Noroviruses belong to a genus of genetically diverse viruses within the family Caliciviridae and cause acute gastroenteritis in humans and animals. They are subdivided into genogroups, each of which further segregates into genotypes. Until recently, a new genotype was based on a defined pairwise distance cutoff of complete VP1 sequences, but with the increasing number of available norovirus sequences, this cutoff is no longer accurate, and sequences in the public database have been misclassified. In this paper, we demonstrate that the pairwise distance cutoff method can no longer be used and outline a phylogenetic approach to classify noroviruses. Further-more, we propose a dual nomenclature using both ORF1 and VP1 sequences, as recombination is common and recognizing recombinant viruses may be relevant. With the continuing emergence of new norovirus lineages, we propose to coordinate nomenclature of new norovirus genotypes through an international norovirus working group.

Norovirus (NoV) genogroups I and II (GI and GII) are now recognized as the predominant worldwide cause of outbreaks of acute gastroenteritis in humans. Three recombinant NoV GII isolates were identified and characterized, 2 of which are unrelated to any previously published recombinant NoV. Using data from the current study, published sequences, database searches, and molecular techniques, we identified 23 recombinant NoV GII and 1 recombinant NoV GI isolates. Analysis of the genetic relationships among the recombinant NoV GII isolates identified 9 independent recombinant sequences; the other 14 strains were close relatives. Two of the 9 independent recombinant NoV were closely related to other recombinants only in the polymerase region, and in a similar fashion 1 recombinant NoV was closely related to another only in the capsid region. Breakpoint analysis of recombinant NoV showed that recombination occurred in the open reading frame (ORF)1/ORF2 overlap. We provide evidence to support the theory of the role of subgenomic RNA promoters as recombination hotspots and describe a simple mechanism of how recombination might occur in NoV.

Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.

Noroviruses are the leading cause of outbreaks of gastroenteritis in the world. At present, norovirus genogroup II, genotype 4 (GII/4), strains are the most prevalent in many countries. In this study we investigated 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food handlers in foodcatering settings between 10 November 2005 and 9 December 2006 in Japan. Stool specimens were collected from both symptomatic and asymptomatic individuals and were examined for norovirus by real-time reverse transcription-PCR; the results were then confirmed by sequence analysis. Norovirus was detected in 449 of 2,376 (19%) specimens. Four genogroup I (GI) genotypes and 12 GII genotypes, including one new GII genotype, were detected. The GII/4 sequences were predominant, accounting for 19 of 55 (35%) outbreaks and 16 of 35 (46%) sporadic cases. Our results also showed that a large number of asymptomatic food handlers were infected with norovirus GII/4 strains. Norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI, i.e., 3.81 ؋ 10 8 versus 2.79 ؋ 10 7 copies/g of stool. Among norovirus GI strains, GI/4 had the highest mean viral load, whereas among GII strains, GII/4 had the highest mean viral load (2.02 ؋ 10 8 and 7.96 ؋ 10 9 copies/g of stool, respectively). Importantly, we found that asymptomatic individuals had mean viral loads similar to those of symptomatic individuals, which may account for the increased number of infections and the predominance of an asymptomatic transmission route.The positive-sense polyadenylated single-stranded RNA virus family Caliciviridae contains four genera: Norovirus, Sapovirus, Lagovirus, and Vesivirus (1). The prototype strain of human norovirus is the Norwalk virus (NV/Human/US/1968), which was first discovered in an outbreak of gastroenteritis in an elementary school in Norwalk, OH, in 1968 (15). Noroviruses are the leading cause of outbreaks of gastroenteritis in the world; they cause outbreaks in various settings, including hospitals, cruise ships, schools, and restaurants (2,9,12,15,23,24,29). In addition, noroviruses have been detected in environmental samples (e.g., treated and untreated sewage) as well as in contaminated foods such as oysters, shellfish, sandwiches, salads, raspberries, and even ice (7,18,19,26). Numerous molecular epidemiological studies have revealed a global distribution of these viruses (25,27,31).The most widely used method of detecting noroviruses is reverse transcription-PCR (RT-PCR), which has high sensitivity; also, the products can be used for further genetic analysis. Real-time RT-PCR assays have also been developed; they are sensitive, broadly reactive, and rapid for the detection of human noroviruses in clinical stool specimens and environmental samples (13,14,21).As the detection methods become more and more sensitive, the numbers of genogroups and genotypes are expected to increase. One emerging characteristic is that strains have been found to persist in one geographical region, only to disapp...

Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10-and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal ␣fucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical ␣fucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule-and antibody-mediated neutralization of GII norovirus.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Stool specimens from hospitalized infants with sporadic gastroenteritis in Chiang Mai, Thailand, between July 2000 and July 2001 were examined for norovirus and sapovirus by reverse transcription-PCR and sequence analysis. These viruses were identified in 13 of 105 (12%) specimens. One strain was found to be a recombinant norovirus

IMPORTANCEOur data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can accommodate numerous HBGA types. Human noroviruses are responsible for most epidemic outbreaks of gastroenteritis. There are still no antivirals or vaccines approved, despite the discovery of these viruses over 4 decades ago (1). Noroviruses are genetically and antigenically diverse (2), yet a single genetic cluster (genogroup II, genotype 4 [GII.4]) has dominated over the past decade (3). The GII.4 noroviruses evolve ϳ5% every year and are believed to have a mechanism that allows them to evade the immune system or alter receptor binding profiles (4-6). However, immunity to noroviruses is still poorly understood (7).Human noroviruses interact with histo-blood group antigens (HBGAs), and this is thought to be important for viral infections (8-11). A recent report showed for the first time that human noroviruses infect B cells and that HBGAs (synthetic or from HBGAexpressing enteric bacteria) can enhance the infection (12). HBGAs are also found as soluble antigens in saliva and are expressed on epithelial cells. Genetic polymorphisms in genes that control their synthesis are known to provide intraspecies diversity (13). To date, based on the ABH and Lewis HBGA types, at least nine different HBGAs have been found to interact with human noroviruses. Individuals expressing the O type are thought to have a significantly higher infection rate than those for individuals with other blood types (11). The GII noroviruses are thought to have preferences for HBGAs in a strain-dependent manner (14)(15)(16)(17)(18)(19).Expression of the norovirus capsid protein in insect cells results in the formation of virus-like particles (VLPs) that are antigenically similar to native virions. The X-ray crystal structure of prototype (GI.1) norovirus VLPs identified two domains: the shell (S) and protruding (P) domains (20). The S domain forms a scaffold surrounding the viral RNA, whereas the P domain is thought to contain the determinants for cell attachment and strain diversity. The P domain can be further subdivided into P1 and P2 subdomains, and each subdomain likely has unique functions. In this study, we determined the X-ray crystal structures of P domains from three epidemic GII.4 variants, from 2004GII.4 variants, from , 2006GII.4 variants, from , and 2012, in complex with a panel of HBGAs in order to elucidate HBGA binding mechanisms. Our data showed that the GII.4 noroviruses bound numerous HBGA types and that bindi...