Magic™ Anti-Norovirus VP1 Monoclonal antibody

Background: Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. Objective: To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. Study design: An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Results: Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. Discussion and conclusions: The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons. (C) 2011 Elsevier B.V. All rights reserved.

Noroviruses (NoVs) are increasingly recognized as the leading cause of acute non-bacterial gastroenteritis worldwide. To screen for NoV-specific monoclonal antibodies (mAbs) with wide spectrum binding activities that could be used for the development of NoV-related detection reagents, we immunized mice with a combination of virus like particles (VLPs) derived from eight different genotypes (two from genogroup I and six from genogroup II), of which two (GI.7 and GII.2) were newly produced VLPs. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that two mAbs (8D8 and 10B11) bound to all eight major capsid proteins (VP1) with varied binding abilities. Epitope mapping using short peptides covering the N-terminal half of GII.3 VP1 indicated that the binding site of mAb 8D8 was located between amino acid 31 and 60. Multiple amino acid sequence alignment of VP1 suggested that this site harbors conservative sequences across all genogroups. Indirect and sandwich ELISA indicated that mAb 8D8 was unable bind intact VLPs. In summary, we successfully produced GI.7 and GII.2 VLPs using recombinant baculovirus expression system and a cross-reactive mAb by immunizing mice with eight different VLPs that might be useful in the studying and detecting NoVs.