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Journal of Clinical Microbiology, September 2006, p. 3440-3442, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00939-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Genotype 1 Hepatitis E Virus in Samples from Swine in Cambodia

M. Caron,1* V. Enouf,1 S. C. Than,2 L. Dellamonica,2 Y. Buisson,3 and E. Nicand1

Centre National de Référence du Virus de l'Hépatite E, HIA Val de Grâce, Paris, France,1 Institut Pasteur, Phnom Penh, Cambodia,2 Institut de Médecine Tropicale du Service de Santé des Armées, Marseille, France3

Received 5 May 2006/ Returned for modification 25 June 2006/ Accepted 4 July 2006


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ABSTRACT
 
Hepatitis E virus (HEV) is a major enterically transmitted pathogen in many developing countries, where it causes outbreaks and sporadic cases of acute hepatitis. A study conducted with pigs from several livestock farms in Cambodia identified one swine genotype 1 HEV isolate as being associated with prevalent swine genotype 3 HEV.


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TEXT
 
Hepatitis E virus (HEV) is an important causative agent of large-scale epidemics and sporadic cases of acute hepatitis in many areas of Asia, Africa, and Central America, where it is endemic. Genotype 1 of human HEV is most prevalent in Asian countries. Nevertheless, several genotypes circulate in the neighboring countries, such as China, Thailand, or Vietnam, where genotypes 3 and 4 have also been identified from human and swine infections (3, 6, 9).

Pigs are believed to be a major animal reservoir, being involved in natural cross-species infections (7). A swine HEV isolate from the United States (swine US) was the first animal HEV strain found to belong to genotype 3, and it is closely related to the U.S. human HEV strains. Interspecies transmission involving pigs has been demonstrated for HEV genotypes 3 and 4 but not for genotypes 1 and 2 (8).

The circulation of hepatitis E in Cambodia was difficult to evidence before 2000 because of the political instability and civil war. Then, a serological survey found no significant difference in anti-HEV seroprevalence between pig breeders (6.7%), slaughterhouse workers (11.5%), and the unexposed population (12.8%) (1). The aim of the present study is to report the circulation of HEV strains in a potential swine reservoir in Cambodia.

Background. A total of 181 specimens (92 serum and 89 fecal samples) were collected from 2- to 6-month-old pigs on livestock farms near Phnom Penh, Cambodia. During this study, serum samples were tested for detection of anti-HEV immunoglobulin G (IgG) with an Abbott HEV enzyme immunoassay kit after adaptation of horseradish peroxidase-conjugated goat anti-swine IgG. The seroprevalence of anti-HEV IgG antibodies was 5.6% (L. Dellamonica, personal data). Subsequent work was undertaken to identify and characterize swine HEV strains from these Cambodian specimens.

Specimens. Serum and stool samples collected for the previous survey were stored at –80°C in the Institute Pasteur, Phnom Penh, Cambodia. Then, specimens were shipped in dry ice to the Reference National Centre for Hepatitis E Virus, Paris, France.

Molecular investigation. RNA was extracted with a MagNA Pure LC RNA isolation HP kit according to the manufacturer's instructions (MagNA Pure LC instrument; Roche Diagnostics, Basel, Switzerland). The open reading frame 1 (ORF1) and ORF2 regions of the HEV genome were detected by reverse transcription-PCR as previously described (4, 6). PCR products of 487 bp from ORF1 and 145 bp from ORF2 were sequenced with an automated DNA sequencer (CEQ 8000; Beckman-Coulter Inc., Fullerton, California). The swine HEV sequences were aligned using ClustalX (version 1.82) and compared to other human and swine HEV sequences available in the GenBank database. For phylogenetic analysis within the ORF1 region, designations used for the HEV isolates (the first two are the Cambodian isolates) and their GenBank accession numbers (in parentheses) are as follows: CAM-3F13 (DQ145798), CAM-3F15 (DQ145799), Burma (M73218), Nepal (AF051830), Pakistan Sar-55 (M80581), Chad (AY204877), Mexico (M74506), US1 (AF060668), US2 (AF060669), swine US (AF082843), and China (AJ272108). The assays were repeated in duplicate in three independent runs to avoid contamination. A phylogenic tree was produced using the neighbor-joining method of the PHYLIP 3.6 program. The bootstrap method was used with 1,000 replications and values of >70% to generate a consensus tree.

HEV RNA was detected in 3 of the 92 serum samples and 19 of the 89 fecal samples. One HEV strain (the CAM-3F13 isolate) was characterized in both the ORF1 and ORF2 regions, 20 strains in only the ORF2 region, and one strain (the CAM-3F15 isolate) in only the ORF1 region (Table 1).


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  		TABLE 1. Sequencing of ORF1 and ORF2 regions of swine HEV strains isolated in Cambodia

The CAM-3F13 isolate, amplified in both the ORF1 and ORF2 regions, showed a high association with the human HEV strain US1, with 93.5% to 92.3% identity within the ORF1 and ORF2 regions. This isolate was also close to the swine US strain, with 93.1% to 87.6% identity, respectively, within the ORF1 and ORF2 regions. The CAM-3F13 isolate and the 20 other isolates amplified in the ORF2 region were close to each other, with 86.2% to 100% identity. These isolates demonstrated 87.6% to 94.5% identity with the human HEV strain US1 and 84.1% to 92.4% with the swine US strain. Phylogenetic analysis showed that these Cambodian isolates were grouped into genotype 3.

In contrast, the CAM-3F15 isolate shared 77.1% to 77.4% identity with the human US1 and swine US strains but 94.1% identity with the Burma strain. This single isolate was classified into genotype 1 (Fig. 1; Table 2).


Figure 1
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  		FIG. 1. Unrooted phylogenic tree depicting the ORF1 analysis of the CAM-3F13 and CAM-3F15 isolates with referenced HEV strains. The scale bar indicates distance between HEV strains. GenBank database accession numbers are given in parentheses. The Cambodian swine HEV strains are indicated in boldface type.


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  		TABLE 2. Percentages of identity between pairwise comparisons of the 487-bp ORF1 fragmenta of HEV

Genetic analysis of the 21 swine isolates confirmed the circulation in Cambodia of HEV belonging to genotype 3. Pigs may serve as a major viral reservoir. Genotype 3 was recently detected in pigs in Thailand and Mexico, although genotypes 1 and 2 are prevalent among patients from these countries, where HEV is endemic (2). The zoonotic potential of genotypes 3 and 4 has been well-documented in Asian countries. Until now, neither genotype 1 nor genotype 2 HEV has been able to infect pigs experimentally (8). Interestingly, the present data suggest that genotype 1 is not restricted to human infections but can also infect pigs. Although the region of HEV ORF2 in CAM-3F15 could not be characterized, a correlation between genotyping results for the ORF1 and ORF2 regions has been demonstrated previously (10). This would be the first report of a genotype 1 HEV infection in pigs.

Prior exposure to HEV has been described for both domestic and wild animal species with the detection of anti-HEV antibodies. However, HEV sequences are available for only a few animal species. Circulation of genotype 1 has been reported to occur only among rodents in Nepal. These HEV isolates from rodents shared at least 95% identity with human isolates in the same area of HEV endemicity (5). The identification of genotype 1 in peridomestic animals suggests cross-species transmission. The identification of both genotypes 1 and 3 in pigs may even suggest multiple sources of infection for these animals. Pigs are omnivorous, and a possible source of contamination could be food. These data raise further concerns about potential zoonosis and food safety. They also confirm the value of investigating HEV spread.

Nucleotide sequence accession numbers. CAM-3F13 and CAM-3F15 sequences have been deposited in GenBank under accession numbers DQ145798 and DQ145799, respectively.


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ACKNOWLEDGMENTS
 
We thank the Institute Pasteur in Phnom Penh, Cambodia, for its technical support.

This work was supported by the French Ministry of Health (National Reference Centre of HEV) and the French Ministry of Defense.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centre National de Référence du Virus de l'Hépatite E, HIA Val de Grâce, Laboratoire de Biologie, 74 bd. Port Royal, Cedex 05, Paris 75230, France. Phone: 33140515116. Fax: 33140514298. E-mail: mcaronfr{at}yahoo.fr. Back


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Journal of Clinical Microbiology, September 2006, p. 3440-3442, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00939-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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  • Feagins, A. R., Opriessnig, T., Guenette, D. K., Halbur, P. G., Meng, X.-J. (2007). Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. J. Gen. Virol. 88: 912-917 [Abstract] [Full Text]  

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