Comparisons of nucleotide and deduced amino acid sequences of NSP4 genes of virulent and attenuated pairs of group A and C rotaviruses

The NSP4 protein of rotavirus is a nonstructural glycoprotein and has a cru cial function in virus morphogenesis during infection of host cells. It was recently reported that NSP4 may also function as a viral enterotoxin in th e induction of rotavirus diarrhea by causing Ca++ influx in the cytoplasm o f the infected cells. We sequenced and analyzed two (Wa and M strains) pair s of NSP4 genes of virulent (v) and attenuated (a) (after 30 to 40 passages in cell culture) human group A rotaviruses and a pair of NSP4 genes of vir ulent and attenuated porcine group C rotavirus (Cowden strain). These strai ns were previously identified as virulent (induce diarrhea) or attenuated ( no diarrhea) in a gnotobiotic pig model of rotavirus infection [Bohl et al. (4), Saif et al. (13), Ward et al. (17)]. The NSP4 genes of the Wa, M and Cowden strains were amplified with RT-PCR using a proof reading polymerase (Tli) and the RT-PCR product was sequenced directly. Analysis of the NSP4 d educed amino acid sequences showed that only 3 (Wa) and 2 (M and Cowden) am ino acids differed between the virulent and attenuated strains. For the Wa strain, the changes from the virulent to attenuated strain were in amino ac ids 13 (V to A), 16 (L to S) and 34 (P to L); in the M strain, the differen ce was in amino acids 53 (T to I) and 104 (K to E), and in the Cowden strai ns, amino acids 50 (L to F) and 97 (D to N) differed between virulent and a ttenuated strains. To our knowledge, this is the first sequence comparison between NSP4 of a virulent and attenuated pair of group C rotaviruses. The potential impact of these few amino acid changes on the pathogenesis of the NSP4 protein for piglets is unclear, relative to previous findings in mice (1), but requires further study using purified recombinant NSP4 proteins o r peptides.