We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

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"Norwalk-like viruses" (NLV), a member of the family Caliciviridae, are the major causative agents of acute gastroenteritis and are genetically divided into two groups, genogroup I (GI) and genogroup II (GII). We have determined the complete nucleotide sequences of 10 new NLV strains. Using this information together with eight known NLV sequences, the criteria to further classify genotypes of NLV were investigated. Validation of the topological error based on the bootstrap value and the branch length (distance) allowed us to identify two potential subgenomic regions suitable for the genotyping. They were the putative 3D-like RNA-dependent RNA polymerase (polymerase) and the capsid N-terminal/Shell domains (capsid N/S domain). When the distance distribution analysis was performed, the polymerase-based classification did not separate the strains into internal clusters within the genogroup. Furthermore, a diversity plot analysis of the complete nucleotide sequences of WUG1, a NLV GI strain, and Saitama U1, a NLV GII strain, indicated that the genotype was different between the polymerase and capsid N/S domain, suggesting that these strains are the genetic recombinants. Therefore, polymerase is not suitable for genotyping. On the other hand, the clustering based on the capsid N/S domain successfully distinguished the NLV as well as the grouping based on the antigenicity, as determined by both antigen and antibody ELISAs with recombinant virus-like particles. As the nucleotide sequences of the primers for the capsid N/S domain are highly conserved among the NLV, the amplification of the unknown genotype can be easily performed. This method will facilitate global surveying as well as epidemiologic study on NLV.

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The system of registering stroke patients was begun in 1963 in Ikawa Town, Akita Prefecture, Japan. The town is located in the northeastern part of the country and in 1975 had a population of 6,427. From 1975 to 1981, 109 patients who suffered their first stroke were registered and were monitored for 5 years. The average annual incidence rates of stroke were 2.8 and 2.0 per 1,000 population in males and females, respectively. Mean age at stroke onset was 63-3 and 71.4 years in males and females, respectively (p<0.01). According to the clinical classification of stroke, 76 patients suffered cerebral infarction, 21 cerebral bleeding, and six subarachnoid hemorrhage; six strokes were unclassified. The survival rates were compared by sex, age, and clinical stroke type using Cox's proportional hazards model. The survival rate of female stroke patients was lower than that of males, but not significantly so. The survival rate of stroke patients >65 years old was significantly lower (p<0.01) than that of younger patients. Moreover, the survival rate of patients with cerebral bleeding was significantly lower (p<0.01) than that of patients with cerebral infarction. In the analysis of self-care among the survivors, performance of the activities of daily living in older patients indicated significantly less independence (/><0.01) in younger patients. Follow-up of new stroke cases showed that age and clinical stroke type were significantly associated with survival and that age was also related to disability of the survivors. (Stroke 1990-^1:72-77) M ortality due to cerebrovascular disease (stroke) in Japan is much higher than in other developed countries, and from 1951 to 1980 stroke was the leading cause of death in this country. The geographic pattern of stroke mortality in Japan varies from high in eastern to low in western Japan and high in more rural areas to low in more urban ones.1 Furthermore, in Akita Prefecture, stroke mortality has been among the highest in the 47 prefectures of Japan.The incidence of and mortality due to stroke are decreasing; however, the age at stroke onset and the percentage of infarction are increasing in Japan. Stroke is a major cause of disability and dementia in the elderly, and related problems in health care have become more important in recent years.The system of registering stroke patients was begun in 1963 in Ikawa Town, Akita Prefecture, Japan. The Address for reprints: Saburo Kojima, MD, Akita Prefectural Institute of Public Health, 6-6, Sensyu-Kubota-Machi, Akita, 010 Japan.Received July 15, 1988; accepted September 14, 1989. stroke incidence rates during [1975][1976][1977][1978][1979] and factors related to stroke onset have been reported. 2 Although a number of studies have described the natural history of stroke and the factors influencing survival, it is uncommon to find a study that combines data on survival and disability of stroke patients over a long period.The purpose of our study is to determine the differences in survival rates according to the variables sex, age at s...

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3 genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer-and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.Norovirus (NV), a member of the family Caliciviridae, is the leading cause of epidemic acute, nonbacterial gastroenteritis. NV infection causes nausea, vomiting, low-grade fever, and diarrhea, which can be severe in infants and young children. For these reasons, it is a major public health concern (7). An effective vaccine or other therapeutic agent would be valuable for preventing the significant morbidity and potential mortality associated with NV infections. However, the lack of an efficient culture system has hampered the biochemical characterization of the NV proteins, and thus far, molecular biological techniques have been the most useful tools for the study of NV.The NV virion contains a polyadenylated plus-strand RNA genome of ϳ7.7 kb (11, 15). The structures of the full-length genome, phylogenetic trees, and genetic recombination among distinct genogroups have been analyzed in detail (13). Based on sequence similarities with other single-stranded RNA viruses, the NV open reading frame 1 (ORF1) is predicted to encode a large polyprotein that is cleaved into several viral proteins, including NTPase, proteinase, and RNA-dependent RNA polymerase (RdRp) (11,15).The RdRp encoded by the 3D region has a conserved amino acid motif, glycine-aspartic acid-aspartic acid (GDD), which is found in the active site of many viral RdRps (14), and thus might have an important role in NV replication. As in other positive-strand RNA viruses (2), NV genomic RNA likely acts as a template for the synthesis of minus-strand RNA. The minus-strand RNA then, in turn, serves as a template for the synthesis of progeny genomic plus-strand RNA molecules. Thus, RdRp is central to the synthesis of both plus-and minusstrand RNA molecules. Among the Caliciviridae, Rabbit hemorrhagic disease virus (RHDV) and Feline calicivirus (FCV) express an enzymatically active RdRp protein (27, 28). However, caliciviruses that infect humans have not been examined for RdRp activity.The aims of this study were to develop a cell-free system that permits the identification of RdRp activity in vitro and to charac...

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