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In this study, we addressed the question whether cholesterol is important for transmissible gastroenteritis virus (TGEV), a porcine coronavirus, in the initiation of an infection. We found that cholesterol depletion from the cellular membrane by methyl-beta-cyclodextrin (MbetaCD) significantly impaired the efficiency of TGEV infection. Infectivity was also reduced after depleting cholesterol from the viral envelope. This finding is surprising because coronaviruses bud from a pre-Golgi compartment which is expected to be low in cholesterol compared to the plasma membrane. Addition of exogenous cholesterol resulted in a restoration of the infectivity confirming our conclusion that efficient TGEV infection requires cholesterol in both the viral and the cellular membranes. Our data raise the possibility that the viral and cellular proteins involved in the entry process may be associated with cholesterol-rich membrane microdomains.

Transmissible gastroenteritis coronavirus (TGEV) and Escherichia coli K99 are both enteropathogenic for pigs with infections being most severe in neonate animals. For both microorganisms, a sialic acid binding activity has been shown to be an essential pathogenicity factor. Here we demonstrate with haemagglutination and haemagglutination-inhibition assays that TGEV and E. coli K99 differ in their sialic acid binding activities with respect to the type and amount of sialic acid residues required on the erythrocytes surface as well as with respect to the type of sialoglycoconjugate preferentially recognized. Intestinal mucins from piglets (12-14 days old) and adult animals were shown to inhibit TGEV to the same extent. From our results we conclude that E. coli K99 and TGEV interact with different sialoglycoconjugates to establish an intestinal infection. The implications for the enteropathogenicity of TGEV are discussed.

The influenza C glycoprotein HEF was analyzed for acetylesterase activity after SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. Using a histological esterase assay, the glycoprotein was detected as a colored band indicating that it is enzymatically active. The enzyme activity was not affected by low pH, but was abolished after denaturation by SDS as well as after breaking the disulfide bonds by reducing agents. Glycoprotein inactivated by SDS regained its enzyme activity if the ionic detergent was displaced by either bovine serum albumin or a nonionic detergent. The stability of the enzyme combined with the color assay provides a convenient tool to study the acetylesterase activity of the influenza C virus glycoprotein.

The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.