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Journal of Clinical Microbiology, September 2004, p. 3950-3957, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.3950-3957.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutation in a Lordsdale Norovirus Epidemic Strain as a Potential Indicator of Transmission Routes

Kate E. Dingle^1,2* Norovirus Infection Control in Oxfordshire Communities Hospitals,

Nuffield Department of Clinical Sciences, Oxford University,¹ Department of Microbiology, Oxford Radcliffe NHS Trust, John Radcliffe Hospital, Oxford, United Kingdom²

Received 6 April 2004/ Returned for modification 11 May 2004/ Accepted 22 May 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
An increase in norovirus outbreaks was reported internationally during 2002 and 2003 and was also observed in Oxfordshire (United Kingdom) hospitals. To understand their epidemiological relationships, viruses from 22 outbreaks (15 from one hospital) were subjected to nucleotide sequencing. The 3'-terminal 3,255 nt or complete genomes were determined for 49 viruses. All outbreaks were caused by a genogroup II norovirus related to the Lordsdale virus (GII 4), common in healthcare settings. The norovirus mutation rate was sufficiently high that the 3,255-nucleotide sequences allowed separate and potentially connected outbreaks to be identified, since all outbreaks with identical sequences were temporally or geographically linked. The high mutation rate was further indicated by four mutations and three microheterogeneities in 3,255 nucleotides during 17 days of norovirus shedding by an immunocompromised patient. The data suggested that multiple virus introductions from the community, occasional transmission among wards, and one instance of ongoing environmental contamination had occurred. The accumulation, or lack, of mutations within an outbreak was also used to indicate the predominant transmission route. In an outbreak where person-to-person spread was thought to predominate, six mutations were detected throughout the genome, whereas one mutation was detected when point source infection was suspected. This norovirus epidemic strain differed from its closest previously described relative by 11.4 to 13.6% in the outer P2 domain of the capsid, which also had a single-amino-acid insertion. Alterations to the capsid structure compared to previous noroviruses may explain the increased number of outbreaks during 2002 and 2003.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Noroviruses are recognized as a worldwide cause of epidemic acute nonbacterial gastroenteritis. They are members of the family Caliciviridae, having a single-stranded RNA genome with positive polarity 7.5 kb long. Noroviruses frequently cause outbreaks of gastroenteritis in settings where people congregate, such as hospitals, hotels, cruise ships, and schools (4, 12, 19, 21). Transmission occurs fecal-orally via aerosols, fomites, food, or water, and the attack rate is high, with the virus affecting all age groups (5). Outbreak control is hampered by the low infectious dose and environmental persistence of the virus (14). It is estimated that in the United States 23,000,000 infections, 50,000 hospitalizations, and 300 deaths are caused by noroviruses each year (23).

An unusual increase in the number of norovirus outbreaks was reported in Europe and the United States during the winter of 2002-2003 (2, 3, 16, 32). Outbreaks are a significant problem in healthcare institutions (8, 18), and 68% of the 2002-2003 increase in the United Kingdom involved elderly patients in such a setting (3). Hospitals and care homes accounted for 79% of 1,877 reported general outbreaks in England and Wales from 1992 to 2000 (18).

Nucleotide sequence data for norovirus strains collected over 30 years have demonstrated high levels of genetic diversity. Two genogroups infect humans (genogroup I [GI] and GII) (9, 15), and each can be subdivided into up to 10 phylogenetic clades (1, 30). In contrast to the wider community, outbreaks in healthcare settings are usually caused by a small number of GII strains (18), with a Lordsdale virus-like GII strain (GII clade 4 [GII 4]) predominating (7, 8, 10, 20). Lordsdale virus was identified following a United Kingdom hospital outbreak in March 1993 (6). Studies to track transmission among hospital outbreaks have often used short fragments of nucleotide sequence (150 to 300 bp) from the relatively conserved RNA polymerase region. However, the predominance of the Lordsdale virus-like strain limits their utility, as many hospital outbreak strains collected over a short period are identical in this region (20).

Since noroviruses have an RNA genome and replicate rapidly using a polymerase that lacks proofreading activity, they would be expected to mutate quickly. This was demonstrated by Nilsson et al. (24), who found 32 amino acid changes in the capsid protein of a norovirus shed chronically over 1 year by an immunocompromised patient. Longer nucleotide sequences could therefore allow the relationships among outbreaks in healthcare settings to be determined by detecting the small numbers of mutations which occur over short periods of time. The objectives of this study were, first, to obtain sufficient sequence data to clarify the relationships among multiple outbreaks in a single hospital during 2002 and 2003 and to compare the viruses to those in outbreaks from six surrounding hospitals and, secondly, to find a possible explanation for the increase in outbreaks during 2002 and 2003.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Noroviruses. Stool samples were obtained from 15 suspected norovirus outbreaks that occurred from 2 October 2002 to 19 February 2003 in hospital A in the United Kingdom. Outbreaks were defined as two or more patients on a ward who experienced norovirus gastroenteritis within 2 days of each other. If two wards were affected in the same hospital simultaneously, these were classified as separate outbreaks (following criteria used by infection control staff). For comparison, strains from outbreaks during the same season at six additional hospitals were included (Table 1). These were from two outbreaks in the nearby hospital B and one each in hospitals C, D, E, F, and G, located 12, 14, 14, 20, and 28 miles from hospital A. The stool samples were stored at 4°C. They (and the norovirus they contained) were numbered by batch (B) and sample (S), for example, B1S1.

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TABLE 1. Locations, dates, samples, and sequences obtained from norovirus outbreaks

RNA extraction and cDNA synthesis. A 20% fecal suspension was made in molecular biology grade water (Sigma), vortexed briefly, and clarified by centrifugation at 1,000 rpm for 10 min. Total RNA was extracted from 70 µl of supernatant using a silica column-based kit (Viral RNA minikit; Qiagen Ltd., Crawley, United Kingdom) according to the manufacturer's instructions. The RNA was eluted from the column in 40 µl of AVE buffer (Qiagen Ltd.). Reverse transcription was performed in a 20-µl reaction mixture containing 10.5 µl of RNA and 0.5 µl (250 ng) of random hexamers (Promega UK, Southampton, United Kingdom) or 50 ng of specific primer. The RNA and primers were denatured at 70°C for 10 min, followed by immediate transfer to ice, to which the remainder of the reaction mixture was added: 4 µl of first-strand buffer (Invitrogen Ltd., Paisley, United Kingdom), 2 µl of 0.1 M dithiothreitol (Invitrogen Ltd.), 1 µl of deoxynucleoside triphosphates (10 mM) (Invitrogen Ltd.), 1 µl of RNasin (Promega UK), and 1 µl of Moloney murine leukemia virus reverse transcriptase (Invitrogen Ltd.). Incubation was performed at 37°C for 1 h, followed by inactivation at 70°C for 10 min.

Identification of positive stool samples. Stool samples containing norovirus were identified by reverse transcription-PCR using previously described PCR primers (JV12Y, 5'-ATACCACTATGATGCAGAYTA-3', and JV13I, 5'-TCATCATCACCATAGAAIGAG-3') that were designed to detect the majority of known strains (29). This assay was chosen because it performed optimally when a panel of different assays were compared (31). To identify the strain associated with each outbreak, the nucleotide sequence of the 327-bp amplicon was determined using the same primers as for amplification.

Amplification and nucleotide sequencing of 3'-terminal half-genomes and complete genomes. To determine the sequences of the 3'-terminal 3,255 nucleotides (nt) of 49 strains and the complete genome sequences of eight of these strains, randomly primed cDNA was used in PCR amplifications to produce overlapping amplicons 500 to 700 bp in length which spanned the required sequences. Since all the viruses were members of GII and closely related to Lordsdale virus, the amplifications were performed using oligonucleotide primer pairs designed from conserved regions of the sequences of Lordsdale virus (GenBank accession no. X86557 [6]) and three GII Lordsdale-like viruses. These were Camberwell virus (GenBank accession no. AF145896 [27]), human calicivirus Hu/NLV/GII/MD145-12/1987/US (GenBank accession no. AY032605 [8]), and Saitama virus (GenBank accession no. AB039775 [11]).

To determine the 3'-terminal sequence adjacent to the poly(A) tail of each strain, cDNA synthesis was primed using 50 ng of oligonucleotide NVT₂₀linker: 5'-ATCATTCGATTGATGACGAGC(T)₂₀VN-3'. Within this oligonucleotide, N refers to any of the four bases and V refers to any base other than T. This was intended to force the primer to bind at the start of the virus sequence preceding the poly(A) tail. PCR amplification was performed using the linker oligonucleotide, 5'-ATCATTCGATTGATGACGAGC-3', versus an internal virus-specific primer 500 nt from the 3' terminus. Since the 5' terminus of the genome is highly conserved within each genogroup, a 5' GII sequence-specific primer, JR45 (5'-GTGAATGAAGATGGCGTCTAAC-3'), was designed to bind to this sequence and was used in amplifications with internal primers.

Each 50-µl amplification reaction mixture was assembled on ice and comprised 5 µl of cDNA, 0.2 µM (each) PCR primer, 1x PCR DyNAzyme EXT buffer (Finnzymes Oy, •, Finland), 0.2 mM deoxynucleoside triphosphates, and 1.25 U of DyNAzyme EXT polymerase (Finnzymes Oy). This polymerase mixture was chosen because it contains a 3'-to-5' proofreading exonuclease activity which removes misincorporated nucleotides to improve fidelity 5- to 10-fold over standard Taq polymerases. Proofreading polymerases introduce base substitutions in fidelity assays on the order of 3 x 10^–5, or 1 in 33,333 (22). The reaction conditions were denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 20 s, 50°C for 20 s, and 72°C for 1 min or 1 min 30 s, depending on the amplicon size.

The amplification products were purified by precipitation with 20% polyethylene glycol (molecular weight, 8,000) and 2.5 M NaCl, and their nucleotide sequences were determined at least two times on each DNA strand using the amplification primers and BigDye Ready Reaction Mix (Applied Biosystems, Warrington, United Kingdom) in accordance with the manufacturer's instructions. The reaction conditions were 30 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 2 min. Unincorporated dye terminators were removed by precipitation of the termination products with 2 volumes of ethanol and 1/10 volume of 3 M sodium acetate, pH 5.2, and centrifugation, followed by washing the resultant pellet with 70% ethanol. The reaction products were separated and detected with a Prism 3730 automated DNA sequencer (Applied Biosystems). Sequences were assembled from the resultant chromatograms with the STADEN suite of computer programs (28). Care was taken to exclude the sequences determined by the primer sequences at the 5' and 3' termini of the overlapping PCR amplicons.

Phylogenetic analysis. The relationships among the strains and with previous strains were determined using the multiple alignment program CLUSTAL W (version 1.8) and the program MEGA (Molecular Evolutionary Genetic Analysis version 2.1, available at http://www.megasoftware.net/).

Nucleotide sequence accession numbers. The complete genome sequences (7,558 nt) of eight norovirus strains were submitted to GenBank. The accession numbers are AY581254 and AY587983 to AY587989. The 3'-terminal 3,255-nt sequences extending from the RNA polymerase within open reading frame 1 (ORF1) through ORF2 and ORF3 to the 3' terminus were also submitted for 41 additional strains. These correspond to accession numbers AY587990 to AY588030.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Outbreaks, samples, and strain identification. Noroviruses from a total of 22 outbreaks in seven different Oxfordshire (United Kingdom) hospitals were included in the study (Table 1). Most outbreaks selected (n = 15) occurred in a single hospital, referred to as hospital A. The outbreaks lasted between 3 and 20 days, and 9 to 48 people were affected (Table 1).

Norovirus-positive stools were identified by reverse transcription-PCR using previously published oligonucleotide primers that bind conserved sequences within the RNA polymerase region to amplify a 327-bp fragment (29). A total of 75 positive stool samples were identified among 130 tested. Nucleotide sequencing of this amplicon demonstrated that all the viruses were very closely related, many having identical sequences within an RNA polymerase fragment (variant 1) (Table 1 and Fig. 1). Four additional closely related sequences were found (Fig. 1). Comparison to previously described viruses revealed that the five variants were closely related to Lordsdale virus, a member of GII (GII 4). However, they were distinct from previously described variants.

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FIG. 1. Nucleotide sequences (285 nt) of the five closely related norovirus variants identified among 22 outbreaks (Table 1) and Lordsdale virus. The sequence is located within the RNA polymerase between primer pairs JV12Y and JV13I. Dots indicate sequence identity with variant 1. The 42 nt of primer sequence have been deleted from the 327-bp PCR amplicon. The locations of this sequence within the genome of strain Hu/NLV/Oxford/B5S22/2003/UK (GenBank accession no. AY581254) are indicated. The sequence equivalent to the 6-nt motif thought to indicate a new norovirus variant (16) is highlighted and boxed.

Norovirus mutation as a tool for defining outbreaks and transmission routes. The relationships among the majority of outbreaks could not be determined from the short fragments of RNA polymerase sequence, so longer sequences were obtained to increase the resolution. The 3'-terminal 3,255-nt sequences of 49 noroviruses from 18 of the 22 outbreaks were determined (Table 1). This sequence included part of the RNA polymerase gene, the entire capsid, ORF3, and the 3' untranslated region. Where possible, more than one sample was included from each outbreak to provide a control. A total of 86 variable nucleotide sites (2.6%) were identified in the 49 viruses, 80 (93%) of which were transitions and 6 (7%) of which were transversions. Four of the transversions were located in ORF3, and one each was in ORF1 and -2. Transitions occurred throughout the 3,255-nt sequence, but there were only four in a 550-nt region extending 350 nt 5' and 200 nt 3' of the junction between ORF1 and ORF2. There were no mutations in the 3'-terminal 136 nt and no indication of multiple changes at particular sites. The same change was frequently present in more than one outbreak.

Sequence identity was considered a likely indicator that outbreaks (defined in Materials and Methods) could be connected. The level of identity between outbreaks was assessed using a neighbor-joining tree constructed from the 49 sequences (Fig. 2). Eight outbreaks with unique virus sequences were identified, indicating that they were not connected. The remaining 10 outbreaks formed three clusters. Cluster 1 included three outbreaks from late September and October 2002. Two occurred in hospital A and differed by one point mutation, and a third occurred in nearby hospital B. Cluster 2 included an outbreak in hospital A, ward 7C (26 October to 4 November 2002), and this sequence recurred 18 days later on the same ward plus three additional wards (Fig. 2). A concurrent outbreak on ward 7F was distinct. Cluster 3 involved concurrent outbreaks on two hospital A wards. Therefore, the outbreaks that had identical virus sequences were temporally or geographically linked, i.e., part of larger outbreaks involving more than one ward.

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FIG. 2. Neighbor-joining tree showing relationships among 49 strains from 18 outbreaks within the 3'-terminal 3,255 nt. The strains and outbreaks are indicated as follows: hospital (Hosp), plus ward where more than one outbreak from one hospital was included; date of sample collection (month/day/year); and sample number. *, variants shed over 17 days by an immunocompromised patient.

To examine the predominant transmission routes within two different outbreaks, the accumulation of virus mutations was investigated in different patients infected over the course of each outbreak. If person-to-person spread predominated, many accumulated mutations would be expected, as they are inherited by consecutive hosts. Conversely, in a common-source outbreak, fewer accumulated mutations would be expected, since each patient develops his or her own mutations in the virus received from the common source. The two outbreaks were chosen on the basis of observations made by infection control staff. Patients tended to develop symptoms consecutively in one outbreak (hospital B, ward L; 29 September to 5 October 2002), but more cases occurred together in the other outbreak (hospital A, ward 7C; 26 October to 4 November 2002) (Table 1). Both outbreaks affected 25 people; the former lasted 7 days with 6 positive fecal samples available, and the latter lasted 10 days with 10 samples available.

The complete genome sequences of the viruses in the six samples from the hospital B, ward L, outbreak were determined. A total of six mutations occurred gradually during the 7-day outbreak (Table 2). In the hospital A, ward 7C, outbreak, eight viruses were identical in the 3'-terminal 3,255 kb and two differed at the same position. The complete genome sequences of viruses collected at the start and end of the outbreak demonstrated no further differences. These data support the idea that prolonged person-to-person transmission may be indicated by the accumulation of mutations during an outbreak, as they are inherited by consecutive hosts.

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TABLE 2. Mutations accumulated in virus genome in six patients infected during an outbreak

The rapid norovirus mutation rate was further indicated by 3'-terminal 3,255-nt sequences obtained 17 days apart from an immunocompromised patient who had received a bone marrow transplant. Four mutations and three nucleotide microheterogeneities (the presence of two different nucleotides at the same position within a sequence chromatogram) were detected (Fig. 2, hospital A, ward 5E outbreak).

Identification of a new norovirus strain. The relationship of the GII strain identified in the present study to previous strains was assessed using capsid sequences obtained from GenBank (Fig. 3). The Oxfordshire viruses formed a unique cluster within the Lordsdale virus-like group (GII 4). Previous strains in this group formed two additional clusters. One occurred from 1987 to 1994, and the other occurred from 1995 to 2001. The three clusters did not overlap temporally. This observation was repeated when neighbor-joining trees were constructed for ORF1 and ORF3 (data not shown), so no evidence of recombination was detected.

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FIG. 3. Relationships among capsid nucleotide sequences of eight of the Oxfordshire viruses, previous GII Lordsdale virus-like viruses, and other prototype noroviruses shown using a neighbor-joining tree. The input alignment file was generated using Clustal W version 1.8. The isolation years of the strains within the Lordsdale virus-like group are indicated with their corresponding clusters of variants. Bootstrap values are given at appropriate nodes. The GenBank accession numbers of the strains used are as follows: Dillingen/259/01/Germany, AF425766; Berlin/495/2000/Germany, AF427123; Koenigswusterhaus/120/2000/Germany, AF427121; 379/96019984/1996/Az, AF080556; 373/96019743/1996/SC, AF080555; Burwash Landing/331/1995/US, AF414425; 004/95 M-14/1995/AU, AF080551; Dijon171/1996/France, AF472623; Miami Beach/326/1995/US, AF414424; Berlin/238/98/Germany, AF425764; Beeskow/124/2000/Germany, AF427120; Oberschleissheim/112/1999/DE, AF427113; 416/97003156/1996/LA, AF 080559; Mora/1997/Sweden, AY081134; Parkroyal/1995/UK, AJ277613; Symgreen/1995/UK, AJ277619; Efurt/007/2000/Germany, AF427117; Hu/NV/Oxford/B5S22/2003/UK (a representative of the Oxfordshire viruses), AY581254; Bristol/1993/UK, X76716; UK3-17/12700/192/GB, AF414417; Lordsdale/1993/UK, X86557; Camberwell/1994/AU, AF145896; MD145-12/1987/US, AY032605; MD134-7/1987/US, AY030098; Toronto virus, U02030; Hawaii virus, U07611; Hillingdon virus, AJ277607; Snow Mountain virus, L23831; Melksham virus, X81879; Mexico virus, U22498; Desert Shield virus, U04469; Norwalk virus, M87661; and Southampton virus, L07418.

The norovirus most closely related to the Oxfordshire variant was identified by a BLAST search of the GenBank database. It was 004/95 M-14/1995/AU (GenBank accession number AF080551), which occurred in Australia during 1995 (25). An alignment was constructed using the capsid sequences of (i) a representative of the Oxfordshire viruses (Hu/NLV/Oxford/B5S22/2003/UK), (ii) strain 004/95 M-14/1995/AU, and (iii) Norwalk virus, the only norovirus for which the capsid crystal structure is known (26) (Fig. 4). The regions of the Lordsdale virus-like capsids which corresponded to the Norwalk virus capsid were estimated from the alignment. The majority of capsid amino acid differences between the Oxford virus and its closest relative from 1995 occurred within the outer P2 domain (11.4% divergence), followed by the P1 domain (2.2% divergence), the S domain (1.1% divergence), and the amino-terminal arm (0% divergence) (Fig. 4). A single-amino-acid insertion (glycine) was present in the P2 regions of all the Oxfordshire viruses sequenced.

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FIG. 4. Alignment of the capsid amino acid sequences of the representative Oxfordshire variant Hu/NV/Oxford/B5S22/2003/UK (GenBank AY581254), its closest previously described relative (004/95 M-14/1995/AU [GenBank AF080551]), and Norwalk virus (GenBank M87661). The different subdomains of the capsids, estimated using the previously described crystal structure of the Norwalk virus capsid (26), are indicated. The majority of amino acid sequence changes between the new strain and its previous closest relative occurred in the outer P2 subdomain of the capsid and are highlighted in boxes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gastroenteritis outbreaks in healthcare settings are often caused by Lordsdale virus-like noroviruses (18), also referred to as GII 4 strains. Their relatively high level of genetic identity makes the epidemiological relationships between outbreaks difficult to discern, particularly over the short term. The reported increase in norovirus outbreaks during the winter of 2002-2003 (2, 3, 16, 32) also occurred in Oxfordshire hospitals. Twenty-two of these outbreaks formed the basis of the present study. All the outbreaks were caused by a Lordsdale virus-like strain, and most outbreaks could not be distinguished using a short fragment of RNA polymerase sequence (Table 1 and Fig. 1). However, noroviruses mutate rapidly (24), and this characteristic, together with longer sequences from each outbreak, was exploited to identify those that were potentially linked.

Nucleotide sequencing of the 3'-terminal 3,255 nt of 49 strains was applied successfully to determine the relationships of 18 outbreaks representing seven Oxfordshire hospitals. The definition of outbreaks on a per-ward basis by infection control staff was verified as correct by molecular data in 8 of the 18 outbreaks. However, the remaining 10 outbreaks occurred in three clusters of two or more outbreaks with identical sequences (Fig. 2). The outbreaks in each of these clusters were temporally or geographically linked, being (i) concurrent in wards of the same or a nearby hospital, i.e., areas sharing frequent patient and staff movements (clusters 1 and 3), or (ii) in the same location as a previously identical strain but 18 days later (cluster 2). The latter suggests ongoing environmental contamination by the virus, since no mutations occurred over the interval between outbreaks. The temporal and/or spatial links between identical viruses suggest that the norovirus mutation rate is sufficiently high for sequence identity in the 3' 3,255 nt to indicate potentially connected outbreaks.

To determine whether mutation accumulation could be used to infer the predominant transmission route within an outbreak, two outbreaks were studied in greater detail, using 3'-terminal 3,255-nt and complete genome sequences. In one outbreak where person-to-person transmission was considered likely, six mutations gradually accumulated in the virus genome over time (Table 2). In contrast, a single point mutation was detected in an outbreak where many patients developed symptoms closer together, suggesting a greater role for simultaneous infection.

Combined with precise epidemiological data, this approach could allow the relative importance of different transmission routes in particular healthcare settings to be determined and could assist infection control procedures. The data described here suggest that the 18 outbreaks variously involved (i) multiple virus introductions, (ii) transmission among wards, (iii) person-to-person transmission, (iv) simultaneous infection of a large number of patients, and (v) environmental contamination over an extended period.

The Oxfordshire outbreaks included in the present study were part of a global increase which took place during 2002 and 2003, beginning in January 2002, with an unusually high number of outbreaks continuing into the summer (17). A national epidemic of Lordsdale virus at this time was reported in the United Kingdom (32). A new strain associated with the Europe-wide outbreak increase has also been identified as a Lordsdale virus-like strain on the basis of short RNA polymerase sequences containing a motif (AATCTG) which differed by 2 nt from certain previous GII 4 strains (16). All five of the RNA polymerase variants in the present study contained this hexamer (Fig. 1).

The mutations detected within the 3'-terminal 3,255 nt of 49 viruses included in this study were predominantly transitions (93%). Only four mutations were observed in 550 nt spanning the junction between ORF1 and ORF2, and none were observed in the 3'-terminal 136 nt of 49 viruses. The absence of mutations from certain regions of the genome may indicate areas where conservation of secondary structure is particularly important.

A comparison of the Oxfordshire strain capsid amino acid sequence to those of other Lordsdale-like viruses indicated that it differed by 11.4 to 13.6% in the outer P2 domain, in which it also had a single-amino-acid insertion. This strain may have been sufficiently distinct antigenically to evade previous host immunity, leading to the increased number of outbreaks. Capsid structural changes have been predicted as a result of only eight cumulative amino acid changes in the P2 domain (24), and the Oxfordshire viruses differed from their closest relative by 16 amino acids within this region.

The sequences obtained in the present study were combined with data obtained from GenBank to allow three clusters to be identified within the Lordsdale virus-like group (Fig. 4). None of the three had cocirculated, and all of the groups had similar levels of divergence within the capsid P2 domain. This further confirms that the predominant Lordsdale virus-like strain is replaced periodically by a new, antigenically distinct variant (13, 25).

The P2 domain was the region of the capsid in which the majority of amino acid changes occurred during chronic norovirus shedding by an immunocompromised patient (24). During the present study, an immunocompromised patient (a bone marrow transplant recipient) who was infected nosocomially shed norovirus for at least 17 days. Four mutations occurred within the 3,255-nt sequence determined, and there were three microheterogeneities. Long-term shedders may be the source of new human norovirus strains which are pathogenic to others (24), since an animal reservoir has not been identified. The demonstration of norovirus shedding for extended periods in immunocompromised patients has important implications for hospital infection control. Our data suggest that the effectiveness of infection control procedures can be monitored using molecular data, which can also indicate the predominant transmission routes.

 
This work was funded by the Sir Jules Thorn Charitable Trust, London, United Kingdom, with additional support for complete genome sequencing from the Oxford University Medical Research Fund.

Norovirus Infection Control in Oxfordshire Communities and Hospitals (NICOCH) comprised Ian Bowler, Chris Hall, Sandy Clayton-Kent, Karen Webb, Luisa Goddard, Leanne Collins, and Kathy Topley (Hospital Infection Control, Oxford Radcliffe National Health Service Trust, Oxford, United Kingdom) and Kyle Knox, Elena Terol Sabino, Candy Pulling, Janette Mills, Liza Sherry, Dona Foster, and Carol Hodges (Thames Valley Local Health Protection Unit [Oxfordshire Team], Oxford, United Kingdom).

We acknowledge Audrey Parsons, who provided laboratory assistance in organizing specimens; Katie Jeffery, who identified the severely immunocompromised patient who shed norovirus for at least 17 days; and Ian Clarke and Paul Lambden (Southampton University) for helpful comments on the manuscript.

 
* Corresponding author. Mailing address: Nuffield Department of Clinical Sciences, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom, OX3 9DU. Phone: 44 1865 220870. Fax: 44 1865 764192. E-mail: kate.dingle{at}ndcls.ox.ac.uk.

The contributing members of Norovirus Infection Control in Oxfordshire Communities and Hospitals (NICOCH) are listed in Acknowledgments.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, September 2004, p. 3950-3957, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.3950-3957.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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