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PubMed2004

Cross-reactivity among sapovirus recombinant capsid proteins.

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.

Animals↗

PubMed2025

Genotypic Analysis and Clinical Findings of Sapovirus-Associated Acute Gastroenteritis in Mie Prefecture, Japan, 2010-2022.

Sapovirus (SaV) is one of the major viruses causing acute gastroenteritis. Of the 1981 fecal specimens collected through sentinel pediatric acute gastroenteritis pathogen surveillance in Mie Prefecture, Japan (2010-2022), 236 were positive for SaV, according to PCR screening. Whole or near-whole genome sequences were determined for 158 strains by next-generation sequencing. Genotype GI.1 was the most common of the nine SaV genotypes detected, followed by GII.3 and GII.1. Phylogenetic analysis showed that SaVs of these three genotypes separated into three different clusters depending on the year of detection, suggesting continuous genetic changes in the same genotype. Coinfections involving different SaV genotypes, as well as reinfections with SaV in the same individual, were observed in this study. The main clinical manifestations were diarrhea (68.4%) and vomiting (61.6%), with an increased rate of emesis, particularly in patients over 3 years of age. In addition, 18.1% of the children had fever. This study clarified the prevalence of viral genotypes as well as clinical findings of SaV-positive gastroenteritis in children, and revealed trends by age.

Humans↗

PubMed2004

Human astrovirus, norovirus (GI, GII), and sapovirus infections in Pakistani children with diarrhea.

Fecal specimens from 517 infants and young children admitted to the Civil Karachi Hospital, Dow Medical College, Karachi city, Pakistan with acute gastroenteritis from 1990 to 1994 were collected and screened by RT-PCR for human astrovirus (AstV), norovirus (NV), and sapovirus (SV). The specific epidemiological data for illness caused by these viruses in Pakistan are not available. AstV, NV, and SV were detected in 58, 51, and 17 of 517 fecal specimens, and this represented 11.2, 9.9, and 3.2%, respectively. An outbreak of gastroenteritis attributable to AstV serotype 1 was identified during September and October 1990. Moreover, one specimen with a triple mixed infection between AstV (serotypes 1 and 3) and NV GII was found. NV and SV were subjected to molecular analysis by sequencing. One of the sequenced specimens positive for SV turned out to be similar to a strain tentatively called a genogroup IV. The result underscores the importance of these viruses in association with acute gastroenteritis in Karachi city, Pakistan.

Astroviridae Infections↗

PubMed2006

Characterization of sapoviruses collected in the United Kingdom from 1989 to 2004.

A fecal archive containing 115 sapovirus (SaV) strains detected in samples collected from 15 outbreaks and 98 sporadic cases of gastroenteritis between 1989 and 2004 in the UK were characterized in order to determine the genomic diversity within SaV co-circulating in the human population. Strains were characterized by partial sequencing of the genes encoding the RNA-dependent RNA polymerase (RdRp) region and/or the polymerase/capsid (Pol/Cap) junction of the open reading frame (Orf) 1. Overall, SaV of genogroup I genotype 1 (GI 1) were the predominant strains circulating in the UK in each year between 1989 and 2004. During 2004, GII 1 was the predominant strain. These two SaV types accounted for 89.5% of the sporadic cases and outbreaks in the UK. The remaining cases were caused by six other SaV genotypes. On the basis of partial sequencing of the RdRp and capsid encoding genes of strains, which did not show sufficient homology to any of the currently recognized genotypes, we propose the inclusion of a presumptive fourth genotype within genogroup I (GI 4).

Adult↗

PubMed2004

Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam.

This report describes norovirus (NoV) and sapovirus (SaV) infections in hospitalized children with acute sporadic gastroenteritis in Ho Chi Minh City, Vietnam. Stool specimens collected between December 1999 and November 2000 were examined for NoV and SaV using reverse transcription-PCR and phylogenetic analysis. NoVs were detected in 72 of 448 rotavirus-negative specimens, counted as part of an overall annual detection rate of 5.4% (72 of 1,339 children). This included four NoV genogroup I (GI) strains and 68 NoV GII strains. Only one SaV GI strain was detected in the rotavirus-negative specimens. Over 73% of the NoV sequences belonged to GII/4 (Lordsdale cluster) and were detected in all months except March. We also detected GII/3 strains (Saitama U201 cluster), a naturally occurring recombinant NoV, between January 2000 and March 2000 but not after this period. Other NoV strains belonging to GI/4, GI/8, GII/1, and GII/7 were also detected but were infrequent. In addition, two almost identical NoV GII strains (strains 026 and 0703) collected six months apart were classified into a new genotype that includes the Mc37 strain, which was previously shown to be a recombinant NoV. During this one-year study, the NoV prevailed at the end of the rainy season and the beginning of the dry season. Further epidemiological studies may be necessary to determine whether the GII/4 strains continue to dominant in this region.

Adolescent↗

PubMed2005

Cleavage activity of the sapovirus 3C-like protease in Escherichia coli.

We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 x His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E(1055) and A(1056). In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.

Amino Acid Sequence↗

PubMed2005

Deletion analysis of the sapovirus VP1 gene for the assembly of virus-like particles.

Human sapovirus (SaV) strains are agents of gastroenteritis. They cannot be grown in cell culture. In this study, constructs containing SaV N- and C-terminal-deleted recombinant capsid proteins (rVP1) were expressed in a baculovirus expression system to allow us to better understand the sequence requirements for the formation of virus-like particles (VLPs). Only proteins derived from N-terminal-deleted rVP1 constructs that began 49 nucleotides downstream assembled into VLPs, which included both small and native-size VLPs. Our results were similar to those reported in a rabbit hemorrhagic disease virus (RHDV) N- and C-terminal-deleted rVP1 expression study but were distinct from those reported in a norovirus N- and C-terminal-deleted rVP1 expression study, suggesting that SaV and RHDV may have similar expression requirements.

Baculoviridae↗

PubMed2005

Expression of sapovirus virus-like particles in mammalian cells.

Sapovirus (SaV) is an etiological agent of acute gastroenteritis in human and swine. SaV can be divided into five genogroups, GI to GV. Virus-like particles (VLPs) morphologically similar to native SaV have been expressed for GI, GII, GIII and GV strains in insect cells, although only low expression levels were observed for GII strains. In this study, we report the successful expression of SaV GII VLPs using cultured mammalian COS-7 and 293T cells. Our results demonstrated that this mammalian expression system was able to express and form SaV VLPs.

Animals↗

PubMed2005

Development of an antigen ELISA to detect sapovirus in clinical stool specimens.

Human sapovirus (SaV) strains are etiological agents of mild and/or acute gastroenteritis in children and adults. In this study, we describe the development of a novel antigen enzyme-linked immunosorbent assay (ELISA) detection system that was based on hyperimmune rabbit and guinea pig antisera raised against SaV genogroup I (GI) virus-like particles. The ELISA had 100% specificity, and sensitivities of 60% and 25% when compared to single-round PCR and nested PCR, respectively. Our results have shown the ELISA was useful in detecting SaV GI antigens in clinical stool specimens collected two days after the onset of illness.

Animals↗

PubMed2006

The detection of human sapoviruses with universal and genogroup-specific primers.

Sapovirus (SV) causes gastroenteritis in humans and comprises genetically divergent viruses. A nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid-protein-coding region was developed using universal and genogroup-specific primer sets. The universal primers were capable of detecting human SV genogroups I, II, IV and V. Genetic analysis of the amplified products enabled us to phylogenetically determine the genotypes of the viruses. In addition, genogroup-specific primers that amplified different lengths of the amplicon depending on the genogroup were developed. These genogroup-specific primers were also used as inner primers for the nested PCR. These two simple RT-PCR methods are powerful tools for both detection and epidemiological studies of human SV.

Caliciviridae Infections↗

PubMed2006

Binding activity of norovirus and sapovirus to histo-blood group antigens.

Noroviruses (NoVs) and sapoviruses (SaVs) are causative agents of human gastroenteritis. There is increasing evidence that certain human NoV strains bind to histo-blood group antigens (HBGAs). We found that several NoV virus-like particles (VLPs) showed binding activity to HBGAs, while neither SaV genogroup I (GI) VLP nor SaV GV VLP showed such activity.

Blood Group Antigens↗

PubMed2006

Genetic variability in the sapovirus capsid protein.

Sapovirus (SV), which causes gastroenteritis in humans, is composed of genetically divergent viruses classified into 5 genogroups. In this study, 2.2-kb nucleotide sequences of the 3' terminus of the genome of 15 SV strains detected in Japan were determined. The 15 SV strains could be classified into four genogroups (GI, GII, GIV and GV), and in two of these, GI and GII, 10 genotypes were identified. The amino acid sequences of the central variable region of the capsid protein showed less than 81% identity when strains belonging to different genotypes were compared. It was therefore supposed that antigenic variety exists between different genotypes. These results will be useful for further genetic and antigenic analyses of SV.

Antigenic Variation↗

PubMed2006

Enhancement of sapovirus recombinant capsid protein expression in insect cells.

Human sapovirus (SaV) is uncultivable, but expression of the recombinant capsid protein (rVP1) in insect cells results in the formation of virus-like particles (VLPs) that are morphologically similar to the native viruses. However, the SaV rVP1 expression levels are considerably low. We have found that inclusions of short foreign nucleotide sequences inserted directly upstream from the predicted rVP1 AUG start codon lead to increased yield of VLPs. This method allowed us to express a SaV rVP1, which could not have been expressed to measurable or practical levels otherwise.

Animals↗

PubMed2005

Development of a new microwell hybridization assay and an internal control RNA for the detection of porcine noroviruses and sapoviruses by reverse transcription-PCR.

Recently, genetically diverse porcine noroviruses (NoV) and sapoviruses (SaV) were identified from field pig fecal samples. Reverse transcription (RT)-PCR is the primary method used for detection of human NoVs and SaVs. However, RT-PCR inhibitors frequently cause false-negative results. In this study, a competitive internal control (IC) RNA, specific for use in the SaV RT-PCR assay, was developed to monitor inhibition of RT-PCR; primers for detection of genetically diverse porcine NoVs and SaVs were designed; and microwell hybridization assays to confirm the specific RT-PCR products were developed. The primer pairs and the RT-PCR-hybridization combinations were compared using representative porcine NoV and SaV strains, positive pig fecal samples and a panel of 30 field pig fecal samples. Extracted RNA from 3 of 30 samples failed to amplify the IC RNA. However, this inhibition was not present after a 10-fold dilution of the extracted RNA. The five different RT-PCR-hybridization combinations developed specifically detected all three genotypes of porcine NoVs, all GIII porcine SaVs, unclassified JJ681-like, QW19 and LL26-like porcine SaVs, respectively. These RT-PCR-hybridization assays are specific, less time consuming and economical and particularly applicable to testing large number of samples for porcine NoVs and SaVs.

Animals↗

PubMed2006

Sapovirus detection by quantitative real-time RT-PCR in clinical stool specimens.

Sapovirus (SV) is one of the major causative agents of viral gastroenteritis affecting all age groups worldwide. A new method for the quantitative detection of SV from clinical stool specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR) based on TaqMan MGB technology was described. Primers and probe were designed to target the RNA-dependent RNA polymerase/capsid genes junction. Performance of the newly developed assay was validated against a panel of 244 clinical stool specimens collected for patients with gastroenteritis. SV was detected in eight (3.3%) specimens. Phylogenetic analysis of the positive isolates suggested that the assay could detect at least SV genogroups I, II and IV. In addition, the assay had an increased detection rate compared with a widely used conventional RT-PCR assay. Quantitative analysis showed that the assay could detect as low as 10 copies of viral cDNA per reaction. No cross-reactivity with norovirus and rotavirus was observed. In conclusion, the assay is a sensitive and specific method for the detection of SV from clinical stool specimens.

Adolescent↗

PubMed2006

Identification of the cleavage sites of sapovirus open reading frame 1 polyprotein.

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: NH(2)-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 E(1055)/A(1056) 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 E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). 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.

Binding Sites↗

PubMed2006

Development of an enzyme immunoassay for detection of sapovirus-specific antibodies and its application in a study of seroprevalence in children.

Sapoviruses (SVs) are an important cause of acute pediatric gastroenteritis. Due to the lack of appropriate diagnostic methods, the epidemiology of SV-associated illness remains poorly understood. Baculovirus and Escherichia coli expression systems were evaluated for the development of antibody detection enzyme immunoassays (EIA). Age-related antibody prevalence in children was studied using the new EIA. Because of the low yield of the baculovirus system, the E. coli-expressed SV capsid proteins were used to develop the EIA. The antigenic specificities of the E. coli-expressed SV capsid proteins were demonstrated using hyperimmune antisera raised in animals and sera collected from patients. A high prevalence (>90%) of antibodies to both SV (strain Mex340) and norovirus (strain VA387) was observed in children involved in a birth cohort at 20 to 24 months of age; however, at 1 to 3 months of age, <25% of the children possessed anti-SV antibodies versus >90% with anti-NV antibodies. The E. coli-derived SV proteins are an excellent source of antigens for the EIA. SV infection is common in the first 2 years of life. The low prevalence of maternal antibodies detected in Mexican children against SVs in this study is unique and needs to be addressed in future studies.

Aging↗

PubMed2006

Prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcription-PCR and microwell hybridization assays.

Noroviruses (NoVs) and sapoviruses (SaVs) are emerging enteric pathogens that cause diarrhea in humans and animals. Porcine genogroup II (GII) NoVs replicate in pigs, but their pathogenesis is undefined. The porcine SaV/GIII/Cowden/80/US strain causes diarrhea and intestinal lesions in pigs. Recently, genetically diverse porcine NoVs (genotypes 11, 18, and 19 within GII) and SaVs comprising at least two genogroups (GIII and GVI?/JJ681-like) and two unclassified strains (G?/QW19 and G?/LL26) were identified; however, their prevalence has not been reported. To investigate the prevalence of porcine NoVs and SaVs, 621 fecal samples were collected from swine of various ages from seven swine farms and one slaughterhouse in three states in the United States. Fecal samples were tested by reverse transcription-PCR and microwell hybridization assays with porcine NoV- and SaV-specific primers and probes, respectively. Porcine GII NoVs were detected exclusively from finisher pigs with an overall prevalence of 20%. Porcine GIII SaVs were detected in 62% of pigs, with the highest prevalence in postweaning pigs and lowest in nursing pigs. Porcine GVI?/JJ681-like SaVs and the G?/QW19-like SaVs were detected infrequently in pigs. The G?/LL26-like SaVs were detected mainly in younger pigs. Because some porcine NoVs and SaVs are genetically or antigenically related to human strains and recombinants within NoVs or SaVs occur for human and pig strains, the high prevalence and subclinical infection rate of these viruses in pigs raise questions of whether pigs may be reservoirs for human strains or for the emergence of new human and porcine recombinants.

Abattoirs↗