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Volume 16, Number 1–January 2010
Research
Norovirus Gastroenteritis Outbreak with a Secretor-independent Susceptibility Pattern, Sweden
Johan Nordgren, Elin Kindberg, Per-Eric Lindgren, Andreas Matussek, and Lennart Svensson
Author affiliations: University of Linköping, Linköping, Sweden (J. Nordgren, E. Kindberg, P.-E. Lindgren, L. Svensson); National Board of Forensic Medicine, Linköping (E. Kindberg); County Hospital Ryhov, Jönköping, Sweden (P.-E. Lindgren, A. Matussek); and Capio St Görans Hospital, Stockholm, Sweden (A. Matussek)

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Abstract
Norovirus (NoV) is recognized as the commonest cause of acute gastroenteritis among adults. Susceptibility to disease has been associated with histo-blood group antigens and secretor status; nonsecretors are almost completely resistant to disease. We report a foodborne outbreak of GI.3 NoV gastroenteritis that affected 33/83 (40%) persons. Symptomatic disease was as likely to develop in nonsecretors as in secretors (odds ratio [OR] 1.41, 95% confidence interval [CI] 0.46–4.36 vs. OR 0.71, 95% CI 0.23–2.18, p = 0.57). Moreover, no statistical difference in susceptibility was found between persons of different Lewis or ABO phenotypes. The capsid gene of the outbreak strain shares high amino acid homology with the Kashiwa645 GI.3 strain, previously shown to recognize nonsecretor saliva, as well as synthetic Lewis a. This norovirus outbreak affected persons regardless of secretor status or Lewis or ABO phenotypes.

Norovirus (NoV) is the leading cause of nonbacterial, acute gastroenteritis among adults and is responsible for numerous outbreaks worldwide (1–4). The virus is frequently associated with contaminated food, causing >50% of all food-related outbreaks (5). Several studies (6–11) have associated norovirus susceptibility with the presence of an α1,2-linked fucose on histo-blood group antigens (HBGAs), which is determined by the FUT2 gene (12,13). Persons carrying >1 functional FUT2 allele, and thus expressing α1,2 fucosyltransferase 2 (FucT-II), are termed secretor positive (secretors) and can express the A and B blood group antigens as well as H-type 1 and Lewis b (Leb) antigens on mucosa and in secretions. Persons lacking FucT-II are termed secretor negative (nonsecretors) and have been shown to be highly protected from infections with several NoV genotypes, including the common GII.4, as well as the Norwalk virus prototype strain (GI.1) (6–11).

Saliva-binding studies have demonstrated that different NoV strains exhibit different binding patterns (14–16), with the Norwalk virus (GI.1) mainly recognizing saliva from secretors with blood groups A and O, while exhibiting low or no binding to saliva to nonsecretors and carriers of blood group B, suggesting protection against infection among the latter 2 groups. Virus-like particles (VLPs) of the common GII.4 strains have been found to mainly bind saliva from secretors irrespective of blood group (16), although binding to nonsecretor saliva has been described for VLPs of some GII.4 strains (17).

Although NoV infections of secretors are well documented (18) and a few cases of infected nonsecretors have been reported (19,20), no virus has been identified in authentic outbreaks that is completely secretor or Lewis antigen independent, where homozygous carriers of the nonsense G428A mutation in FUT2 are at similar or higher risk for infection than are secretors. We describe a foodborne NoV outbreak in which persons were infected regardless of secretor status or Le phenotypes; and no difference was observed between nonsecretor (Lea+b-) persons and secretors regarding risk of symptomatic norovirus infection. Our data provide new knowledge about susceptibility factors and NoV genotypes and suggest that additional studies of host genetic receptor factors and NoV are needed.

Materials and Methods
Outbreak Data and Sample Collection
In October 2007, a NoV gastroenteritis outbreak occurred in Jönköping, Sweden, at a seminar for healthcare improvement (October 25–27), attended by 112 healthcare workers from different parts of Sweden. The healthcare workers were asked to take part in this case–control study, and 83 persons, including 4 employees of the restaurant that provided food service, decided to participate. Thirty-three of these 83 persons acquired acute gastroenteritis during or shortly after the seminar. Saliva samples were collected from all 83 participants in the study and stored at –20°C until further use. Stool samples (n = 4) were obtained from the cook, 2 employees, and 1 participant of the conference with symptoms of NoV gastroenteritis. Epidemiologic investigations indicated that the lunch on the first day was contaminated with NoV and was subsequently the cause of the outbreak. The cook was ill 4 days before the outbreak started, and 3 days later other employees of the restaurant became ill, suggesting the restaurant employees as the probable source of NoV contamination in the food. NoV disease was identified by at least 1 of the following signs or symptoms: vomiting, diarrhea, or nausea combined with stomach ache ≈12–60 hours after ingesting the meal. Description of symptoms was obtained through a questionnaire sent to all participants in the study. The study was approved by the local ethics committee (M205-04 T48-08).

DNA Extraction from Saliva
Genomic DNA from 200 μL saliva was extracted by using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer (Blood and Body Fluid Spin Protocol). Extracted DNA was stored in AE buffer (QIAGEN) at –20°C until PCR amplification.

PCR Amplification of FUT2 and Determination of FUT2 428 Genotype
The FUT2 gene amplification by PCR was performed as previously described (6). Genotyping of the G428A mutation in the FUT2 gene was performed as previously described (6,7,21). These methods can distinguish between carriers of the homozygous wild-type, heterozygous, and homozygous mutated genotype.

Detection of Histo-Blood Group Antigens in Saliva
The ABO histo-blood group phenotype of secretor-positive persons and the Lewis phenotype of all 83 persons were determined by a saliva-based ELISA, essentially as described by Bucardo et al. (6) and Rydell et al. (22). Protein concentration was determined in boiled (5 min) and centrifuged (5 min, 10,000 rpm) saliva by means of a Bradford assay. ELISA plates (NUNC 96F Maxisorp; Thermo Fisher Scientific, Roskilde, Denmark) were coated with saliva, diluted to a final protein concentration of 1 μg/mL in coating buffer (0.1 M carbonate–bicarbonate buffer, pH 9.6); plates were incubated for 2 h at 37°C followed by 4°C overnight. The following day, the plates were washed 4 times with washing buffer (0.9% NaCl, 0.05% Tween 20 [Sigma-Aldrich, St. Louis, MO, USA]), and then incubated for 1.5 h at 37°C with antibodies α-A (ABO1 clone 9113D10), α-B (ABO2 clone 9621A8) (Diagast, Loos Cedex, France), α-Lea (Seraclone, LE1 clone 78FR 2.3), and α-Leb (Seraclone LE2 clones LM129-181 and 96 FR2.10) (Biotest AG, Dreieich, Germany). Antibodies were diluted 1:5000 in phosphate-buffered saline with 10% fetal bovine serum (Invitrogen AB, Lidingö, Sweden) and 0.05% Tween 20 (Sigma-Aldrich). After 4 washes, horseradish peroxidase–conjugated goat anti-mouse IgG (heavy plus light chain) (Bio-Rad Laboratories, Hercules, CA, USA), diluted 1:7,500, was added, and plates were incubated for another 1.5 h at 37°C and subjected to 4 final washes. The reaction was developed using 3´,3´,5´,5-tetramethylbenzidine (DakoCytomation, Carpinteria, CA, USA) and stopped by addition of 2M H2SO4. The plate was read at 450 nm in a spectrophotometer. The cutoff value was twice the mean level of 6 known negative samples. The α-Leb antibody cross-reacted weakly with Lea; this signal was subtracted from the Leb values read in Lea-positive persons.

Virus RNA Extraction and Reverse Transcription
RNA extraction from the 4 collected stool specimens was performed by using the EZ1 robot (QIAGEN) according to the manufacturer's instructions and stored at –80°C until used for reverse transcription. Reverse transcription was performed as previously described (6,23), by using random hexamer primers (GE Healthcare, Uppsala, Sweden) and Illustra Ready-To-Go RT-PCR beads (GE Healthcare).

Norovirus Detection with Real-Time PCR
NoV detection and quantification were performed with a real-time PCR specific for the open reading frame (ORF) 1–ORF2 junction, as described by Nordgren et al. (24). This real-time PCR assay can semiquantify and distinguish between NoVs GI and GII (24). PCR amplification of the N-terminal and shell (N/S) region was performed on a PTC-100TM thermal cycler (MJ Research Inc., South San Francisco, CA, USA) in a 50-μL mixture composed of 1.33 U of Expand High Fidelity polymerase (Boehringer Mannheim GmbH, Mannheim, Germany), 5 μL of the supplied buffer (including 1.5 mmol/L MgCl2; Boehringer Mannheim GmbH), 100 μM GeneAmp dNTP mixture with dTTP (Applied Biosystems, Branchburg, NJ, USA), 200 nM forward primer NVG1f1b (5´-CGY TGG ATG CGN TTC CAT GA-3´) (24), 200 nM reverse primer G1SKR (5´-CCA ACC CAR CCA TTR TAC A-3´) (25), and 5 μL template DNA.

Nucleotide Sequencing of the Norovirus N/S Region and Virus Genotyping
Nucleotide sequencing of the N/S region was performed by Macrogen Inc. (Seoul, South Korea). The sequencing reaction was based on BigDye chemistry; NVG1f1b forward primer (24) and G1SKR reverse primer (25) were used as sequencing primers. The amplicons were sequenced twice in each direction. Sequence alignment of the Jönköping (JKPG) strain and reference NoV genotypes was performed by using the ClustalW algorithm, version 1.8 (www.ebi.ac.uk/clustalw), with default parameters, on the European Bioinformatics Institute server. We performed phylogenetic analysis using the MEGA 4.0 software package (www.megasoftware.net), and the phylogenetic tree was constructed using the neighbor-joining and Kimura 2-parameter methods. Significance of the taxonomic relationships was obtained by bootstrap resampling analysis (1,000 replications). Assignment of genotypes used reference strains described by Zheng et al. (26).

PCR Amplification of the Norovirus Capsid Gene
To amplify the gene encoding the NoV capsid, we set up a PCR mixture containing 2.5 μL 10× native Pyrococcus furiosus (pfu) polymerase buffer (Invitrogen AB, Lidingö, Sweden), 200 μM GeneAmp dNTP mix with dTTP (Applied Biosystems), 200 nM forward primer CapGI3fw (5´-GAT CTC CTG CCC GAT TAT GTA AAT GAT GAT G-3´, this study), targeting the end of ORF1 and beginning of ORF2, 200 nM reverse primer CapGI3rv (5´-CAT TAT GAT CTC CTA ATT CCA AGC CTA CGA GC-3´, this study), specific for the end of ORF2 and beginning of ORF3, 5 μL cDNA, 2.5 U native pfu DNA polymerase (Stratagene, La Jolla, CA, USA), and 36 μL RNAse-free water. After initial denaturation at 94°C for 5 min, PCR amplification was performed with 40 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min, and thereafter a final elongation at 72°C for 10 min. The PCR products were visualized by electrophoresis on a 2% agarose gel, using staining with ethidium bromide and UV transillumination.

Cloning of the Norovirus Capsid Gene and Nucleotide Sequencing
The capsid fragment was cloned into a pPCR-Script Amp SK(+) vector and transformed into XL10-Gold Kan ultracompetent cells, using the Stratagene PCR-Script Amp Cloning Kit (Stratagene) according to the manufacturer's instructions. After overnight incubation of 2 separate colonies from each transformation reaction, plasmid DNA was extracted and purified, using the Plasmid Miniprep Kit (QIAGEN) according to the manufacturer's instructions. Nucleotide sequencing was performed on 2 separate plasmid extractions from each sample (n = 2) by Macrogen Inc., by using the BigDye chemistry with M13 forward and reverse primers. The nucleotide sequences for the N/S region or the complete capsid gene of the JKPG isolates are available under GenBank accession nos. FJ711163, FJ711164, and FJ711165.

Statistical Analysis
Categorical data were analyzed using the Fisher exact test with 2-tailed significance. Unadjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using SPSS 14.0 for Mac (SPSS Inc., Chicago, IL, USA).

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