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Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.

Noroviruses are leading cause of acute gastroenteritis worldwide. In our previous study, we established an in vitro histo-blood group antigens (HBGAs) binding blockade assay against GII.3 Norovirus virus like particles (VLPs) with trypsin digestion. In this study, we characterized the blocking antibody binding site and epitope type (linear or conformational) by using hyperimmune sera produced against different antigens. VP1 from Jingzhou402 (GII.3, JZ402) strain was expressed by using pGEX-6p-1 expression vector and the insoluble proteins were purified for immunization in rabbit. Previously characterized chimeric VP1-assembled VLPs (GII.4-VP1/GII.3-P2) were used to immunize guinea pig. Peptides reactive with hyperimmune serum against VLPs derived from the VP1 of JZ402 strain were conjugated with BSA and used to immunize rabbits. Hyperimmune sera against above antigens and JZ402 and JZ403 strain-derived VLPs were used to compare their HBGAs blocking effects. Rabbit anti-GST-VP1 and BSA-peptide conjugated hyperimmune sera demonstrated no blocking effects against the binding of GII.3 and GII.4 NoV VLPs to salivary HBGAs. Guinea pig anti-GII.4-VP1/GII.3-P2 hyperimmune serum blocked the binding of trypsin cleaved GII.3 VLPs to salivary HBGAs with no or very weak blocking effects against the binding of GII.4 VLPs to salivary HBGAs. Our data indicated that HBGAs blocking antibodies primarily bound the P2 domain of GII.3 NoV VP1 and their binding epitopes were most probably conformation-dependent.

Genogroup II, genotype 3 noroviruses (GII.3 NoVs) are secondary to GII.4 NoVs in causing acute non-bacterial gastroenteritis worldwide. In our previous study, we found that virus-like particles (VLPs) derived from a GII.3 NoV strain exhibited no binding activity to any salivary and synthetic histo-blood group antigens (HBGAs) tested. In this study, the nucleotide sequence encoding the major capsid protein of another documented GII.3 NoV strain was codon-optimized and synthesized, and the major capsid protein was expressed using recombinant baculovirus virus expression system. The assembly of VLPs was verified by electron microscopy, and the binding profiles of the assembled VLPs to salivary HBGAs were determined, and in vitro VLP-salivary HBGAs binding blockade assay was used to test the cross-blocking effects of hyperimmune sera produced against different genotypes (GI.2, GII.3, and GII.4). The expression of the major capsid proteins led to the successful assembly of VLPs, and in vitro VLP-salivary HBGAs binding assay indicated that the assembled VLPs bound to salivary HBGAs from blood type A, B, AB, and O individuals, with the highest binding capacity to type A salivary HBGAs. In vitro VLP-salivary HBGAs binding blockade assay demonstrated the absence of blocking activities for hyperimmune sera produced against GI.2and GII.4 VLPs and the presence of blocking activity for that against GII.3 VLPs. Our results suggest the absence of cross-blocking activities among different genotypes and the presence of blocking activities between GII.3 NoVs from different clusters, which might have implications for the design of multivalent NoV vaccines.

A newly emerged pandemic Sydney GII.4-like norovirus (NoV) (Jingzhou GII.4) was isolated in Jingzhou, China in April, 2013, demonstrating the rapid spread of the variant to China. The complete nucleotide sequence was compared with the prototype Sydney 2012 variant and its VP1 gene with that of Huzhou strain (isolated in January 2013 in Huzhou, China). The result demonstrates that the new variant has evolved rapidly, including mutations in the hypervariable P2 domain of the major capsid protein VP1. Our study also shows that the new Jingzhou GII.4 variant co-circulated with GII.3 and GI.2 at the same time, supporting further monitoring of the evolution of the new NoV variant in China.