Swine as a Principal R e servoir of Hepatitis E Virus That Infects ...-2

NOTE.

　　HEV strains with distinct nucleotide sequences were isolated from swine or humans in Huzhou City, Shanghai, and Deqing in eastern China, 2002–2005. Nucleotide sequences of these isolates can be accessed in GenBank. Phylogenetic analysis was based on alignments of a 150-nt segment of open reading frame 2 of HEV.

Figure 1.

　　Phylogenetic tree of human and swine hepatitis E virus (HEV) isolates, eastern China , 2002–2005. The phylogenetic tree was produced with a 150-nt open reading frame (ORF) 2 sequence alignments of HEV isolated from swine (white circles), healthy human subjects (white squares), or patients with HEV infection (black squares) and prototypes of genotype I strains from China, Burma, and India; a genotype II strain ( Mexico ); genotype III strains isolated from humans or swine in the United States ; and prototype genotype IV strains isolated from swine or humans in Japan or China . Genotypes are underlined, and subtypes are circled. The scale bar indicates a genetic distance of 0.02% nucleotide substitution per position. Values for various branches are percentages of the tree obtained from 1000 resamplings of the dat a. 0.040 %) were essentially the same as the corresponding values determined for the human and swine isolates combined(0.037% and 0.040%).

　　It is noted in table 2 that subgroup A was prevalent in humans in 2004 and 2005 and in swine herds in Deqing and Shanghai in 2004. Subgroup D virus was prevalent in humans in 2004 and 2005 and in swine herds in 2002 and 2004. This confirms that the 2 subgroups cocirculated in humans and

　　swine herds in 2004 in eastern China . Subgroup B and C isolates were detected in only human samples, although it is possible that these subgroups are prevalent in swine from other areas.

Seroepidemiologic analysis.

　　A cross-sectional seroepidemiologic study was conducted in Deqing to assess the risk of　infection attributable to swine farming (table 4). The seroprevalence of anti-HEV IgG was highest for persons engaged in swine farming–related occupations, and seroprevalence increased with the duration of occupational exposure to swine. Among the control subjects who were not engaged in swine farming–related occupations, seroprevalence was higher in those residing downstream of the swine farms than in those residing upstream. The seroprevalence of anti-HEV IgG also increased with age and was higher in male than female subjects (data not shown). Multivariate analysis identified occupational exposure to swine, proximity of residence to a swine farm, age, and sex to be independent determinants of anti-HEV IgG positivity. Compared with control subjects residing upstream of the swine-farming district, the analysis estimated that the risk of infection was increased by 74% for those engaged in swine farming–related occupations (odds ratio [OR], 1.74 [95% confidence interval {CI} 1.24–2.44]). The risk increased with an increasing duration of occupational swine exposure, with ORs increasing from 0.82 for those working for ! 5 years to 1.84 and 2.82 for those working for 5–14 and _ 15 years, respectively. The risk of infection was also higher for persons residing downstream of the swine-farming district (OR, 1.29 [95% CI, 1.02–1.64]). These results suggest that the virus might be transmitted to workers in the swine-farming industry by direct contact with swine and that the virus present in effluents from swine farms can spread via waterways to communities downstream.

DISCUSSION

Study design.

　　Genotype IV HEV has been the dominant cause of hepatitis E disease in China since 2000 [27]. The virus is also prevalent in swine, and isolates from swine are genetically closely related to isolates from humans [13, 38]. To study

NOTE.

　　Genetic distance is given as the percentagenucleotide substitution. Subgroup A, ; subgroup B, n p 12; subgroup C, ; subgroup D, . n p 3 n p 2 n p 10

 　whether swine are a source of human infection, we estimated the viral burden of humans and swine, analyzed the genetic makeup of the respective virus populations, and assessed the risk of infection attributable to swine farming. The virologic study was conducted in 2 swine-farming districts of eastern China . Swine fecal samples were obtained from swine herds in both districts, and bile samples were obtained from abattoirs in Deqing. The human serum samples used for virological study were obtained from patients with hepatitis E disease who were admitted to hospitals in Shanghai and Deqing, healthy subjects residing in Deqing, and blood donors residing in Huzhou City ( 120 km from Deqing). Thus, whereas isolates from swine re-flect the virus population circulating in swine herds of the 2farming districts, isolates from humans are more broadly representative of the virus population circulating in eastern China. Our study aimed to assess whether HEV might be transmitted from swine to humans by occupational contact with the animals and whether the discharge of swine wastes into waterways could disseminate infection to neighboring communities.

Methodology.

　　The primers used for virus detection had been optimized for genotypes I and IV, which are the prevalent genotypes in China [26]. Only these genotypes were detected in the present study, although the primers also afford sensitive detection for prototype genotype II and III strains. The detection rate—68.8% for serum samples from patients with hepatitis E disease, 9.6% for swine fecal samples, and 3.1% for swine bile samples—was higher than that achieved by other methods [38]. Genetic analysis was based on alignments of a 150-nt ORF2 sequence. On the basis of previous studies by Geet al. [26] and Schlauder and Mushahwar [39], the phylogenetic relationship thus established is expected to be similar to that based on analysis of the entire viral genome. The anti-HEVassays used in the present study are produced with a recombinant peptide of HEV structural protein that naturally occurs as a homodimer [40]. The 3-dimensional structure of the latter models certain features of HEV neutralization sites and other important antigenic determinants located on the protrusions projecting from the virus shell [40, 41]. The antigenic specificity of the assays produced with this peptide is correlated with protective immunity [4, 42]. Previous studies have shown that the IgM antibody detected by the assay is a reliable marker of recent HEV infection and that the seroprevalence of IgG antibody reflects the cumulative exposure of a study population to HEV [43, 44]. Multivariate unconditional logistic-regression analysis was used to identify independent determinants of the seroprevalence of anti-HEV IgG and to estimate the risk of infection attributable to the independent variables

Zoonosis involved in HEV infection.

　　The results of our study showed that 9.6% of swine on farms excreted HEV in stool. The detection rate from swine in slaughter houses was lower, presumably because they were older than those on farms. The HEV burden of human subjects was estimated to be 0.3%. Except for the few patients with hepatitis E disease, all human subjects and swine showed no or a minimum of symptoms of the infection. Genetic analysis showed that 4 human virus isolates were genotype I and 17 were genotype IV, whereas all 25 swine isolates were genotype IV viruses. The human genotype

NOTE.

　　Serum samples were obtained from subjects working in swine farming–related occupations (occupational exposure) for indicated times and from those not working with swine but who resided at least 6 km downstream or at least 30 km upstream of a swine-farming district and were tested for anti-HEV IgG. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using multivariate unconditional logistic-regression analysis and adjusted for sex and age.

　　IV isolates were further separated into 4 subgroups, designated A–D. It was shown that subgroup A and D isolates cocirculate in swine herds in Deqing and Shanghai . Multivariate unconditional logistic-regression analysis of the prevalence of anti-HEV IgG showed that persons engaged in occupations related to swine farming have a 74% higher risk of HEV infection than control subjects and that the risk increases with the duration of work. Among subjects who did not have occupational exposure to swine, it was shown that those residing downstream of the swine-farming district have a 29% higher risk than those residing upstream.

　　Taken together, the results show that HEV infection commonly occurs in humans and swine in eastern China and that virtually all of the infections are asymptomatic. The virus population consists of genotype I and IV virus, with the latter being by far the dominant genotype. Genotype I viruses circulate only in humans, and the principal source of infection by genotype I virus is healthy subjects with asymptomatic infection. Therefore, contact with patients who have hepatitis E disease, in contrast to hepatitis A disease, is not associated with a significant risk of infection [34]. Moreover, detection of HEV in blood donors confirms previous findings [45] that suggested that the infection might also be acquired through blood transfusion.

　　Genotype IV viruses, on the other hand, are freely transmissible between humans and swine. We showed that subgroups A and D cocirculate in humans and among the swine herds in Deqing and Shanghai , and it is likely that subgroups B and C are prevalent in swine herds in other areas. Because the HEV burden in swine is much larger than that in humans (as is the population size), transmission of infection by genotype IV virus would most likely be from swine to humans. This contention is supported by our seroepidemiologic findings, which suggest that humans can acquire infection by contact with swine and their wastes. Evidently, humans are exposed, through trafficking in swine, to viruses from different swine herds; therefore, the genotype IV virus population prevalent in humans is genetically more diverse than that prevalent in the swine herds that we studied. Therefore, we concluded that swine constitute a principal source of genotype IV HEV for human infection in eastern China .

　　Genotype IV HEV has evolved rapidly as a result of zoonosis. Since its first detection in humans in 1993 [24], the virus has replaced genotype I as the dominant cause of hepatitis E disease in China . The largest majority of infections caused by these viruses to date have been asymptomatic, but it is possible that virulent strains might evolve in the future, and this would have an important health and economic impact in Asia , where the virus is prevalent. Genotype III HEV has also been implicated as a possible zoonotic agent [14]. Although the virus rarely infects humans, a recent study showed that human infection has become more common in the United Kingdom , like genotype IV HEV did in China in the past decade [14, 27]. Unlike genotype IV HEV, genotype III HEV is widely distributed, and evolution of virulent strains of this genotype would have more far-reaching consequences.

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　　Received 11 December 2005; accepted 10 February 2006; electronically published 10 May 2006.

　　Potential conflicts of interest: none reported.Financial support: Fudan University Fund for Young Scientists (grant JKF201001);National High Technology Research and Development Program of China (grant 2005AA2Z3H20); Key Science and Technology Project of Fujian Province (grant 2004YZ01-1).

　　a Y.Z. and S.G. contributed equally to the work.

　　Reprints or correspondence: Pr. Qingwu Jiang, Department of Epidemiology, School of Public Health, The Key Laboratory on Public Health Safety, Ministry of Education, Fudan University, Shanghai 200032, China (jiangqw@shmu.edu.cn); Pr. Ning-Shao Xia, Centre for Research on Medical Molecular Virology of Fujian Province, Xiamen University, Xiamen 361005, China (nsxia@xmu.edu.cn).

　　The Journal of Infectious Diseases 2006; 193:1643–9_ 2006 by the Infectious Diseases Society of America . All rights reserved.0022-1899/2006/19312-0006$15.00

　　Hepatitis E Virus Is a Zoonotic Agent ? JID 2006:193 (15 June) ? 1643