Advances in Laboratory Methods for Detection and Typing of Norovirus

Vinjé, Jan

doi:10.1128/jcm.01535-14

Cited by 680 publications

(606 citation statements)

References 38 publications

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“…Noroviruses are genetically diverse and can be divided into seven genogroups (GI-GVII) that are further subdivided into numerous genotypes [7]. The GII genotype 4 (GII.4) includes most epidemic and pandemic strains, while GII.17 was recently attributed with major outbreaks in East Asia [8].…”

Section: Introductionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. PLOS Author summaryWe determined the binding sites of six novel human norovirus specific Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies (Nano-4, Nano-26, Nano-27, and Nano-42) bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding regio...

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Section: Introductionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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“…The expression of the capsid protein in insect cells leads to the self-assembly of virus-like particles (VLPs) that are morphologically and antigenically similar to the native virions (1). Based on the capsid gene sequences, at least seven genogroups (GI to GVII) have been assigned (2,3). The genogroups can be further subdivided into numerous genotypes, and an association between genetic clusters and antigenicity is evident (1).…”

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confidence: 99%

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Koromyslova

Hansman

2015

J Virol

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Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ϳ10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCEWe characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.

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“…Norovirus GI.2 was identified in the four stool samples. This strain of human origin [11], was possibly introduced by one person or more of the team preparing the path or by participants in the first waves. In spite of the negative tests for norovirus in the muddy water, we cannot exclude that it was contaminated with such viruses [12].…”

Section: Discussionmentioning

confidence: 99%

Outbreak of diarrhoeal illness in participants in an obstacle adventure race, Alpes-Maritimes, France, June 2015

et al. 2016

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An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Côte d’Azur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bacteria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers.

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Advances in Laboratory Methods for Detection and Typing of Norovirus

Cited by 680 publications

References 38 publications

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Outbreak of diarrhoeal illness in participants in an obstacle adventure race, Alpes-Maritimes, France, June 2015

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