Detection and genetic characterization of sporadic noroviruses in Nara Prefecture between April 2006 and September 2007.

*Corresponding author: Mailing address: Virology and Bacteriology Division, Nara Prefectural Institute for Hygiene and Environment, 57-6 Ohmori-cho, Nara city, Nara 630-8131, Japan. Tel: +81-742-20-2887, Fax: +81-742-27-0634, E-mail: y.kitahori @ihe.pref.nara.jp Norovirus (NoV) is usually prevalent in the early winter season and causes symptoms of acute gastroenteritis such as diarrhea or vomiting upon infection. According to a surveillance report by the Forborne Viruses in Europe Network, two new GII/4 variants (E2006a and E2006b) appeared during the 2005/2006 season, and these variants were globally observed (1,2). Siebenga et al. reported in detail the genetic changes in NoV GII/4 capsid proteins from 1995 to 2006 (3). From November to December in 2006 in Japan, extensive outbreaks of gastroenteritis caused by NoV GII/4 strains were recorded in hospitals, hotels, schools, nursing homes, and other sites. To clarify the genetic characteristics of sporadic NoV in Nara Prefecture, Japan, from April 2006 to September 2007, we phylogenetically analyzed nucleotide sequences in regions of the ORF2 obtained from surveillance specimens. Between April 2006 and September 2007, a total of 289 stool specimens were collected from patients with acute gastroenteritis in Nara Prefecture. Viral RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, Calif., USA), and then reverse transcription was performed with ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). PCR was performed with the COG1/2F and SKG1/2R primer pair. The NoV GII genotype was detected in 84 (29.0%) of these specimens, and no NoV GI-positive specimens were detected. The 74 NoV GII-positive specimens selected were sequenced using a sequencing kit (Thermo Sequenase Cy5.5 Dye Therminator Cycle Sequencing Kit; GE Healthcare UK, Ltd., Buckinghamshire, UK). Based on these results, a BLAST search was carried out, and then phylogenetic analysis was performed using the neighbor-joining method with the Kimura two-parameter model. For the phylogenetic analysis of the capsid sequences (282 bp), sequences from GenBank were included, and the GenBank accession numbers for the reference strains are: X76716 (Bristol), AJ004864 (Grimsby), AY502023 (Farmington Hills), DQ078794 (Hunter284E), EF126964 (Terneuzen70), EF126966 (Nijmegen115), AB291542 (Kobe034), AB220925 (Chiba), AB240187 (Hokkaido), and DQ095875 (Nagano). The distribution of NoV-associated gastroenteritis patients during April 2006 and September 2007 are shown in Fig. 1. According to our surveillance data, the number of NoVpatients reached a peak in November 2006 (43/84, 51.1%),