Comparative analysis of South African norovirus GII.4 strains identifies minor recombinant variants

Recombination within the norovirus (NoV) GII.4 genotype is well documented as a mechanism by which novel variants evolve. Norovirus GII.4 has been the predominant NoV genotype detected in South Africa (SA) in recent years and putative NoV recombinants were previously identified in SA based on partial regions of the viral genome. The objective of this study was to determine the complete genome sequence of representative NoV GII.4 variants that have circulated in SA between 2009 and 2013 and to compare major and minor GII.4 variants based on nucleotide sequence. The complete genomes of 11/27 GII.4 strains could be amplified in three or five overlapping segments, these included major variants New_Orleans_2009 and Sydney_2012 as well as three types of minor GII.4 variants. Phylogenetic and recombination analysis identified GII.4 recombinants with breakpoints located at or near the ORF1/2 junction. Apart from recombinants already recognised as major variants (GII.P4 New_Orleans_2009/GII.4 Sydney_2012 (n=2) and GII.Pe/GII.4 Sydney_2012 (n=2)) four further recombinant strains were detected (GII.P4 New_Orleans_2009/GII.4 Hunter_2004 (n=1) and GII.P4 Yerseke_2006a/GII.4 Apeldoorn_2007 (n=3)) that were attributed to three distinct minor variants. The encoded proteins with the highest diversity were p48 (Nterm), p22, VP1 and VP2. Analysis of antigenic sites in VP1 revealed mutations at epitopes A, B, C, D and E, with epitopes A and D being most variable. The high variation at epitope D was reflected in structural differences in models of GII.4 variants in the epitope D loop region (aa 393-395). Major and minor variants could not be distinguished based on specific sequence differences. HBGA-binding studies will be necessary to assess the effect of the observed amino acid differences in the P2 domain of these GII.4 strains.