Human sapovirus in clams, Japan.Human sapovirus was detected in 4 of 57 clam packages by reverse transcription-PCR and sequence analysis. This represents the first finding of sapovirus contamination in food. Closely matching sequences have been detected in stool specimens from patients with gastroenteritis gastroenteritis: see enteritis.
Acute infectious syndrome of the stomach lining and intestines. Symptoms include diarrhea, vomiting, and abdominal cramps. in Japan, which indicates a possible food-to-human transmission link.
Sapoviruses and noroviruses are etiologic agents of human gastroenteritis. Human noroviruses are the most important cause of outbreaks of gastroenteritis worldwide and can be transmitted by a variety of routes, including food (1). Sapovirus infections are mostly associated with sporadic gastroenteritis in young children; however, foodborne transmission routes are yet to be determined. The most widely used method of detection is reverse transcription--PCR (RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. ), which has a high sensitivity and can also be used for genetic analysis. Sapovirus strains can be divided into 5 genogroups; GI-GV infect humans; sapovirus Gill infects porcine porcine /por·cine/ (por´sin) pertaining to swine.
pertaining to pig. See also hog (1), swine.
porcine circovirus 1
a nonpathogenic virus. species. Phylogenetic studies have also designated sapovirus clusters or genotypes to further describe strains.
The purpose of this study was to detect sapovirus in the clam Corbicula For the pollen holding structure on the posterior tibiæ of some hymenopterans, see .
Corbicula is a genus of clams. Best known is Corbicula fluminea which is an invasive species in many areas of the world. japonica japonica (jəpŏn`əkə): see quince; camellia. (bivalve bivalve, aquatic mollusk of the class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed body and a shell consisting of two valves, or movable pieces, hinged by an elastic ligament. mollusk mollusk: see Mollusca.
Any of some 75,000 species of soft-bodied invertebrate animals (phylum Mollusca), many of which are wholly or partly enclosed in a calcium carbonate shell secreted by the mantle, a soft ) and describe the genetic diversity of the strains. A total of 57 clam packages (30-60 clams per package) were collected from supermarkets or fish markets from 6 different areas in western Japan from December 8, 2005, to September 6, 2006. The samples were shucked, and the digestive diverticulum diverticulum
Small pouch or sac formed in the wall of a major organ, usually the esophagus, small intestine, or large intestine (the most frequent site of problems). was removed by dissection and weighed. One gram of digestive diverticulum (10-15 clam/package) was homogenized ho·mog·e·nize
v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es
1. To make homogeneous.
a. To reduce to particles and disperse throughout a fluid.
b. with an Omini-mixer (Sorvall Inc., Newtown, CT, USA) in 10 mL phosphate-buffered saline. After centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal at 10,000x g for 30 min at 4[degrees]C, the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. was centrifuged at 100,000x g for 2 h (SW41 Rotor, Beckman Instruments, Inc., Fullerton, CA, USA). The pellet was resuspended in 140 gL distilled water and stored at -80[degrees]C until use.
RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic extraction and nested RT-PCR were performed as described (2). Briefly, for the first PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) , F13, F14, R13, and R14 primers were used; for the nested PCR, F22 and R2 primers were used. All RT-PCR products were analyzed by 2% agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). and visualized by ethidium bromide staining. RT-PCR products were excised from the gel and purified by the QIAquick gel extraction kit (QIAGEN, Hilden, Germany). Nucleotide sequences were prepared with the terminator cycle sequence kit (version 3.1, Applied Biosystems, Warrington, England) and determined with the ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.
(Application Binary Interface) A specification for a specific hardware platform combined with the operating system. 3130 sequencer See MIDI sequencer.
(music) sequencer - Any system for recording and/or playback of music via a programmable memory which stores music not as audio data, but as some representation of notes. (ABI, Boston, MA, USA). Nucleotide sequences were aligned with ClustalX, and the distances were calculated by Kimura's 2-parameter method, as described elsewhere (2). Nucleotide sequence data determined in this study have been deposited in GenBank under accession nos. EF104251-EF104254.
Four (7%) of 57 clam packages were contaminated with sapovirus (termed Shijimi1, Shijimi2, Shijimi3, and Shijimi4). Genetic analysis of the partial capsid capsid /cap·sid/ (kap´sid) the shell of protein that protects the nucleic acid of a virus; it is composed of structural units, or capsomers.
n. gene showed that these 4 sequences shared >98% nucleotide similarity and >97% amino acid identity. Phylogenetic analysis grouped these 4 sequences in the same genotype, i.e., GI/1 (Figure). Similar sequences were found on the database (Figure). Strains from this cluster likely represent the dominant genotype worldwide (3). Three of 4 sapovirus-positive clam packages were collected from different areas and at different times (Figure). The clam packages that were contaminated with Shijimi1 and Shijimi3 were collected from the same area, but 6 weeks apart, which indicates an ongoing sapovirus contamination or resistance in the natural environment. The seasonality of sapovirus infection in Japan is unknown; however, as with norovirus, sapovirus infections may also peak during winter, although further epidemiologic and environmental studies are needed.
In a recent study, we detected sapovirus strains in 7 of 69 water samples, which included untreated wastewater, treated wastewater, and a river in Japan (4). Three of 7 sapovirus sequences detected in the water samples belonged to GI/1 and shared >97% nucleotide similarity with the sapovirus sequences detected in the clam packages. Additionally, sapovirus sequences belonging to GI/1 and sharing >99% nucleotide similarity, for example, Chiba/010598F strain (Figure), have been detected in stool specimens from children with sporadic gastroenteritis in Japan (5,6). The closely matching sapovirus sequences detected in the water, clams, and patients suggest that sapovirus contamination in the natural environment can lead to foodborne infections in humans, although direct evidence is lacking. More important, a recent study found animal sapovirus in oysters and suggested that coinfection with human and animal sapovirus strains could result in genomic recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. and the emergence of new strains (7). At the same time, we recently described the first human sapovirus intergenogroup recombinant strain (8). Phylogenetic analysis of the nonstructural region (i.e., genome start to capsid start) grouped this sapovirus strain in GII GII Global Information Infrastructure
GII Getty Information Institute
GII Gasherbrum II (26,360 ft. mountain near Pakistan-China)
GII Government Information Infrastructure
GII Ghana Integrity Initiative , while the structural region (i.e., capsid start to genome end) grouped this strain in GIV GIV Gasherbrum IV (26,000 ft. mountain near Pakistan-China)
GIV Geological Information Visualization .
A large number of studies have detected norovirus in oysters. In 2 recent studies, norovirus was detected in oysters (Crassosterea gigas) harvested from geographically isolated areas in Japan (9,10). We also screened the same oyster samples for sapovirus; however, all of the samples were negative for sapovirus. That sapovirus was detected in the clam samples, but not in the oyster samples, is of interest. In the past several years, increasing evidence has emerged that human noroviruses bind to histo-blood group antigens (HBGAs) (11). These carbohydrate epitopes are present in mucosal secretions and throughout many tissues of the human body, including the small intestine, and in oyster digestive tissues. A number of studies have found that different norovirus strains exhibit different binding patterns to HBGAs and oyster digestive tissues (12,13). In a recent study, we found that sapovirus GI and GV strains showed no such binding activity to HBGAs (14). These results suggest that human norovirus and sapovirus strains have different binding receptors or that human sapovirus may not concentrate in detectable levels in oysters.
Foodborne diseases are a major problem worldwide. We report what is, to the best of our knowledge, the first account of sapovirus contamination in food destined des·tine
tr.v. des·tined, des·tin·ing, des·tines
1. To determine beforehand; preordain: a foolish scheme destined to fail; a film destined to become a classic.
2. for human consumption. The report may represent a possible food-to-human transmission link, although direct evidence is lacking. In Japan, clams are usually boiled before they are consumed in soups. However, boiling to open the clam may not inactivate in·ac·ti·vate
1. To render nonfunctional.
2. To make quiescent.
in·acti·va the virus (15); in addition, some areas in Japan do not boil clams before eating them. Further studies are needed to determine if boiling inactivates sapovirus and if the contaminated clams are indeed infectious. In conclusion, these novel results highlight the importance of sapovirus, in particular the GI/1 strains. A new awareness of sapovirus transmission routes is necessary and may help reduce sapovirus infections.
Dr Hansman is a scientist at the National Institute of Infectious Diseases, Japan. He studies viruses that cause gastroenteritis in humans, namely sapovirus and norovirus. His research interests include epidemiology, virus expression, and cross-reactivity.
(1.) Koopmans M, Vinje J, de Wit M, Leenen I, van der Poel W, van Duynhoven Y. Molecular epidemiology of human enteric enteric /en·ter·ic/ (en-ter´ik) within or pertaining to the small intestine.
1. Of, relating to, or within the intestine.
2. caliciviruses in The Netherlands. J Infect Dis. 2000;181 (Suppl 2):S262-9.
(2.) Hansman GS, Takeda N, Katayama K, Tu ET, McIver CJ, Rawlinson WD, et al. Genetic diversity of sapovirus in children, Australia. Emerg Infect Dis. 2006;12:141-3.
(3.) Hansman GS, Oka T, Katayama K, Takeda N. Human sapoviruses: genetic diversity, recombination, and classification. Rev Med Virol. 2007;17:133-41.
(4.) Hansman GS, Sano D, Ueki Y, Imai T, Oka T, Katayama K, et al. Sapovirus in water, Japan. Emerg Infect Dis. 2007;13:133-5.
(5.) Okada M, Yamashita Y, Oseto M, Shinozaki K. The detection of human sapoviruses with universal and genogroup-specific primers. Arch Virol. 2006;151:2503-9.
(6.) Okada M, Shinozaki K, Ogawa T, Kaiho I. Molecular epidemiology and phylogenetic analysis of Sapporo-like viruses. Arch Virol. 2002;147:1445-51.
(7.) Costantini V, Loisy F, Joens L, Le Guyader FS, Saif LJ. Human and animal enteric caliciviruses in oysters from different coastal regions of the United States. Appl Environ Microbiol. 2006;72:1800-9.
(8.) Hansman GS, Yakeda N, Oka T, Oseto M, Hedlund KO, Katayama K. Intergenogroup recombination in sapoviruses. Emerg Infect Dis. 2005;11:1916-20.
(9.) Ueki Y, Sano D, Watanabe T, Akiyama K, Omura T. Norovirus pathway in water environment estimated by genetic analysis of strains from patients of gastroenteritis, sewage, treated wastewater, river water and oysters. Water Res. 2005;39:4271-80.
(10.) Nishida T, Nishio O, Kato M, Chuma T, Kato H, Iwata H, et al. Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan. Microbiol Immunol. 2007;51:177-84.
(11.) Hutson AM, Atmar RL, Graham DY, Estes MK. Norwalk virus infection and disease is associated with ABO ABO
See: Accumulated Benefit Obligation histo-blood group type. J Infect Dis. 2002;185:1335-7.
(12.) Le Guyader F, Loisy F, Atmar RL, Hutson AM, Estes MK, Ruvoen-Clouet N, et al. Norwalk virus-specific binding to oyster digestive tissues. Emerg Infect Dis. 2006;12:931-6.
(13.) Tan M, Jiang X. Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle. Trends Microbiol. 2005;13:285-93.
(14.) Shirato-Horikoshi H, Ogawa S, Wakita T, Takeda N, Hansman GS. Binding activity of norovirus and sapovirus to histo-blood group antigens. Arch Virol. 2007;152:457-2006.
(15.) Myrmel M, Berg EM, Rimstad E, Grinde B. Detection of enteric viruses in shellfish from the Norwegian coast. Appl Environ Microbiol. 2004;70:2678-84.
Address for correspondence: Grant S. Hansman, Department of Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, 208-0011, Japan; email: firstname.lastname@example.org
Grant S. Hansman, * Tomoichiro Oka, * Reiko Okamoto, ([dagger]) Tomoko Nishida, ([dagger]) Shoichi Toda, ([dagger]) Mamoru Noda, ([double dagger]) Daisuke Sano, ([section]) You Ueki, ([paragraph]) Takahiro Imai, ([section]) Tatsuo Omura, ([section]) Osamu Nishio, * Hirokazu Kimura, * and Naokazu Takeda *
* National Institute of Infectious Diseases, Tokyo, Japan; ([dagger]) Yamaguchi Prefectural Research Institute of Public Health, Yamaguchi, Japan; ([double dagger]) Hiroshima City Institute of Public Health, Hiroshima, Japan; ([section]) Tohoku University, Sendai, Japan; and ([paragraph]) Miyagi Prefectural Institute of Public Health and Environment, Sendai, Japan