Identification of Genogroup I and Genogroup II broadly reactive epitopes on the norovirus capsid

Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.     

Identification of a Broadly Cross-Reactive Epitope in the Inner Shell of the Norovirus Capsid

Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods. Here, we report the characterization of a broadly cross-reactive monoclonal antibody (MAb) raised against the capsid protein of a GII.3 norovirus strain. The MAb reacted with VLPs and denatured VP1 protein from GI, GII, GIV and GV noroviruses, and mapped to a linear epitope located in the inner shell domain. An alignment of all available VP1 sequences showed that the putative epitope (residues 52–56) is highly conserved across the genus Norovirus. This broadly cross-reactive MAb thus constitutes a valuable reagent for the diagnosis and study of these diverse viruses.     

Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle

Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.     

Characterization of norovirus infections in Seoul,Korea

The present study has determined the detection rate of norovirus (NoV) with acute gastroenteritis (AGE) in hospitalized children and describes the molecular epidemiology of NoV circulating in Seoul, Korea. Six hundred and eighty‐three (9.8%) of samples were positive for NoV. Of these, the NoV GII genogroup was the most commonly found, with a prevalence of 96.2% (683 of 710). Only 27 samples were positive for the NoV GI genogroup. Ten kinds of GI genotype (GI/1, GI/2, GI/3, GI/4, GI/5, GI/6, GI/7, GI/9, GI/12, and GI/13) and eight kinds of GII genotype (GII/2, GII/3, GII/4, GII/8, GII/14, GII/15, GII/16, and GII/17) were identified in children with AGE during the years 2008–2011.     

Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ～80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.     

Characterization of Blockade Antibody Responses in GII.2.1976 Snow Mountain Virus-Infected Subjects

Snow Mountain virus (GII.2.1976) is the prototype strain of GII.2 noroviruses (NoVs), which cause an estimated 8% of norovirus outbreaks, yet little is known about the immunobiology of these viruses. To define the human immune response induced by SMV infection and the antigenic relationship between different GII.2 strains that have circulated between 1976 and 2010, we developed a panel of four GII.2 variant virus-like particles (VLPs) and compared their antigenicities by enzyme immunoassay (EIA) and surrogate antibody neutralization (blockade) assays. Volunteers infected with GII.2.1976 developed a mean 167-fold increase in blockade response against the homotypic VLP by day 8 postchallenge. Blockade extended cross-genotype activity in some individuals but not cross-genogroup activity. Polyclonal sera from GII.2.1976-infected volunteers blocked GII.2.1976 significantly better than they blocked GII.2.2002, GII.2.2008, and GII.2.2010, suggesting that blockade epitopes within the GII.2 strains have evolved in the past decade. To potentially map these epitope changes, we developed mouse monoclonal antibodies (MAbs) against GII.2.1976 VLPs and compared their reactivities to a panel of norovirus VLPs. One MAb had broad cross-genogroup EIA reactivity to a nonblockade, linear, conserved epitope. Six MAbs recognized conformational epitopes exclusive to the GII.2 strains. Two MAbs recognized GII.2 blockade epitopes, and both blocked the entire panel of GII.2 variants. These data indicate that the GII.2 strains, unlike the predominant GII.4 strains, have undergone only a limited amount of evolution in blockade epitopes between 1976 and 2010 and indicate that the GII.2-protective component of a multivalent norovirus vaccine may not require frequent reformulation.     

Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase.

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.     

Genogroup II noroviruses efficiently bind to heparan sulfate proteoglycan associated with the cellular membrane

Norovirus (NV), a member of the family Caliciviridae, is one of the important causative agents of acute gastroenteritis. In the present study, we found that virus-like particles (VLPs) derived from genogroup II (GII) NV were bound to cell surface heparan sulfate proteoglycan. Interestingly, the VLPs derived from GII were more than ten times likelier to bind to cells than were those derived from genogroup I (GI). Heparin, a sulfated glycosaminoglycan, and suramin, a highly sulfated derivative of urea, efficiently blocked VLP binding to mammalian cell surfaces. The reagents known to bind to cell surface heparan sulfate, as well as the enzymes that specifically digest heparan sulfate, markedly reduced VLP binding to the cells. Treatment of the cells with chlorate revealed that sulfation of heparan sulfate plays an important role in the NV-heparan sulfate interaction. The binding efficiency of NV to undifferentiated Caco-2 (U-Caco-2) cells differed largely between GI NV and GII NV, whereas the efficiency of binding to differentiated Caco-2 (D-Caco-2) cells did not differ significantly between the two genogroups, although slight differences between strains were observed. Digestion with heparinase I resulted in a reduction of up to 90% in U-Caco-2 cells and a reduction of up to only 50% in D-Caco-2 cells, indicating that heparan sulfate is the major binding molecule for U-Caco-2 cells, while it contributed to only half of the binding in the case of D-Caco-2 cells. The other half of those VLPs was likely to be associated with H-type blood antigen, suggesting that GII NV has two separate binding sites. The present study is the first to address the possible role of cell surface glycosaminoglycans in the binding of recombinant VLPs of NV.     

Crystal structures of GI.8 Boxer virus P dimers in complex with HBGAs,a novel evolutionary path selected by the Lewis epitope

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le^b) and Le^y antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.     

Noroviruses distinguish between type 1 and type 2 histo-blood group antigens for binding

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.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.     

Detection and phylogenetic analysis of norovirus in Corbicula fluminea in a freshwater river in Japan

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 10(4) copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters.     

Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity

Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.     

Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.     

An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum

Among nonneutralizing HIV-1 envelope antibodies (Abs), those capable of mediating antibody-dependent cellular cytotoxicity (ADCC) activity have been postulated to be important for control of HIV-1 infection. ADCC-mediating Ab must recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fcγ receptor (FcR) of the effector cell population. However, the precise targets of serum ADCC antibody are poorly characterized. The human monoclonal antibody (MAb) A32 is a nonneutralizing antibody isolated from an HIV-1 chronically infected person. We investigated the ability of MAb A32 to recognize HIV-1 envelope expressed on the surface of CD4(+) T cells infected with primary and laboratory-adapted strains of HIV-1, as well as its ability to mediate ADCC activity. The MAb A32 epitope was expressed on the surface of HIV-1-infected CD4(+) T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.     

Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A+ and two H+). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A+ or H+ pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A+ pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A+ and H(+) pigs (51%) than non-A+ and non-H+ pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A+ or H+ pigs (66%) than non-A+ or H+ pigs (25%) seroconverted.     

Production of a monoclonal antibody specific to Escherichia coli H33 flagellin by using predetermined polypeptides

The Escherichia coli H serogroup is determined by flagellin, which has both H-type-specific and cross-reactive epitopes. The cross-reactive epitopes are responsible for the cross-reaction found in agglutination. To identify the specific epitope in H33 flagellin, the H33 flagellin gene was sequenced and the encoded central variable region (CVR) was determined. Four overlapping fragments of the CVR were prepared and their specificity was verified using different H-type antisera. Short fragments carrying potential H-type-specific determinants were selected, and monoclonal antibodies (MAbs) against these fragments were prepared. A murine MAb of subtype IgG1 showing specificity to H33 flagellin was produced. The epitope of the MAb was mapped to amino acid residues 250-260.     

Molecular epidemiology of norovirus in asymptomatic food handlers in Busan,Korea, and emergence of genotype GII.17

The molecular epidemiology of norovirus infections was studied in food handlers without any symptoms from January to December 2015 in Busan city, Korea. A total of 2,174 fecal specimens from asymptomatic food handlers were analyzed, and 2.3% (49/2,174) were norovirus-positive. Fourteen of 335 samples (4.2%) were positive in January; fifteen of 299 samples (5.0%) in February, and seven of 189 samples (3.7%) in December. However, norovirus was rarely detected in other months. From sequencing analysis, 11 genotypes (five GI and six GII genotypes) were detected. Among the 42 capid gene sequences identified, 14 were from the GI genogroup, while 28 were from the GII genogroup. The most commonly detected genotype was GII.17, comprising 15 (35.7%) of positive samples. From January 2012 to December 2015, 5,138 samples were collected from gastroenteritis patients and outbreaks in Busan. The most detected genotype in 2012, 2013, and 2014 was GII.4 (121, 24, and 12 cases, respectively), but in 2015, GII.17 (25 cases) was the most common. The GII.4 genotype was the major cause of acute gastroenteritis from 2012 to 2014, but the GII.17 genotype became the most prevalent cause in 2015. Continued epidemiological surveillance of GII.17 is needed, together with assessment of the risk of norovirus infection.     

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG₁/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.