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Porcine transmissible gastroenteritis virus (TGEV) has been shown to agglutinate erythrocytes using a2,3-linked sialic acid on the cell surface as binding site. The hemagglutinating activity requires the pretreament of virus with neuraminidase. We obtained evidence that TGEV recognizes not only N-acetylneuraminic acid but also N-glycoloylneuraminic acid, a sialic acid present on many porcine cells.

Bovine coronavirus (BCV), human coronavirus OC43 (HCV-OC43) and hemagglutinating encephalomyelitis virus (HEV) are serologically related viruses that all have hemagglutinating activity. The receptor determinant for attachment to erythrocytes has been shown to be N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). We compared the ability of the three coronaviruses to recognize 9-O-acetylated sialic acid and found that they all bind to Neu5,9Ac2 attached to galactose in either A2,3 or A2,6-linkage. There are, however, some differences in the minimum amount of sialic acid that is required on the cell surface for agglutination by these viruses. Evidence is presented that HCV-OC43 uses Neu5,9Ac2 as a receptor determinant not only for agglutination of erythrocytes but also for attachment to and infection of a cultured cell line, MDCK I cells.

The designation myxoviruses has been chosen historically for a group of viruses comprising influenza viruses, Newcastle disease virus, and mumps virus, because they were able to interact with mucins (see Chapter 5). A characteristic feature of these viruses was the presence of two activities that appeared to counteract each other. On the one hand, the viruses bind to receptors present on erythrocytes, resulting in a hemagglutination reaction. On the other hand, they contain a receptor-destroying enzyme rendering erythrocytes resistant to the viral agglutinating activity. The receptor determinant recognized by this group of viruses turned out to be sialic acid, and the receptor-destroying enzyme has been characterized as a sialidase and a sialate O-acetylesterase. Despite the similarities, members of the myxovirus group differ in several fundamental aspects, e.g., the presence of a segmented or a nonsegmented genome. Therefore, they have been grouped into two taxonomic families. Influenza A, B, and C viruses belong to the family Orthomyxoviridae. Viruses such as mumps virus, Newcastle disease virus, Sendai virus, and other parainfluenza viruses are members of the family Paramyxoviridae. In addition to the receptor-binding and the receptor-destroying activity, these viruses have a fusion activity. They differ, however, in the distribution of the three activities on the viral surface glycoproteins. With influenza A and B viruses, receptor-binding and fusion activity are functions of the hemagglutinin (HA). A second glycoprotein (NA) is responsible for the sialidase activity. In the case of paramyxoviruses, the receptor-binding and the sialidase activity are combined on one type of glycoprotein designated HN, whereas the fusion activity is localized on a separate glycoprotein (F). Influenza C viruses have only a single glycoprotein that is responsible for all three activities. They also differ from the other viruses in that they recognize N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) rather than N-acetylneuraminic acid (Neu5Ac) as receptor determinant. Furthermore, the receptor-destroying enzyme of influenza C viruses is a sialate 9-O-acetylesterase. In this chapter, the interaction of viral proteins with sialic acid and the biological significance of the receptor-binding and the receptor-destroying activity are discussed.

Epithelial cells are highly polarized cells divided into an apical and a basolateral plasma membrane. The two domains are composed of a distinct set of proteins and lipids. Concerning virus infection of epithelial cells, the polarity of host cell receptor distribution defines the domain from which infection may be mediated. We were interested to analyze the infection of polarized cells by bovine coronavirus (BCV). The entry of BCV into MDCK I cells was investigated by growing the cells on a permeable support. Cell were infected with BCV from either the apical or basolateral domain. The efficiency of infection was determined my measuring the hemaglutinating activity of the virus released into the apical compartment. Virus replication was only detectable after inoculation from the apical surface. Therefore, infection of MDCK I cells with BCV is restricted to the apical side.

Porcine transmissible gastroenteritis virus (TGEV) was found to resemble avian infectious bronchitis virus (IBV) in its interaction with erythrocytes. Inactivation of the receptors on erythrocytes by neuraminidase treatment and restoration of receptors by reattaching N-acetylneuraminic acid (Neu5Ac) to cell surface components indicated that alpha 2,3-linked Neu5Ac serves as a receptor determinant for TGEV as has been reported recently for IBV. Similar to IBV, the haemagglutinating activity of TGEV is evident only after pretreatment of virus with neuraminidase indicating that inhibitors on the virion surface have to be inactivated in order to induce the HA-activity of these viruses. A model is presented to explain why the HA-activity of untreated virus is masked and how neuraminidase treatment results in the unmasking of this activity.

The S protein of hemagglutinating encephalomyelitis virus is shown to be a hemagglutinin requiring N-acetyl-9-O-acetylneuraminic acid as a receptor determinant on the surface of erythrocytes. The ability of bovine coronavirus to recognize 9-O-acetylated sialic acid was used to establish a binding assay for the detection of glycoproteins containing this type of sugar. The assay is very fast, because it uses the acetylesterase of the viral HE protein to localize bound virus.

Receptor-destroying enzymes have long been thought to be present only on influenza viruses and paramyxoviruses but not on other animal viruses. Paramyxoviruses as well as influenza A and B viruses are able to inactivate their own receptors by virtue of a neuraminidase which cleaves terminal sialic acid from glycoproteins or gangliosides (Drzeniek, 1972). The corresponding enzyme of influenza C virus is a sialate 9-0-acetylesterase which releases acetyl residues from position C-9 of N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2) (Herrler et al., 1985). Recently, sequencing of the genome of mouse hepatitis virus revealed an open reading frame which surprisingly showed some similarity to the glycoprotein HEF of influenza C virus (Luytjes et al., 1988). This finding prompted a search which resulted in the discovery that bovine Coronavirus (BCV) contains a sialate O-acetylesterase similar to the receptor-destroying enzyme of influenza C virus (Vlasak et al., 1988a). This enzyme is inhibited by diisopropylfluorophos-phate (DFP), which binds covalently to serine in the active-site of serine proteases and esterases. By using a radioactive form of this inhibitor protein E3 of BCV was identified as esterase (Vlasak et al., 1988b). The same glycoprotein has been shown previously to be involved in the hemagglutinating activity of BCV (King et al., 1985). Therefore, E3 is designated HE.

Only little information is available about the initial events in the infectious cycle of coronaviruses. Attachment of strain A59 of mouse hepatitis virus to the cell surface has been shown to be mediated by a protein of about 110 kDalton (Boyle et al., 1988). Recently, bovine Coronavirus (BCV) and human corona-virus OC43 (HCV-OC43) have been shown to recognize receptors on erythrocytes similar to influenza C virus. The evidence is based on the finding that both influenza C virus and BCV contain a sialate O-acetylesterase, which functions as a receptor-destroying enzyme (Vlasak et al, 1988). Incubation of chicken red blood cells with the esterase of either virus renders the erythrocytes resistant to agglutination by both viruses as well as by HCV-OC43. This result indicates that N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) is a receptor determinant for attachment of BCV and HCV-OC43 to erythrocytes as has been shown previously for influenza C virus (Rogers et al., 1986). Here we confirm the importance of Neu5,9Ac2 for the binding of BCV to the cell surface by resialylation of erythrocytes. We present evidence that strain Johannesburg/1/66 of influenza C virus is more efficient in recognizing Neu5,9Ac2-containing receptors than BCV.