Am. J. Trop. Med. Hyg., 76(1), 2007, pp. 170-172
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT
DETECTION OF HEPATITIS E VIRUS RNA FROM THE BIVALVE YAMATO-SHIJIMI (CORBICULA JAPONICA) IN JAPAN
TIAN-CHENG LI*,
TATSUO MIYAMURA, AND
NAOKAZU TAKEDA
Department of Virology II, National Institute of Infectious Diseases, Tokyo Japan
ABSTRACT
To evaluate whether bivalves are contaminated with hepatitis E virus (HEV), samples of a bivalve called Yamato-Shijimi (Corbicula japonica) were examined for HEV by reverse transcription–polymerase chain reaction. Genotype 3 HEV was detected from 2 of 32 packages of Yamato-Shijimi obtained from Japanese rivers, which indicated that HEV contaminates river water in Japan.
Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus recently classified as the sole member of the genus Hepevirus in the family Hepeviridae.1,2 This virus causes human hepatitis E and is transmitted primarily by the fecal-oral route through contaminated drinking water.3,4 However, recent studies have demonstrated that various animal species have serum antibodies to HEV, and its viral genome has been detected in swine, wild deer, wild boar, and mongoose, which suggests that hepatitis E is a zoonotic disease.5–10 Because HEV is excreted into feces,11,12 there is a risk of HEV contamination in environmental water. In fact, HEV has been detected in sewage from industrialized countries, including Spain, the United States, and France.13–15 However, HEV contamination of river water has not been examined. In the present study we detected the HEV genome from a bivalve called Yamato-Shijimi (Corbicula japonica), which suggested that river water in Japan is contaminated with HEV.
A total of 32 packages of Yamato-Shijimi were obtained at a fish market in December 2005 and March 2006. All samples were harvested at areas A, B, C, D, E, F, G, and H in western Japan. The package numbers and collection days are shown in Table 1
. The samples were shucked, and the digestive diverticulum were removed by dissection and weighed. One gram of digestive diverticulum obtained from 10–15 Yamato-Shijimi was homogenized with an Omni-mixer (OCI Instruments, Waterbury, CT) in 10 mL of phosphate-buffered saline, pH 7.4, for two 30-second intervals at a maximum speed of 18,000 rpm. After centrifugation at 10,000 x g for 30 minutes at 4°C, the supernatant was centrifuged at 100,000 x g for 2 hours in an SW41 rotor (Beckman Instruments, Inc., Fullerton, CA). The pellet was resuspended in 140 µL of distilled water and stored at –80°C until use.
Total RNA was extracted with the QIAmp viral RNA mini kit (Qiagen, Hilden, Germany) and resuspended in 20 µL of DNase-, RNase-, and proteinase-free water. Reverse transcription (RT) was performed at 42°C for 50 minutes, followed by 70°C for 15 minutes in a 20-µL reaction mixture containing 1 µL of SuperscriptTM II RNase H– reverse transcriptase (Invitrogen, Carlsbad, CA), 1 µL of oligo (dT) primer, 1 µL of RNaseOUTTM (Invitrogen), 2 µL of 0.1 M dithiothreitol, 4 µL of 5x RT buffer, 1 µL of 10 mM deoxynucleoside triphosphates, 5 µL of RNA, and 5 µL of distilled water. An RT–polymerase chain reaction was performed to amplify part of the open reading frame 2 (ORF2) as described previously.8,10 Two microliters of the cDNA was used for the first PCR in a 50-µL reaction mixture with external forward primer HEV-F1 (5'-GGBGTBGCNGAGGAGGAGGC-3') and external reverse primer HEV-R2 (5'-TGYTGGTTRT-CRTARTCCTG-3'), which corresponded to nucleotide residues 5903–5922 and 6486–6467, respectively, of the G1 Myanmar strain (D10330). Each cycle consisted of denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds, followed by final extension at 72°C for 7 minutes. Two microliters of the first PCR product was used for a nested PCR with internal forward primer HEV-F2 (5'-TAYCGHAAYCAAGGHTG-GCG-3'; nucleotide residues 5939–5958) and internal reverse primer HEV-R1 (5'-CGACGAAATYAATTCTGTCG-3', nucleotide residues 6316–6297) under the same conditions.
Two packages, B4 and B6, collected in area B on February 7, 2006, and March 1, 2006, were positive for HEV RNA by RT-PCR. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and cloned into TA cloning vector pCR2.1 (Invitrogen). The nucleotide sequence of each of 10 clones was determined. Most of the sequences formed a single genotype 3 cluster. The exceptions (B4-13) formed a different cluster along with Sakai-9 detected from a wild boar in 2004 in Japan (Figure 1). We found large numbers of small different nucleotide sequences among the clones with 88.9–100% identity, even when they were derived from the same package, which indicated that multiple HEV strains were accumulated in the digestive diverticulum of Yamato-Shijimi.
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FIGURE 1. Phylogenetic trees of hepatitis E virus (HEV) constructed with avian HEV as an outgroup. A partial open reading frame 2 ORF2 (338 basepairs) of each of 10 clones of B4 (B4-13, B4-16, B4-17, B4-18, B4-19, B4-20, B4-22, B4-25, B4-26, and B4-27) and B6 (B6-31, B6-32, B6-33, B6-36, B6-38, B6-40, B6-41, B6-43, B6-45, and B6-46) were analyzed by the neighbor-joining method. The bootstrap values correspond to 1,000 replications. All nucleotide sequences determined in this study are shown in bold. Other HEV sequences were obtained from GenBank.
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To further analyze the HEV RNA detected in the Yamato-Shijimi, the entire ORF2 of B4 RNA was amplified as overlapping segments, and the nucleotide sequences were determined. The full-length ORF2 consisted of 1,980 basepairs and were phylogenetically classified into genotype 3. High amino acid identities (97.57–98.87%) were observed with HEV strains detected from hepatitis E patients, swine, wild boar, and wild deer in Japan. This is the first report on the detection of HEV from a bivalve.
The HEV sequences were detected from Yamato-Shijimi (Corbicula japonica) harvested on February 7, 2006, and March 1, 2006, in western Japan. The Yamato-Shijimi, a brackish-water bivalve, grows in sandy mud in or near rivers, ponds, and lakes. During breathing and feeding, this bivalve filters a large amount of water. When the water is contaminated with HEV, the virus is ultimately concentrated in the digestive diverticula of the bivalves.
The source of HEV in this organism is not known. Since Japan was considered not to be endemic for this virus, and disposal of sewage is in this country is efficient, the risk of HEV contamination from human stool was believed to be low. There have been no outbreaks of hepatitis E in Japan from drinking water. However, HEV has been detected in wild deer, wild boar, and mongoose in Japan, and HEV shed in the feces of these animals may pollute environmental water. Wild deer and wild boar are controlled in Japan to eliminate their ability to damage agriculture and forestry; hunting is the main control strategy. Hunters usually wash killed animals in river water and this would increase the risk of HEV contamination in river water. These wild animals presumably play an important role in the contamination of environmental water.
In Japan, many outbreaks caused by bivalves contaminated with hepatitis A virus and noroviruses have been reported. Fortunately, Yamato-Shijimi is generally eaten as an ingredient in hot miso soup in Japan, and the heat, usually at 100°C for nearly 10 minutes, decreases the risk of HEV transmission from Yamato-Shijimi to humans. However, more efforts are needed to determine the infectivity and stability of HEV in the natural environment, including that in Yamato-Shijimi.
Received June 19, 2006.
Accepted for publication September 10, 2006.
Acknowledgments: We thank Tomoko Mizoguchi and Satoko Ogawa for secretarial work.
Financial support: This study was supported in part by grants for Research on Emerging and Re-emerging Infectious Diseases, Research on Hepatitis, and Research on Food Safety from the Ministry of Health, Labor and Welfare, Japan.
* Address correspondence to Tian-Cheng Li, Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan. E-mail: litc{at}nih.go.jp 
Authors’ address: Tian-Cheng Li, Tatsuo Miyamura, and Naokazu Takeda, Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan, Telephone: 81-42-561-0771, Fax: 81-42-561-4729, E-mails: litc{at}nih.go.jp, tmiyam{at}nih.go.jp, and ntakeda{at}nih.go.jp.
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