Information about the secretor status in the Swedish population was obtained from 104 unselected plasma donors genotyped by PCR-restriction fragment length polymorphism for the FUT2
gene 428G→A nonsense mutation (8
). A majority, 54%, were heterozygous secretors (Sese428
), 26% homozygous secretors (SeSe), and 20% homozygous nonsecretors (se428
), consistent with previous reports that about 20% of Europeans are genetically nonsecretors.
On 20 September 2002, a patient with gastroenteritis was transferred to an internal medicine ward (ward A). Two days later, an assistant nurse in the ward fell ill with acute vomiting and diarrhea. On the following 5 days, 14 additional medical staff and 11 patients on the ward got sick with gastroenteritis. Thus, all together, 15 medical staff and 12 patients had gastroenteritis within 8 days (Fig. , Ward A). The symptoms and the outbreak were in agreement with a norovirus infection (i.e., acute onset of gastroenteritis that lasts for a few days) (12
). A stool sample taken from one symptomatic patient contained norovirus as determined by electron microscopy and norovirus-specific PCR (12
). Genotyping revealed that the virus belonged to the genogroup II cluster and was Lordsdale-like (LDV), a genotype most commonly found in nosocomial outbreaks in Europe (14
Time course of the nosocomial outbreaks. Ward A included 15 medical staff and 12 patients with symptomatic norovirus infection; ward B included 19 medical staff and 12 patients; ward C included 28 medical staff and 14 patients.
Saliva samples were obtained from 41 of the medical staff and from 19 of the patients, including 10 of the 12 patients and 13 of the 15 medical staff with gastroenteritis. DNA was obtained from 50 of the 60 saliva samples and examined for mutations partially or completely inactivating FUT2
(385A→T, 571C→T, and 428G→A) by PCR-SSP, a technique previously used for Lewis secretor and ABH genotyping (3
) (Fig. ). Of the symptomatic individuals from this outbreak, 47% were homozygous secretors (SeSe), 53% heterozygous secretors (Sese428
), and most interestingly, none were secretor negative (Fig. ), in contrast to 19% (se428
) who either were not exposed or were resistant to infection. Three of the six nonsecretors were medical staff (ages 39, 45, and 59), and three were patients (ages 88, 89, and 89). Interestingly, two of the three secretor-negative patients were nursed together in the same rooms as symptomatic patients with gastroenteritis, of which one was the index patient. In spite of the potential exposure from a symptomatic roommate, none of these two secretor-negative patients had any clinical symptoms of gastroenteritis. To investigate if the outbreak strain could bind to saliva from infected individuals, a saliva-based ELISA was established. As shown in Fig. , saliva from secretor-positive and ill individuals but not from secretor-negative individuals bound the LDV associated with the outbreak (LDV, Ward A).
FIG. 2. Example of FUT2 gene polymorphism characterization. The PCR-SSP patterns from two patients identify their secretor or nonsecretor genotype. Genotypes were characterized by detecting the presence or absence of PCR products indicating wild-type (Wt) or (more ...)
FIG. 3. Allelic distribution of the nonsense mutation (428G→A) in the FUT2 gene and resistance to symptomatic nosocomial (wards A to C) norovirus infections caused by genogroup II Lordsdale-like strains. A total of 38 saliva samples from symptomatic patients (more ...)
FIG. 4. Secretor-positive salivas from symptomatic individuals recognize the outbreak virus strain by ELISA. LDV (Ward A), virus and saliva from outbreak A; LDV (Ward C), virus and saliva from outbreak C; GGII, virus from a sporadic outbreak, with Lordsdale-like (more ...)
The second nosocomial outbreak with norovirus occurred in a pediatric ward (Fig. , Ward B) on 22 November 2002 with an index patient with acute gastroenteritis. Within the following 6 days, 12 patients and 19 medical staff fell ill with gastroenteritis (Fig. , Ward B). In fecal samples collected from ill patients, norovirus were identified by electron microscopy and PCR for three patients. Genotyping by reversed line blotting (25
) revealed that all viruses again belonged to genogroup II and were Lordsdale-like. Saliva samples were collected from 38 of the medical staff, of which 12 had symptoms. Unfortunately, saliva could not be collected from patients. Of the 38 saliva samples investigated, secretor status could be established in 28. None of the 7 symptomatic medical staff was secretor negative, in contrast to 43% (9/21) secretor-negatives among the medical staff that remained without clinical symptoms (Fig. ).
A third nosocomial outbreak affected orthopedic patients (Fig. , Ward C) and started on 8 December 2002 with the introduction of a patient with gastroenteritis. One day later, another patient in the ward had similar symptoms, and thereafter, patients and medical staff fell ill during the following 10 days. In all, 14 patients and 28 medical staff experienced gastroenteritis. The epidemic and symptoms were in agreement with norovirus infection. Norovirus was also confirmed by PCR in stool samples from four patients. Once again the norovirus identified belonged to genogroup II and the Lordsdale-like cluster. Saliva samples were obtained from 26 of the medical staff, of which 16 had gastroenteritis. Saliva samples could not be collected from patients. Secretor status was established from 18/26 salivas, and none of the 12 symptomatic individuals was a nonsecretor, in contrast to 50% (3/6) among those who remained asymptomatic. To better understand the phenotype correlation between secretor status and resistance to norovirus infection, saliva samples from secretor and nonsecretor individuals were tested in a saliva ELISA. As shown in Fig. , saliva from ill secretor individuals but not from nonsecretors recognized the virus strain responsible for the outbreak, strongly suggesting that receptors for norovirus GGII are present in saliva from secretors but not from nonsecretors.
To obtain further information about the role of FUT2 in norovirus infections, fecal and saliva samples were collected from community outbreaks. Of salivas collected from 19 individuals associated with 3 community outbreaks with noroviruses belonging to genotypes GGII/Melksam, GGI/Sindlesham, and another GGII strain, 4 individuals were asymptomatic and 15 symptomatic with vomiting and diarrhea. Of the asymptomatic individuals, two were Sese428 and two SeSe, and of the 15 symptomatic individuals, 47% (7/15) were SeSe and 53% (8/15) Sese428. Thus, none of the 15 symptomatic individuals was a nonsecretor.
Disease studies have given different results on whether individual ABO (H) blood groups are related to an increased risk of norovirus infection or not. Hutson et al. (10
) first reported that individuals with the O phenotype were more likely to be infected with Norwalk virus belonging to genogroup I viruses, whereas individuals with an A or B histo-blood group antigen had a decreased risk of infection. A similar observation was made by Hennessy et al. (7
) in investigating a norovirus outbreak at a British military field hospital, finding a reduced susceptibility of blood group B individuals to symptomatic norovirus infections. These observations are in contrast to those of Meyer et al. (18
), who reported that persons with the O phenotype were significant less affected than expected from the normal distribution. Furthermore, Rockx et al. (22
) recently reported that individuals secreting type B antigens in saliva were significantly protected against infection with genogroup I virus (2 of 22 individuals) and were also less likely to acquire norovirus-specific immunoglobulin G.
Possible explanations for these different observations may be that the secretor status was not determined in all cases and, secondly, whether it was determined by phenotyping rather than by genotyping (3
). Norwalk virus have been found to bind to gastrointestinal cells in vitro independently of ABO (H) blood groups but with dependence on secretor status (17
) and found to correlate to resistance against genogroup I viruses (16
), suggesting that the FUT2
gene and secretor status are susceptibility markers for Norwalk virus infection. The fact that nonsecretors do not express blood group A or B in saliva or other body fluids could suggest a coincidental correlation between the ABO phenotype in blood and infection of the intestinal mucosa. Indeed, individuals can have the A or B phenotype in the blood, but if secretor negative, they do not express the A or B antigen in saliva or mucosa. However, it cannot be ruled out that certain noroviruses are secretor independent, since multiple receptor specificities have been reported from in vitro studies (5
Our studies, the first to include a host genetic approach and to examine secretor status for nosocomial and sporadic outbreaks, revealed several novel observations. We report for the first time that a nonsense mutation (428G→A) in the FUT2 gene is strongly associated (P < 0.00001) with resistance to winter vomiting disease. In fact, all (100%) individuals with a confirmed norovirus infection studied have so far been secretor positive (Table ).
Association between a FUT2 nonsense mutation (428G→A) and resistance to symptomatic nosocomial and sporadic norovirus infections in Sweden
Furthermore, we also show that only homozygous nonsecretors (se428se428) are protected. The inactivating mutation examined in this study is by far the most common in Europe and was found to occur homozygously in 20% among Swedish plasma donors (Table ). However, it should been mentioned that protective immunity might very well play a role, although correlates of protection remains to be identified.
In vitro studies have shown that binding of Norwalk-like viruses to ABO, Lewis, and secretor histo-blood group antigens are strain specific and that different attachment mechanisms exist (4
). Since cells transfected with the FUT2
gene enhance norovirus binding to nonpermissive cells (17
), it is reasonable to believe that H type 1 or related blood group antigens act as receptors for norovirus. Our study supports this hypothesis, and we observed that the saliva ELISA by Marionneau et al. (17
) was successfully applied to show that the virus causing disease also could be recognized by the saliva of the patient. This new observation, together with our secretor polymorphism data, provide biochemical and genetic explanations for resistance to noroviruses belonging to the common GGII Lordsdale-like cluster.