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Histo-blood group antigen (HBGA) recognition by norovirus (NoV) has been studied using various techniques. Enzyme-linked immunosorbent assays (ELISAs) using virus-like particles (VLPs) have enabled us to visualize the last step of HBGAs-NoV binding with a total reaction time of approximately 8 h. Herein, we describe two ELISA-based methods to detect and quantify NoV VLP attachment to HBGAs: saliva-VLP binding assay and carbohydrate-VLP binding assay.

Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains.

Histo-blood group antigen (HBGA) recognition by norovirus (NoV) has been studied using various techniques. Enzyme-linked immunosorbent assays (ELISAs) using virus-like particles (VLPs) have enabled us to visualize the last step of HBGAs-NoV binding with a total reaction time of approximately 8 h. Herein, we describe two ELISA-based methods to detect and quantify NoV VLP attachment to HBGAs: saliva-VLP binding assay and carbohydrate-VLP binding assay.

G12 rotaviruses are globally emerging rotavirus strains causing severe childhood diarrhea. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed, of which only one G12P[4] and one G12P[6] are from Africa. In this study, we sequenced and characterized the complete genomes of three G12 strains (RVA/Human-tc/KEN/KDH633/2010/G12P[6], RVA/Human-tc/KEN/KDH651/2010/G12P[8], and RVA/Human-tc/KEN/KDH684/2010/G12P[6]) identified in three stool specimens from children with acute diarrhea in Kenya, Africa. On whole genomic analysis, all three Kenyan G12 strains were found to have a Wa-like genetic backbone: G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strains KDH633 and KDH684) and G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (strain KDH651). Phylogenetic analysis showed that most genes of the three strains examined in this study were genetically related to globally circulating human G1, G9, and G12 strains. Of note is that the NSP4 genes of strains KDH633 and KDH684 appeared to be of porcine origin, suggesting the occurrence of reassortment between human and porcine strains. Furthermore, strains KDH633 and KDH684 were very closely related to each other in all the 11 gene segments, indicating derivation of the two strains from a common origin. On the other hand, strain KDH651 consistently formed distinct clusters of 10 of the 11 gene segments (VP1-2, VP4, VP6-7, and NSP1-5), indicating a distinct origin of strain KDH651 from that of strains KDH633 and KDH684. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Kenya. Our observations will provide important insights into the evolutionary dynamics of emerging G12 rotaviruses in Africa.

In order to identify the repertoire of antibodies generated on natural infection of norovirus (NoV) in humans, and to characterize the human monoclonal antibodies against NoV, three phage-displayed antibody libraries originating from healthy person(s) were screened using purified virus-like particles (VLPs) of strain Narita 104 (r104, genogroup II, genotype 4) or strain Chiba 407 (rCV, genogroup I, genotype 4) as antigens. On screening with r104, 62 clones were isolated. Among these antibodies, two clones, 12A11 and 12B10, showed intra-genogroup cross-reactivity to genotypes 1, 3-7, 12, and 14, and genotypes 1, 4, 6, and 7 of genogroup II, respectively. In addition, antibodies belonging to the same group were isolated from two different libraries. On screening with rCV, five clones were isolated, two of which were cross-reactive. One, CV-2F5, reacted to genotypes 1-4, and 8 of genogroup I, and the other, CV-1A5, showed inter-genogroup cross-reactivity to all the VLPs employed in this study. The blocking activities of the monoclonal antibodies against the interaction of homotypic VLPs (VLPs used in the panning procedure) with histo-blood group antigens were also assessed as an alternative to neutralization assay. Although the blocking activity of 12A11 was partially limited 12B10 prevented the binding of r104 to histo-blood group antigens that had been reported to bind r104. The blocking activity of CV-2F5 against the attachment of rCV to suitable histo-blood group antigens was weak, but the blocking activity of CV-1A5 was well recognized. Thus, 12B10 and CV-1A5 were suggested to be cross-reactive monoclonal antibodies with neutralizing activity.

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le a ) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le a , Le b , H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le a antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le a antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le a antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le a antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.

Infection of insect cells with recombinant baculoviruses carrying the VP1 gene from Chiba strain norovirus resulted in the production of 57 and 50 kDa proteins, and the assembly of a smaller, 23 nm form of the virus-like particles (VLPs), together with the normal, 38 nm form of the VLPs. The N-terminal residues of the 57 and 50 kDa proteins were Ala4 and Thr45, respectively. When the tripeptide Leu43-Ala44-Thr45 was changed to Ala-Pro-Val, only 38 nm VLPs were assembled. The 38 nm VLPs showed essentially the same pattern of carbohydrate binding as the 23 nm VLPs, despite the significant difference in the degree of Lewis b antigen binding.

Norovirus (NoV), a member of the family Caliciviridae, is a major cause of acute water- and food-borne nonbacterial gastroenteritis and forms antigenically diverse groups of viruses. Human NoVs are divided into at least three genogroups, genogroups I (GI), GII, and GIV, which contain at least 15, 18, and 1 genotypes, respectively. Except for a few genotypes, all NoVs bind to histo-blood group antigens (HBGAs), namely ABH antigens and Lewis antigens, in which carbohydrate core structures, (types 1 and 2) constitute antigenically distinct phenotypes. Volunteer challenge studies have indicated that carbohydrate binding is essential for genogroup I genotype 1 (GI/1) infection. Non-secretors who do not express FUT2 fucosyltransferase, and consequently do not express H type 1 or Lewis b antigens in the gut, are not infected after challenge with GI/1. NoV virus-like particles (VLPs), which are recombinant particles that are morphologically and antigenically similar to the native virion, display different ABH and Le carbohydrate-binding profiles in vitro. Epidemiological studies have shown that individuals with different ABH phenotypes are infected with NoV strains in a genotype-specific manner. On the other hand, an in vitro binding assay using NoV VLPs showed a uniform recognition pattern against type 1 and 2 core structures, and bind more tightly to type 1 carbohydrates than to type 2. Type 1 carbohydrates are expressed at the surface of the small intestine and are presumably targeted by NoV. This property may afford NoV tissue specificity. GII/4 includes global epidemic strains and binds to more HBGAs than other genogroups. This characteristic may be linked to the worldwide transmission of GII/4 strains. Although it is still unclear whether HBGAs act as primary receptors or enhance NoV infectivity, they are important factors in determining tissue specificity and the risk of transmission.

Noroviruses (NoVs) and sapoviruses (SaVs) are causative agents of human gastroenteritis. There is increasing evidence that certain human NoV strains bind to histo-blood group antigens (HBGAs). We found that several NoV virus-like particles (VLPs) showed binding activity to HBGAs, while neither SaV genogroup I (GI) VLP nor SaV GV VLP showed such activity.

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.

“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.

As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vaccine (IPV) derived from the Sabin strains (sIPV) as an option for an affordable polio vaccine, especially in low-income countries. The Japan Poliomyelitis Research Institute (JPRI) inactivated three serotypes of the Sabin strains and made sIPV preparations, including serotypes 1, 2 and 3 D-antigens in the ratio of 3:100:100. The National Institute of Infectious Diseases, Japan, assessed the immunogenic stability of these sIPV preparations in a rat potency test, according to an evaluation method recommended by the WHO. The immunogenicity of the three serotypes was maintained for at least 4 years when properly stored under -70°C. Based on these data, the sIPV preparations made by JPRI have been approved as national reference vaccines by the Japanese national control authority and used for the quality control of the tetracomponent sIPV-containing diphtheria-tetanus-acellular pertussis combination vaccines that were licensed for a routine polio immunization in Japan.

Norovirus (NoV), a member of the family Caliciviridae, is a major cause of acute water- and food-borne nonbacterial gastroenteritis and forms antigenically diverse groups of viruses. Human NoVs are divided into at least three genogroups, genogroups I (GI), GII, and GIV, which contain at least 15, 18, and 1 genotypes, respectively. Except for a few genotypes, all NoVs bind to histo-blood group antigens (HBGAs), namely ABH antigens and Lewis antigens, in which carbohydrate core structures, (types 1 and 2) constitute antigenically distinct phenotypes. Volunteer challenge studies have indicated that carbohydrate binding is essential for genogroup I genotype 1 (GI/1) infection. Non-secretors who do not express FUT2 fucosyltransferase, and consequently do not express H type 1 or Lewis b antigens in the gut, are not infected after challenge with GI/1. NoV virus-like particles (VLPs), which are recombinant particles that are morphologically and antigenically similar to the native virion, display different ABH and Le carbohydrate-binding profiles in vitro. Epidemiological studies have shown that individuals with different ABH phenotypes are infected with NoV strains in a genotype-specific manner. On the other hand, an in vitro binding assay using NoV VLPs showed a uniform recognition pattern against type 1 and 2 core structures, and bind more tightly to type 1 carbohydrates than to type 2. Type 1 carbohydrates are expressed at the surface of the small intestine and are presumably targeted by NoV. This property may afford NoV tissue specificity. GII/4 includes global epidemic strains and binds to more HBGAs than other genogroups. This characteristic may be linked to the worldwide transmission of GII/4 strains. Although it is still unclear whether HBGAs act as primary receptors or enhance NoV infectivity, they are important factors in determining tissue specificity and the risk of transmission.

In order to identify the repertoire of antibodies generated on natural infection of norovirus (NoV) in humans, and to characterize the human monoclonal antibodies against NoV, three phage-displayed antibody libraries originating from healthy person(s) were screened using purified virus-like particles (VLPs) of strain Narita 104 (r104, genogroup II, genotype 4) or strain Chiba 407 (rCV, genogroup I, genotype 4) as antigens. On screening with r104, 62 clones were isolated. Among these antibodies, two clones, 12A11 and 12B10, showed intra-genogroup cross-reactivity to genotypes 1, 3-7, 12, and 14, and genotypes 1, 4, 6, and 7 of genogroup II, respectively. In addition, antibodies belonging to the same group were isolated from two different libraries. On screening with rCV, five clones were isolated, two of which were cross-reactive. One, CV-2F5, reacted to genotypes 1-4, and 8 of genogroup I, and the other, CV-1A5, showed inter-genogroup cross-reactivity to all the VLPs employed in this study. The blocking activities of the monoclonal antibodies against the interaction of homotypic VLPs (VLPs used in the panning procedure) with histo-blood group antigens were also assessed as an alternative to neutralization assay. Although the blocking activity of 12A11 was partially limited 12B10 prevented the binding of r104 to histo-blood group antigens that had been reported to bind r104. The blocking activity of CV-2F5 against the attachment of rCV to suitable histo-blood group antigens was weak, but the blocking activity of CV-1A5 was well recognized. Thus, 12B10 and CV-1A5 were suggested to be cross-reactive monoclonal antibodies with neutralizing activity.

Norovirus (NoV) is the major causative agent of acute viral gastroenteritis worldwide. Based on genetic analyses, human NoV strains have been classified into at least three genogroups: genogroup I (GI), GII, and GIV, which contain at least 15, 18, and 1 genotypes, respectively (Kageyama et al., 2004). Notably, these NoV genotypes are morphologically similar to one another but differ antigenically (Kapikian, 1996; Estes et al., 1997; Hansman et al., 2006). Since no cell culture system has been developed yet, molecular analyses to elucidate the mechanisms underlying infection or productive replication have been stalled until recently. The primary NoV replication site in humans is unknown, but intestinal biopsy from volunteer who became ill following oral administration of NoV exhibited histopathologic lesions, in which the villi of the proximal small intestine were blunted (Agus et al., 1973). More than 30 years ago, the initial NoV challenge studies conducted in volunteers found that a subset of individuals was repeatedly susceptible to NoV infection, whereas a second subset was repeatedly resistant to infection (Parrino et al., 1977). A genetic factor, possibly a receptor, was hypothesized to affect the susceptibility of an individual to NoV infection. Recently, a mechanism that explains the susceptibility or resistance to NoV infection has been identified; namely, NoV attaches to histo-blood group antigens (HBGAs) on potential host cells in the gut (Lindesmith et al., 2003).

Norovirus (NoV) is a causative agent of acute gastroenteritis. NoV binds to histo-blood group antigens (HBGAs), namely, ABH antigens and Lewis (Le) antigens, in which type 1 and type 2 carbohydrate core structures constitute antigenically distinct variants. Norwalk virus, the prototype strain of norovirus, binds to the gastroduodenal junction, and this binding is correlated with the presence of H type 1 antigen but not with that of H type 2 antigen (S. Marionneau, N. Ruvoen, B. Le Moullac-Vaidye, M. Clement, A. Cailleau-Thomas, G. Ruiz-Palacois, P. Huang, X. Jiang, and J. Le Pendu, Gastroenterology 122 :1967-1977, 2002). It has been unknown whether NoV distinguishes between the type 1 and type 2 chains of A and B antigens. In this study, we synthesized A type 1, A type 2, B type 1, and B type 2 pentasaccharides in vitro and examined the function of the core structures in the binding between NoV virus-like particles (VLPs) and HBGAs. The attachment of five genogroup I (GI) VLPs from 5 genotypes and 11 GII VLPs from 8 genotypes, GI/1, GI/2, GI/3, GI/4, GI/8, GII/1, GII/3, GII/4, GII/5, GII/6, GII/7, GII/12, and GII/14, to ABH and Le HBGAs was analyzed by enzyme-linked immunosorbent assay-based binding assays and Biacore analyses. GI/1, GI/2, GI/3, GI/4, GI/8, and GII/4 VLPs were more efficiently bound to A type 2 than A type 1, and GI/8 and GII/4 VLPs were more efficiently bound to B type 2 than B type 1, indicating that NoV VLPs distinguish between type 1 and type 2 carbohydrates. The dissociation of GII/4 VLPs from B type 1 was slower than that from B type 2 in the Biacore experiments; moreover, the binding to B type 1 was stronger than that to B type 2 in the ELISA experiments. These results indicated that the type 1 carbohydrates bind more tightly to NoV VLPs than the type 2 carbohydrates. This property may afford NoV tissue specificity. GII/4 is known to be a global epidemic genotype and binds to more HBGAs than other genotypes. This characteristic may be linked with the worldwide transmission of GII/4 strains. GI/2, GI/3, GI/4, GI/8, GII/4, and GII/7 VLPs bound to Le a expressed by nonsecretors, suggesting that NoV can infect individuals regardless of secretor phenotype. Overall, our results indicated that HBGAs are important factors in determining tissue specificity and the risk of transmission.

Norovirus (NOV), a member of the family Caliciviridae, is a major cause of water and food-borne acute nonbacterial gastroenteritis, and forms many morphologically similar but antigenically diverse groups of viruses. The virus-like particles (VLPs) derived from the prototype strain of NoV, Norwalk virus (NV/68), bind to histo-blood group antigens (HBGAs). HBGAs are carbohydrates that contain structurally related saccharide moieties, and are found in saliva and mucosal secretions from intestinal epithelial cells of secretor individuals who have FUT2 gene encoding a fucosyltransferase. From volunteer challenge studies, there is strong evidence that the carbohydrate-binding is essential for the NV/68 infection. Non-secretors, who do not express FUT2 fucosyltransferase and consequently do not express H type 1 or Leb in the gut, were not infected after the challenge with NV/68. However, other NoV VLPs display different ABH and Lewis carbohydrate-binding profiles, and indeed epidemiological studies showed that some NoV strains could infect individuals with another ABH phenotypes. GII/4 is known to be global epidemic strain and bound more HBGAs when compared with other strains. The strength of the transmission of GII/4 strains may be linked with their wide recognition of HBGAs. It is obvious that HBGAs are important factors to determine the host specificity, although it is still unclear whether the HBGAs act as the primary receptor or enhance NoV infectivity. Further investigation is needed.

Human noroviruses (NoVs), members of the genus Norovirus in the family Caliciviridae, are the leading agents of nonbacterial acute gastroenteritis worldwide. Human NoVs are currently divided into at least two genogroups, genogroup I (GI) and genogroup II (GII), each of which contains at least 14 and 17 genotypes. To explore the genetic and antigenic relationship among NoVs, we expressed the capsid protein of four genetically distinct NoVs, the GI/3 Kashiwa645 virus, the GII/3 Sanbu809 virus, the GII/5 Ichikawa754 virus, and the GII/7 Osaka10-25 virus in baculovirus expression system. An antigen enzyme-linked immunosorbent assay (ELISA) with hyperimmune serum against the four recombinant capsid proteins and characterized previously three capsid proteins derived from GI/1, GI/4, and GII/12 was developed to detect the NoVs antigen in stools. The antigen ELISA was highly specific to the homotypic strains, allowing assignment of a strain to a Norovirus genetic cluster within a genogroup. J. Med. Virol. 76:129–136, 2005. © 2005 Wiley-Liss, Inc.

A highly sensitive biosensor to detect norovirus in environment is desired to prevent the spread of infection. In this study, we investigated a design of surface plasmon resonance (SPR)-assisted fluoroimmunosensor to increase its sensitivity and performed detection of norovirus virus-like particles (VLPs). A quantum dot fluorescent dye was employed because of its large Stokes shift. The sensor design was optimized for the CdSe-ZnS-based quantum dots. The optimal design was applied to a simple SPR-assisted fluoroimmunosensor that uses a sensor chip equipped with a V-shaped trench. Excitation efficiency of the quantum dots, degree of electric field enhancement by SPR, and intensity of autofluorescence of a substrate of the sensor chip were theoretically and experimentally evaluated to maximize the signal-to-noise ratio. As the result, an excitation wavelength of 390nm was selected to excite SPR on an Al film of the sensor chip. The sandwich assay of norovirus VLPs was performed using the designed sensor. Minimum detectable concentration of 0.01ng/mL, which corresponds to 100 virus-like particles included in the detection region of the V-trench, was demonstrated.