Transition of genotypes associated with norovirus gastroenteritis
outbreaks in a limited area of Japan, Hiroshima Prefecture,
during eight epidemic seasons
Received: 4 June 2009 / Accepted: 28 September 2009 / Published online: 1 December 2009
Ó Springer-Verlag 2009
Abstract The transition of genotypes implicated in 102
NoV gastroenteritis outbreaks in Hiroshima Prefecture,
Japan, during eight epidemic seasons was investigated.
Eighteen genotypes were implicated in the outbreaks, with the
chronological characteristics as in GII.3, GII.4, GII.5 and
GII.12. In GII.4 variants, amino acid changes and positively
selected sites were of note and signiﬁcantly concentrated in
the surface-exposed P2 subdomain of the VP1 protein.
Notably, variant-speciﬁc epitopes at which positively selected
sites are located may be signiﬁcant for distinguishing a new
GII.4 variant. The interaction of these genetic changes with
developing immunity seems to inﬂuence NoV epidemics.
Noroviruses (NoVs), which belong to the family Calicivi-
ridae, are classiﬁed into ﬁve genogroups (GI to GV), of
which GI, II, and IV cause acute gastroenteritis in humans
, and further divided into more than 30 genotypes .
In particular, GII.4 has emerged in recent years. Although
genetic variants of GII.4 such as \1996, 1996, and 2002
had been identiﬁed in the past , in 2004 and 2005, a
new GII.4 variant, termed Hunter virus, became prevalent
. Then, in 2006 and 2007, the variants 2006a and 2006b
became epidemic [3, 11, 16, 22]. In Japan, there was more
than a fourfold increase in NoV infections compared to the
previous year during the 2006/2007 epidemic season, and
the increase was attributed to the emergence of the 2006b
variant, with a small role played by the 2006a variant .
Before 2004, genotypes such as GII.3 and GII.5 as well as
GII.4 were associated with outbreaks of gastroenteritis in
Japan [19, 24]. Here, we describe the chronological chan-
ges of NoV genotypes and the genetic characterization of
GII.4 implicated in outbreaks of gastroenteritis in Hiro-
shima Prefecture, Japan from the 2000/2001 to the 2007/
2008 epidemic season.
A total of 121 strains detected from patients of 102 NoV
gastroenteritis outbreaks in Hiroshima Prefecture, Japan,
between the 2000/2001 and 2007/2008 epidemic seasons
were used. Of the 121 strains, 9, 14, 8, 10, 19, 6, 38, and 17
were obtained in the 2000/2001, 2001/2002, 2002/2003,
2003/2004, 2004/2005, 2005/2006, 2006/2007, and 2007/
2008 epidemic seasons, respectively.
For phylogenetic analysis, PCR amplicons of 330 and
344 bp for GI and GII, respectively, targeting from the C
terminus of the RNA-dependent RNA polymerase gene to
the N-terminal/Shell domain region [13, 17] were
sequenced directly as described previously . Genotyping
was carried out according to the scheme described by
Kageyama et al. , and the GII.4 variants were typed
using the 5
end of capsid sequences according to the
cluster of Motomura et al. .
Furthermore, full-length amplicons (1,623 bp) of the VP1
region of 25 GII.4 strains, which were randomly selected in
each epidemic season, were made using the primers G2SKF
 and LVCAPEND . Nucleotide sequencing was
conducted after cloning into pDrive cloning vectors using a
PCR cloning kit (Qiagen, Valencia, CA) . Three clones
were independently analyzed for sequence determination.
The following primers were used: M13 FWD(-29) (M&S
TechnoSystems, Osaka, Japan), LV5375 (5
), LV5862 (5
), and LV6362 (5
) for forward sequencing and the anti-sense
S. Fukuda (&) Á S. Takao Á N. Shigemoto Á Y. Tanizawa Á
Center for Public Health and Environment,
Hiroshima Prefectural Technology Research Institute,
Minami-machi 1-6-29, Minami-ku, Hiroshima 734-0007, Japan
Arch Virol (2010) 155:111–115