In this study, we used real-time RT-PCR plus electron microscopy to screen NV in stool specimens from 66 outbreaks in the Saitama area. The real-time RT-PCR method greatly saved the time required for selecting stool specimens for further analysis. From 156 NV-positive specimens, we obtained 368 capsid N/S gene sequences after cloning the RT-PCR products that were amplified with primer sets GIFF/GISKR and G2FB/G2SKR (18
). Genotyping was performed by phylogenetic analyses according to the scheme described previously (19
We note that all shellfish-related outbreaks were caused by multiple NV genotypes (Table ). With their filter-feeding mechanisms, shellfish, such as oysters, can concentrate NV from an environment contaminated by multiple genotypes. In fact, oysters in the markets were found to contain several different genotypes (data not shown). Furthermore, some outbreaks displayed multiple genotypes with relatively high frequencies; the outbreaks occurring in 67% of private homes, 56% of restaurants, 33% of hotels, and 13% of catered lunches were strongly suspected to be due to shellfish. For example, shellfish were the common source for outbreak 199917 (Table ). In this outbreak, specimens were obtained from four patients, and each specimen contained multiple genotypes, but the genotypes did not coincide with each other except for GII/15. Also, for example, in outbreak 200126, three specimens contained multiple genotype strains, but a common genotype strain did not exist (Table ).
Among outbreaks which were not directly related to shellfish, there were some in which multiple genotypes were detected. In outbreak 199921 involving a restaurant but not shellfish, multiple genotypes were identified. One specimen (KU105) contained three genotypes (GI/4, GII/4, and GII/6), two specimens (KU109 and KU111) contained two genotypes (GI/4 and GII/6), and another specimen (KU112) contained only one genotype (GI/4). In this outbreak, the four individuals had eaten dinner together. Since no common foods, such as oysters, were identified, the cook, from whom KU115 was collected, was presumed to be the source. KU115 contained one genotype, GI/4, that was a common genotype in this outbreak. Other genotypes were not detected from this specimen. However, the cook was tested for NV long after the other patients were tested. Possibly, in the early stage of the disease, the cook had shed at least all three genotypes (GI/4, GII/4, and GII/6) and transmitted them to the other individuals by poor handling of cooked food. At a later stage, perhaps the virus titers in KU115 were lower, and only the predominant genotype GI/4 was detected. Also, in outbreak 200138, at school, multiple genotypes (GII/4 and GII/5) were also identified in one of three specimens. The other two specimens contained only a common genotype (GII/5) (Table ).
On the other hand, stool specimens from outbreaks in semiclosed communities contained only single genotypes, with the exception of outbreak 200138. Fourteen of 66 outbreaks occurred in semiclosed communities (schools, nursery schools, nursing homes, and dormitory), and only seven genotypes (i.e., GI/3, GI/4, GII/2, GII/3, GII/4, GII/5, and GII/6) were found. In each outbreak, one genotype was likely transmitted through the fecal-oral route.
In NV infection, individual patients seem to differ in their susceptibility to each genotype. We confirmed that the genotypes which we identified were also antigenically distinct by an antigen enzyme-linked immunosorbent assay with hyperimmune sera against virus-like particles (unpublished data). Thus, susceptibility to each genotype seems to differ in each individual, perhaps due to differences in acquired immunity from previous NV infections. The different susceptibilities may also be due to specific ABO histo-blood group antigens in each individual, as described in recent studies (12-14, 25).
Furthermore, in the case of person-to-person infection with NV, selection of strains may occur during sequential passages in the outbreak due to the factors on the agent side, such as pathogenicity, reproductive rate in the host, and/or stability in the environment. When a person is infected first by multiple genotype strains, a strain that replicates faster and has greater stability may eventually become predominant later in the outbreak. Further epidemiological investigation may be necessary to clarify the mechanism of selection.
In the present study, we classified NV into 31 distinct genotypes (Fig. ). This analysis added five GI and seven GII genotypes to the previously published list (19
), and all of these new genotypes, except for GII/9 and GII/13, were detected in the Saitama area. GII/10 and GII/14 were isolated exclusively in Germany and the United States. In the Saitama area during the study period, a total of 26 of the 31 genotypes, including 10 new genotypes, were found. Saitama Prefecture is only 3,800 km2
and ≈1% of the total area of Japan. It is surprising that this small region contained such a diversity of genotypes, including ones found in North and South America, Europe, Oceania, and Asia (Fig. ). The extensive diversity in the Saitama area suggests that many genotypes were imported from and exported to other countries with NV-contaminated foods and travelers afflicted with NV. Various genotypes of NV may be circulating around the world, and more new genotypes are likely to be discovered in the future.
With a combination of screening by real-time RT-PCR and genotyping by phylogenetic analysis, detection of NV in sewage, rivers, seawater, and foods may improve our understanding in the epidemiology of NV and, in turn, help us to prevent and control future NV outbreaks.