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Substitution and reactivity of NV aa 514 and 472. Deletion mutant NV 454-520 was changed at the indicated positions using site-directed mutagenesis, and mutant proteins were analyzed by Western blotting with anti-GST antiserum (top panel), anti-NV VLP antiserum (middle panel), or MAb NV3901 (bottom panel). GST, purified GST protein; rNV VLPs, purified Norwalk VLPs.
Source publication
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
reagen...
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Context 1
... shows that the lysine at position 514 can interact with the glutamic acid at position 472, potentially forming a salt bridge. The glutamic acid at position 472 is conserved in both GI and GII viruses. A series of point muta- tions at these two positions was generated to test the impor- tance of this interaction for MAb NV3901 and NV3912 bind- ing (Fig. 4). An alanine substitution at position 472 abolished MAb NV3901 binding. The substitution of a conservative ar- ginine at position 514 was sufficient to restore binding; how- GST, purified GST protein; rNV VLPS, purified Norwalk ...
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Snow Mountain virus (GII.2.1976) is the prototype strain of GII.2 noroviruses, 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...
Application of reverse transcription (RT)-PCR to detect small round-structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polyme...
Citations
... Every 2 to 3 years, new GII.4 variants emerge and replace the previously predominant strains (Chhabra et al., 2019;Kroneman et al., 2013). In 1995, GII.4 pandemic first documented by GII.4 US95_96 variant, followed by the emergence of GII.4 Farmington Hills in 2002, GII.4 Hunter in 2004, GII.4 Yerseke and GII.4 Den Haag in 2006, GII.4 New Orleans in 2009, and GII.4 Sydney in 2012(Kroneman et al., 2013Vinjé, 2015;Tohma et al., 2019) as well as several local epidemics of GII.4 variants such as Grimsby 1995, Henry 2001, Japan 2001, Asia 2003, 2006a, Cairo 2007, Apeldoorn 2008, and Japan 2008(Tohma et al., 2019Tu et al., 2008;Parker et al., 2005;Seto et al., 2005;Belliot et al., 2010;Kamel et al., 2009;Motomura et al., 2010). Recombination, as a major driving force of norovirus diversity, may further increased the emergence of novel strains (Chhabra et al., 2019). ...
The present study was aimed to both detect emerging noroviruses and investigate RdRp and VP1-based dual typing of circulating noroviruses in hospitalized children less than 5 years of age with acute gastroenteritis (AGE) in Iran. For this purpose, a total of 200 stool specimens were screened during 2021-2022 by real-time RT-PCR for genogroup I and II (GI and GII) and dual-typed by sequence analysis of PCR products, using a web-based norovirus Typing Tool and phylogenetic analysis. The GI and GII noroviruses were detected in 20% of 200 specimens. The GII.4 norovirus was found to be the most common VP1 genotype (53%) followed by GII.8 (32%), GII.7 (6%), GII.17 (6%), and GII.3 (3%). The GII.P16 norovirus was also found as the predominant RdRp type (53%) followed by GII.P8 (32%), GII.P7 (6%), GII.P17 (6%), and GII.P31 (3%). To our knowledge, this is the first report that highlights the dominancy of recombinant norovirus GII.4Sydney[P16] and newly emerging of norovirus GII.8 [P8], GII.17 [P17] and GII.3 [P16] in Iran. These findings further indicate inter-genotype recombinant strains of noroviruses.
... The results obtained from such analysis provide a valuable profile for the development of vaccines. At present, the search for the B-cell linear epitope of NoV is mainly conducted using recombinant virus-like particles (VLPs) and VLP-derived monoclonal antibodies [19][20][21][22]. Anti-HNoV monoclonal antibodies purified from human blood are used in the epitope analysis of HNoV [23]. ...
... Structural analysis of GVI FNoV VP1 showed that the peptide containing epitope A1 formed a prominent loop region. Shanker et al. [20] reported that human IgA monoclonal antibody (IgA 5I2) with binding inhibitory activity against GI HNoV and HBGA recognize three loop structures (Loop T, Loop Q, and Loop U) exposed on the surface of the P2 subdomain of VP1.We speculate that the Loop containing P-10 is homologous to these Loop T. In addition, epitope D, one of the blockade epitopes of GII HNoV, is equivalent to a part of Loop T. Given this information, it is suggested that steric hindrance or allosteric HBGA blockade epitope of FNoV is likely to be present in P-10. ...
Norovirus (NoV) infection remains a major public health concern worldwide. Appropriate animal models are essential for the development of effective NoV vaccines. We previously established the feline NoV (FNoV)-cat model as a surrogate animal model for human NoV infection. In the present study, we analyzed the B-cell linear epitope in the P domain of FNoV to confirm the basic immunological features of the FNoV-cat model. B-cell linear epitopes were present in the P2 subdomain. We compared antibody levels to peptides containing the B-cell linear epitope (P-10) in three FNoV-infected cats with time-course changes in viral load and symptom scoring. After FNoV infection, viral shedding and clinical symptoms were shown to improve by elevated levels of antibodies against P-10 in the plasma. This report provides important information for understanding NoV infections in humans and cats.
... All mAbs recognising these epitopes (previously produced from viruslike particles (VLPs) or recombinant NoV capsid protein) have been shown to be broadly reactive (Li et al., 2009;Parra et al., 2013). Most cross-reactive mAbs that recognise various NoV genogroups have been reported to target the S domain and P1 domain (Crawford et al., 2015;Hansman et al., 2012;Huang et al., 2014;Li et al., 2009;Parker et al., 2005;Parra et al., 2013;Zheng et al., 2018). However, since the S domain is localised at the capsid interior, parts of it can be less accessible to antibodies. ...
Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 10⁵ gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.
Graphic Abstract
... Each protein sample was mixed with an equal volume of 2 × sodium dodecyl sulfate (SDS) loading buffer (0.125 M of Tris-HCl pH 6.8, 4% w/ v of SDS, 20% v/v of glycerol, 0.01% of bromophenol blue, and 0.01% w/v of 2-mercaptoethanol) and heated at 100 • C. Samples were separated on 12% SDS-polyacrylamide gels and stained with CBB before transfer to the polyvinylidene difluoride membrane (Merck, Tokyo, Japan). After transfer, the membrane was blocked with 5% (w/v) skim milk (Wako, Tokyo, Japan) in TBS + 0.1% Tween 20 (TBS-T) followed by anti-NoV-VP1 (NS14) mouse monoclonal antibody (mAb) [39] as a primary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (MBL, Tokyo, Japan) as a secondary antibody. The specific protein bands were visualized by applying Immobilon Western Chemiluminescent HRP substrate (Merck, Tokyo, Japan) on the membrane. ...
Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 54.4% (equivalent to 930 μg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.
... In fact, the outermost region of the capsid protein sequence is the most variable segment of the norovirus genome [2]. This creates a challenge when conventional ligands, such as monoclonal antibodies, are used because the antibodies generally lack cross-reactivity with all viral strains [4][5][6]. Alternatively, carbohydrates known as "histo-blood group antigens" have been used as ligands for the virus, as these carbohydrates are suspected to be co-receptors for a number of human norovirus strains. ...
... This may be expected, as the VPg protein present in infectious capsids is needed for replication and typically more conserved than the variable major capsid protein targeted by many traditional detection ligands ( Figure S5) [22,25,37]. This is promising news for both detection and therapeutic applications, as one of the major obstacles to detection for these viruses has been the ability to generate a ligand with broad specificity for the entire diverse group of noroviruses [4][5][6]. Strong binding to both a VPg from the genus Norovirus as well as the proposed genus Recovirus further suggests the promise of the VPg protein as both a detection target and a therapeutic target for public health. Further work should be conducted to better elucidate the degree to which these aptamers bind the VPgs of other caliciviruses, including murine norovirus (another cultivable surrogate in genogroup V) and other genotypes of human noroviruses. ...
Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G < −5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative > 5.0), and all but two of the candidates displayed very strong binding (positive:negative > 10.0), significantly higher than binding to the negative control protein (p < 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application.
... VP1 consists of a shell (S) domain and a protruding (P) domain. The S domain is the most highly conserved region in VP1, and forms a shell surrounding the RNA genome, while the P domain of VP1 contains the most variable sequence [1][2][3]. HuNoVs are divided into 5 genogroups based on the VP1, including genogroup I (GI), II (GII), IV (GIV), VIII (GVIII), and IX (GIX) [4]. GI and GII HuNoVs are the major epidemic strains circulating worldwide [5]. ...
Background
Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks.
Results
In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 10 ⁶ genomic copies per gram of stool sample (gc/g) and 4.4 × 10 ⁵ gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6–84.8%), 100.0% (95% CI: 98.5–100.0%) and 87.7% (95% CI: 85.6–89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.
Conclusions
This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.
... Therefore, MAb 8C7 and the antibodies from rabbit sera may recognize similar sequences (50). In addition, the epitopes for MAbs 3901 and 3912 have been mapped within the residue 461 to 479 region, suggesting that the MAbs also recognize the epitope found in the rabbit sera (54). It should also be noted that, although we have described five epitopes for the rabbit sera, this is a conservative estimate. ...
NoV infections are a leading cause of gastroenteritis in the United States. Human NoVs exhibit extensive genetic and antigenic diversity, which makes it challenging to design a vaccine that provides broad protection against infection. Antibodies developed during the immune response play an important role in the control of NoV infections. Neutralizing antibodies that act by sterically blocking the site on the virus used to bind human cells have been identified. Identification of other antibody binding sites associated with virus neutralization is therefore of interest. Here, we use a high-resolution method to map multiple antibody binding sites simultaneously from complex serum samples. The results show that a relatively small number of sites on the virus bind a large number of independently generated antibodies, suggesting that immunodominance plays a role in the humoral immune response to NoV infections.
... Structural analysis of the antibody Fab fragments in complex with the P domain of GII.4 (2002 Farmington Hills strain) revealed cross GI and GII reactive antibodies to target a site on the P domain that would be completely buried in the context of the intact viral particle 17 . Similar antibodies (herein referred to as occluded-site antibodies) have been previously observed after immunization, with norovirus VLPs in mice [18][19][20] . Two questions arise: how can such antibodies be elicited, and can vaccine performance be improved by preventing their elicitation? ...
... Conversely, the blocking antibody 512 could be found in complex with both intact particles and VP1 dimers. This suggests a potential route for the elicitation of cross-reactive but nonneutralizing antibodies, previously isolated from humans and mice immunized with norovirus VLPs [17][18][19] . When animals are immunized with VLPs, the partially dissociated particles expose highly conserved sites (mostly located at the base of the P domain and Notice that the sizes of the intact particles increase by~7 nm, consistent with the presence of a layer of Fabs bound to each particle. ...
Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.
... VP1 consists of a shell (S) domain and a protruding (P) domain. The S protein, the most highly conserved region in VP1, forms a shell surrounding the RNA genome, while the P protein contains the most variable sequence [1][2][3]. HuNoVs are divided into 5 genogroups based on the VP1, including genogroup I (GI, n=9), GII (n=27), GIV (n=2), GVIII (n=1), and GIX (n=1) [4]. GI and GII HuNoVs are the epidemic strains circulating worldwide [5]. ...
Background: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all ages worldwide. As the replication of HuNoVs in vitro is immature, the detection of HuNoVs is depended on molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced operators to perform, and extended time to get results. In addition, the genotypes of HuNoVs are broad. Therefore, a method for rapidly, broad-range, and effective approach for HuNoVs would be more applicable for screening infected people when outbreaks occur.
Results: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for highly effective detection of HuNoVs in clinical samples. Monoclonal antibodies (McAbs) against the shell (S) domain in the major capsid protein of HuNoV were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2×10⁶ genomic copies per gram (gc/g) and 4.4×10⁵ gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 samples were tested for HuNoVs by ICA and compared against that by RT-qPCR. The relative sensitivity, specificity and agreement of the ICA was 84.2 % (95% CI: 83.6-84.8 %), 100.0 % (95 % CI: 98.5-100.0 %) and 87.7 % (95% CI: 85.6-89.8 %), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA could detect a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.
Conclusions: Our results demonstrated that ICA targeting the S domain protein is a promising candidate for effectively improve identifying different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.
... VP1 consists of a shell (S) domain and a protruding (P) domain. The S domain, the most highly conserved region in VP1, forms a shell surrounding the RNA genome, while the P domain of VP1 contains the most variable sequence [1][2][3]. HuNoVs are divided into 5 genogroups based on the VP1, including genogroup I (GI), II (GII), IV (GIV), VIII (GVIII), and IX (GIX) [4]. GI and GII HuNoVs are the epidemic strains circulating worldwide [5]. ...
Background: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all ages worldwide. As the replication of HuNoVs in vitro is immature, the detection of HuNoVs is depended on molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced operators to perform, and extended time to get results. In addition, the diversity of viral genotypes is high. Therefore, a method for rapidly, broad-range, and effective detectionf of HuNoVs was desiderated for screening the excrement or vomit from infected people when outbreaks occur.
Results: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for highly effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2×10⁶ genomic copies per gram of stool sample (gc/g) and 4.4×10⁵ gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against that by RT-qPCR. The relative sensitivity, specificity and agreement of the ICA was 84.2 % (95% CI: 83.6-84.8 %), 100.0 % (95 % CI: 98.5-100.0 %) and 87.7 % (95% CI: 85.6-89.8 %), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA could detect a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.
Conclusions: Our results demonstrated that ICA targeting the S domain of VP1 is a promising candidate for effectively improve identifying different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.
