D10330, was amplified using primers E1 ( 5 ' -CTGTTTAAYCTTGCTGACAC -3 ' ) and E5 ( 5 '-WGARAGCCAAAGCACATC -3 ' ) for the first round of PCR and primers E2 ( 5 ' -GACAGAATTGATTTCGTCG -3 ' ) and E4 ( 5 ' -TGYTGGTTRTCRTAATCCTG-3) for the second round. PCR cycling conditions for both rounds consisted of 35 cycles of denaturation for 30 s at 94 ℃ annealing for 30 s at 53 ℃ , and extension for 40 s at 72 ℃ .
Phylogenetic analysis.
PCR products were purified and sequencedin a DNA analyzer (Applied Biosystems 3730 DNA Analyzer; Invitrogen). The viral sequences were aligned using Lasergene (version 5.03; DNAstar). Genetic distances between pairs of virus isolates were calculated using the Kimura 2-parameter method. A phylogenetic tree was constructed using the neighbor-joining method and evaluated using the interior branch test method with MEGA software (version 3.0; available at: http://www.megasoftware.net/). Prototype HEV strains used as references in the analysis and their GenBank accession numbers are as follows: Burma 1, M80581; Burma 2, D10330; China 1, D11092; China 2, AJ272108; India 1, U22532; India 2, AF124407;India 3, AF459438; Mexico 1, M74506; US 1, AF060668; swUS 1, AY575857; Japan 1, AB082545; Japan 2, AB074915; and swJapan 1, AB097811.
Statistical analysis.
Multivariate unconditional logistic-regression analysis was performed using SAS software (version 8.2; SAS) and was used to identify independent determinants of the prevalence of anti-HEV IgG and to estimate the level of associated risk [35].
RESULTS
Occurrence of HEV.
Table 1 shows a comparison of the occurrence of HEV in samples from swine and humans obtained between 2002 and 2005. The patient serum samples were reactive for anti-HEV IgM but not for anti–hepatitis A or anti–hepatitis B core antigen IgM. Samples from healthy subjects and blood donors were screened first for anti-HEV IgM, and reactive samples were used for virus detection. Virus detection was effected by RT-PCR amplification of a 150-nt segment of the ORF2 sequence. The method had been optimized for genotype I and IV HEV, which are prevalent in China , and it affords sensitive detection of prototype genotype III strains [26].
Table 1 shows that 9.6% of swine excreted HEV in stool, and this probably approximates the HEV burden of animals on swine farms in eastern China . Swine in abattoirs were older than those on farms, and their viral burdens were lower. All serum samples from the 16 patients with confirmed hepatitis E disease were reactive for anti-HEV IgM, and 11 (68.8%) of them tested positive for viral RNA. A total of 4767 healthy subjects and blood donors were screened for anti-HEV IgM, and reactive samples were further tested, by RT-PCR, for the presence of the viral RNA. The results showed that 127 individuals (2.7%) were having an episode of current infection (i.e.,they were reactive for anti-HEV IgM), and 10% (10/97) of these episodes were accompanied by viremia (i.e., they were positive for viral RNA). The overall viral burden of the study subjects was estimated to be 0.3%. This probably approximates the viral burden of the general human population in eastern China , whereas the number of cases of overt HEV infection would be comparatively too small to significantly contribute to the viralburden of the population.
Phylogenetic analysis of HEV isolates.
The 150-nt PCRamplified products of 46 isolates were sequenced. The results identified 31 isolates with distinct nucleotide sequences (table2). Phylogenetic analysis was conducted using the sequence alignments of these isolates (figure 1). Results showed that 4 were genotype I isolates, and the others were genotype IV isolates. The genotype I isolates were phylogenetically related to the prototype genotype I strains previously isolated from Burma [5], India [6–8], and China [9]; the genotype IV isolates were phylogenetically related to prototype genotype IV strains isolated from China [36] and Japan [28, 29, 37]. The genotype IV isolates could be further divided into 4 subgroups, designated A–D, which were separate from the prototype Japanese and Chinese strains. The mean pairwise genetic distances between isolates within each subgroup ranged from 0.034% to 0.041% nucleotide substitutions per position, and the corresponding values between different subgroups were higher, ranging from 0.07% to 0.144% (table 3). As shown in figure 1, subgroups A and D were represented by both human and swine isolates, and the mean genetic distances between pairs of human and swine isolates within the respective subgroups (0.025% and
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