*2.1. Recombinant Proteins Production and Purification*

Production and purification of NoV genotypes GI.3 (reference strain Genebank accession no: AF414403) (Figure 1a), GII.4-1999 (AF080551) (Figure 1b), GII.17 (BAR42289.1) (Figure 1c, and GII.4 SYD-2012 (AFV08795.1) (Figure 1d), as well as RV VP6 (GQ477131), used for immunizations, were produced in Sf9 insect cells by a recombinant baculovirus technology and purified in our laboratory as described in details previously [5,36]. VLPs used for analytical methods were produced either in a baculovirus expression system (GI.1-2001 and GII.4 NO-2010) [5,36] or in Nicotiana benthamiana plants (GI.4 and GII.4-2006 VLPs), as previously described [4]. The purity, integrity, and morphology of the VLPs were determined by SDS-polyacrylamide gel electrophoresis, immunoblotting, densitometric analysis, and electron microscopy (Figure 1a–d), as described elsewhere [5]. Protein concentration was determined by using a Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

**Figure 1.** Electron microscopy images of the purified norovirus (NoV) genotypes: (**a**) GI.3, (**b**) GII.4, (**c**) GII.17, and (**d**) GII.4 SYD virus-like particles (VLPs). VLPs were examined by FEI Tecnai F12 electron microscope (Philips 487 Electron Optics, Holland) after negative staining with 3% uranyl acetate, pH 4.6. Bar, 200 nm.
