The rotavirus spike protein domain VP8* is essential for recognition of cell surface carbohydrate receptors, notably those incorporatingN-acylneuraminic acids (members of the sialic acid family). N-Acetylneuraminic acids occur naturally in both animalsand humans, whereas N-glycolylneuraminic acids are acquired only through dietary uptake in normal human tissues. The preferenceof animal rotaviruses for these natural N-acylneuraminic acids has not been comprehensively established, and detailedstructural information regarding the interactions of different rotaviruses with N-glycolylneuraminic acids is lacking. In thisstudy, distinct specificities of VP8* for N-acetyl and N-glycolylneuraminic acids werrevealed using biophysical techniques. VP8* protein from the porcine rotavirus CRW-8 and the bovine rotavirus Nebraska clf diarrhea virus (NCDV) showed a preferencefor N-glycolyl-over N-acetylneuraminic acids, in contrast to results obtained with rhesus rotavirus (RRV). Crystallographicstructures of VP8* from CRW-8 and RRV with bound methyl-N-glycolylneuraminide revealed the atomic details of their interactions.We examined the influence of amino acid type at position 157, which is proximal to the ligand's N-acetyl or N-glycolylmoiety and can mutate upon cell culture adaptation. A structure-based hypothesis derived from these results could account forrotavirus discrimination between the N-acylneuraminic acid forms. Infectivity blockade experiments demonstrated that the determinedcarbohydrate specificities of these VP8* domains directly correlate with those of the corresponding infectious virus.This includes an association between CRW-8 adaption to cell culture, decreasedcompetition by N-glycolylneuraminic acid forCRW-8 infectivity, and a Pro157-to-Ser157 mutation in VP8*that reduces binding affinity for N-glycolylneuraminic acid. © 2012, American Society for Microbiology.