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Letter
Novel Recombinant Norovirus
in China
Tung Gia Phan,* Hainian Yan,* Yan Li,† Shoko Okitsu,* Werner E.G.
Müller,‡ and Hiroshi Ushijima*
*The University of Tokyo, Tokyo, Japan; †Kunming Medical College, Kunming,
People’s Republic of China; and ‡Universität Mainz, Mainz, Germany
Suggested
citation for this article
To the Editor: Norovirus (NoV), the distinct genus within the
family Caliciviridae, is a major cause of sporadic cases and outbreaks
of acute gastroenteritis in humans (1). NoV possesses
a positive-sense, single-stranded RNA genome surrounded by an icosahedral
capsid. The NoV genome contains 3 open reading frames (ORFs). ORF1 encodes
nonstructural proteins, ORF 2 encodes capsid protein (VP1), and ORF3 encodes
a small capsid protein (VP2). NoV is still uncultivable by standard culture
with different cell lines. However, expression of either VP1 or both VP1
and VP2 with recombinant baculoviruses formed viruslike particles that
are morphologically and antigenically similar to the native virion (2).
A fecal specimen was collected from an infant hospitalized with acute
gastroenteritis in Kunming, China, in November 2004 and was tested for
diarrheal viruses in a cooperative laboratory in Japan. The viral genome
was extracted by using a Qiagen kit (Qiagen, Hilden, Germany). Polymerase
chain reaction with specific primers resulted in the identification of
astrovirus, rotavirus, sapovirus, adenovirus, and NoV genogroup I (GI)
and GII (3). NoV polymerase was also amplified
to identify recombinant NoV with primers Yuri22F and Yuri22R (4).
Products were sequenced directly, and sequence analysis was performed
by using ClustalX and SimPlot.
The fecal specimen was positive for NoV GII. The Figure
shows that the 146/Kunming/04/China sequence clustered into the distinct
GII genotype 7 (Leeds/90/UK cluster). 146/Kunming/04/China was classified
into the Saitama U4 cluster (GI/6) when polymerase-based grouping was
performed. Altogether, 146/Kunming/04/China was expected to be the recombinant
NoV with GII/7 capsid and GII/6 polymerase.
To eliminate the possibility of co-infection with 2 different NoV genotypes,
to localize the potential recombination site, and to clarify a possible
recombination mechanism, the ORF1/ORF2 overlap and flanking polymerase
and capsid regions of 146/Kunming/04/China was amplified with primers
Yuri22F and GIISKR to produce a 1,158-bp amplicon (3,4).
When the sequence of 146/Kunming/04/China was compared with that of Saitama
U4 by using SimPlot, a recombination site was found at the ORF1/ORF2 overlap.
Before this junction, 146/Kunming/04/China and Saitama U4 were homologous.
After the ORF1/ORF2 overlap, however, the homology was notably different.
SimPlot showed a sudden drop in the nucleotide identity for 146/Kunming/04/China.
ClustalX showed that 146/Kunming/04/China shared a high identity (93%)
in the polymerase region and a low identity (78%) in the capsid region
with Saitama U4. In contrast, high identity (95%) in the capsid region
was found between 146/Kunming/04/China and Leeds/90/UK. Since Leeds/90/UK
polymerase was not available in GenBank, the polymerase homology between
146/Kunming/04/China and Leeds/90/UK was unknown. Polymerase of 146/Kunming/04/China
was almost identical with that of Saitama U4, but the capsids of 146/Kunming/04/China
and Leeds/90/UK were distinctly different from that of Saitama U4. This
genetic pattern of 146/Kunming/04/China implied a novel, naturally occurring
recombinant NoV with GII/7 capsid and GII/6 polymerase.
RNA recombination is a mechanism for virus evolution (5).
Literature documenting recombination in NoV is fairly rich, but none is
from China (6). Therefore, 146/Kunming/04/China
was not only the first but also the first recombinant NoV from China.
This isolate shared the closest sequences of polymerase and capsid with
Saitama U4 and Leeds/90/UK, respectively. Strain Saitama U4 was detected
in 1997 in Japan (7), whereas strain Leeds/90/UK
was detected in 1990 in the United Kingdom (8).
Quite possibly, Saitama U4 and Leeds/90/UK were parental strains of 146/Kunming/04/China.
However, the distant geographic relationship of these strains obscured
evidence of where and when the recombination event occurred. This phenomenon
also suggested that these parent strains or this progeny strain might
be more prevalent than is often assumed.
Recombination depends on various immunologic and intracellular constraints.
Recombinant viruses are all alike in that they successfully pass through
5 stages: 1) successful co-infection of a single host, 2) successful co-infection
of a single cell, 3) efficient replication of both parental strains, 4)
template switching, and 5) purifying selection (9).
In this study, 146/Kunming/04/China was recovered from a patient with
diarrhea, fever, and vomiting. This observation indicated that this strain
theoretically fulfilled all prerequisites for recombination.
The NoV capsid is predicted to be well suited for genotype classification
(10). In this study, 146/Kunming/04/China belonged
to 2 distinct genotypes, 7 and 6, by capsid- and polymerase-based groupings,
respectively. Moreover, the recent demonstration of recombination in an
increasing number of NoVs suggests that it is a more widespread event
than was previously realized. Consequently, the phylogenetic classification
of NoV on the basis of on capsid sequence is questionable. We suggest
that classification of NoV strains should rely on not only capsid sequence
but also polymerase sequence.
In conclusion, our results described the genetic characterization of
novel, naturally occurring recombinant NoV and increased evidence for
the worldwide distribution of recombinant NoV. This report is the first
to describe acute gastroenteritis caused by recombinant NoV in China and
warns of the threat it poses.
This study was supported
by grants-in-aid from the Ministry of Education and Sciences and the
Ministry of Health, Labor and Welfare, Japan. This study was also supported
by the Heiwa Nakajima Foundation, the Mishima-Kaiun Foundation, and
the Sumitomo Foundation, Japan.
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Suggested citation
for this article:
Phan TG, Yan H, Li
Y, Okitsu S, Müller WEG, Ushijima H. Novel recombinant norovirus in China
[letter]. Emerg Infect Dis [serial on the Internet]. 2006 May [date
cited]. Available from http://www.cdc.gov/ncidod/EID/vol12no06/05-1566.htm
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