Arch Virol (2007) 152: 1075–1086
DOI 10.1007/s00705-007-0938-9
Printed in the Netherlands
Norovirus mixed infection in an oyster-associated outbreak:
an opportunity for recombination
S. J. Symes
1
, I. C. Gunesekere
1
, J. A. Marshall
2
, and P. J. Wright
1
1
Department of Microbiology, Monash University, Clayton, Victoria, Australia
2
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Received September 15, 2006; accepted January 8, 2007; published online February 28, 2007
# Springer-Verlag 2007
Summary
We describe an outbreak of gastroenteritis in which
the nucleic acid of three distinct noroviruses was
amplified from the same fecal sample. To enable
the separate amplification of each virus, an inclu-
sion=exclusion RT-PCR primer design strategy was
developed. This paired a virus-specific exclusion
primer (designed with the exact sequence of one
virus in a region displaying low conservation
among the three viruses) with a virus-nonspecific
inclusion primer (designed in a conserved region).
Thus, in each reaction the exclusion primer provid-
ed specificity for a single virus, and the inclusion
primer increased the sensitivity and allowed hybri-
dization in a region of unknown sequence. Analysis
of the partial genomic sequences of the three vi-
ruses (3.6–3.8 kb) indicated that each virus be-
longed to a separate genogroup II cluster, and each
displayed evidence of a potential recombination
event when the sequences were compared with other
published norovirus sequences. Our results, which
show a mixed norovirus infection in a single individ-
ual, confirm the need to be aware of the possibility
of mixed norovirus infections, and of the possibility
of genomic recombination causing anomalies in
phylogenetic analyses in such instances.
Introduction
Noroviruses, family Caliciviridae, are increasingly
recognized as etiological agents of acute nonbac-
terial gastroenteritis in humans. Noroviruses are
nonenveloped positive sense, single-stranded RNA
viruses with a gemome of approximately 7.5 kb
[19]. The genomes have three open reading frames
(ORFs). ORF1 encodes the nonstructural polypro-
tein, ORF2 the capsid protein, and ORF3 a putative
minor structural protein.
Infections or outbreaks involving more than one
norovirus strain (mixed infections=outbreaks) have
been reported. These typically involve contami-
nation of food or water with sewage. They usually
present as either waterborne outbreaks [2, 3, 11, 16,
28, 34] or shellfish (especially oyster)-associated
outbreaks [20, 21, 24, 31, 37, 42].
An important aspect of mixed norovirus infec-
tions is the opportunity they provide for recombina-
tion to occur between different norovirus strains,
leading to an increase in the genetic diversity of
Author’s address: S. J. Symes, Centre for Equine Virology,
School of Veterinary Science, The University of Melbourne,
North Melbourne, Victoria 3010, Australia. e-mail: ssymes@
unimelb.edu.au