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A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258–268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. AnN-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (∼2 × 107virions) and 2 × 106 cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 μM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3′- and 6′-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9.84 μM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).

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. On the basis of these results, enterocytes 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 monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100°C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

Hepatocellular vacuolation can be a diagnostic challenge since cytoplasmic accumulations of various substances (lipid, water, phospholipids, glycogen, and plasma) can have a similar morphology. Cytoplasmic accumulation of phospholipids following administration of cationic amphiphilic drugs (CAD) can be particularly difficult to differentiate from nonphosphorylated lipid accumulations at the light microscopic level. Histochemical methods (Sudan Black, Oil Red-O, Nile Blue, etc.) can be used to identify both nonphosphorylated and/or phosphorylated lipid accumulations, but these techniques require non-paraffin-embedded tissue and are only moderately sensitive. Thus, electron microscopy is often utilized to achieve a definitive diagnosis based upon the characteristic morphologic features of phospholipid accumulations; however, this is a low throughput and labor intense procedure. In this report, we describe the use of immunohistochemical staining for LAMP-2 (a lysosome-associated protein) and adipophilin (a protein that forms the membrane around non-lysosomal lipid droplets) to differentiate phospholipidosis and lipidosis, respectively in the livers of rats. This staining procedure can be performed on formalin-fixed paraffin embedded tissues, is more sensitive than histochemistry, and easier to perform than ultrastructural evaluation.

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