Different genotypic sapoviruses detected in two simultaneous outbreaks of gastroenteritis among schoolchildren in the same school district in Hokkaido, Japan.

Norovirus (NV) and sapovirus (SV) are members of the family Caliciviridae and are etiologic agents for acute gastroenteritis in humans (1,2). Presently, NV is the more causative of the two for the outbreak of gastroenteritis, whereas only a few outbreaks of SV have been reported. Acute gastroenteritis due to SV occurs mainly in infants and young children (2). Previously, we reported on outbreaks of SV-associated gastroenteritis in Hokkaido, Japan (3). Here, we describe two outbreaks that occurred almost simultaneously among schoolchildren in the same district in Hokkaido. The SV genomes were detected and differentiated into a genogroup I genotype 2 (GI/2) and a GI/3. The first outbreak (Outbreak 1) occurred at an elementary school between March 8 and 23 in 2009. A total of 48 schoolchildren developed gastroenteric symptoms such as vomiting and diarrhea. The second outbreak (Outbreak 2) occurred at a junior high school between March 11 and 23 in 2009. Finally, a total of 74 students suffered from gastroenteritis. These two outbreaks occurred in the same school district. Nine stool specimens from Outbreak 1 were collected from 1 to 13 days of illness, while 11 stool specimens from Outbreak 2 were collected from 1 to 8 days of illness. The epidemic curves of these two outbreaks are shown in Figs. 1 and 2. Each stool specimen was prepared as 10% emulsions with nuclease-free distilled water (Nippon Gene Co., Tokyo, Japan) and was centrifuged at 3,000 rpm for 20 min at 4°C. The supernatant was collected and transferred to microtubes, followed by centrifugation at 10,000 rpm for 10 min at 4°C. Extraction of viral RNA and reverse transcription (RT) were carried out as described previously (4). The cDNA was used for the polymerase chain reaction (PCR) and real-time PCR. Two sets of primers, P1/P3 and NV82/SM82/NV81, and another two sets of primers, COG1F/G1SKR and COG2F/ G2SKR, were used to amplify the polymerase and the capsid protein regions of the NV genome, respectively (5–8). To amplify the polymerase and capsid protein regions of the SV genome, the sets of primers SV-r-c/sapp36 and SV-F13/SVF14/SV-R13/SV-R14 were used. The conditions of PCR amplification were performed as described previously (4,9, 10). The PCR products were purified and directly sequenced using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif., USA). Nucleotide sequences determined in this study have been submitted to Genbank/ EMBL/DDBJ (accession no. AB518056 and AB518057). Multiple alignment and phylogenetic analyses were carried out based on partial capsid nucleotide sequences (approxiFig. 1. Number of patients and virus detection for norovirus (NV) and/ or sapovirus (SV) of gastroenteritis in Outbreak 1 from March 8 to 23, 2009. Specimens which were positive for NV and/or SV genomes were indicated. Two specimens which were collected in March 13 or 16 were positive for both NV and SV genomes.