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Background/purpose Sapoviruses (SaVs) become important pathogens causing both sporadic and outbreaks of acute gastroenteritis (AGE) after rotavirus vaccination era worldwide. SaVs were included in AGE screening items when norovirus and rotavirus were negative in Taiwan CDC since 2008. However, no complete SaV genome sequence of any genotype detected in Taiwan was determined. This study aimed to investigate SaVs infection and complete genome sequences detected in Taiwan. Methods This prospective survey, SaVs samples with untyped or weak PCR result were selected for testing the new design qRT-PCR assay from AGE hospitalized children during 2008–2011, 2016–2017 and AGE outbreak in 2012–2014. Those were genetically characterized using long RT-PCR with different primer combinations as well as primer independent deep sequencing and with 5′ RACE and 3′ terminal region targeting RT-PCR. Results Overall, 14 SaV-AGE hospitalized children and 4 SaV-AGE outbreaks were enrolled in this study. In addition to the AGE symptoms, 6 children also showed URI symptoms (cough, pharyngitis, rhinorrhea and nasal congestion). The detected 19 SaVs were classified as eight genotypes (GI.1, GI.2, GI.3, GII.2, GII.3, GII.5, GII.8, and GIV.1) and the complete genome sequence of representative strain for each genotype were determined except GI.3. The GII.3 was the most major genotype following GI.1 and GIV.1. Conclusion Our result confirmed that SaV is one of the pathogens detected from Taiwanese AGE patients. Multiple SaV genotype strains would associate with AGE as similar to those detected in different countries/areas. The whole genome of SaV strains detected including rarely reported GII.8 was firstly determined.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log(10)-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

Calicivirus infection in patients and animals is associated with the production of multiple inflammatory cytokines, including tumor necrosis factor α (TNF-α). Here we studied the feline calicivirus (FCV) non-structural proteins and found that the FCV protease was a key factor for TNF-α gene expression in cultured cells. The expression of the TNF-α gene in cells expressing FCV, human norovirus, and rabbit hemorrhagic disease virus protease was compared, revealing that the induction of TNF-α could be a common phenomenon during the infection by the viruses in the Caliciviridae. The level of TNF-α mRNA in the cells expressing mutant proteases that lacked the active site was measured. These data indicate that the protease activity is crucial for TNF-α expression. These findings provide new insight into the induction of inflammation during calicivirus infection.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Sapoviruses (SaVs) are enteric viruses and have been detected in various mammals. They are divided into multiple genogroups and genotypes based on the entire major capsid protein (VP1) encoding region sequences. In this study, we determined the first complete genome sequences of two genogroup V, genotype 3 (GV.3) SaV strains detected from swine fecal samples, in combination with Illumina MiSeq sequencing of the libraries prepared from viral RNA and PCR products. The lengths of the viral genome (7494 nucleotides [nt] excluding polyA tail) and short 5'-untranslated region (14 nt) as well as two predicted open reading frames are similar to those of other SaVs. The amino acid differences between the two porcine SaVs are most frequent in the central region of the VP1-encoding region. A stem-loop structure which was predicted in the first 41 nt of the 5'-terminal region of GV.3 SaVs and the other available complete genome sequences of SaVs may have a critical role in viral genome replication. Our study provides complete genome sequences of rarely reported GV.3 SaV strains and highlights the common 5'-terminal genomic feature of SaVs detected from different mammalian species.

Sapovirus (SaV) is a causative agent of gastroenteritis in humans in both sporadic cases and outbreaks. During the period from January 2005 to August 2014, SaV was detected in 30 (5.9%) of 510 gastroenteritis outbreaks in Osaka City, Japan using real-time RT-PCR. Seasonal distribution of SaV-associated outbreaks revealed an increase during the 2011-2012 season and the highest frequency of outbreaks during the 2012-2013 season. Genotyping analysis based on the capsid region demonstrated that the most common genotype was GI.2 (36.7%), in which the strains were closely related. The comparison of complete capsid gene sequences with 18 GI.2 strains (7 strains in this study and 11 from GenBank) between 1990 and 2013 showed that GI.2 strains were classified into at least three genetic clusters (1990-2000, 2004-2007, and 2008-2013) with chronologically unique amino acid residues and accumulation of mutations in the predicted P domain, suggesting the one of the causes of emergence and spread of GI.2 strains. This study will also be helpful for understanding the evolutionary mechanism of the SaV genome.

Highlights • The pathogenesis of porcine genogroup (G) III Sapovirus (SaV) in neonatal gnotobiotic pigs. • Epidemiology of different genogroups of porcine SaVs. • Genetic classification of porcine SaVs. • Diagnosis of porcine SaVs., The first porcine Sapovirus (SaV) Cowden strain was discovered in 1980. To date, eight genogroups (GIII, V-IX) and three genogroups (GIII, GV, and GVI) of porcine SaVs have been detected from domestic pigs worldwide and wild boars in Japan, respectively based on the capsid sequences. Although GIII Cowden strain replicated in the villous epithelial cells and caused intestinal lesions in the proximal small intestines (mainly in duodenal and less in jejunum), leading to mild to severe diarrhea, in the orally inoculated neonatal gnotobiotic pigs, the significance of porcine SaVs in different ages of pigs with diarrhea in the field is still undetermined. This is due to two reasons: 1) similar prevalence of porcine SaVs was detected in diarrheic and non-diarrheic pigs; and 2) co-infection of porcine SaVs with other enteric pathogens is common in pigs. Diagnosis of porcine SaV infection is mainly based on the detection of viral nucleic acids using reverse transcription (RT)-PCR and sequencing. Much is unknown about these genetically diverse viruses to understand their role in pig health and to evaluate whether vaccines are needed to prevent SaV infection.

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirus and therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

SUMMARYSapoviruses cause acute gastroenteritis in humans and animals. They belong to the genusSapoviruswithin the familyCaliciviridae. They infect and cause disease in humans of all ages, in both sporadic cases and outbreaks. The clinical symptoms of sapovirus gastroenteritis are indistinguishable from those caused by noroviruses, so laboratory diagnosis is essential to identify the pathogen. Sapoviruses are highly diverse genetically and antigenically. Currently, reverse transcription-PCR (RT-PCR) assays are widely used for sapovirus detection from clinical specimens due to their high sensitivity and broad reactivity as well as the lack of sensitive assays for antigen detection or cell culture systems for the detection of infectious viruses. Sapoviruses were first discovered in 1976 by electron microscopy in diarrheic samples of humans. To date, sapoviruses have also been detected from several animals: pigs, mink, dogs, sea lions, and bats. In this review, we focus on genomic and antigenic features, molecular typing/classification, detection methods, and clinical and epidemiological profiles of human sapoviruses.

Noroviruses (NoVs) and sapoviruses (SaVs) are important human pathogens. Although the involvement of porcine NoVs in disease in pigs is unclear, they are genetically and antigenically closely related to human NoVs. Human NoV-like strains have been detected in pigs, raising public health concerns of potential interspecies transmission. Porcine SaVs are highly diverse and emerging in swine populations. Recently, at least three new genogroups of porcine SaVs have been proposed. In this study, we tested 413 pooled fecal samples collected from apparently healthy finisher pigs in North Carolina swine farms during 2009. Reverse transcription (RT)-PCR coupled hybridization assays were performed to detect known porcine NoVs. The overall prevalence of porcine NoVs determined was 18.9% based on this method. Samples were then tested by RT-PCR targeting the 5′ end of the capsid region for genogroup II (GII) NoVs, a group which includes human NoVs, followed by sequence analysis. All NoVs identified belonged to typical porcine NoV genotypes, and no human NoV-like strains were detected in specimens from these pigs. Porcine NoV-negative samples ( n = 335) were subsequently screened using universal calicivirus primers, and 17 SaV strains were confirmed by sequencing. Based on the partial RNA-dependent RNA polymerase (RdRp) region, they clustered with GIII, GVII, and GVIII and with currently unclassified SaVs. According to analysis of the complete capsid sequences, 7 representative strains clustered with GVII, GVIII, and GIX? SaVs. We tentatively classified SaVs into 14 genogroups based on the complete capsid protein VP1. In summary, porcine NoVs and highly divergent SaVs were present in North Carolina finisher pigs.

Caliciviruses are contagious pathogens of humans and various animals. They are the most common cause of viral gastroenteritis in humans, and can cause lethal diseases in domestic animals such as cats, rabbits and immunocompromised mice. In this study, we conducted cytopathic effect-based screening of 2080 selected compounds from our in-house library to find antiviral compounds against three culturable caliciviruses: feline calicivirus, murine norovirus (MNV) and porcine sapovirus (PoSaV). We identified active six compounds, of which two compounds, both related to theaflavins, showed broad antiviral activities against all three caliciviruses; three compounds (abamectin, a mixture of avermectin B1a and B1b; avermectin B1a; and (-)-epigallocatechin gallate hydrate) were effective against PoSaV only; and a heterocyclic carboxamide derivative (BFTC) specifically inhibited MNV infectivity in cell cultures. Further studies of the antiviral mechanism and structure-activity relationship of theaflavins suggested the following: (1) theaflavins worked before the viral entry step; (2) the effect of theaflavins was time- and concentration-dependent; and (3) the hydroxyl groups of the benzocycloheptenone ring were probably important for the anti-calicivirus activity of theaflavins. Theaflavins could be used for the calicivirus research, and as potential disinfectants and antiviral reagents to prevent and control calicivirus infections in animals and humans.

We isolated a novel murine norovirus (MNV), MT30-2 strain, from feces of conventional mice in Japan to evaluate the virucidal activity of four antiseptics. The MNV MT30-2 strain was inactivated by as little as 0.2% (w/v) povidone-iodine (PVP-I) and 0.1% (w/v) sodium hypochlorite (NaOCl) treatment as determined by a novel plaque assay. Importantly, PVP-I reduced the MNV titer by 4 log(10) within 15 s of exposure. The other two antiseptics, benzethonium chloride (BEC) and chlorhexidine gluconate (CHG), did not reduce the MNV titer even when treatment lasted for 60 s. When the virus titer was reduced by PVP-I or NaOCl treatment, the amount of MNV RNA was not reduced, indicating that the presence of viral RNA was not related to the virucidal activity of the antiseptics. PVP-I and NaOCl will be useful in controlling the spread of MNV, which is a common problem in mice colonies. In this study, we isolated a novel MNV and newly revealed that two antiseptics (PVP-I and NaOCl) were able to inactivate MNV at low concentrations and in a short contact time.

The genetically diverse sapoviruses (SaVs) are a significant cause of acute human gastroenteritis. Human SaV surveillance is becoming more critical, and a better understanding of the diversity and distribution of the viral genotypes is needed. In this study, we analyzed 106 complete human SaV capsid nucleotide sequences to provide a better understanding of their diversity. Based on those results, we propose a novel standardized classification scheme that meets the requirements of the International Calicivirus Scientific Committee. We believe the classification scheme and strains described here will be of value for the molecular characterization and classification of newly detected SaV genotypes and for comparing data worldwide.

Murine norovirus (MNV) is a viral agent newly identified in laboratory mice and a large number of genetically diverse MNV strains have been reported to date. A broadly reactive TaqMan-based real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay was developed for MNVs. Novel primers and a TaqMan MGB probe were designed targeting highly conserved sequences among MNV strains, which are located in the open reading frames 1 (ORF1)–ORF2 junction region. The quantitative range of this assay was determined as 1.0 × 102–1.0 × 108 copies/PCR tube based on a 10-fold serial dilution of plasmid DNA containing the target sequences. Viral RNA in eight murine stool specimens positive by nested RT-PCR assay was measured, and the highest viral RNA load was calculated at 4.7 × 106 copies/g-stool. MNV was inoculated into RAW 264.7 cells, and the viral RNA was monitored to validate assay sensitivity. MNV-RNA in the supernatant was detected during in vitro replication, which increased substantially from 5 to 30 h post-infection (hpi) and reached more than 1.0 × 1010 copies/mL at 96 hpi. This real-time RT-PCR assay is a useful tool to detect and quantify MNV-RNA in in vivo and in vitro studies.

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 approximately 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.

SaV, a pathogen of acute gastroenteritis, is divided into five genogroups, GI to GV. However, the relation between SaV antigenicity and genetic clusters is not fully understood. We have recently identified two GII SaV strains, Mc10 and C12, which are grouped into the same cluster based on the polymerase but are grouped into distinct clusters based on the capsid. To evaluate the difference in antigenicity between these two strains, VLP were expressed in mammalian cells. An antigen ELISA demonstrated for the first time that strains in the same GII SaV genogroup, but within different clusters, have distinct antigenicities.

We expressed full-length sapovirus genome constructs in insect cells and analyzed their products. The capsid protein was cleaved from the ORF1 polyprotein from a native-like genome construct and two full-length genome constructs with mutations in an active polymerase motif, whereas the capsid protein was not cleaved from a full-length genome construct with a mutation in an active protease motif. Our results showed that the sapovirus protease-polymerase precursor protein cleaved the capsid protein from the polyprotein at the putative conserved capsid start. Importantly, the cleaved capsid protein formed empty virus-like particles that were morphologically and antigenically similar to native sapovirus.

Correspondence between virus antigenicity and capsid (VP1) genogrouping and genotyping is likely., Sapovirus (SaV) is a causative agent of gastroenteritis. On the basis of capsid protein (VP1) nucleotide sequences, SaV can be divided into 5 genogroups (GI–GV), of which the GI, GII, GIV, and GV strains infect humans. SaV is uncultivable, but expression of recombinant VP1 in insect cells results in formation of viruslike particles (VLPs) that are antigenically similar to native SaV. In this study, we newly expressed SaV GII and GIV VLPs to compare genetic and antigenic relationships among all human SaV genogroups. Hyperimmune antiserum samples against VLPs reacted strongly with homologous VLPs. However, several antiserum samples weakly cross-reacted against heterologous VLPs in an antibody ELISA. Conversely, an antigen ELISA showed that VLPs of SaV in all human genogroups were antigenically distinct. These findings indicate a likely correspondence between SaV antigenicity and VP1 genogrouping and genotyping.

Sapovirus (SaV) is a causative agent of gastroenteritis in humans in both sporadic cases and outbreaks. During the period from January 2005 to August 2014, SaV was detected in 30 (5.9%) of 510 gastroenteritis outbreaks in Osaka City, Japan using real-time RT-PCR. Seasonal distribution of SaV-associated outbreaks revealed an increase during the 2011-2012 season and the highest frequency of outbreaks during the 2012-2013 season. Genotyping analysis based on the capsid region demonstrated that the most common genotype was GI.2 (36.7%), in which the strains were closely related. The comparison of complete capsid gene sequences with 18 GI.2 strains (7 strains in this study and 11 from GenBank) between 1990 and 2013 showed that GI.2 strains were classified into at least three genetic clusters (1990-2000, 2004-2007, and 2008-2013) with chronologically unique amino acid residues and accumulation of mutations in the predicted P domain, suggesting the one of the causes of emergence and spread of GI.2 strains. This study will also be helpful for understanding the evolutionary mechanism of the SaV genome.

Feline calicivirus (FCV) is an important veterinary pathogen that causes acute upper respiratory tract diseases and, occasionally, highly contagious febrile hemorrhagic syndrome in cats. Many viruses have adopted mechanisms for evading IFN-α/β signaling, particularly by directly or indirectly suppressing activation of IRF-3. In this study, we investigated whether nonstructural proteins of FCV possess these mechanisms. When p39, a nonstructural protein of FCV, was transiently expressed in 293T cells, it suppressed IFN-β and ISG15 mRNA production induced by dsRNA. Expression of p39 also suppressed phosphorylation and dimerization of IRF-3 induced by dsRNA. These results suggest that p39 suppresses type 1 IFN production by preventing IRF-3 activation. This may become an important factor in understanding the pathogenesis and virulence of FCV.

Noroviruses are a major health burden and are responsible for the majority of outbreaks of gastroenteritis in the world. Human noroviruses can be genetically divided into two main genogroups (GI and GII) and subdivided into many genotypes. In this study, stool specimens collected from 12 outbreaks of gastroenteritis in Taiwan were screened for viral agents between the 23rd of November 2004 and 9th of March 2005. Noroviruses were detected in all outbreaks. We detected six different norovirus genotypes: GI/11, GI/14, GII/3, GII/4, GII/6, and GII/18. Noroviruses belonging to GII/4 were dominant, 50 of 60 (83%) sequences, and were detected in 10 of 12 outbreaks. Furthermore, the norovirus GII/4 strains were detected throughout Taiwan, demonstrating their widespread distribution. We also found that three outbreaks had noroviruses from multiple genotypes. Our results have shown for the first time that noroviruses are an important cause of gastroenteritis in Taiwan.

Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI–GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 × 107 to 2.5 × 101 copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses. J. Med. Virol. 78:1347–1353, 2006. © 2006 Wiley-Liss, Inc.

To the Editor: Sapovirus is an etiologic agent of human gastroenteritis. Although many of the previously reported cases were of mild, sporadic infections in young children (1–3), several recent sapovirus-associated gastroenteritis outbreaks have affected adults, which suggests that the virus’s virulence, prevalence, or both, may be increasing (4–6). In this study, we describe a sapovirus-associated outbreak of gastroenteritis that occurred during May 4–8, 2007, and involved college students in northern Taiwan. A total of 55 students had clinical symptoms of gastroenteritis, including diarrhea (45), vomiting (22), abdominal cramps (17), and fever (2). The clinical symptoms continued for up to 10 days (mean 4.7 days). Stool specimens were collected from 8 of 55 students on May 8 (Table). Initially, the specimens were screened for bacteria, rotavirus, and norovirus, but all specimens were negative for these pathogens. The 8 stool specimens were then examined by electron microscopy (EM), and 1 was positive for calicivirus-like particles. Table Clinical symptoms and laboratory diagnosis results for sapovirus-related outbreak among college students, northern Taiwan, May 2007*† To confirm the EM results, we performed reverse transcription–PCR (RT-PCR), real-time RT-PCR, and sequence analysis as previously described (7). Briefly, purified RNA (10 μL) was reverse transcribed by using SuperScript III reverse transcriptase according to the manufacture’s instructions (Invitrogen, Carlsbad, CA, USA). PCR was carried out by using the SV-F11 and SV-R1 primer set directed against the conserved N terminal capsid region (8). The PCR products were analyzed with 2% agarose gel electrophoresis and visualized after ethidium bromide staining. The PCR-generated amplicons (≈780 bp) 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) and determined with the ABI 3130 sequencer (Applied Biosystems, Foster City, CA, USA.). Nucleotide sequences were aligned by using ClustalX (www.clustal.org), and the distances were calculated by using the Kimura 2-parameter method. A phylogenetic tree was generated by the neighbor-joining method as described previously (1,8). Of the 8 specimens, 7 were positive by RT-PCR and real-time RT-PCR (Table). SaV124F, SaV1F, SaV5F, and SaV1245R primers as well as SaV124TP and SaV5TP minor-groove binding probes were used for real-time RT-PCR diagnosis, which targets the sapovirus RdRp-capsid junction region as described (7). The number of sapovirus cDNA copies ranged from 2.86 × 107 to 1.72 × 1010 copies/g of stool specimen; mean was 2.71 × 109 copies/g of stool specimen (Table). Sequence analysis of the 7 positive specimens showed 100% nucleotide identity (nt 5098–5878), indicating that the outbreak was caused by 1 sapovirus strain. To better classify the sapovirus, we reamplified the 3′ end of the genome from 1 positive specimen and sequenced ≈2,400 nt (nt 5074-3′) (Hu/SaV/9–5/Taipei/07/TW; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU124657","term_id":"158929628"}}EU124657). PCR was performed with SV-F13, SV-F14, and TX30SXN primers as described (1). Database searches found a closely matching sapovirus sequence (99%) that was detected in a patient with gastroenteritis in Japan, in 2004 (Chiba041413 strain; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB258427","term_id":"118442701"}}AB258427). The next closely matching sequence was detected in an outbreak of gastroenteritis among adults in the United States in 1994 (Parkville strain; {"type":"entrez-nucleotide","attrs":{"text":"HCU73124","term_id":"1916653"}}HCU73124) (6). Phylogenetic analysis clustered these 3 sapovirus sequences into genogroup I/genotype 2 (GI/2) (Appendix Figure). Sapovirus was reported in Japan in water samples (untreated wastewater, treated wastewater, and a river) and in clam samples intended for human consumption (1). Apart from these 2 environmental studies, little is known about reservoir of sapovirus or its route of infection in the natural environment. The source of contamination in this current outbreak was not determined; however, none of the food handlers associated with the college reported symptoms of gastroenteritis. However, in a recent molecular epidemiologic study in Japan, a large number of symptomatic and asymptomatic food handlers were found to be infected with noroviruses (9). Several seroprevalence studies also indicated high prevalence rates of antibodies to sapovirus in adults and children (10). All of these findings highlight the need to collect stool specimens from asymptomatic persons and indicate possible “silent” transmission through an asymptomatic route. Symptoms of sapovirus infection are thought to be milder than symptoms of norovirus infections. However, in this study approximately one third (17) of the 55 students reported symptoms of abdominal pain and 22 (40%) reported symptoms of vomiting. Many of the earlier sapovirus studies described sapovirus GI/1 infections in young Japanese children (1), which indicated that infecting virus had a different genotype than the virus detected in this study (GI/2). In addition, the viral load in this study appeared to be comparatively high. These results suggest that some sapovirus genotypes are more virulent than others. Similar findings were obtained with norovirus infections around the world; strains belonging to norovirus GII/4 were the most prevalent in many countries. Although several recombinant sapovirus strains have been identified and found to be the cause of increased numbers of infections in some countries (1,5), they were not observed in this study. Increased sapovirus surveillance and reporting are needed to shed some more light on this poorly understood virus.

To the Editor: Sapovirus and norovirus are causative agents of gastroenteritis in children and adults. Norovirus is the most important cause of outbreaks of gastroenteritis, whereas only a few outbreaks of sapovirus have been reported (1,2). On the basis of complete capsid gene sequences, sapovirus can be divided into 5 genogroups, among which GI, GII, GIV, and GV infect humans, whereas sapovirus GIII infects porcine species. We report 2 outbreaks of gastroenteritis in Hokkaido, Japan. The first outbreak (A) occurred at a college from May 29 to June 2, 2000. A total of 12 persons (11 students and 1 teacher) reported symptoms of gastroenteritis (nausea, vomiting, stomachache, diarrhea, and fever); 11 stool specimens were collected from days 1 to 7 after onset of illness (Table). These specimens were negative for norovirus (data not shown), but 5 were positive for sapoviruslike viruses by electron microscopy (Table). Table Analysis of 18 stool specimens for sapovirus during 2 outbreaks of gastroenteritis, Japan* The 11 specimens were then examined for sapovirus by using nested reverse transcription–PCR (RT-PCR) as described (3). A total of 9 (82%) of 11 specimens were positive for sapovirus. Sequence analysis showed that these 9 viruses had 100% nucleotide identity and likely represented the same sapovirus strain (termed Yak2 strain, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB046353","term_id":"148717098","term_text":"AB046353"}}AB046353). To determine the number of cDNA copies per gram of stool, we performed real-time RT-PCR as described (4). The number of sapovirus cDNA copies ranged from 5.36 × 105 to 7.47 × 109/g stool (median 5.49 × 109 copies/g stool) (Table). The second outbreak (B) occurred at a kindergarten from February 1 to 22, 2005. A total of 23 persons (15 children and 8 adults) reported symptoms of gastroenteritis (nausea, vomiting, stomachache, diarrhea, and fever); 7 stool specimens were collected (Table). These specimens were negative for norovirus (data not shown), but all were positive for sapovirus by nested RT-PCR. The 7 sequences from this outbreak had 100% nucleotide identity and likely represented the same sapovirus strain (termed Nay1 strain, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF213768","term_id":"126565998","term_text":"EF213768"}}EF213768). The number of sapovirus cDNA copies ranged from 1.14 × 109 to 5.41 × 1010/g stool (median 2.50 × 1010 copies/g stool) (Table). One positive sapovirus specimen from each outbreak was subjected to further sequence analysis in which a single overlapping PCR fragment covering the partial polymerase gene and capsid gene was amplified. The Yak2 and Nay1 sequences shared ≈71% nucleotide identity for this fragment and likely represented different sapovirus strains. The Yak2 sequence closely matched sapovirus GIV Ehime1107 and SW278 sequences (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"DQ058829","term_id":"70799531","term_text":"DQ058829"}}DQ058829 and {"type":"entrez-nucleotide","attrs":{"text":"AY237420","term_id":"45545440","term_text":"AY237420"}}AY237420, respectively) and had 98% and 97% nucleotide identity for the entire fragment, respectively (5). The Nay1 sequence closely matched the sapovirus GII C12 sequence ({"type":"entrez-nucleotide","attrs":{"text":"AY603425","term_id":"51243518","term_text":"AY603425"}}AY603425) and had 91% nucleotide identity for the entire fragment. The Nay1 sequence closely matched the C12 sequence, which was detected in Osaka, Japan, in 2001 (6), whereas the Yak2 sequence closely matched the Ehime1107 sequence, which was detected in Matsuyama, Japan, in 2002 (5) and the SW278 sequence, which was detected in Sweden in 2003 (1). We recently described the C12 strain as intragenogroup recombinant sapovirus strain (6), whereas the Ehime1107 and SW278 strains were described as intergenogroup recombinant sapovirus strains (5). Our results indicate that recombination sites in intragenogroup and intergenogroup recombinant sapovirus strains were at the polymerase and capsid junction (5,6). Sapovirus Sydney53 ({"type":"entrez-nucleotide","attrs":{"text":"DQ104360","term_id":"308196023","term_text":"DQ104360"}}DQ104360) and Sydney3 strains ({"type":"entrez-nucleotide","attrs":{"text":"DQ104357","term_id":"308196021","term_text":"DQ104357"}}DQ104357), which were detected in Australia from August 2001 to August 2004 (7), closely matched C12 and Ehime1107/SW278 sequences, respectively. These results showed that recombinant sapovirus strains are stable in the environment and may be globally distributed. Our findings also suggest a changing distribution of sapovirus-associated gastroenteritis in Hokkaido because different sapovirus GI strains were predominant sapovirus strains that caused causing outbreaks of gastroenteritis in Hokkaido (8,9). In a recent study, the number of norovirus cDNA copies per gram of stool specimen was analyzed and a discrepancy was found between the different norovirus genogroups (10). Chan et al. found that noroviruses GI and GII showed medians of 8.4 × 105 and 3.0 × 108 copies/g of stool specimen, respectively, and speculated that increased viral loads were caused by higher transmissibility of norovirus GII strains (10). Our results showed that sapovirus GII Nay1 and GIV Yak2 strains showed higher viral loads than norovirus GII strains. These results suggest that a high degree of shedding of sapovirus GII Nay1 and GIV Yak2 strains may have caused the outbreak of gastroenteritis. However, to elucidate this suggestion, further studies are needed with other sapovirus strains.

Norovirus GII.4 is a major etiological agent of acute viral gastroenteritis worldwide. We examined GII.4 evolution using 277 near-full-length GII.4 genome sequences from human stool specimens collected at 20 sites in Japan between May 2006 and March 2010. We found outbreaks of 8 monophyletic GII.4 subtypes, among which a single subtype, termed 2006b, had continually predominated (222/277: 80.7%). Four of the 8 GII.4 subtypes were chimera viruses of recently prevalent GII.4 subtypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF) 1 and ORF 2 (P0.0001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII.4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsid were preferentially positioned on the outer surface loops of the protruding P2 domain. These data and computer-assisted structural study of NoV capsid protein are compatible with a model of antigenic drift with tuning of the structure-functions of multiple proteins for the survival strategy of GII.4 2006b variant.

The prevalence of porcine sapoviruses (SaVs) and noroviruses (NoVs) in nursing piglets on three pig farms in Ohio was studied. Fecal samples (n = 139) were collected from individual pigs and screened for caliciviruses by RT-PCR. Phylogenetic analysis was conducted using partial sequences of the RNA polymerase region. Three different SaV genogroups, including a newly emerging one (DO19 Korea-like) were detected. No NoVs were detected. Kobuviruses, emerging members of the family Picornaviridae, were detected by primers designed for SaV. To our knowledge, this is the first report of porcine DO19 Korea-like SaV and kobuvirus in the United States.

Sapovirus (SaV) is an important pathogen that causes acute gastroenteritis in humans. Human SaV is highly diverse genetically and is classified into multiple genogroups and genotypes. At present, there is no clear evidence for gastroenteritis cases caused by re-infection with SaV. We found that two individuals were sequentially infected with SaVs of two different genogroups and had gastroenteritis after each infection, although in one of the subsequent cases, both SaV and norovirus were detected. We also found a genetic shift in SaVs from gastroenteritis outpatients in the same geographical location. Our results suggest that protective immunity may be at least genogroup-specific for SaV.

SaV sequences which are either genetically identical or similar were detected from oysters, feces from gastroenteritis patients, and domestic wastewater samples in geographically close areas. This is the first report of the detection of SaV in oysters which meet the legal requirements for raw consumption in Japan.

Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3Cpro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10−3 nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5). Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53). Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3Dpol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events. To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.

Norovirus (NoV) and sapovirus (SaV) are important pathogens of human gastroenteritis. Compared to NoV, the transmission route of SaV is unclear. An outbreak of gastroenteritis occurred at a restaurant in June 2008, and SaV and NoV were detected in fecal specimens from 17 people who ate at the restaurant and one asymptomatic food handler and also in stripped shellfish and liquids remaining in the shellfish packages by reverse transcription-polymerase chain reaction (RT-PCR) and/or real-time RT-PCR. Nucleotide sequencing analysis of the RT-PCR products corresponding to the partial capsid region revealed 99.3-100% identities for SaV and 98.6-99.3% identities for NoV among the digestive diverticulum of the frozen stripped shellfish (Ruditapes philippinarum), "Asari," the package liquid, and feces from symptomatic or asymptomatic guests. These results suggested a link between the consumption of contaminated shellfish and clinical features in the patients. While the transmission of NoV by shellfish has been reported, this report shows that SaV can also be transmitted by shellfish.

Sapovirus (SaV) is an important pathogen of human acute gastroenteritis. A gastroenteritis outbreak occurred at a wedding hall in October 2007 in Ehime Prefecture, Japan. One hundred nine people who had either attended wedding parties or had eaten a box lunch at a conference held at the same hall complained of gastroenteritis symptoms. Among these 109 people, stool specimens from 56 patients were available for pathogen screening, and 20 (35.7%) of these specimens were positive for SaV, of whom 18 showed symptoms. The numbers of cDNA copies of the specimens ranged from 2.36 x 10(6) to 3.03 x 10(10) for symptomatic patients, and 2.19 x 10(6) and 1.18 x 10(9) per gram of stool for two asymptomatic food handlers. The incubation periods of the 18 symptomatic patients ranged from 14.5 to 99.5 hr. Identical nucleotide sequence types of SaV; that is, a single synonymous nucleotide difference (transition) or microheterogeneity, was detected in stool specimens from the symptomatic patients and the asymptomatic food handlers, with the direct nucleotide sequence of approximately 2.3 kb 3' end of the genome. Based on the phylogenetic analysis with the complete capsid nucleotide sequence, these strains were clustered into genogroup IV. This outbreak was thought to be caused by a single source, and underscores the importance of proper hygiene in the environment and/or in food-handling practices to control SaV outbreaks.

Infectious acute gastroenteritis is an important public health problem worldwide. A total of 639 stool specimens were tested for the presence of diarrhea pathogens. The specimens were from outpatients with acute gastroenteritis who consulted the pediatric clinic in Kumamoto Prefecture, Japan, from June 2002 to December 2007. Of these, 421 (65.9%) were positive for diarrhea pathogens. Among them were norovirus (NoV) in 260 (61.8%), sapovirus (SaV) in 81 (19.2%), rotavirus in 49 (11.6%), adenovirus in 19 (4.5%), enterovirus in 13 (3.1%), astrovirus in 9 (2.1%), kobuvirus in 1 (0.2%), and bacterial pathogens in 11 (2.6%). Mixed infection (co-infection of viruses) was found in 22 (5.2%) of the 421 pathogen-positive stool samples. NoV was the most prevalent pathogen throughout the study period; however, the SaV detection rate was unexpectedly high and was found to be the secondary pathogen from 2005 to 2007. Genetic analysis of SaV with 81 strains demonstrated that SaV strains belonging to genogroup IV emerged in 2007, and dynamic genogroup changes occurred in a restricted geographic area. This study showed that SaV infection is not as rare as thought previously.

Sapovirus (SaV) is an important pathogen causing gastroenteritis in humans. Quantitative analysis of the viral loads in feces collected from two SaV outbreaks was performed. Our results showed that SaV excretion generally decreased to an undetectable level in 2 weeks; however, some individuals excrete SaV in feces at high concentrations for 2-4 weeks after the onset of illness. In addition, we identified for the first time nucleotide changes in the capsid region during prolonged excretion.

Noroviruses are the leading cause of outbreaks of gastroenteritis in the world. At present, norovirus genogroup II, genotype 4 (GII/4), strains are the most prevalent in many countries. In this study we investigated 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food handlers in food-catering settings between 10 November 2005 and 9 December 2006 in Japan. Stool specimens were collected from both symptomatic and asymptomatic individuals and were examined for norovirus by real-time reverse transcription-PCR; the results were then confirmed by sequence analysis. Norovirus was detected in 449 of 2,376 (19%) specimens. Four genogroup I (GI) genotypes and 12 GII genotypes, including one new GII genotype, were detected. The GII/4 sequences were predominant, accounting for 19 of 55 (35%) outbreaks and 16 of 35 (46%) sporadic cases. Our results also showed that a large number of asymptomatic food handlers were infected with norovirus GII/4 strains. Norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI, i.e., 3.81 × 10 8 versus 2.79 × 10 7 copies/g of stool. Among norovirus GI strains, GI/4 had the highest mean viral load, whereas among GII strains, GII/4 had the highest mean viral load (2.02 × 10 8 and 7.96 × 10 9 copies/g of stool, respectively). Importantly, we found that asymptomatic individuals had mean viral loads similar to those of symptomatic individuals, which may account for the increased number of infections and the predominance of an asymptomatic transmission route.

The family Caliciviridae contains four genera Sapovirus, Norovirus, Lagovirus and Vesivirus, which include Sapporo virus (SaV), Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV) and Feline calicivirus (FCV), respectively. SaV is a causative agent of gastroenteritis in children and adults. SaV can be divided into five genogroups (GI-GV), among which GI, GII, GIV and GV are known to infect humans, whereas SaV GIII infects porcine species. Detection methods include ELISA, RT-PCR and real-time RT-PCR. Since few SaV studies have been conducted, it is difficult to draw correlations between or conclusions about rates of incidence, detection and overall prevalence. Nevertheless, most studies agree that SaV infection is more frequent in young children than adults and that infection in children almost always occurs by 5 years of age. In addition, children at day-care centres and institutions are at greatest risk of SaV-associated infection and transmission. Recently, a number of important findings concerning human SaV were discovered. SaV strains were detected in water samples, which included untreated wastewater specimens, treated wastewater samples and river samples. SaV strains were also detected in shellfish samples destined for human consumption, and recombinant SaV strains were identified in a number of different countries. The purpose of this review was to highlight the current knowledge of human SaV, which appears to be an increasingly important virus causing gastroenteritis in humans.

Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI–GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH2–p11–p28–p35 (NTPase)–p32–p14 (VPg)–p70 (Pro–Pol)–p60 (VP1)–COOH. The cleavage site between p14 (VPg) and p70 (Pro–Pol) was identified as E1055/A1056 by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription–translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E69/G70, Q325/G326, Q666/G667, E940/A941, E1055/A1056 and E1722/G1723. The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1′ position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.

Stool specimens collected between November 2002 and April 2003 from hospitalized infants with acute gastroenteritis from four distinct geographical regions in Thailand were examined for norovirus (NoV) and sapovirus (SaV) by reverse transcription-PCR and sequence analysis. Of the 80 specimens examined, we identified 11 NoV and 9 SaV single infections, and 3 NoV/SaV mixed infections. The majority of NoV strains (64%) belonged to genogroup II/ genotype 4 (GII/4; Lordsdale cluster). Other NoV strains co-circulating belonged to GII/1, GII/3, GII/6, and one new genotype cluster (GII/New). The majority of SaV strains (83%) were from the Manchester cluster. One isolated SaV strain represented a recently discovered novel genogroup within the SaV genus (SG-V), and another isolated SaV strain represented a novel SaV genogroup II cluster.

Norovirus (NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology 299: 225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol. 41: 1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences. Human SaV strains are noncultivable, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV. In this study, rVP1 constructs of SaV GI, GII, and GV strains were expressed in a baculovirus expression system. The structures of the GI, GII, and GV VLPs, with diameters of 41-48 nm, were morphologically similar to those of native SaV. However a fraction of GV VLPs were smaller, with diameters of 26-31 nm and spikes on the outline. This is the first report of GII and GV VLP formation and the first identification of small VLPs. To examine the cross-reactivities among GI, GII, and GV rVP1, hyperimmune rabbit antisera were raised against Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1. Western blotting showed the GI antisera cross-reacted with GV rVP1 but not GII rVP1; GII antisera cross-reacted weakly with GI rVP1 but did not cross-react with GV rVP1; and GV antisera reacted only with GV rVP1. Also, hyperimmune rabbit and guinea pig antisera raised against purified GI VLPs were used to examine the cross-reactivities among GI, GII, and GV VLPs by an antigen enzyme-linked immunosorbent assay (ELISA). The ELISA showed that the GI VLPs were antigenically distinct from GII and GV VLPs.

An outbreak of gastroenteritis occurred at a kindergarten in Yokote City, Japan, between February 2006 and March 2006. Sapovirus was identified in 19 of 26 stool specimens by reverse transcription-PCR. A high viral shedding pattern was found for this strain, which was shown to be antigenically distinct from other genogroups.

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 in Japan, which indicates a possible food-to-human transmission link.