Abstract
A study was undertaken to investigate the diversity of noroviruses (NVs) in fecal samples from patients from 529 outbreaks and 141 sporadic cases of gastroenteritis in the North of England from September 1998 to August 2001. NV strains were detected by electron microscopy and characterized by a combination of the Grimsby virus antigen enzyme-linked immunosorbent assay, reverse transcriptase PCR, the heteroduplex mobility assay, and DNA sequencing. Twenty-one distinct NV strains, including several novel or variant strains not seen previously, were found circulating in the population studied. Genogroup II NVs were responsible for 83% of the outbreaks. Several strains cocirculated at any one time. The Bristol (Grimsby/Lordsdale) and Hawaii (Girlington) genotypes were the most prevalent among the NVs identified, detected in 49 and 20% of the outbreaks, respectively. A limited number of other genogroup II and I strains were cocirculating. The virus populations detected in hospitals and nursing homes were distinct from those found in community-based outbreaks. Outbreaks in hospitals and nursing homes were more likely to be caused by genogroup II strain Grimsby or Girlington (P < 0.0001) than by other genogroup II or I strains.
Noroviruses (NVs) are members of the Caliciviridae family (9, 31) that were previously known as Norwalk-like viruses or small round structured viruses. NVs are the most common cause of outbreaks of acute nonbacterial gastroenteritis worldwide (21). Outbreaks due to NVs have been associated with closed or semiclosed institutions such as hospitals (7) and homes for the elderly (19). NV outbreaks also occur in other settings, including cruise ships (15), eating establishments (33), and schools (23). The transmission of NVs is usually person to person (22), although food (24), water (4), shellfish (26), and environmental or airborne contamination (7, 29) have all been implicated in transmission. The lack of a susceptible cell culture system or animal model for NVs has hindered research and development; however, since the cloning of the prototype Norwalk virus genome in the early 1990s (17), molecular detection and characterization of NVs have expanded our knowledge of this group of viruses (5, 6, 11).
Recently, detailed studies on the molecular epidemiology of NVs have been published by various groups (3, 28, 37). The genomic diversity of NVs includes two genogroups (I and II) (2, 37), possibly a third, and a number of genotypes, which have yet to be formally agreed upon. The genogroup I strains include Norwalk virus (GI-1), Southampton virus (SOV; GI-2), Desert Shield virus (DSV; GI-3), and Valetta virus (VLV; GI-4), and the genogroup II strains include Hawaii virus (GII-1), Melksham virus (GII-2), Mexico virus (GII-3), and Grimsby virus (GRV; GII-4) (see Table 1) (8).
TABLE 1.
NV strains associated with outbreaks and sporadic cases of gastroenteritis
| NV strain designation
|
Geno group and I-typeg | No. (%) of strains
|
GenBank no. or reference | ||
|---|---|---|---|---|---|
| GenBank | EVUf | Outbreak | Sporadic caseh | ||
| Lordsdale/1995/UK | Grimsby/1995/UK | GII-4 | 259 (49) | 63 (45) | X86557 |
| Pfaffenhofen028/2000/DE | Girlington/1993/UKa | GII-1 | 104 (20) | 4 (3) | AF31259 |
| 12C/92/UK | GII-2 | 8 | 6 | L25111 | |
| Bad Berleberg/477/01 | Harrow/2001/UKb,c,i | GII-1 & GII-3 | 9 | 12 | AF40906 |
| Berlin385/2000/DE | GII-2? | 8 | 3 | AF31254 | |
| Hillingdon/1994/UKd | GII-5 | 2 | 0 | 37 | |
| 96-34007F/JP | GII-5? | 1 | 0 | AB04438 | |
| Mexico/1989/MX | GII-3 | 2 | 0 | U22498 | |
| Mur1/1997/JP | GII-4? | 1 | 2 | AB01929 | |
| H/Calv/94E/NO | GII-7? | 1 | 0 | X89033 | |
| Halle445/1999/DE | GII-7 | 40 (8) | 10 (7) | AF31253 | |
| Lancaster/2000/UKe,i | GII-2? | 2 | 1 | ||
| Stepping Hill/2001/UKb,i | GII-1? | 3 | 3 | ||
| Norwalk/1968/US | GI-1 | 0 | 0 | M87661 | |
| Hak1d/2000/JP | Valetta/1995/MAa | GI-4 | 21 (4) | 7 (5) | AB04639 |
| Desert Shield/1990/SA | Birmingham/1993/UKa | GI-3 | 18 | 5 | U04469 |
| Southampton/1991/UK | GI-2 | 11 | 0 | L07418 | |
| KY89/89/JP | GI-1 | 0 | 1 | L23828 | |
| Fin/H/Vesi/G1 | GI-6? | 1 | 0 | AJ243787 | |
| 12121/89/UK | GI-6 | 1 | 0 | S71765 | |
| Rossendale/2000/UKb,i | GI-1 | 1 | 0 | ||
| Fort Lauderdale/1998/US | GIII-1 | 1 | 1 | AF414426 | |
| Sapporo-like virus | 2 | 4 | |||
| No virus detected (untyped) | 33 (6) | 19 (13) | |||
| Total | 529 | 141 | |||
See reference 37.
Novel strain (unpublished data).
Strain has been shown to be a recombinant NV (unpublished data).
Recently published as White River/290/1994/US (GenBank no. AF 414423).
Novel strain (only 90% identity with Snow Mountain/1976/US).
Enteric Virus Unit, Central Public Health Laboratory.
?, capsid sequence not known.
Sporadic strains occurred between November 1999 and August 2001 (outbreaks were from September 1998 to August 2001).
Sequence of novel strains may be obtained by contacting the corresponding author.
The diversity among NVs is maintained through the accumulation of point mutations associated with the error-prone nature of RNA replication, and genetic recombination involving the exchange of sequences between two RNA viruses with nonsegmented genomes was first described for poliovirus by Hirst (14) and has been reviewed by Lai (25). Several features of RNA viruses (antigenic variation, genotypic diversity, and immune escape) have been attributed to the lack of proofreading of the RNA-dependent RNA polymerases (35). The error-prone nature of template copying by RNA polymerases (1) can also lead to point mutations when the viruses replicate, some of which may be silent and others which can lead to amino acid changes.
Recombinant NV particles have been produced for several strains representing a number of NV genotypes, including Norwalk virus (20), Snow Mountain agent (18), Hawaii virus (10), and GRV (13), and have enabled the development of genotype-specific enzyme-linked immunosorbent assays (ELISAs), allowing the rapid screening of large numbers of fecal samples.
This paper describes the diversity of a representative population of NVs circulating in a defined region of the United Kingdom between 1998 and 2001 and compares the diversity of NVs in two particular geographical locations within that region and in different outbreak settings.
MATERIALS AND METHODS
Specimens and outbreaks.
The area in the North of England used in this study has a population of approximately 3.5 million, with the majority living in the metropolitan areas of Leeds, Bradford (West Yorkshire), Newcastle, and Sunderland (Tyne and Wear). The counties or areas covered by this study include Northumberland, Tyne and Wear, Cumbria, Durham, Cleveland, North Yorkshire, West Yorkshire, and East Riding or North Lincolnshire. The distance between Leeds or Bradford and Newcastle or Sunderland is approximately 110 miles. A total of 989 fecal samples were selected from patients from 529 outbreaks of gastroenteritis and 141 sporadic cases, all confirmed by electron microscopy (EM) to be due to NVs. Details of the locations were available for 452 (85%) of the outbreaks. Of these, 188 outbreaks (46%) occurred in hospitals, 195 (43%) in nursing homes (including rest homes), 30 in schools and nurseries, 16 in restaurants and hotels, 7 within families, and 16 in other settings.
The outbreaks occurred between September 1998 and August 2001, and the sporadic cases were identified between November 1999 and August 2001, which together represent the majority of NV incidents identified in this health care region.
A 20% fecal suspension was prepared for EM (undertaken at the Leeds Laboratory), and an aliquot of this suspension was sent to the Enteric, Respiratory, and Neurological Virus Laboratory at the Central Public Health Laboratory, Colindale, London, for testing by an ELISA specific for GRV antigen (GRV-ELISA), reverse transcriptase (RT)-PCR, and the heteroduplex mobility assay (HMA) and for cloning and DNA sequencing. All samples were stored at 4°C prior to testing.
Testing algorithm.
The GRV-ELISA was used to screen the EM-positive samples for GRV antigen. The samples negative by ELISA were then tested by RT-PCR. PCR amplicons of suitable quality were characterized by HMA as Grimsby/1995/UK-like, Mexico/1989/MX-like, Southampton/1995/UK-like, Valetta/1995/UK-like, or Desert Shield/1990/SA-like strains. HMA was not performed on weak PCR amplicons or when PCR products were generated with the SG1-D1 and SG2-D1 primer sets (11). DNA sequencing was used to characterize PCR amplicons not identified or tested by HMA and to confirm representative HMA profiles.
EM.
Fecal suspensions were made by mixing 20% (wt/vol) feces in 0.1 M phosphate-buffered saline (pH 7.2) and vortexing the mixture for 20 s. Suspensions were then centrifuged at 2,500 × g for 30 min. Virus pellets were produced by centrifuging 1 ml of supernatant at 45,000 × g for 1 h (Optima TL; Beckman Coulter, High Wycombe, United Kingdom). A 100-μl cushion of 30% (wt/vol) sucrose was injected into the bottom of each tube before centrifugation. The pellet was resuspended in 50 μl of phosphate-buffered saline, and 25 μl of 1,1,2-trichlorotrifluoroethane was added before the mixture was vortexed for 10 s. The suspension was clarified by centrifugation at 2,500 × g for 10 min. To prepare grids, a copper grid with a hydrophilic carbon-Formvar support film was floated for 1 h on 25 μl of the final supernatant and then stained with 2% sodium phosphotungstate, pH 6.5. The grids were examined for 5 min with a JEOL 1200EX electron microscope at a magnification of ×50,000.
Antigen ELISA.
The GRV-specific ELISA was carried out as described previously (13).
Nucleic acid extraction and RT-PCR.
Fecal samples for RT-PCR were processed as 10% suspensions and extracted by the guanidinium isothiocyanate-silica method (6). The primers used in this study were broadly reactive primers amplifying a region of open reading frame 1 (ORF1) and have been previously described; they included primer sets Ni-E3 (5), Ando-E3 (28), and SG1-D1 and SG2-D1 (11). These primers amplify regions of 113 to 150 bases of the RNA polymerase gene of NVs. Samples that were NV negative by RT-PCR were further screened for sapoviruses (SVs) by use of primer set SR80-JV33 (32, 36).
HMA.
PCR amplicons generated from fecal samples were analyzed by the HMA technique (30). Briefly, for an HMA of Ni-E3 amplicons, two reaction volumes were prepared. The first contained 4.5 μl of PCR amplicon with 4.5 μl of reference amplicon A (Grimsby-like virus DNA), and the second contained 4.5 μl of PCR amplicon with 4.5 μl of reference amplicon B (Mexico-like virus DNA). Each reaction volume was mixed with 1 μl of annealing buffer, denatured at 95°C for 5 min (Perkin-Elmer 9600 thermocycler), and allowed to cool to 4°C. For Ando-E3 amplicons, three reference strains were used: Southampton-like virus, Desert Shield-like virus, and Valetta-like virus (VLLV). The assay used VLLV and Southampton-like virus or VLLV and Desert Shield-like virus, depending on the prevalence of the two non-VLLV strains at the time of testing. The HMA reaction mixtures were then electrophoresed with a mutation detection enhancement polyacrylamide vertical gel (Biowhittaker Molecular Applications, Wokingham, United Kingdom) (30). The gel was stained with Gelstar (Biowhittaker Molecular Applications) and photographed with a Polaroid camera.
The presence of a homoduplex profile for one reference strain and a heteroduplex profile for the other reference strain in an HMA was taken to indicate the characterization of that strain. PCR products giving heteroduplex profiles for both reference strains were further characterized by DNA sequencing. Pattern matching of strains, which resulted in heteroduplex profiles identical to either reference strain in a single gel, negated the requirement to sequence all strains not characterized at the HMA stage. For genogroup II characterization, the strains used in the HMA were GRV-like (Carousel/1998/UK; GenBank accession no. AF439539), with 100% identity to Grimsby/1995/UK, and Mexico virus-like (Driffield/1999/UK; GenBank accession no. AF439540), with 95% identity to Mexico/1989/MX. For genogroup I characterization, the following were used: SOV-like (Ramridge/1998/UK; GenBank accession no. AF439543), with 93% identity to Southampton/1995/UK; DSV-like (Windlesham/1998/UK; GenBank accession no. AF439542), with 96% identity to Desert Shield/1990/SA; and VLV-like (Luton/1999/UK; GenBank accession no. AF439541), with 99% identity to Valetta/1995/MA. Reference strains were determined with primer sets Ni-E3 and Ando-E3 to have 74 and 72% sequence identity, respectively, over the region of the polymerase amplified by these primers. Strains not typed by HMA were characterized by DNA sequencing of a cloned amplicon.
Cloning and DNA sequencing.
PCR amplicons of NVs were cloned with a TOPO TA cloning system (Invitrogen, Paisley, United Kingdom) prior to sequencing as previously described (27). Contiguous sequences and pairwise alignments of the 76-bp interprimer region (for Ni-E3 and Ando-E3) and the 109-bp interprimer region (for SG1-D1 and SG2-D1) of the NV ORF1 sequences were generated with the Genebuilder and Clustal programs of Bionumerics version 2.5 (Applied Maths, Kortrij, Belgium).
Virus nomenclature.
As NV genotypes are defined by their capsid sequences (8), the viruses identified in this study, through the amplification of a region of the RNA-dependent RNA polymerase gene, have been designated strains. The strains are then related to an NV genotype based on published polymerase and capsid sequences of prototype NV strains (37).
Statistical analysis.
The odds of an outbreak being caused by a predominant strain (Grimsby/1995/UK or Girlington/1993/UK) versus any other strain were also stratified by outbreak setting. Odds ratios were compared by the χ2 test with 1 df.
RESULTS
Detection of caliciviruses.
The majority of outbreak samples were tested by GRV-ELISA (521 of 529; 98%). GRV antigen was detected in 243 (46%) of the outbreak samples by GRV-ELISA. The 286 (54%) outbreak samples which tested negative by GRV-ELISA were tested by RT-PCR, and 16 of these samples were identified as GRV by HMA or DNA sequencing (259 of 529 GRV-positive outbreak samples or 49%; see Table 1). Of the remaining 270 GRV-ELISA-negative outbreak samples, NVs were detected in 235 (87%). Two outbreak samples were associated with SV, and NVs were not detected in 33 (6%) of the outbreak samples by ELISA or RT-PCR and were termed NV untyped. All 33 were previously reported to be EM positive, but 17 were EM negative on retesting and 16 had insufficient sample volumes for retesting.
HMA was able to type 39 of 235 (16%) NVs, which included the GRV, SOV, DSV, and VLV genotypes. Of 235 NV strains, 196 (83%) were not tested by HMA but rather were characterized by DNA sequencing.
The GRV-ELISA was used to examine fecal samples from patients with sporadic cases of NV gastroenteritis. GRV was detected by ELISA in 57 of 141 (40%) sporadic-case samples. Of the ELISA-negative samples, six were RT-PCR positive for GRV (63 of 141 GRV positives or 45%). The remaining 55 (39%) sporadic-case samples were non-GRV NVs, and in 19 (13%), no virus was detected by RT-PCR. Seven samples were retested by EM and found to be negative. The remaining 12 were not tested. Four SVs were detected by RT-PCR in the remaining samples from patients with sporadic cases.
Characterization of caliciviruses.
Enteric viruses from 496 outbreaks and 122 sporadic cases of gastroenteritis were characterized. NV was found in 494 outbreaks and 118 sporadic cases. SV was found in two outbreaks and four sporadic cases. The genotypes found and their frequencies are shown in Table 1.
Two outbreak strains originally identified as NV by EM were confirmed by RT-PCR as containing SVs (32, 36). The strain from one outbreak was characterized as Houston/1990/US-like (GenBank accession no. U67859), and the other was Lyon/1998/FR-like (GenBank accession no. AJ251991). Also, four sporadic-case strains were shown to be associated with SVs, including one Lyon/1998/FR-like strain, one strain designated Leeds/2000/UK (which had only 76% identity to Lyon/1998/FR), a third strain designated Leeds/2001/UK (which had only 78% identity to London/1992/UK; GenBank accession no. U67858), and a fourth strain which was London/1992/UK-like.
Distribution of NV strains by geographical location within the North region.
A comparative analysis of strains circulating in two metropolitan areas within the North region is shown in Fig. 1. Overall, in the period of September 1998 to August 2001 in the West Yorkshire area, 53% of the NV outbreaks were due to Grimsby/1995/UK, 15% were due to Girlington/1993/UK, 17% were due to other genogroup II strains, and 15% were due to genogroup I strains. In Tyne and Wear, 40% of the NV outbreaks were due to Grimsby/1995/UK, 30% were due to Girlington/1993/UK, 17% were due to other genogroup II strains, and 13% were due to genogroup I strains. In the remaining areas, 55% of the NV outbreaks were due to Grimsby/1995/UK, 22% were due to Girlington/1993/UK, 15% were due to other genogroup II strains, and 9% were due to genogroup I strains.
FIG. 1.
Temporal distribution of NV strains in two metropolitan areas of the North of England from 1998 to 2001. A, Girlington/1993/UK; B, Grimsby/1995/UK; C, Halle445/1999/DE; D, Berlin385/2000/DE; E, Hillingdon/1994/UK; F, Harrow/2001/UK; G, 12C/92/UK; H, Stepping Hill/2001/UK; I, H/Calv/94E/NO; J, Mexico/1989/MX; K, Lancaster/2000/UK; L, Desert Shield/1990/SA; M, Southampton/1991/UK; N, Hak1d/2000/JP (Valetta/1995/MA); O, 12121/1989/UK; P, Fort Lauderdale/1998/US. Genogroup II strains are indicated by dark gray shading; genogroup I strains are indicated by light gray shading. Untyped strains are not shown but are included in the numbers of outbreaks.
Distribution of NV strains by outbreak setting.
Hospital and nursing home outbreaks were more frequently caused by Grimsby/1995/UK or Girlington/1993/UK strains than by other genogroup II or I strains (P < 0.0001) (Fig. 2). There were significant differences in the odds of outbreaks caused by Grimsby/1995/UK and Girlington/1993/UK strains in hospitals (odds = 5.7) compared to those in nursing homes (odds = 2.1) and in hospitals compared with those in all other settings (odds = 0.8).
FIG. 2.
Distribution of NV strains circulating in outbreaks in hospitals, nursing homes, and other community settings. Percentages of NVs for each group are shown for a three-year period, from 1998 to 2001 (see Table 1 for details); untyped strains are not included. Black, Grimsby virus; white, Girlington virus; dark gray, other genogroup II strains; light gray, genogroup I strains.
DISCUSSION
The North region of the United Kingdom includes the more rural areas of Northumberland, Cumbria, and parts of North Yorkshire and the more metropolitan areas of West Yorkshire and Tyne and Wear. The study clearly shows the predominance of the GII-4 Grimsby/1995/UK strain of NV between September 1998 and August 2001, except for the 1998-1999 winter season in which the GII-1 strain Girlington/1993/UK was predominant. These two strains are both genogroup II NVs, which along with the other 11 genogroup II strains accounted for 440 (83%) of the 529 outbreaks between 1998 and 2001. Six distinct genogroup I strains were responsible for 53 (10%) of the 529 outbreaks. Of the remaining outbreaks, 33 (6%) were negative by RT-PCR for NVs and were classified as untyped.
The GRV-ELISA proved successful in detecting NV genotypes antigenically related to GRV (12), with 243 of 529 (43%) outbreaks being characterized at this stage. However, there were 16 (3%) outbreak strains that were negative by GRV-ELISA but positive by RT-PCR, demonstrating the increased sensitivity of PCR compared to ELISA. Of 529 RT-PCR-positive NV outbreaks, 251 (48%) were not detected by the GRV-ELISA (including the 16 GRVs). The GII-1 strain was responsible for 104 outbreaks, predominantly between August 1998 and October 1999, with 93 of 104 (89%) occurring between these dates. Only 11 GII-1 outbreaks have occurred in this region since November 1999.
Only small regions of the polymerase gene were used for phylogenetic analysis, as the primers used had to be broadly reactive in order to amplify the cDNA of a wide range of genogroup I and II NV strain types. Although the sequence data generated from a small region of the RNA polymerase should be interpreted with caution, previous phylogenetic studies using related primers have revealed a robust relationship between this small amplified region and more extensive regions of the polymerase gene (6). The use of other primer pairs, which amplify a larger region of the polymerase gene, compromises the sensitivity of the PCR and would have led to far fewer strains being detected and characterized.
A novel NV strain was defined as having 90% or less identity at the nucleotide level with published sequences. This is a figure based on the analysis of other RNA genomes, as there are no clear guidelines defining what constitutes a novel strain of NV based on a partial sequence of the RNA-dependent RNA polymerase-encoding region. Several of the NV strains detected appear to be novel strains that have not previously been reported in the United Kingdom.
One outbreak was associated with Fort Lauderdale/1998/US (Alphatron/1998/NL-like; GenBank accession no. AF195847), a putative genogroup III NV (8) with less than 70% homology at the nucleotide level to all genogroup I and II strains.
Several outbreaks and sporadic cases were associated with the Harrow/2001/UK strain not seen previously in our laboratory, although an identical strain, Bad Berleberg/477/01 (GenBank accession no. AF409066), was detected in Germany also in 2001. It has been shown to be a recombinant NV associated with at least two different capsids (GII-1 Girlington and GII-3 Mexico) (unpublished data).
The Stepping Hill/2001/UK strain, determined by sequence analysis of the ORF1-ORF2 region to be a variant of Hawaii/1971/US (unpublished data); the Rossendale/2000/UK strain, a variant of Norwalk/1968/US, its nearest neighbor at 89% identity; and the Lancaster/2000/UK strain, a variant of Snow Mountain/1976/US, its nearest neighbor at 90% identity, were detected in outbreaks and/or sporadic cases.
The distributions of NV strains of both genogroups were very similar in sporadic cases of gastroenteritis in adults and children, although a KY/89/JP-like strain, a variant of Norwalk/1968/US, was not seen in any outbreaks during this study.
In West Yorkshire, the distribution of NVs was similar to the overall pattern in the North region. However, in the Tyne and Wear area, there was an absence of GII-4 in the 1998-1999 season, with GII-1 strains being predominant. In the West Yorkshire area, GII-4 and GII-1 strains were evenly distributed in the 1998-1999 season; however, GII-1 strains were predominant in 1998 to 1999 in the North region as a whole.
The cocirculation of diverse NV strains in a particular location can lead to the genesis of naturally occurring recombinant strains through recombination events during dual infection (16, 37).
The temporal distribution of NV strains (Fig. 1) shows that in a particular geographical area of the North region in a single month (December 2000), three genogroup II strains were cocirculating, i.e., Grimsby/1995/UK, 12C/92/UK, and Stepping Hill/2001/UK. In September 1999, three genogroup I strains, Southampton/1991/UK, Desert Shield/1990/SA, and Hak1d/2000/JP, were cocirculating.
Genogroup II strains were responsible for the majority (83%) of NV outbreaks. GII-4, the predominant genogroup and -type in the United Kingdom, was the predominant NV genogroup and -type worldwide during this time (3, 12). NV strains capable of causing epidemics have previously been identified. In the 1993-1994 season in the United Kingdom, a GII-3 strain, Mexico/1989/MX, was associated with epidemic spread (12), and this phenomenon was repeated in the 1998-1999 season with the GII-1 strain Girlington/1993/UK. This occurrence demonstrates that the GII-4 strain, although predominant in the United Kingdom and worldwide, can be displaced, even if for only one or two seasons. This finding has implications for virus detection, particularly with the increased use of commercial antigen enzyme immunoassays (34).
The results of this study strengthen the reported association between the Grimsby/Lordsdale cluster of NV strains and outbreaks of gastroenteritis in semiclosed institutions (3, 12). The Grimsby/1995/UK strain was associated with 49% of the outbreaks that occurred in 27 of the 36 and 30 of the 36 months of the study period in nursing homes and hospitals, respectively.
The data presented describe in detail the diversity and temporal distribution of NV strains cocirculating in this region of the United Kingdom between 1998 and 2001, the establishment or presence of a predominant endemic type, and the introduction and subsequent disappearance of an epidemic type.
Although NV diversity due to the accumulation of point mutations and genetic recombination has been described, the differing properties of endemic and epidemic NV strains and the role played by immunity in the selection of NV strains are poorly understood.
Several factors may play a role in determining the prevalent strains circulating in the population, including viral factors such as infectious dose, environmental stability, and virulence, which may have an important role in determining transmissibility in this setting.
The possibilities for investigating biological differences between NV genotypes in the absence of an animal model and tissue culture system are limited, but the findings presented here are consistent with the Grimsby/Lordsdale virus cluster having distinct characteristics. Several other factors may have contributed to the pattern seen, including herd immunity, population mobility and density, and the level of hygiene in institutional settings.
Future approaches to the control and prevention of NV-associated outbreaks, particularly in the hospital setting, will require a better understanding of the importance and interactions of these different factors.
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