A variety of studies using time ordered human outbreak sera and mouse monoclonal antibodies support the hypothesis that the GII.4 noroviruses are undergoing antigenic variation and that this variation contributes to the emergence of new outbreak strains over time [13, 22, 33, 34] . However, the lack of a cell culture or small animal model for human norovirus cultivation restricts study of neutralization antibodies and epitopes. To circumvent this problem, highly informative in vitro assays have been developed that measure the ability of an antibody to ''block'' binding of a VLP to a carbohydrate ligand [13, 35, 36] . This assay is highly sensitive, as it differentiates between norovirus strains too similar to be distinguished by enzyme immunoassay (EIA). The clinical relevance of the blockade assay, as a surrogate neutralization assay, has been confirmed in both infected chimpanzees [37] and Norwalk virus-infected humans [10, 38] . For NoV strains that hemagglutinate RBCs, high blockade antibody titers that prevent hemagglutination also correlate with protection from disease in humans [39] . Using human norovirus outbreak sera, VLPimmunized mouse sera, and mouse mAbs [13, 33, 34] , the early GII. 4 strains (1987 and 1997) were antigenically indistinguishable from each other by EIA and surrogate neutralization assays. VLPs of strains circulating post 2002 had significantly less reactivity with sera directed against earlier strains and no reactivity to mouse mAbs directed to GII.4.1987 . Conversely, select mouse mAbs generated against GII.4.2006 reacted with VLPs that circulated only from 2002 or later. No blockade epitopes were found to be in common between GII. 4.1987 and GII.4.2006. Prior to this work, we and others had predicted GII.4 antibody epitopes using bioinformatic techniques. As expected, discreet amino acids are repeatedly identified as potential evolving epitopes. In particular, residues 296-298 and 393-395 are consistently identified as putative epitopes that change between epidemic GII.4 strains. Additional surface residues at 333, 340, 356, 368, 372, 407 and 412-413 were also predicted as potential antibody epitopes [13, 18, 19, 22, 28, [40] [41] [42] . These amino acids tend to cluster on loops and ridges of the P2 subdomain where antibody interaction would be most accessible. Beyond bioinformatics predictions, only a few studies have shown empirical evidence mapping GII.4 antigenic change. Allen et al. [42] compared the reactivity of five monoclonal antibodies against a pre and post-2002 epidemic GII.4 strain, and identified conformational epitopes composed of residues 294-296 and 393-395. The carbohydrate blockade potential of these antibodies was not reported, so the role of these sites in escape from herd immunity was unclear. However, the finding that residues 393-395 were antigenically important supported our previously published work identifying amino acid 395 as an antigenic determinant in the GII.4.2002 Farmington Hills strain [13] . Using mouse mAbs and molecular biology approaches to exchange predicted epitopes between GII.4 strain backbones, we have clearly identified amino acids 294, 296-298, 368 and 372 to comprise an evolving blockade epitope, as exchange of these amino acids from GII.4.1987 into GII.4.2006 conferred binding of mAbs that recognize GII.4.1987 but not GII.2006 [43] . Extending this approach, we have also confirmed amino acids 407, 412 and 413 to constitute a GII.4.2002 Farmington Hills-specific blockade epitope [17] . These empirical studies support the validity of using computational analysis to guide norovirus epitope studies.