Abstract
BackgroundThe human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion. The noroviruses are classified on the basis of nucleotide sequence diversity in the VP1-encoding ORF2 gene, which divides the majority of human noroviruses into two genogroups (GI and GII). GII-4 noroviruses are the major aetiological agent of outbreaks of gastroenteritis around the world. During a winter season the diversity among the GII-4 noroviruses has been shown to fluctuate, driving the appearance of new virus variants in the population. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B) in the hypervariable P2 domain. We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants.ResultsIn this paper, we describe the construction of recombinant baculoviruses to express VLPs representing one pre-epidemic and one epidemic variant of GII-4 noroviruses, and the production of monoclonal antibodies against them. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding.ConclusionAs predicted by our earlier study, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that are antibody escape mutants.
Highlights
The human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion
Due to the lack of a tissue culture system [17] and suitable animal models in which to study noroviruses, we synthesised recombinant virus-like particles (VLPs) using a baculovirus expression system based on previously described methods [18,19]. These VLPs were used to generate monoclonal antibodies in order to test the functionality of site at positions 886-894 (site A) and site B. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding, and that as predicted, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that represent antibody escape mutants
Construction of Recombinant Baculoviruses Expressing GII-4 Norovirus VLPs Analysis of sequence data for the strains used in this study showed >88% nucleotide identity in the ORF2 gene between strains GII-4v0, GII-4v2 and the reference strain Lordsdale virus (LV), and >90% nucleotide identity between GII-4v0 and GII-4v2 sequences
Summary
The human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B) in the hypervariable P2 domain We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants. The genogroup II-genotype 4 (GII-4) noroviruses have been the dominant circulating strain since the early 1990s [11], and in 2002 a variant GII-4 norovirus emerged that caused unusually high numbers of outbreaks of gastroenteritis in the summer of 2002, and epidemic gastroenteritis around the world in the winter of 2002/2003 [12] This variant possessed a 3 nucleotide (nt) insertion in the hypervariable P2 domain of the VP1 protein at position 6265. This epidemiological pattern was repeated in 2006 when another novel GII-4 norovirus variant emerged, no insertions or deletions were observed in the genome of this virus (J Gray, personal communication)
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