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Identification of sapovirus infection among Japanese infants in a day care center.

A total of 921 fecal specimens collected from 44 infants in a day care center in Tokyo, Japan during June 1999 to July 2000 were tested for the presence of sapovirus by reverse transcription-polymerase chain reaction (RT-PCR). Of 88 fecal specimens from infants with acute gastroenteritis, 2.3% (2) were found to be positive for sapovirus. Twenty-two of 833 (2.6%) fecal specimens collected from asymptomatic infants were also infected with this virus. Another interesting feature was the demonstration of high incidence of sapovirus infection (95.5%, 21 of 22) identified in a single day care center, which was not due to viral shedding after the latest acute gastroenteritis. Sapovirus was subjected to genetic analysis by sequencing. Up to 4 of 24 sapoviruses (16.7%) were clustered into genogroup IV known as a rare group. Remarkably, majority (79.2%, 19 of 24) of sapovirus isolates detected in this study turned out to present one novel sapovirus genotype tentatively called GI/8. It was noteworthy to point out that the GI/8 sapovirus infection was apparently confined only within the period of 9 weeks (44th through 52nd weeks). This pattern of infection implied the outbreak of asymptomatic GI/8 sapovirus infection in these subjects. The findings clearly indicate genogroup I sapovirus can be classified into eight genotypes. This is the first report to underscore that sapovirus pathogen causes not only clinical manifestations of acute gastroenteritis but also asymptomatic infection in infants in day care centers in Japan.

Acute Disease↗

Emergence of rare sapovirus genotype among infants and children with acute gastroenteritis in Japan.

A total of 1,154 fecal specimens from infants and children with acute gastroenteritis in five cities in Japan (Maizuru, Tokyo, Sapporo, Saga, and Osaka), collected from July 2003 to June 2005, were tested for the presence of diarrheal viruses by reverse transcriptase multiplex PCR. Overall, 469 of 1,154 (40.6%) were positive for diarrheal viruses, of which 49 (10.4%) were positive for sapovirus. The peak of sapovirus infection shifted from April-June in 2003-2004 to October-December in 2004-2005. The observations show that maximum sapovirus prevalence can occur during warmer seasons. Sapovirus was subjected to molecular genetic analysis by sequencing. The results indicated that sapovirus genogroup I was a dominant group (100%). Sapovirus strains detected in this study were further classified into four genotypes (GI/1, GI/4, GI/6, and GI/8). Of these, sapovirus GI/1 was the most predominant, followed by sapovirus GI/6; these accounted for 93% (13 of 14) and 7% (1 of 14), respectively, in 2003-2004. However, it was noteworthy that sapovirus GI/6 suddenly emerged to become the leading genotype, accounting for 77% (27 of 35) of isolates in 2004-2005. This is believed to be the first report of the changing distribution of sapovirus genotypes and of the emergence of the rare sapovirus GI/6.

Adolescent↗

Outbreak of sapovirus infection among infants and children with acute gastroenteritis in Osaka City, Japan during 2004-2005.

One hundred and twenty five fecal specimens were collected from sporadic cases of acute gastroenteritis in a pediatric clinic in Osaka City, Japan from July 2004 to June 2005 and tested for the presence of rotavirus, norovirus, sapovirus, astrovirus, and adenovirus by RT-multiplex PCR. Among diarrheal viruses detected, norovirus was the most prevalent (19.2%, 24 of 125), followed by group A rotavirus (18.4%, 23 of 125), astrovirus (1.6%, 2 of 125), and adenovirus (0.8%, 1 of 125), respectively. Interestingly, sapovirus infection was identified with high incidence of 17.6% (22 of 125). Sapovirus was subjected to molecular genetic analysis by sequencing. It was found that sapovirus detected in this study was classified into four genotypes (GI/1, GI/4, GI/6, and GI/8), and sapovirus GI/6 was predominant, followed by GI/8 and accounted for 81.8% (18 of 22) and 9.1% (2 of 22), respectively. It was noteworthy that sapovirus GI/6 infection was apparently confined within the period of 5 months (August 2004 through December 2004). This pattern of infection implied the outbreak of sapovirus GI/6 in these patients, which was the first outbreak of acute gastroenteritis attributed to sapovirus in Osaka City. Another interesting feature of the study was the appearance of two novel, naturally occurring recombinant sapoviruses circulating in the Japanese population studied. This report confirmed the presence as well as the importance of sapovirus causing acute gastroenteritis among Japanese infants and children.

Aging↗

Outbreak of acute gastroenteritis associated with group A rotavirus and genogroup I sapovirus among adults in a mental health care facility in Japan.

An outbreak of acute gastroenteritis consisting of 57 cases occurred in a mental health care facility in Takasaki city, Japan during 6th February and 27th March 2002. A total of 18 fecal specimens collected from 17 residents and one member of the medical staff during this outbreak were tested for the presence of viral enteropathogens by RT-PCR and latex agglutination. Group A rotavirus and sapovirus were detected in 5 out of 18 fecal specimens (55.6%). To our knowledge, this is the first finding of an outbreak of gastroenteritis associated with co-circulation of different kinds of viruses such as group A rotavirus and sapovirus. All of group A rotaviruses were typed further as P[4]G2 strains. Both rotavirus and sapovirus were subjected to molecular analysis by sequencing. It was noteworthy that all rotaviruses and sapoviruses had high homologies, respectively, to each other and sapoviruses presented a potential novel sapovirus genogroup I (GI) genotype, which was obviously different from any GI genotypes (GI-a, b, c, and d). The outbreak associated with these viruses spread gradually from dormitory to dormitory, suggesting a spread by person-to-person contact, although investigation on the route of transmission of the outbreak is lacking. The findings confirm the presence of group A rotavirus and sapovirus are important in acute gastroenteritis among adults in Japan.

Adult↗

Structural and functional characterization of sapovirus RNA-dependent RNA polymerase.

Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3D(pol)-like protein. Enzymatically active sapovirus 3D(pol) and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3D(pol) was determined by X-ray crystallography to 2.32-A resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3D(pol) prefers Mn(2+) over Mg(2+) but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3D(pol) synthesizes a double-stranded RNA or labels the template 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3D(pol) an attractive target for developing drugs to control calicivirus infection in humans.

Amino Acid Sequence↗

Genetic diversity of sapovirus in fecal specimens from infants and children with acute gastroenteritis in Pakistan.

A total of 517 fecal specimens collected from infants and children with acute gastroenteritis in Karachi city, Pakistan during 1990-1994 were examined for the presence of sapovirus by RT-PCR and sequence analysis methods. Sapovirus was identified in 17 of 517 (3.2%) specimens. Sapovirus was further clustered into three distinct genogroups (I, II and IV) and these presented 70.6%, 23.5% and 5.9%, respectively. Our results clearly indicated that sapovirus could be classified into 7 GI and 4 GII genotypes. It was noteworthy to point out that sapovirus detected among Pakistani infants and children with acute gastroenteritis demonstrated the great genetic diversity and presented novel sapovirus genotypes.

Acute Disease↗

Low effectiveness of highly active antiretroviral therapy and high mortality in the Greenland HIV-infected population.

Greenland is a high-income country with free access to human immunodeficiency virus (HIV) care, including highly active antiretroviral therapy (HAART). We aimed to examine the HIV prevalence, age and gender distribution, and the effectiveness of HAART on CD4 positive cell count, viral load (VL), and mortality in the Greenland HIV-infected population. In this population-based cohort study we collected demographic, clinical and biochemical data on all HIV-infected patients followed in health clinics since 1995. For each case, we identified 100 age- and gender-matched population controls. The HIV cohort included 103 patients of whom 91% were Inuit; 81% were infected heterosexually. Only 40% of the patients had a VL <400 copies/ml at 48 weeks after starting HAART, and patients on HAART had a substantial excess relative mortality compared with the general population (mortality rate ratio [MRR]: 10.6; 95% confidence interval [CI]: 6.9-16.4). After the introduction of HAART, the mortality decreased (MRR: 2.5; 95% CI 0.9-6.8), but remained high with a mortality rate of 62 per 1000 person-years (py) (95% CI 29-129). Our findings underline the difficulties of implementing successful HIV treatment even with unlimited economic resources and free access to health care.

Adult↗

Novel intragenotype recombination in sapovirus.

Based on the genetic analysis, a novel, naturally occurring recombination between two distinct sapovirus subtypes (subtype a and subtype b) within genogroup I genotype 1 was identified. Breakpoint analysis of recombinant sapovirus showed that the recombination site was at the polymerase-capsid junction. This is the first report of the existence of acute gastroenteritis caused by intragenotype recombinant sapovirus. The results also provided evidence that the natural recombination occurs not only in sapovirus genogroup II but also in sapovirus genogroup I.

Base Sequence↗

Etiologic agents of acute gastroenteritis among Japanese infants and children: virus diversity and genetic analysis of sapovirus.

A total of 371 fecal specimens from infants and children with acute gastroenteritis in Maizuru, Tokyo and Osaka, Japan from July 2002 to June 2003 were tested for the presence of diarrheal viruses by reverse transcription-polymerase chain reaction (RT-PCR), reverse passive hemagglutination (PRHA), RNA-polyacrylamide gel electrophoresis (PAGE), latex agglutination and sequence analysis methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (42.2%) followed by norovirus (28.9%), group C rotavirus (8.4%), sapovirus (6.7%), adenovirus (5.3%) and astrovirus (0.9%), respectively. There was the high rate (7.6%) of viral mixed infections. Sapovirus was classified into 6 genotypes (GI/1, GI/4, GI/5, GI/6 and GII/1 and one novel tentatively called GII/5). It is noteworthy that genogroup II sapovirus can be classified into 5 genotypes. Our findings confirmed the presence of many diarrheal viruses co-circulating among Japanese infants and children and showed the great genetic diversity among sapoviruses.

Acute Disease↗

Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.

A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.

Adolescent↗

Intergenogroup recombination in sapoviruses.

Sapovirus, a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. Analyses of the complete genome sequences led us to identify the first sapovirus intergenogroup recombinant strain. Phylogenetic analysis of the nonstructural region (i.e., genome start to capsid start) grouped this strain into genogroup II, whereas the structural region (i.e., capsid start to genome end) grouped this strain into genogroup IV. We found that a recombination event occurred at the polymerase and capsid junction. This is the first report of intergenogroup recombination for any calicivirus and highlights a possible route of zoonoses because sapovirus strains that infect pig species belong to genogroup III.

Base Sequence↗

[Noroviruses and sapoviruses in man and farm animals].

Noro- and Sapoviruses belong to the virus family Caliciviridae and are important causative agents of acute epidemic gastroenteritis in man. In many cases transmission of Noro- and Sapoviruses occurs via contaminated food or water and the respective diseases are therefore designated as food borne. Recently, the presence of Noro- and Sapoviruses in farm animals has attracted increasing attention. Clinical symptoms were observed after experimental infection of cattle and swine with members of the two virus groups. Thus far it is not known, whether virus transmission from animals to man does occur.

Acute Disease↗

Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction.

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 x 10(7) to 2.5 x 10(1) 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.

Australia↗

Genetic diversity among sapoviruses.

Norovirus and Sapovirus are two genera of the family Caliciviridae that contain viruses that can cause acute gastroenteritis in humans. Noroviruses (NOR) are genetically highly diverse but limited studies of the genetic diversity of sapoviruses (SAP) have been reported. In this study we characterized twenty-five SAP detected in our laboratory from outbreaks or sporadic cases of acute gastroenteritis in children from different geographical locations and in adults involved in a cruise ship outbreak investigation and a nursing home outbreak. Based on significant differences of partial RNA polymerase sequences (278-286 nt), the 25 strains were grouped into 12 genetic clusters, including 9 potential new clusters. Extended sequence analysis of the capsid gene of selected strains representing five potential new clusters supported this grouping. Four strains (Hou7-1181/90, Mex340/90, Cruise ship/00 and Argentina39) had <84% amino acid (aa) identity to each other and to the published sequences in the GenBank. Mex14917/00 was almost identical to Stockholm/97/SE whose RNA polymerase sequence was unknown. Phylogenetic and distance analyses of the capsid region of the four new strains showed that Hou7-1181/90 and Argentina39 represent two new genogroups and Mex340/90 and Cruise ship/00 belong to two new clusters within the London/92 genogroup. Thus, based on the capsid sequences we propose to classify the currently known SAP into nine genetic clusters within five genogroups, including one genogroup that is represented by an animal calicivirus, the porcine enteric calicivirus (PEC).

Adult↗

Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains were recently divided into five genogroups (GI to GV). We characterized novel polyclonal antibodies raised against SaV GV virus-like particles (VLPs) by Western blot analysis, and both antibody and antigen enzyme-linked immunosorbent assays (ELISAs). Our results have indicated SaV GI and GV VLPs were antigenically distinct by Western blotting and ELISAs. These reagents may be useful for genogroup specific detection of SaV.

Antibodies, Viral↗

Novel recombinant sapovirus.

We determined the complete genome sequences of two sapovirus strains isolated in Thailand and Japan. One of these strains represented a novel, naturally occurring recombinant sapovirus. Evidence suggested the recombination site was at the polymerase-capsid junction within open reading frame one.

Capsid↗

Genetic diversity of Sapovirus in children, Australia.

Sapovirus was detected in 7 of 95 stool specimens from children with gastroenteritis of unknown etiology in Sydney, Australia, from August 2001 to August 2002 and from February 2004 to August 2004, by using reverse transcription-polymerase chain reaction. Sequence analysis of the N-terminal capsid region showed all human sapovirus genogroups.

Australia↗

[Detection of sapoviruses in children with acute gastroenteritis in Moscow in 2002-2003].

Sapoviruses were found, for the first time, to be circulating in children with acute gastroenteritis in the city of Moscow. On the basis of a genetic analysis, they were classified as belonging to genotypes 1 and 2. Two groups of sapoviruses that are essentially different from the strains presented now at the GenBank NCBI were described within the case study.

Adolescent↗