Human monoclonal antibodies clearly identified two evolving epitopes on the surface of the GII.4 VLP. Epitopes A and D were both confirmed as evolving GII.4 antibody blockade epitopes using chimeric VLPs containing a mixture of epitopes derived from early or contemporary strains. We recognize that Epitopes A and D have not been structurally defined as epitopes, supporting the need for structural studies to define the exact mAb binding site on the VLPs. Moreover, we focused on discrete regions of varying residues and all of the residues within 8 Å (representing approximately 201 Å 2 ) to the primary sites that may be influenced directly by an amino acid replacement ( Figure 9B ). However, Ab binding sites have been reported to be much larger (700-800 Å 2 ), suggesting that the epitopes that we have predicted may actually work in concert to form larger Ab recognition sites. By expanding the putative epitopes, we identified other residues that were less variable; however, the exact role that these varying residues play in evolution is less clear. In some cases variation may be required to encode changes that are necessary for the replacement at a primary site. In addition, all of our structural analyses have been conducted using models of the P dimer, representing about 1/90 th of the VLP structural surface. Interactions between the epitopes in the context of the superstructure have not been determined. Therefore, these observations indicate that the complex nature of the NoV Ab epitope requires further research to define the specific boundaries and residues that regulate Ab binding.