Identification and characterization of an enterocyte receptor for group A porcine rotavirus

Rotaviruses are the most common agents of viral diarrhea in the young of many species, including piglets. A virus-host cell binding assay was developed and used to investigate specific binding between Group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. Enterocyte gangliosides were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialylganglioside (GMX) that displayed a thin-layer chromatographic mobility between that of GM2 and GM3. Inhibition of blocking activity by treatment of GMX with neuraminidase and ceramide glycanase, but not by treatment with protease or heat (100$\sp\circ$C) supported the identification of GMX as a ganglioside. Further purification of GMX by HPLC resulted in the resolution of two monosialylgangliosides, GNM I and GMX II. These gangliosides possess essentially equivalent blocking activity in binding assays. Biochemical characterization of GMX I and GMX II by acid hydrolysis followed by HPAEC-PAD revealed that these gangliosides contain glucose and galactose and either N-acetylneuraminic acid or N-glycolylneuraminic acid in equal molar ratios. These results suggest that GMX I is NeuAc-GM3 and GMX II is NeuGc-GM3. Analysis of the ganglioside content of enterocytes from pigs from newborn to 4 months of age demonstrated that NeuGc-GM3 is present in high concentrations in newborn piglets. The concentration rapidly decreases with advancing age. The concentration of NeuAc-GM3 is lower and remains relatively constant with increasing age. Preincubation of group A porcine rotavirus with 0 to 43.5 nmol/ml (as sialic acid) of NeuGc-GM3 or NeuAc-GM3 results in a dose dependent reduction in infectivity in MA-104 cells by up to 90%. These data suggest NeuGc-GM3 and NeuAc-GM3 may be in vivo relevant receptors for Group A porcine rotavirus.U of I OnlyETDs are only available to UIUC Users without author permissio