Emergence of a New Norovirus Genotype II.4 Variant
http://www.100md.com
微生物临床杂志 2006年第2期
ABSTRACT
Norovirus (NoV) is highly infectious and is the major cause of outbreak gastroenteritis in adults, with pandemic spread of the virus being reported in 1995 and 2002. The NoV genome is genetically diverse, which has hampered development of sensitive molecular biology-based methods. In this study we report on a nested reverse transcriptase PCR (nRT-PCR) that was designed to amplify the highly conserved 3' end of the polymerase region and the 5' end of the capsid gene of NoV genogroup II (GII). The nRT-PCR was validated with strains isolated from sporadic and outbreak cases between 1997 and 2004 in New South Wales, Australia. Phylogenetic analysis identified six genotypes circulating in New South Wales, GII.1, GII.3, GII.4, GII.6, GII.7, and GII.10, with GII.4 being the predominant genotype. In 2004, there was a marked increase in NoV GII activity in Australia, with a novel GII.4 variant being identified as the etiological agent in 18 outbreaks investigated. This novel GII.4 variant, termed Hunter virus, differed by more than 5% at the amino acid level across the capsid from any other NoV strain in the GenBank and EMBL databases. The Hunter virus was subsequently identified as the etiological agent in large epidemics of gastroenteritis in The Netherlands, Japan, and Taiwan in 2004 and 2005.
INTRODUCTION
Gastroenteritis is one of the leading causes of death by an infectious disease (38). Norovirus (NoV), a member of the Caliciviridae family, is considered the major cause of acute gastroenteritis in adults (8, 37, 45) and is estimated to be responsible for 93% of food-related outbreaks of gastroenteritis in the United States (8). NoV is associated with sporadic and outbreak cases of gastroenteritis in individuals of all ages, with a distinct seasonality linked to the winter months (40). Infection is characterized by the acute onset of nausea, vomiting, abdominal cramps, and diarrhea, which generally last for about 48 h (22). Transmission occurs predominantly through ingestion of contaminated water; food (particularly oysters); and person to person by the fecal-oral route, airborne transmission, and contact with contaminated surfaces (14, 21, 30, 37, 42). The ease with which NoV is transmitted and the low infectious dose required to establish an infection results in extensive outbreaks in numerous environments, including hospitals, hotels, schools, nursing homes, and cruise ships (5, 33, 34, 53).
NoV is a small round virion of 27 to 35 nm in diameter and possesses a single-stranded, positive-sense, polyadenylated RNA genome of 7,400 to 7,700 nucleotides (2). The genome is divided into three open reading frames (ORFs). ORF1 encodes a large polyprotein which undergoes proteolytic cleavage to produce viral proteins that are homologous to proteins fromother single-stranded RNA viruses, including nucleoside triphosphatase, 3C-like protease, and RNA-dependent RNA polymerase (RdRp) (17). The two structural proteins, VP1, the major capsid protein, and VP2, the minor capsid protein, are encoded by ORF2 and ORF3, respectively (15, 43). The region from the C-terminal end of ORF1 to the N-terminal region of ORF2 is the most highly conserved region across all known NoV strains (27). We have recently identified this region as the breakpoint in recombinant NoVs (3).
NoV is divided into five genogroups based on the genome sequence of the RdRp and the capsid regions (50). However, only NoV genogroup I (GI), GII, and GIV have been associated with human gastroenteritis (1, 23, 28). NoV GI is further subdivided into 7 genotypes, and NoV GII is further subdivided into 12 genotypes (50). However, simple genotyping has been complicated by the emergence of recombinant NoV that have polymerase and capsid regions derived from separate ancestral strains. Nine NoV recombinant types with an assortment of polymerase and capsid genotypes have been identified (3).
A rapid and definitive diagnosis is important in outbreak settings to prevent the further dissemination of the virus in the population. Older detection methods like electron microscopy have been replaced by reverse transcriptase (RT) PCR and, more recently, enzyme immunoassays (EIAs). While EIA is faster and less labor-intensive than RT-PCR, recent evaluations of commercial EIAs show that they have poor sensitivities and are unable to detect all NoV GII genotypes (4, 18, 44).
Until recently, the majority of NoV GII RT-PCRs have targeted ORF1, in particular, the middle of the RdRp-encoding region, as ORF1 is overall more highly conserved than ORF2 (24). However, Katayama et al. illustrated, using multiple alignments of 18 full-length NoV genomes, that highly conserved sequence motifs are located at the 3' end of the RdRp and the adjacent 5' end of ORF2 (24). Primers targeting these highly conserved sequences were able to detect a broader range of NoV GII strains than primers targeting the central region of RdRp (19, 27). We have adopted this approach in the current study.
Due to the large genomic and antigenic diversity of NoV, it is essential that there be a comprehensive understanding of the strains circulating within a population to ensure that sensitive detection methods are being used. Over the last few years, NoV-associated gastroenteritis has reached unprecedented levels (6, 29, 32, 51, 53). The majority of newly emerging NoV outbreak strains belong to GII.4 and have a global presence (6, 29, 32, 51-53). For example, a GII.4 variant, termed US95/96, caused 55% of gastroenteritis outbreaks throughout the United States in 1995 and 1996 (9) and was then later detected in Brazil, Canada, China, Germany, Netherlands, the United Kingdom, and Australia (39). In New South Wales, Australia, this strain was associated with 86% of NoV-associated outbreaks of gastroenteritis during 1997 and 2000 (52). In 2002, another GII.4 strain, reported by the European Food-Borne Viruses Network, termed the European variant in this study, caused 134 (86%) of 153 outbreaks originating in Germany, Holland, Wales, and the United Kingdom (32). In 2002, two other identical GII.4 variants, termed Farmington Hills and b4s6, caused outbreaks in the United States and England, respectively (6, 53). Farmington Hills virus was associated with 64% of cruise ship outbreaks and 45% of land-based outbreaks in the United States in 2002 (53). b4s6 was linked to 100% of 22 outbreaks in Oxfordshire, United Kingdom, during 2002 and 2003 (6).
In this study we developed a new nested RT-PCR (nRT-PCR) for the detection of NoV GII. The assay was used to determine the genetic diversity of circulating strains implicated in sporadic and outbreak gastroenteritis between 1997 and 2004. In Australia, epidemics of gastroenteritis occurred in 2002 (46) and 2004 (48) and were dominated by NoV infections. Furthermore, increases in gastroenteritis outbreaks in 2004 were correlated with the emergence of a novel GII.4 variant.
MATERIALS AND METHODS
Stool specimens. In total, 88 NoV GII-positive stool samples were obtained from the Department of Microbiology, SEALS, Prince of Wales Hospital, Sydney, Australia. Stool specimens were confirmed as NoV GII positive either by the IDEIA NLV enzyme-linked immunosorbent assay (ELISA; Dakocytomation Ltd.) or nRT-PCR (52) (Table 1). The 88 NoV GII-positive samples included 11 samples from NoV-positive sporadic cases (defined as isolated cases with no known related cases) collected between July 1997 and December 1998, 15 samples from NoV-positive sporadic cases collected between August 2001 and August 2002, 6 samples from sporadic cases collected between February 2004 and August 2004, and 56 samples from 28 outbreaks collected between 1997 and 2004 (Table 1). All stool specimens were stored at –80°C. In addition, two adenovirus-positive specimens, one astrovirus-positive specimen, and one rotavirus-positive specimen, detected by ELISA (Dakocytomation Ltd.), were used as negative controls.
Sample preparation and RNA extraction. A 20% (wt/vol) stool suspension with a total volume of 1 ml was prepared in RNase-free water and centrifuged for 1 min at 13,000 x g. The supernatant was removed, centrifuged for a further 7 min at 13,000 x g, and stored at –20°C. Viral RNA was extracted by using a QIAmp viral RNA kit, according to the manufacturer's instructions (QIAGEN, Hilden, Germany). RNA was resuspended in 50 μl of water and stored at –80°C.
nRT-PCR. Reverse transcription was performed with 10 μl of RNA template added to a 10-μl reaction mixture containing a final concentration of 1x reverse transcription buffer (Promega), 10 mM dithiothreitol, 1 mM each deoxynucleoside triphosphate (dNTP; dATP, dCTP, dGTP, dTTP), 5 μM random primers (Promega), and 6 U of avian myeloblastosis virus RT (Promega). Reverse transcription proceeded by incubation at 42°C for 60 min, and the reverse transcriptase was then denatured by increasing the temperature to 72°C for 15 min.
Regions amplified. The primers used in the study are described in Table 2. For amplification of the highly conserved region targeting the 55 bp at the 3' end of the polymerase and the 344 bp at the 5' end of the capsid, an outer primer set, primers NV2oF2 and NV2oR, was used in conjunction with previously designed primers G2F3 and G2SKR (19, 27) (Table 2). Analysis of potential recombinants was performed by amplifying the region across the ORF1-ORF2 overlap. First- and second-round PCRs were performed as described below with the following combination of either primers CB001 and NV2oR for the first round and primers CB003 and 2212R for the second round or primers hep170 and NV2oR for the first round and primers hep172 and 2212R for the second round (Table 2).
The second-round PCR was performed with 5 μl of the first-round PCR product added to 45 μl of the PCR mixture. The reaction mixture and conditions were as described above, with one exception: the annealing temperature was raised to 60°C. Five microliters of PCR product was electrophoresed on 1.5% agarose in 0.5x TBE (Tris-borate-EDTA) buffer and visualized by ethidium bromide staining.
Amplification of the 5' end of the RNA-dependent RNA polymerase. Reverse transcription was performed as described above. Primers GV21 and hep171 were used for the amplification of nucleotides 3368 to 5099, with reference to the sequence numbering of Lordsdale virus (GenBank accession number X98081) (Table 2). PCR was performed with 5 μl of cDNA added to 45 μl of a PCR mixture containing (final concentrations) 1x Expand High Fidelity plus PCR buffer, 1.5 mM MgCl2, 200 μM each dNTP, 0.5 μM each primer, and 2.5 U Expand High Fidelity Taq polymerase (Roche, Basel, Switzerland). PCR was carried out for 95°C for 5 min and then 35 cycles of 95°C, 55°C, and 72°C for 30 s, 30 s, and 90 s, respectively.
DNA sequence analysis. The PCR products were purified by polyethylene glycol precipitation and were washed with 70% ethanol. The products were sequenced directly on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA) by using dye-terminator chemistry. Database searches for related sequences were conducted by using the BLAST program. Pairwise alignments of the DNA sequences were carried out with the GAP program of the Genetics Computer Group, and multiple-sequence alignments were performed with ClustalW (49).
Phylogenetic analysis. Evolutionary distances between sequences were determined by using the Genetics Computer Group program DNAdist (Kimura two-parameter method) (10). The computed distances were used for the construction of phylogenetic trees by use of the neighbor-joining method (10). To gain an internal estimate of how well the data supported the phylogenetic trees produced by the neighbor-joining method, bootstrap resampling (100 data sets) of the multiple-sequence alignments was carried out with the program Seqboot (10). The consensus tree was calculated with Consense (11). Tree branch lengths were determined by analyzing the consensus tree with Puzzle (47). Trees were plotted by using the program TREEVIEW (version 1.6.6) (41).
Nucleotide sequence accession numbers. The GenBank accession numbers for strains sequenced in this study are DQ078793 to DQ078853.
RESULTS
Development of nRT-PCR for detection of NoV GII. An outer primer set, primers NV2oF2 and NV2oR, was designed and used in conjunction with previously published primer set G2F3 and G2SKR (19, 27) to develop a highly sensitive nRT-PCR for the detection of NoV GII. Both NV2oF2 and NV2oR shared 100% identity to an alignment of 44 strains representing the 12 NoV GII genotypes (50). A PCR product of 311 bp was amplified from all 89 NoV GII-positive samples by the nested RT-PCR. Furthermore, the assay was shown to be specific for NoV GII, as no product was amplified with nucleic acid purified from NoV GI, sapovirus, astrovirus, rotavirus, or enteric adenovirus-positive samples (data not shown).
Sporadic NoV GII strains, 1997 to 2004. Of the 89 NoV GII-positive samples tested as described above, 32 (36%) of the samples were from sporadic cases of gastroenteritis. Sequencing and subsequent phylogenetic analysis of nRT-PCR products identified a diverse collection of NoV GII strains in New South Wales between 1997 and 2004 (Fig. 1 and Table 3). In the first cohort of 11 sporadic samples collected from July 1997 to December 1998, three capsid genotypes were identified: GII.3, GII.4, and GII.6. The nucleotide sequences of the three GII.3 strains were all related to the Sydney 2212 virus nucleotide sequence (>97%) in the capsid (Fig. 1). Sydney 2212 is a known recombinant that caused outbreaks of gastroenteritis in several day care centers across Sydney, Australia, in 1998 (3). Amplification and sequence analysis across the NoV recombination breakpoint, the ORF1-ORF2 overlap (3), in these GII.3 strains revealed that they were also closely related to Sydney 2212 in the polymerase region (95%) and were therefore Sydney 2212-like recombinants. The Sydney 2212-like recombinants did not group with other genotypes in the polymerase region, with the closest relatives belonging to GII.4 (3). Another four strains isolated between 1997 and 1998 had GII.4 capsids and were closely related to the US95/96-like strain that was dominant in New South Wales between 1997 and 2000 (Fig. 1) (52). The final four strains identified were closely related to Seacroft virus (GenBank accession number AJ277620) (16) in the capsid and polymerase regions and belonged to GII.6 (Fig. 1).
The second cohort included samples from 15 sporadic cases collected between August 2001 and August 2002. Five genotypes were identified: GII.3, GII.4, GII.6, GII.7, and GII.10. Seven of the 15 sporadic strains isolated between 2001 and 2002 clustered with GII.3 strains in the capsid, thereby making GII.3 the most prevalent genotype in this cohort (Fig. 1). All seven GII.3 strains were closely related in their capsid region (97% nucleotide identity) to the second GII.3 recombinant Sydney C14, recently identified by us (3). Sydney C14 was associated with an outbreak of gastroenteritis in a Sydney children's hospital in 2002 and was one of nine different recombinant types identified (3). Amplification and sequencing across the recombination breakpoint, the ORF1-ORF2 overlap, revealed that all seven GII.3 capsid strains were related to Sydney C14 in both the polymerase and the capsid regions. The polymerase of the Sydney C14-like recombinants was novel, with the closest relative strain belonging to GII.2 (3). GII.4 was the second most prevalent genotype identified in this cohort. One of the GII.4 strains clustered with the 1997-1998 GII.4 strains. The other three GII.4 strains, however, clustered in the Farmington Hills subset (Fig. 1). The GII.6 strain identified was closely related to the 1997-1998 GII.6 strains (>97% nucleotide identity). The two GII.7 strains were closely related to the capsid of Thai strain Mc17 (Fig. 1) (19). The GII.10 strain, Sydney 4264/01S, was closely related to the known Thai recombinant Mc37 (19). Sequence analysis of the amplified ORF1-ORF2 region revealed that Sydney 4264/01S shared 99% identity over 775 bp of the 3' end of ORF1 and the 5' end of ORF2 with Mc37 and was therefore identified as an Mc37-like recombinant.
The third cohort included samples from six sporadic cases of gastroenteritis collected between February 2004 and August 2004. One strain was a GII.3 recombinant that was closely related to the Sydney C14 recombinant (3), and another was a GII.4 strain that grouped with the Farmington Hills cluster (Fig. 1). The remaining four strains were GII.4 and were closely related to each other and formed a novel cluster (Fig. 1).
Outbreak strains, 1997 to 2004. NoV GII.4 strains are recognized as the predominant cause of viral gastroenteritis outbreaks (6, 29, 32, 51-53). Since 1995, four GII.4 strains have been identified as etiological agents in major NoV epidemics: US95/96 (9), European variant (32), Farmington Hills (53), and b4s6 (6). Genetic analysis grouped these four strains into two distinct GII.4 clusters, defined here as the US95/96 cluster and the Farmington Hills cluster. The Farmington Hills cluster included the 2002 U.S. strain Farmington Hills, the 2002 European variant (32; H. Vennema, personal communication), and the 2002 United Kingdom strain b4s6. NoV b4s6 shared 99.5% identity over the complete genome to the U.S. strain, Farmington Hills, revealing the intercontinental spread of the same variant. Sequence alignment between the members of the two clusters (US95/96 and Farmington Hills) demonstrated greater than 6% divergence across the capsid gene. In this study a new variant was defined as a strain that had greater than 5% amino acid divergence across the complete capsid gene.
This study analyzed strains from 28 outbreaks in New South Wales from 1997 to 2004. Between 1997 and 2001 the major outbreak strains in New South Wales clustered with global outbreak strain US95/96 (Fig. 1). In 2002, an increase in NoV-associated gastroenteritis was reported across Europe, the United States, and Sydney (46). In Fig. 1, Sydney 917J/02/AU is a representative of a predominant strain that was the etiological agent of six outbreaks investigated in New South Wales in 2002 (7). Sydney 917J/02/AU shares 98% nucleotide identity over 266 bp in the capsid region with Farmington Hills.
In 2003, two non-GII.4 strains were each associated with an outbreak of gastroenteritis. A GII.6 strain caused an outbreak at a ball that affected 60 people. A GII.1 strain, termed Picton, caused an outbreak in a geriatric hospital and was isolated from 22 patients (Fig. 1). Strain Picton was previously identified as a novel recombinant (3), with a polymerase region that was closely related to that of Sydney C14 (96% nucleotide identity) and a GII.1 capsid.
In 2004, there was a marked increase in NoV-associated gastroenteritis in New South Wales (48). There was a fivefold increase in the number of outbreaks in institutional settings in 2004 compared to the number in 2003 (48). RNA from 22 specimens from 18 NoV-associated gastroenteritis outbreaks in New South Wales in 2004 was amplified by our nRT-PCR assay. Phylogenetic analysis of the 22 capsid sequences indicated that the 2004 New South Wales strains formed their own cluster, termed the Hunter cluster, within the GII.4 subset, distinct from other sequences in the database (Fig. 1). Pairwise sequence analysis of the complete Hunter capsid gene (GenBank accession number DQ078794) revealed greater than 5% nucleotide and amino acid sequence variations to members of the US95/96 and Farmington Hills cluster, therefore defining the Hunter strain as a new GII.4 variant.
Due to numerous reports of NoV recombinants in New South Wales, a representative of the Hunter cluster was genotyped based on its polymerase sequence. A 1,498-bp region incorporating the 3' end of RdRp and the 5' end of the capsid was amplified by RT-PCR from the RNA of Hunter 532D/04/AU. Sequencing and phylogenetic analysis demonstrated that this strain had a GII.4 polymerase that was distinct (>4% variation) from any of the sequences in the database, in particular, the four NoV GII.4 outbreak strains described in Fig. 1. Hunter 532D/04/AU shared 94% identity with the US95/96 strain Sydney 348/97/AU (Fig. 1) and 96% identity with the Farmington Hills virus across the polymerase region. However, the Hunter strain was identical (>99.6% identity) to a GII.4 strain associated with an increase in gastroenteritis outbreaks in The Netherlands in late 2004 (29). Indeed, in The Netherlands the Hunter strain caused 44 of 77 (57%) outbreaks between August and December 2004 (29).
In summary, US95/96 strains were prominent between 1997 and 1998 in New South Wales. The majority of the strains collected between 2001 and 2002 were closely related to members of the Farmington Hills cluster. In contrast, the 2004 New South Wales strain, Hunter, which was associated with both outbreak and sporadic cases, formed its own cluster, suggesting the emergence of a new GII.4 variant.
DISCUSSION
The accurate diagnosis of viral gastroenteritis is essential in reducing its impact on society. Each year in the United States, approximately 5,000 deaths are attributed to gastrointestinal disease of unknown etiology (36). Despite the absence of a current treatment for viral gastroenteritis, the identification of the agent, especially in the case of an outbreak, enables the implementation of precautions that will prevent further dissemination of the disease. Furthermore, it is important to distinguish between a viral agent and a bacterial or parasitic agent, as infections with the last two types of agents can be treated. Due to the inability to culture NoV and the diversity of its genome, it has proven difficult to develop a sensitive method of detection. In this study we report on the development of an nRT-PCR that has been shown to detect a diverse range of NoV GII strains.
The detection limit of primers G2F3 and G2SKR (19, 27) was improved in this study by incorporating them into an nRT-PCR. The detection of viruses present at low viral loads is important for epidemiological studies of outbreaks, especially when the origin of an outbreak is traced retrospectively, when some of the patients' symptoms may have dissipated and levels of excreted NoV are low. Although nested PCR does have several drawbacks, not only does it require more time and consumables than a single-round PCR, but its use also has a higher risk of false-positive results due to contamination.
Over the last 5 years there has been a significant increase in the number of NoV recombinants that have been isolated. In a previous study we identified nine worldwide recombinant types and identified the ORF1-ORF2 junction as the recombination breakpoint (3). Consequently, to identify recombinants in the present study, strains were sequenced across the breakpoint. Phylogenetic analyses of the partial polymerase and the partial capsid regions were performed with suspected recombinants and prevalent NoV strains. Four of the nine recombinant types have been identified in New South Wales over the last 7 years. In 1997, 1998, 2001, and 2002, the two GII.3 recombinants Sydney C14 and Sydney 2212 were the second most prevalent NoV strains identified after the GII.4 strains, causing 31% of sporadic cases and 7% of outbreak cases. This illustrates not only the importance of recombinant analysis in epidemiological studies but also the fact that recombinants may be more prevalent than was originally thought.
Previous studies of the epidemiology of NoV in Australia have reported strains belonging to GII.1, GII.2, GII.3, GII.4, and GII.5, with GII.4 being the most prevalent (25, 35, 52, 54). The nRT-PCR assay detected six genotypes, GII.1, GII.3, GII.4, GII.6, GII.7, and GII.10, in New South Wales between 1997 and 2004. The nRT-PCR also detected NoV GII.5 (data not shown). Other strains were not available for testing; but the inner primer set alone, primers G2F3 and G2SKR, has been shown to detect another genetic cluster, GII.8 (19), indicating that the nRT-PCR assay can detect eight genotypes, if not more.
The majority of the sporadic and the outbreak strains detected in this study belonged to genotypes GII.4 and GII.3, with the former having been previously identified as a dominant genotype in New South Wales (52). The GII.3 strains were a combination of the two recombinant types, Sydney 2212 and Sydney C14, and this was the first epidemiological study to report on the dominance of recombinant GII.3 strains circulating in New South Wales. This is also the first Australian study to report on the isolation of GII.10. While Australia is geographically isolated in the world, phylogenetic analysis revealed that the same strains are circulating in Asia, Europe, and the Americas (12, 13, 19, 31, 55). Modern methods of transport and the multicultural nature of Australia undoubtedly facilitate the importation of global NoV strains.
This study suggests that the majority of the NoV GII outbreak strains isolated in Australia mirror the strains causing outbreaks during similar time frames in the Northern Hemisphere. In the United States, between 1995 and 1996, a single NoV GII.4 variant was isolated as the predominant cause of outbreaks of gastroenteritis (9). This strain, US95/96, was later found to be the etiological agent of a wave of gastroenteritis outbreaks on five continents, including Australia (39, 52). In this study, US95/96-like strains continued to be detected in New South Wales up to 2002 (Fig. 1). Furthermore, in 2002 there was a similar increase in NoV-associated outbreaks of gastroenteritis across the globe, with related strains belonging to the Farmington Hills cluster being identified across Europe (6, 32) and the United States (53). Farmington Hills-like strains were also isolated in New South Wales in 2001 and 2002 (Fig. 1).
In 2004, there was another marked increase in NoV activity in New South Wales (48). NoV was the main cause of gastroenteritis, accounting for 95% of outbreak cases, in which the etiological agent was identified (48). Eighteen NoV GII-positive outbreaks within New South Wales were investigated. A single NoV GII strain, termed Hunter, was isolated from all 18 outbreaks. Phylogenetic analysis of the complete capsid region revealed that the Hunter strain was unrelated (>5% amino acid divergence in the capsid region) to the global pandemic strains Farmington Hills and US95/96 (Fig. 1). The Hunter strain first appeared in New South Wales in February 2004; and matching sequence data from The Netherlands from September 2004, Taiwan from November 2004 and Japan from 2005 were found in the GenBank and EMBL databases (29). While the origin of the Hunter strain is unknown, we speculate that the Hunter strain, a predominant cause of NoV outbreaks in 2004 (20) and 2005, could also continue to cause global epidemics in 2006 and beyond. Identification of novel GII.4 variants may also lead to an increased ability to predict increases in NoV activity.
Strains belonging to the GII.4 cluster are recognized as the predominant genotype worldwide, and this study supports the dominance of GII.4. In the present study, GII.4 strains accounted for 43.7% of the sporadic cases and 85.8% of the outbreaks. Why GII.4 strains have continued to be dominant is unknown and of great interest. Possibilities include more sensitive methods for the detection of GII.4 strains, viral fitness, capsid interaction with human blood antigens, or recombination; or perhaps the same evolving strain is continuing to dominate.
ACKNOWLEDGMENTS
This work was supported by a Sydney Catchment Authority collaborative research grant. R.A.B. was supported by an Australian postgraduate award, and E.T.V.T. was supported by a University of New South Wales postgraduate award.
We gratefully acknowledge the contributions by Grant Hansman from the National Institute of Infectious Diseases, Tokyo, Japan.
REFERENCES
Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of "Norwalk-like viruses." J. Infect. Dis. 181(Suppl.):S336-S348.
Atmar, R. L., and M. K. Estes. 2001. Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin. Microbiol. Rev. 14:15-37.
Bull, R. A., G. S. Hansman, L. E. Clancy, M. M. Tanaka, W. D. Rawlinson, and P. A. White. 2005. Norovirus recombination in ORF1/ORF2 overlap. Emerg. Infect. Dis. 11:1079-1085.
Burton-MacLeod, J. A., E. M. Kane, R. S. Beard, L. A. Hadley, R. I. Glass, and T. Ando. 2004. Evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples. J. Clin. Microbiol. 42:2587-2595.
Cheesbrough, J. S., J. Green, C. I. Gallimore, P. A. Wright, and D. W. Brown. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol. Infect. 125:93-98.
Dingle, K. E. 2004. Mutation in a Lordsdale norovirus epidemic strain as a potential indicator of transmission routes. J. Clin. Microbiol. 42:3950-3957.
Duckworth, M. 2002. Norwalk-like viruses: approaches for detection. Honours thesis. University of New South Wales, Sydney, Australia.
Fankhauser, R. L., S. S. Monroe, J. S. Noel, C. D. Humphrey, J. S. Bresee, U. D. Parashar, T. Ando, and R. I. Glass. 2002. Epidemiological and molecular trends of "Norwalk-like viruses" associated with outbreaks of gastroenteritis in the United States. J. Infect. Dis. 186:1-7.
Fankhauser, R. L., J. S. Noel, S. S. Monroe, T. Ando, and R. I. Glass. 1998. Molecular epidemiology of "Norwalk-like viruses" in outbreaks of gastroenteritis in the United States. J. Infect. Dis. 178:1571-1578.
Felsenstein, J. 1989. PHYLIP—phylogeny inference package (version 3.2). Cladistics 5:164-166.
Felsenstein, J. 1993. PHYLIP inference package, version 3.5c. Department of Genetics, University of Washington, Seattle.
Foley, B., J. O'Mahony, C. Hill, and J. G. Morgan. 2001. Molecular detection and sequencing of "Norwalk-like viruses" in outbreaks and sporadic cases of gastroenteritis in Ireland. J. Med. Virol. 65:388-394.
Gallimore, C. I., J. Green, D. Lewis, A. F. Richards, B. A. Lopman, A. D. Hale, R. Eglin, J. J. Gray, and D. W. Brown. 2004. Diversity of noroviruses cocirculating in the north of England from 1998 to 2001. J. Clin. Microbiol. 42:1396-1401.
Girish, R., S. Broor, L. Dar, and D. Ghosh. 2002. Foodborne outbreak caused by a Norwalk-like virus in India. J. Med. Virol. 67:603-607.
Glass, P. J., L. J. White, J. M. Ball, I. Leparc-Goffart, M. E. Hardy, and M. K. Estes. 2000. Norwalk virus open reading frame 3 encodes a minor structural protein. J. Virol. 74:6581-6591.
Green, J., J. Vinje, C. I. Gallimore, M. Koopmans, A. Hale, D. W. G. Brown, J. C. S. Clegg, and J. Chamberlain. 2000. Capsid protein diversity among Norwalk-like viruses. Virus Genes 20:227-236.
Green, K. Y., R. M. Chanock, and A. Z. Kapikan. 2001. Human calicivirus, p. 841-874. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4 ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.
Gunson, R., J. Miller, and W. F. Carman. 2003. Comparison of primers for NLV diagnosis. J. Clin. Virol. 26:379-380.
Hansman, G. S., K. Katayama, N. Maneekarn, N. Peerakome, P. Khamrin, S. Tonusin, S. Okitsu, O. Nishio, N. Takeda, and H. Ushijima. 2004. Genetic diversity of norovirus and sapovirus in hospitalized infants with sporadic cases of acute gastroenteritis in Chiang Mai, Thailand. J. Clin. Microbiol. 42:1305-1307.
International Society for Infectious Diseases. 2005. ProMED-mail. http://www.promedmail.org. Accessed 13 March 2005.
Iversen, A. M., M. Gill, C. L. Bartlett, W. D. Cubitt, and D. A. McSwiggan. 1987. Two outbreaks of foodborne gastroenteritis caused by a small round structured virus: evidence of prolonged infectivity in a food handler. Lancet ii:556-558.
Kapikan, A. Z. 1994. Norwalk and Norwalk-like viruses, p. 471-519. In A. Z. Kapikan (ed.), Viral infections of the gastrointestinal tract, 2nd ed. Marcel Dekker, Inc., New York, N.Y.
Karst, S. M., C. E. Wobus, M. Lay, J. Davidson, and H. W. Virgin IV. 2003. STAT1-dependent innate immunity to a Norwalk-like virus. Science 299:1575-1578.
Katayama, K., H. Shirato-Horikoshi, S. Kojima, T. Kageyama, T. Oka, F. B. Hoshino, S. Fukushi, M. Shinohara, K. Uchida, Y. Suzuki, T. Gojobori, and N. Takeda. 2002. Phylogenetic analysis of the complete genome of 18 norwalk-like viruses. Virology 299:225-239.
Kirkwood, C. D., and R. F. Bishop. 2001. Molecular detection of human calicivirus in young children hospitalized with acute gastroenteritis in Melbourne, Australia, during 1999. J. Clin. Microbiol. 39:2722-2724.
Kobayashi, S., K. Sakae, Y. Suzuki, H. Ishiko, K. Kamata, K. Suzuki, K. Natori, T. Miyamura, and N. Takeda. 2000. Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen. Microbiol. Immunol. 44:687-693.
Kojima, S., T. Kageyama, S. Fukushi, F. B. Hoshino, M. Shinohara, K. Uchida, K. Natori, N. Takeda, and K. Katayama. 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100:107-114.
Koopmans, M. P. G., C. H. von Bonsdorrf, J. Vinje, D. DeMedici, and S. S. Monroe. 2002. Foodborne enteric viruses. FEMS Microbiol. Rev. 26:187-205.
Kroneman, A., H. Vennema, Y. van Duijnhoven, E. Duizer, and M. Koopmans. 2004. High number of norovirus outbreaks associated with a GGII. 4 variant in The Netherlands and elsewhere: does this herald a worldwide increase? Eurosurveill. Wkly. 8:51-52.
Lahti, K., and L. Hiisvirta. 1995. Causes of waterborne outbreaks in community water systems in Finland—1980-1992. Water Sci. Technol. 31:33-36.
Lau, C. S., D. A. Wong, L. K. Tong, J. Y. Lo, A. M. Ma, P. K. Cheng, and W. W. Lim. 2004. High rate and changing molecular epidemiology pattern of norovirus infections in sporadic cases and outbreaks of gastroenteritis in Hong Kong. J. Med. Virol. 73:113-117.
Lopman, B., H. Vennema, E. Kohli, P. Pothier, A. Sanchez, A. Negredo, J. Buesa, E. Schreier, M. Reacher, D. Brown, J. Gray, M. Iturriza, C. Gallimore, B. Bottiger, K. O. Hedlund, M. Torven, C. H. von Bonsdorff, L. Maunula, M. Poljsak-Prijatelj, J. Zimsek, G. Reuter, G. Szucs, B. Melegh, L. Svennson, Y. van Duijnhoven, and M. Koopmans. 2004. Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant. Lancet 363:682-688.
Marks, P. J., I. B. Vipond, D. Carlisle, D. Deakin, R. E. Fey, and E. O. Caul. 2000. Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant. Epidemiol. Infect. 124:481-487.
Marks, P. J., I. B. Vipond, F. M. Regan, K. Wedgwood, R. E. Fey, and E. O. Caul. 2003. A school outbreak of Norwalk-like virus: evidence for airborne transmission. Epidemiol. Infect. 131:727-736.
Marshall, J. A., M. E. Hellard, M. I. Sinclair, C. K. Fairley, B. J. Cox, M. G. Catton, H. Kelly, and P. J. Wright. 2003. Incidence and characteristics of endemic Norwalk-like virus-associated gastroenteritis. J. Med. Virol. 69:568-578.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the Uinted States. Emerg. Infect. Dis. 5:607-625.
Meakins, S. M., G. K. Adak, B. A. Lopman, and S. J. O'Brien. 2003. General outbreaks of infectious intestinal disease (IID) in hospitals, England and Wales, 1992-2000. J. Hosp. Infect. 53:1-5.
Murray, C. J. L., and A. D. Lopez. 1996. Evidence-based health policy: lessons from the global burden of disease study. Science 274:740-743.
Noel, J. S., R. L. Fankhauser, T. Ando, S. S. Monroe, and R. I. Glass. 1999. Identification of a distinct common strain of "Norwalk-like viruses" having a global distribution. J. Infect. Dis. 179:1334-1344.
O'Neill, H. J., C. McCaughey, P. V. Coyle, D. E. Wyatt, and F. Mitchell. 2002. Clinical utility of nested multiplex RT-PCR for group F adenovirus, rotavirus and Norwalk-like viruses in acute viral gastroenteritis in children and adults. J. Clin. Virol. 25:335-343.
Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.
Parashar, U. D., and S. S. Monroe. 2001. ‘Norwalk-like viruses’ as a cause of foodborne disease outbreaks. Rev. Med. Virol. 11:243-252.
Pletneva, M. A., S. V. Sosnovtsev, and K. Y. Green. 2001. The genome of Hawaii virus and its relationship with other members of the Caliciviridae. Virus Genes 23:5-16.
Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, H. Appleton, C. I. Gallimore, J. J. Gray, and D. W. G. Brown. 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.
Roche, P., L. Halliday, E. O'Brien, and J. Spencer. 2002. The laboratory virology and serology reporting scheme, 1991 to 2000. Communicable Dis. Intell. 26:323-374.
Spencer, J. 9 December 2004, revision date. Communicable diseases surveillance: tables (LabVISE). Communicable Dis. Intell. 27:563. [Online.] http://www.health.gov.au.
Strimmer, K., and A. von Haeseler. 1997. Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc. Natl. Acad. Sci. USA 94:6815-6819.
Telfer, B., and S. Munnoch. 2005. OzFoodnet—enhancing foodborne disease surveillance across Australia. Annual Report. New South Wales and Hunter Area Health Service, Sydney, Australia.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multisequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
Vinje, J., R. A. Hamidjaja, and M. D. Sobsey. 2004. Developmental and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J. Virol. Methods 116:109-117.
Vipond, I. B., E. O. Caul, D. Hirst, B. Carmen, A. Curry, B. A. Lopman, P. Pead, M. A. Pickett, P. R. Lambden, and I. N. Clarke. 2004. National epidemic of Lordsdale Norovirus in the UK. J. Clin. Virol. 30:243-247.
White, P. A., G. S. Hansman, A. Li, J. Dable, M. Isaacs, M. Ferson, C. J. McIver, and W. D. Rawlinson. 2002. Norwalk-like virus 95/96-US strain is a major cause of gastroenteritis outbreaks in Australia. J. Med. Virol. 68:113-118.
Widdowson, M. A., E. H. Cramer, L. Hadley, J. S. Bresee, R. S. Beard, S. N. Bulens, M. Charles, W. Chege, E. Isakbaeva, J. G. Wright, E. Mintz, D. Forney, J. Massey, R. I. Glass, and S. S. Monroe. 2004. Outbreaks of acute gastroenteritis on cruise ships and on land: identification of a predominant circulating strain of norovirus—United States, 2002. J. Infect. Dis. 190:27-36.
Wright, P. J., I. C. Gunesekere, J. C. Doultree, and J. A. Marshall. 1998. Small round-structured (Norwalk-like) viruses and classical human caliciviruses in southeastern Australia, 1980-1996. J. Med. Virol. 55:312-320.
Zintz, C., K. Bok, E. Parada, M. Barnes-Eley, T. Berke, M. A. Staat, P. Azimi, X. Jiang, and D. O. Matson. 2005. Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the united states. Infect. Genet. Evol. 5:281-290.
School of Biotechnology and Biomolecular Sciences, Faculty of Science
1 School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney, NSW 2052, Australia
2 Virology Division, SEALS, Department of Microbiology, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia3(Rowena A. Bull, Elise T. )
Norovirus (NoV) is highly infectious and is the major cause of outbreak gastroenteritis in adults, with pandemic spread of the virus being reported in 1995 and 2002. The NoV genome is genetically diverse, which has hampered development of sensitive molecular biology-based methods. In this study we report on a nested reverse transcriptase PCR (nRT-PCR) that was designed to amplify the highly conserved 3' end of the polymerase region and the 5' end of the capsid gene of NoV genogroup II (GII). The nRT-PCR was validated with strains isolated from sporadic and outbreak cases between 1997 and 2004 in New South Wales, Australia. Phylogenetic analysis identified six genotypes circulating in New South Wales, GII.1, GII.3, GII.4, GII.6, GII.7, and GII.10, with GII.4 being the predominant genotype. In 2004, there was a marked increase in NoV GII activity in Australia, with a novel GII.4 variant being identified as the etiological agent in 18 outbreaks investigated. This novel GII.4 variant, termed Hunter virus, differed by more than 5% at the amino acid level across the capsid from any other NoV strain in the GenBank and EMBL databases. The Hunter virus was subsequently identified as the etiological agent in large epidemics of gastroenteritis in The Netherlands, Japan, and Taiwan in 2004 and 2005.
INTRODUCTION
Gastroenteritis is one of the leading causes of death by an infectious disease (38). Norovirus (NoV), a member of the Caliciviridae family, is considered the major cause of acute gastroenteritis in adults (8, 37, 45) and is estimated to be responsible for 93% of food-related outbreaks of gastroenteritis in the United States (8). NoV is associated with sporadic and outbreak cases of gastroenteritis in individuals of all ages, with a distinct seasonality linked to the winter months (40). Infection is characterized by the acute onset of nausea, vomiting, abdominal cramps, and diarrhea, which generally last for about 48 h (22). Transmission occurs predominantly through ingestion of contaminated water; food (particularly oysters); and person to person by the fecal-oral route, airborne transmission, and contact with contaminated surfaces (14, 21, 30, 37, 42). The ease with which NoV is transmitted and the low infectious dose required to establish an infection results in extensive outbreaks in numerous environments, including hospitals, hotels, schools, nursing homes, and cruise ships (5, 33, 34, 53).
NoV is a small round virion of 27 to 35 nm in diameter and possesses a single-stranded, positive-sense, polyadenylated RNA genome of 7,400 to 7,700 nucleotides (2). The genome is divided into three open reading frames (ORFs). ORF1 encodes a large polyprotein which undergoes proteolytic cleavage to produce viral proteins that are homologous to proteins fromother single-stranded RNA viruses, including nucleoside triphosphatase, 3C-like protease, and RNA-dependent RNA polymerase (RdRp) (17). The two structural proteins, VP1, the major capsid protein, and VP2, the minor capsid protein, are encoded by ORF2 and ORF3, respectively (15, 43). The region from the C-terminal end of ORF1 to the N-terminal region of ORF2 is the most highly conserved region across all known NoV strains (27). We have recently identified this region as the breakpoint in recombinant NoVs (3).
NoV is divided into five genogroups based on the genome sequence of the RdRp and the capsid regions (50). However, only NoV genogroup I (GI), GII, and GIV have been associated with human gastroenteritis (1, 23, 28). NoV GI is further subdivided into 7 genotypes, and NoV GII is further subdivided into 12 genotypes (50). However, simple genotyping has been complicated by the emergence of recombinant NoV that have polymerase and capsid regions derived from separate ancestral strains. Nine NoV recombinant types with an assortment of polymerase and capsid genotypes have been identified (3).
A rapid and definitive diagnosis is important in outbreak settings to prevent the further dissemination of the virus in the population. Older detection methods like electron microscopy have been replaced by reverse transcriptase (RT) PCR and, more recently, enzyme immunoassays (EIAs). While EIA is faster and less labor-intensive than RT-PCR, recent evaluations of commercial EIAs show that they have poor sensitivities and are unable to detect all NoV GII genotypes (4, 18, 44).
Until recently, the majority of NoV GII RT-PCRs have targeted ORF1, in particular, the middle of the RdRp-encoding region, as ORF1 is overall more highly conserved than ORF2 (24). However, Katayama et al. illustrated, using multiple alignments of 18 full-length NoV genomes, that highly conserved sequence motifs are located at the 3' end of the RdRp and the adjacent 5' end of ORF2 (24). Primers targeting these highly conserved sequences were able to detect a broader range of NoV GII strains than primers targeting the central region of RdRp (19, 27). We have adopted this approach in the current study.
Due to the large genomic and antigenic diversity of NoV, it is essential that there be a comprehensive understanding of the strains circulating within a population to ensure that sensitive detection methods are being used. Over the last few years, NoV-associated gastroenteritis has reached unprecedented levels (6, 29, 32, 51, 53). The majority of newly emerging NoV outbreak strains belong to GII.4 and have a global presence (6, 29, 32, 51-53). For example, a GII.4 variant, termed US95/96, caused 55% of gastroenteritis outbreaks throughout the United States in 1995 and 1996 (9) and was then later detected in Brazil, Canada, China, Germany, Netherlands, the United Kingdom, and Australia (39). In New South Wales, Australia, this strain was associated with 86% of NoV-associated outbreaks of gastroenteritis during 1997 and 2000 (52). In 2002, another GII.4 strain, reported by the European Food-Borne Viruses Network, termed the European variant in this study, caused 134 (86%) of 153 outbreaks originating in Germany, Holland, Wales, and the United Kingdom (32). In 2002, two other identical GII.4 variants, termed Farmington Hills and b4s6, caused outbreaks in the United States and England, respectively (6, 53). Farmington Hills virus was associated with 64% of cruise ship outbreaks and 45% of land-based outbreaks in the United States in 2002 (53). b4s6 was linked to 100% of 22 outbreaks in Oxfordshire, United Kingdom, during 2002 and 2003 (6).
In this study we developed a new nested RT-PCR (nRT-PCR) for the detection of NoV GII. The assay was used to determine the genetic diversity of circulating strains implicated in sporadic and outbreak gastroenteritis between 1997 and 2004. In Australia, epidemics of gastroenteritis occurred in 2002 (46) and 2004 (48) and were dominated by NoV infections. Furthermore, increases in gastroenteritis outbreaks in 2004 were correlated with the emergence of a novel GII.4 variant.
MATERIALS AND METHODS
Stool specimens. In total, 88 NoV GII-positive stool samples were obtained from the Department of Microbiology, SEALS, Prince of Wales Hospital, Sydney, Australia. Stool specimens were confirmed as NoV GII positive either by the IDEIA NLV enzyme-linked immunosorbent assay (ELISA; Dakocytomation Ltd.) or nRT-PCR (52) (Table 1). The 88 NoV GII-positive samples included 11 samples from NoV-positive sporadic cases (defined as isolated cases with no known related cases) collected between July 1997 and December 1998, 15 samples from NoV-positive sporadic cases collected between August 2001 and August 2002, 6 samples from sporadic cases collected between February 2004 and August 2004, and 56 samples from 28 outbreaks collected between 1997 and 2004 (Table 1). All stool specimens were stored at –80°C. In addition, two adenovirus-positive specimens, one astrovirus-positive specimen, and one rotavirus-positive specimen, detected by ELISA (Dakocytomation Ltd.), were used as negative controls.
Sample preparation and RNA extraction. A 20% (wt/vol) stool suspension with a total volume of 1 ml was prepared in RNase-free water and centrifuged for 1 min at 13,000 x g. The supernatant was removed, centrifuged for a further 7 min at 13,000 x g, and stored at –20°C. Viral RNA was extracted by using a QIAmp viral RNA kit, according to the manufacturer's instructions (QIAGEN, Hilden, Germany). RNA was resuspended in 50 μl of water and stored at –80°C.
nRT-PCR. Reverse transcription was performed with 10 μl of RNA template added to a 10-μl reaction mixture containing a final concentration of 1x reverse transcription buffer (Promega), 10 mM dithiothreitol, 1 mM each deoxynucleoside triphosphate (dNTP; dATP, dCTP, dGTP, dTTP), 5 μM random primers (Promega), and 6 U of avian myeloblastosis virus RT (Promega). Reverse transcription proceeded by incubation at 42°C for 60 min, and the reverse transcriptase was then denatured by increasing the temperature to 72°C for 15 min.
Regions amplified. The primers used in the study are described in Table 2. For amplification of the highly conserved region targeting the 55 bp at the 3' end of the polymerase and the 344 bp at the 5' end of the capsid, an outer primer set, primers NV2oF2 and NV2oR, was used in conjunction with previously designed primers G2F3 and G2SKR (19, 27) (Table 2). Analysis of potential recombinants was performed by amplifying the region across the ORF1-ORF2 overlap. First- and second-round PCRs were performed as described below with the following combination of either primers CB001 and NV2oR for the first round and primers CB003 and 2212R for the second round or primers hep170 and NV2oR for the first round and primers hep172 and 2212R for the second round (Table 2).
The second-round PCR was performed with 5 μl of the first-round PCR product added to 45 μl of the PCR mixture. The reaction mixture and conditions were as described above, with one exception: the annealing temperature was raised to 60°C. Five microliters of PCR product was electrophoresed on 1.5% agarose in 0.5x TBE (Tris-borate-EDTA) buffer and visualized by ethidium bromide staining.
Amplification of the 5' end of the RNA-dependent RNA polymerase. Reverse transcription was performed as described above. Primers GV21 and hep171 were used for the amplification of nucleotides 3368 to 5099, with reference to the sequence numbering of Lordsdale virus (GenBank accession number X98081) (Table 2). PCR was performed with 5 μl of cDNA added to 45 μl of a PCR mixture containing (final concentrations) 1x Expand High Fidelity plus PCR buffer, 1.5 mM MgCl2, 200 μM each dNTP, 0.5 μM each primer, and 2.5 U Expand High Fidelity Taq polymerase (Roche, Basel, Switzerland). PCR was carried out for 95°C for 5 min and then 35 cycles of 95°C, 55°C, and 72°C for 30 s, 30 s, and 90 s, respectively.
DNA sequence analysis. The PCR products were purified by polyethylene glycol precipitation and were washed with 70% ethanol. The products were sequenced directly on an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA) by using dye-terminator chemistry. Database searches for related sequences were conducted by using the BLAST program. Pairwise alignments of the DNA sequences were carried out with the GAP program of the Genetics Computer Group, and multiple-sequence alignments were performed with ClustalW (49).
Phylogenetic analysis. Evolutionary distances between sequences were determined by using the Genetics Computer Group program DNAdist (Kimura two-parameter method) (10). The computed distances were used for the construction of phylogenetic trees by use of the neighbor-joining method (10). To gain an internal estimate of how well the data supported the phylogenetic trees produced by the neighbor-joining method, bootstrap resampling (100 data sets) of the multiple-sequence alignments was carried out with the program Seqboot (10). The consensus tree was calculated with Consense (11). Tree branch lengths were determined by analyzing the consensus tree with Puzzle (47). Trees were plotted by using the program TREEVIEW (version 1.6.6) (41).
Nucleotide sequence accession numbers. The GenBank accession numbers for strains sequenced in this study are DQ078793 to DQ078853.
RESULTS
Development of nRT-PCR for detection of NoV GII. An outer primer set, primers NV2oF2 and NV2oR, was designed and used in conjunction with previously published primer set G2F3 and G2SKR (19, 27) to develop a highly sensitive nRT-PCR for the detection of NoV GII. Both NV2oF2 and NV2oR shared 100% identity to an alignment of 44 strains representing the 12 NoV GII genotypes (50). A PCR product of 311 bp was amplified from all 89 NoV GII-positive samples by the nested RT-PCR. Furthermore, the assay was shown to be specific for NoV GII, as no product was amplified with nucleic acid purified from NoV GI, sapovirus, astrovirus, rotavirus, or enteric adenovirus-positive samples (data not shown).
Sporadic NoV GII strains, 1997 to 2004. Of the 89 NoV GII-positive samples tested as described above, 32 (36%) of the samples were from sporadic cases of gastroenteritis. Sequencing and subsequent phylogenetic analysis of nRT-PCR products identified a diverse collection of NoV GII strains in New South Wales between 1997 and 2004 (Fig. 1 and Table 3). In the first cohort of 11 sporadic samples collected from July 1997 to December 1998, three capsid genotypes were identified: GII.3, GII.4, and GII.6. The nucleotide sequences of the three GII.3 strains were all related to the Sydney 2212 virus nucleotide sequence (>97%) in the capsid (Fig. 1). Sydney 2212 is a known recombinant that caused outbreaks of gastroenteritis in several day care centers across Sydney, Australia, in 1998 (3). Amplification and sequence analysis across the NoV recombination breakpoint, the ORF1-ORF2 overlap (3), in these GII.3 strains revealed that they were also closely related to Sydney 2212 in the polymerase region (95%) and were therefore Sydney 2212-like recombinants. The Sydney 2212-like recombinants did not group with other genotypes in the polymerase region, with the closest relatives belonging to GII.4 (3). Another four strains isolated between 1997 and 1998 had GII.4 capsids and were closely related to the US95/96-like strain that was dominant in New South Wales between 1997 and 2000 (Fig. 1) (52). The final four strains identified were closely related to Seacroft virus (GenBank accession number AJ277620) (16) in the capsid and polymerase regions and belonged to GII.6 (Fig. 1).
The second cohort included samples from 15 sporadic cases collected between August 2001 and August 2002. Five genotypes were identified: GII.3, GII.4, GII.6, GII.7, and GII.10. Seven of the 15 sporadic strains isolated between 2001 and 2002 clustered with GII.3 strains in the capsid, thereby making GII.3 the most prevalent genotype in this cohort (Fig. 1). All seven GII.3 strains were closely related in their capsid region (97% nucleotide identity) to the second GII.3 recombinant Sydney C14, recently identified by us (3). Sydney C14 was associated with an outbreak of gastroenteritis in a Sydney children's hospital in 2002 and was one of nine different recombinant types identified (3). Amplification and sequencing across the recombination breakpoint, the ORF1-ORF2 overlap, revealed that all seven GII.3 capsid strains were related to Sydney C14 in both the polymerase and the capsid regions. The polymerase of the Sydney C14-like recombinants was novel, with the closest relative strain belonging to GII.2 (3). GII.4 was the second most prevalent genotype identified in this cohort. One of the GII.4 strains clustered with the 1997-1998 GII.4 strains. The other three GII.4 strains, however, clustered in the Farmington Hills subset (Fig. 1). The GII.6 strain identified was closely related to the 1997-1998 GII.6 strains (>97% nucleotide identity). The two GII.7 strains were closely related to the capsid of Thai strain Mc17 (Fig. 1) (19). The GII.10 strain, Sydney 4264/01S, was closely related to the known Thai recombinant Mc37 (19). Sequence analysis of the amplified ORF1-ORF2 region revealed that Sydney 4264/01S shared 99% identity over 775 bp of the 3' end of ORF1 and the 5' end of ORF2 with Mc37 and was therefore identified as an Mc37-like recombinant.
The third cohort included samples from six sporadic cases of gastroenteritis collected between February 2004 and August 2004. One strain was a GII.3 recombinant that was closely related to the Sydney C14 recombinant (3), and another was a GII.4 strain that grouped with the Farmington Hills cluster (Fig. 1). The remaining four strains were GII.4 and were closely related to each other and formed a novel cluster (Fig. 1).
Outbreak strains, 1997 to 2004. NoV GII.4 strains are recognized as the predominant cause of viral gastroenteritis outbreaks (6, 29, 32, 51-53). Since 1995, four GII.4 strains have been identified as etiological agents in major NoV epidemics: US95/96 (9), European variant (32), Farmington Hills (53), and b4s6 (6). Genetic analysis grouped these four strains into two distinct GII.4 clusters, defined here as the US95/96 cluster and the Farmington Hills cluster. The Farmington Hills cluster included the 2002 U.S. strain Farmington Hills, the 2002 European variant (32; H. Vennema, personal communication), and the 2002 United Kingdom strain b4s6. NoV b4s6 shared 99.5% identity over the complete genome to the U.S. strain, Farmington Hills, revealing the intercontinental spread of the same variant. Sequence alignment between the members of the two clusters (US95/96 and Farmington Hills) demonstrated greater than 6% divergence across the capsid gene. In this study a new variant was defined as a strain that had greater than 5% amino acid divergence across the complete capsid gene.
This study analyzed strains from 28 outbreaks in New South Wales from 1997 to 2004. Between 1997 and 2001 the major outbreak strains in New South Wales clustered with global outbreak strain US95/96 (Fig. 1). In 2002, an increase in NoV-associated gastroenteritis was reported across Europe, the United States, and Sydney (46). In Fig. 1, Sydney 917J/02/AU is a representative of a predominant strain that was the etiological agent of six outbreaks investigated in New South Wales in 2002 (7). Sydney 917J/02/AU shares 98% nucleotide identity over 266 bp in the capsid region with Farmington Hills.
In 2003, two non-GII.4 strains were each associated with an outbreak of gastroenteritis. A GII.6 strain caused an outbreak at a ball that affected 60 people. A GII.1 strain, termed Picton, caused an outbreak in a geriatric hospital and was isolated from 22 patients (Fig. 1). Strain Picton was previously identified as a novel recombinant (3), with a polymerase region that was closely related to that of Sydney C14 (96% nucleotide identity) and a GII.1 capsid.
In 2004, there was a marked increase in NoV-associated gastroenteritis in New South Wales (48). There was a fivefold increase in the number of outbreaks in institutional settings in 2004 compared to the number in 2003 (48). RNA from 22 specimens from 18 NoV-associated gastroenteritis outbreaks in New South Wales in 2004 was amplified by our nRT-PCR assay. Phylogenetic analysis of the 22 capsid sequences indicated that the 2004 New South Wales strains formed their own cluster, termed the Hunter cluster, within the GII.4 subset, distinct from other sequences in the database (Fig. 1). Pairwise sequence analysis of the complete Hunter capsid gene (GenBank accession number DQ078794) revealed greater than 5% nucleotide and amino acid sequence variations to members of the US95/96 and Farmington Hills cluster, therefore defining the Hunter strain as a new GII.4 variant.
Due to numerous reports of NoV recombinants in New South Wales, a representative of the Hunter cluster was genotyped based on its polymerase sequence. A 1,498-bp region incorporating the 3' end of RdRp and the 5' end of the capsid was amplified by RT-PCR from the RNA of Hunter 532D/04/AU. Sequencing and phylogenetic analysis demonstrated that this strain had a GII.4 polymerase that was distinct (>4% variation) from any of the sequences in the database, in particular, the four NoV GII.4 outbreak strains described in Fig. 1. Hunter 532D/04/AU shared 94% identity with the US95/96 strain Sydney 348/97/AU (Fig. 1) and 96% identity with the Farmington Hills virus across the polymerase region. However, the Hunter strain was identical (>99.6% identity) to a GII.4 strain associated with an increase in gastroenteritis outbreaks in The Netherlands in late 2004 (29). Indeed, in The Netherlands the Hunter strain caused 44 of 77 (57%) outbreaks between August and December 2004 (29).
In summary, US95/96 strains were prominent between 1997 and 1998 in New South Wales. The majority of the strains collected between 2001 and 2002 were closely related to members of the Farmington Hills cluster. In contrast, the 2004 New South Wales strain, Hunter, which was associated with both outbreak and sporadic cases, formed its own cluster, suggesting the emergence of a new GII.4 variant.
DISCUSSION
The accurate diagnosis of viral gastroenteritis is essential in reducing its impact on society. Each year in the United States, approximately 5,000 deaths are attributed to gastrointestinal disease of unknown etiology (36). Despite the absence of a current treatment for viral gastroenteritis, the identification of the agent, especially in the case of an outbreak, enables the implementation of precautions that will prevent further dissemination of the disease. Furthermore, it is important to distinguish between a viral agent and a bacterial or parasitic agent, as infections with the last two types of agents can be treated. Due to the inability to culture NoV and the diversity of its genome, it has proven difficult to develop a sensitive method of detection. In this study we report on the development of an nRT-PCR that has been shown to detect a diverse range of NoV GII strains.
The detection limit of primers G2F3 and G2SKR (19, 27) was improved in this study by incorporating them into an nRT-PCR. The detection of viruses present at low viral loads is important for epidemiological studies of outbreaks, especially when the origin of an outbreak is traced retrospectively, when some of the patients' symptoms may have dissipated and levels of excreted NoV are low. Although nested PCR does have several drawbacks, not only does it require more time and consumables than a single-round PCR, but its use also has a higher risk of false-positive results due to contamination.
Over the last 5 years there has been a significant increase in the number of NoV recombinants that have been isolated. In a previous study we identified nine worldwide recombinant types and identified the ORF1-ORF2 junction as the recombination breakpoint (3). Consequently, to identify recombinants in the present study, strains were sequenced across the breakpoint. Phylogenetic analyses of the partial polymerase and the partial capsid regions were performed with suspected recombinants and prevalent NoV strains. Four of the nine recombinant types have been identified in New South Wales over the last 7 years. In 1997, 1998, 2001, and 2002, the two GII.3 recombinants Sydney C14 and Sydney 2212 were the second most prevalent NoV strains identified after the GII.4 strains, causing 31% of sporadic cases and 7% of outbreak cases. This illustrates not only the importance of recombinant analysis in epidemiological studies but also the fact that recombinants may be more prevalent than was originally thought.
Previous studies of the epidemiology of NoV in Australia have reported strains belonging to GII.1, GII.2, GII.3, GII.4, and GII.5, with GII.4 being the most prevalent (25, 35, 52, 54). The nRT-PCR assay detected six genotypes, GII.1, GII.3, GII.4, GII.6, GII.7, and GII.10, in New South Wales between 1997 and 2004. The nRT-PCR also detected NoV GII.5 (data not shown). Other strains were not available for testing; but the inner primer set alone, primers G2F3 and G2SKR, has been shown to detect another genetic cluster, GII.8 (19), indicating that the nRT-PCR assay can detect eight genotypes, if not more.
The majority of the sporadic and the outbreak strains detected in this study belonged to genotypes GII.4 and GII.3, with the former having been previously identified as a dominant genotype in New South Wales (52). The GII.3 strains were a combination of the two recombinant types, Sydney 2212 and Sydney C14, and this was the first epidemiological study to report on the dominance of recombinant GII.3 strains circulating in New South Wales. This is also the first Australian study to report on the isolation of GII.10. While Australia is geographically isolated in the world, phylogenetic analysis revealed that the same strains are circulating in Asia, Europe, and the Americas (12, 13, 19, 31, 55). Modern methods of transport and the multicultural nature of Australia undoubtedly facilitate the importation of global NoV strains.
This study suggests that the majority of the NoV GII outbreak strains isolated in Australia mirror the strains causing outbreaks during similar time frames in the Northern Hemisphere. In the United States, between 1995 and 1996, a single NoV GII.4 variant was isolated as the predominant cause of outbreaks of gastroenteritis (9). This strain, US95/96, was later found to be the etiological agent of a wave of gastroenteritis outbreaks on five continents, including Australia (39, 52). In this study, US95/96-like strains continued to be detected in New South Wales up to 2002 (Fig. 1). Furthermore, in 2002 there was a similar increase in NoV-associated outbreaks of gastroenteritis across the globe, with related strains belonging to the Farmington Hills cluster being identified across Europe (6, 32) and the United States (53). Farmington Hills-like strains were also isolated in New South Wales in 2001 and 2002 (Fig. 1).
In 2004, there was another marked increase in NoV activity in New South Wales (48). NoV was the main cause of gastroenteritis, accounting for 95% of outbreak cases, in which the etiological agent was identified (48). Eighteen NoV GII-positive outbreaks within New South Wales were investigated. A single NoV GII strain, termed Hunter, was isolated from all 18 outbreaks. Phylogenetic analysis of the complete capsid region revealed that the Hunter strain was unrelated (>5% amino acid divergence in the capsid region) to the global pandemic strains Farmington Hills and US95/96 (Fig. 1). The Hunter strain first appeared in New South Wales in February 2004; and matching sequence data from The Netherlands from September 2004, Taiwan from November 2004 and Japan from 2005 were found in the GenBank and EMBL databases (29). While the origin of the Hunter strain is unknown, we speculate that the Hunter strain, a predominant cause of NoV outbreaks in 2004 (20) and 2005, could also continue to cause global epidemics in 2006 and beyond. Identification of novel GII.4 variants may also lead to an increased ability to predict increases in NoV activity.
Strains belonging to the GII.4 cluster are recognized as the predominant genotype worldwide, and this study supports the dominance of GII.4. In the present study, GII.4 strains accounted for 43.7% of the sporadic cases and 85.8% of the outbreaks. Why GII.4 strains have continued to be dominant is unknown and of great interest. Possibilities include more sensitive methods for the detection of GII.4 strains, viral fitness, capsid interaction with human blood antigens, or recombination; or perhaps the same evolving strain is continuing to dominate.
ACKNOWLEDGMENTS
This work was supported by a Sydney Catchment Authority collaborative research grant. R.A.B. was supported by an Australian postgraduate award, and E.T.V.T. was supported by a University of New South Wales postgraduate award.
We gratefully acknowledge the contributions by Grant Hansman from the National Institute of Infectious Diseases, Tokyo, Japan.
REFERENCES
Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of "Norwalk-like viruses." J. Infect. Dis. 181(Suppl.):S336-S348.
Atmar, R. L., and M. K. Estes. 2001. Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin. Microbiol. Rev. 14:15-37.
Bull, R. A., G. S. Hansman, L. E. Clancy, M. M. Tanaka, W. D. Rawlinson, and P. A. White. 2005. Norovirus recombination in ORF1/ORF2 overlap. Emerg. Infect. Dis. 11:1079-1085.
Burton-MacLeod, J. A., E. M. Kane, R. S. Beard, L. A. Hadley, R. I. Glass, and T. Ando. 2004. Evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples. J. Clin. Microbiol. 42:2587-2595.
Cheesbrough, J. S., J. Green, C. I. Gallimore, P. A. Wright, and D. W. Brown. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol. Infect. 125:93-98.
Dingle, K. E. 2004. Mutation in a Lordsdale norovirus epidemic strain as a potential indicator of transmission routes. J. Clin. Microbiol. 42:3950-3957.
Duckworth, M. 2002. Norwalk-like viruses: approaches for detection. Honours thesis. University of New South Wales, Sydney, Australia.
Fankhauser, R. L., S. S. Monroe, J. S. Noel, C. D. Humphrey, J. S. Bresee, U. D. Parashar, T. Ando, and R. I. Glass. 2002. Epidemiological and molecular trends of "Norwalk-like viruses" associated with outbreaks of gastroenteritis in the United States. J. Infect. Dis. 186:1-7.
Fankhauser, R. L., J. S. Noel, S. S. Monroe, T. Ando, and R. I. Glass. 1998. Molecular epidemiology of "Norwalk-like viruses" in outbreaks of gastroenteritis in the United States. J. Infect. Dis. 178:1571-1578.
Felsenstein, J. 1989. PHYLIP—phylogeny inference package (version 3.2). Cladistics 5:164-166.
Felsenstein, J. 1993. PHYLIP inference package, version 3.5c. Department of Genetics, University of Washington, Seattle.
Foley, B., J. O'Mahony, C. Hill, and J. G. Morgan. 2001. Molecular detection and sequencing of "Norwalk-like viruses" in outbreaks and sporadic cases of gastroenteritis in Ireland. J. Med. Virol. 65:388-394.
Gallimore, C. I., J. Green, D. Lewis, A. F. Richards, B. A. Lopman, A. D. Hale, R. Eglin, J. J. Gray, and D. W. Brown. 2004. Diversity of noroviruses cocirculating in the north of England from 1998 to 2001. J. Clin. Microbiol. 42:1396-1401.
Girish, R., S. Broor, L. Dar, and D. Ghosh. 2002. Foodborne outbreak caused by a Norwalk-like virus in India. J. Med. Virol. 67:603-607.
Glass, P. J., L. J. White, J. M. Ball, I. Leparc-Goffart, M. E. Hardy, and M. K. Estes. 2000. Norwalk virus open reading frame 3 encodes a minor structural protein. J. Virol. 74:6581-6591.
Green, J., J. Vinje, C. I. Gallimore, M. Koopmans, A. Hale, D. W. G. Brown, J. C. S. Clegg, and J. Chamberlain. 2000. Capsid protein diversity among Norwalk-like viruses. Virus Genes 20:227-236.
Green, K. Y., R. M. Chanock, and A. Z. Kapikan. 2001. Human calicivirus, p. 841-874. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4 ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.
Gunson, R., J. Miller, and W. F. Carman. 2003. Comparison of primers for NLV diagnosis. J. Clin. Virol. 26:379-380.
Hansman, G. S., K. Katayama, N. Maneekarn, N. Peerakome, P. Khamrin, S. Tonusin, S. Okitsu, O. Nishio, N. Takeda, and H. Ushijima. 2004. Genetic diversity of norovirus and sapovirus in hospitalized infants with sporadic cases of acute gastroenteritis in Chiang Mai, Thailand. J. Clin. Microbiol. 42:1305-1307.
International Society for Infectious Diseases. 2005. ProMED-mail. http://www.promedmail.org. Accessed 13 March 2005.
Iversen, A. M., M. Gill, C. L. Bartlett, W. D. Cubitt, and D. A. McSwiggan. 1987. Two outbreaks of foodborne gastroenteritis caused by a small round structured virus: evidence of prolonged infectivity in a food handler. Lancet ii:556-558.
Kapikan, A. Z. 1994. Norwalk and Norwalk-like viruses, p. 471-519. In A. Z. Kapikan (ed.), Viral infections of the gastrointestinal tract, 2nd ed. Marcel Dekker, Inc., New York, N.Y.
Karst, S. M., C. E. Wobus, M. Lay, J. Davidson, and H. W. Virgin IV. 2003. STAT1-dependent innate immunity to a Norwalk-like virus. Science 299:1575-1578.
Katayama, K., H. Shirato-Horikoshi, S. Kojima, T. Kageyama, T. Oka, F. B. Hoshino, S. Fukushi, M. Shinohara, K. Uchida, Y. Suzuki, T. Gojobori, and N. Takeda. 2002. Phylogenetic analysis of the complete genome of 18 norwalk-like viruses. Virology 299:225-239.
Kirkwood, C. D., and R. F. Bishop. 2001. Molecular detection of human calicivirus in young children hospitalized with acute gastroenteritis in Melbourne, Australia, during 1999. J. Clin. Microbiol. 39:2722-2724.
Kobayashi, S., K. Sakae, Y. Suzuki, H. Ishiko, K. Kamata, K. Suzuki, K. Natori, T. Miyamura, and N. Takeda. 2000. Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen. Microbiol. Immunol. 44:687-693.
Kojima, S., T. Kageyama, S. Fukushi, F. B. Hoshino, M. Shinohara, K. Uchida, K. Natori, N. Takeda, and K. Katayama. 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100:107-114.
Koopmans, M. P. G., C. H. von Bonsdorrf, J. Vinje, D. DeMedici, and S. S. Monroe. 2002. Foodborne enteric viruses. FEMS Microbiol. Rev. 26:187-205.
Kroneman, A., H. Vennema, Y. van Duijnhoven, E. Duizer, and M. Koopmans. 2004. High number of norovirus outbreaks associated with a GGII. 4 variant in The Netherlands and elsewhere: does this herald a worldwide increase? Eurosurveill. Wkly. 8:51-52.
Lahti, K., and L. Hiisvirta. 1995. Causes of waterborne outbreaks in community water systems in Finland—1980-1992. Water Sci. Technol. 31:33-36.
Lau, C. S., D. A. Wong, L. K. Tong, J. Y. Lo, A. M. Ma, P. K. Cheng, and W. W. Lim. 2004. High rate and changing molecular epidemiology pattern of norovirus infections in sporadic cases and outbreaks of gastroenteritis in Hong Kong. J. Med. Virol. 73:113-117.
Lopman, B., H. Vennema, E. Kohli, P. Pothier, A. Sanchez, A. Negredo, J. Buesa, E. Schreier, M. Reacher, D. Brown, J. Gray, M. Iturriza, C. Gallimore, B. Bottiger, K. O. Hedlund, M. Torven, C. H. von Bonsdorff, L. Maunula, M. Poljsak-Prijatelj, J. Zimsek, G. Reuter, G. Szucs, B. Melegh, L. Svennson, Y. van Duijnhoven, and M. Koopmans. 2004. Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant. Lancet 363:682-688.
Marks, P. J., I. B. Vipond, D. Carlisle, D. Deakin, R. E. Fey, and E. O. Caul. 2000. Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant. Epidemiol. Infect. 124:481-487.
Marks, P. J., I. B. Vipond, F. M. Regan, K. Wedgwood, R. E. Fey, and E. O. Caul. 2003. A school outbreak of Norwalk-like virus: evidence for airborne transmission. Epidemiol. Infect. 131:727-736.
Marshall, J. A., M. E. Hellard, M. I. Sinclair, C. K. Fairley, B. J. Cox, M. G. Catton, H. Kelly, and P. J. Wright. 2003. Incidence and characteristics of endemic Norwalk-like virus-associated gastroenteritis. J. Med. Virol. 69:568-578.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the Uinted States. Emerg. Infect. Dis. 5:607-625.
Meakins, S. M., G. K. Adak, B. A. Lopman, and S. J. O'Brien. 2003. General outbreaks of infectious intestinal disease (IID) in hospitals, England and Wales, 1992-2000. J. Hosp. Infect. 53:1-5.
Murray, C. J. L., and A. D. Lopez. 1996. Evidence-based health policy: lessons from the global burden of disease study. Science 274:740-743.
Noel, J. S., R. L. Fankhauser, T. Ando, S. S. Monroe, and R. I. Glass. 1999. Identification of a distinct common strain of "Norwalk-like viruses" having a global distribution. J. Infect. Dis. 179:1334-1344.
O'Neill, H. J., C. McCaughey, P. V. Coyle, D. E. Wyatt, and F. Mitchell. 2002. Clinical utility of nested multiplex RT-PCR for group F adenovirus, rotavirus and Norwalk-like viruses in acute viral gastroenteritis in children and adults. J. Clin. Virol. 25:335-343.
Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.
Parashar, U. D., and S. S. Monroe. 2001. ‘Norwalk-like viruses’ as a cause of foodborne disease outbreaks. Rev. Med. Virol. 11:243-252.
Pletneva, M. A., S. V. Sosnovtsev, and K. Y. Green. 2001. The genome of Hawaii virus and its relationship with other members of the Caliciviridae. Virus Genes 23:5-16.
Richards, A. F., B. Lopman, A. Gunn, A. Curry, D. Ellis, H. Cotterill, S. Ratcliffe, M. Jenkins, H. Appleton, C. I. Gallimore, J. J. Gray, and D. W. G. Brown. 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26:109-115.
Roche, P., L. Halliday, E. O'Brien, and J. Spencer. 2002. The laboratory virology and serology reporting scheme, 1991 to 2000. Communicable Dis. Intell. 26:323-374.
Spencer, J. 9 December 2004, revision date. Communicable diseases surveillance: tables (LabVISE). Communicable Dis. Intell. 27:563. [Online.] http://www.health.gov.au.
Strimmer, K., and A. von Haeseler. 1997. Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc. Natl. Acad. Sci. USA 94:6815-6819.
Telfer, B., and S. Munnoch. 2005. OzFoodnet—enhancing foodborne disease surveillance across Australia. Annual Report. New South Wales and Hunter Area Health Service, Sydney, Australia.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multisequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
Vinje, J., R. A. Hamidjaja, and M. D. Sobsey. 2004. Developmental and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J. Virol. Methods 116:109-117.
Vipond, I. B., E. O. Caul, D. Hirst, B. Carmen, A. Curry, B. A. Lopman, P. Pead, M. A. Pickett, P. R. Lambden, and I. N. Clarke. 2004. National epidemic of Lordsdale Norovirus in the UK. J. Clin. Virol. 30:243-247.
White, P. A., G. S. Hansman, A. Li, J. Dable, M. Isaacs, M. Ferson, C. J. McIver, and W. D. Rawlinson. 2002. Norwalk-like virus 95/96-US strain is a major cause of gastroenteritis outbreaks in Australia. J. Med. Virol. 68:113-118.
Widdowson, M. A., E. H. Cramer, L. Hadley, J. S. Bresee, R. S. Beard, S. N. Bulens, M. Charles, W. Chege, E. Isakbaeva, J. G. Wright, E. Mintz, D. Forney, J. Massey, R. I. Glass, and S. S. Monroe. 2004. Outbreaks of acute gastroenteritis on cruise ships and on land: identification of a predominant circulating strain of norovirus—United States, 2002. J. Infect. Dis. 190:27-36.
Wright, P. J., I. C. Gunesekere, J. C. Doultree, and J. A. Marshall. 1998. Small round-structured (Norwalk-like) viruses and classical human caliciviruses in southeastern Australia, 1980-1996. J. Med. Virol. 55:312-320.
Zintz, C., K. Bok, E. Parada, M. Barnes-Eley, T. Berke, M. A. Staat, P. Azimi, X. Jiang, and D. O. Matson. 2005. Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the united states. Infect. Genet. Evol. 5:281-290.
School of Biotechnology and Biomolecular Sciences, Faculty of Science
1 School of Medical Sciences, Faculty of Medicine, the University of New South Wales, Sydney, NSW 2052, Australia
2 Virology Division, SEALS, Department of Microbiology, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia3(Rowena A. Bull, Elise T. )