Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of ␣-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

Bovine coronavirus (BCV) and hemagglutinating encephalomyelitis virus (HEV) from swine were found to grow to high titers in MDCK I cells, a subline of Madin Darby canine kidney cells. Virus grown in these cells was used to isolate and purify the HE-protein. This protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of BCV. Here we show that HEV contains this enzyme, too. The glycoproteins were solubilized by treatment of virions with octylglucoside. Following centrifugation through a sucrose gradient the surface proteins S and HE (hemagglutinin-esterase) were obtained in purified form. After removal of the detergent by dialysis, HE formed rosettes as shown by electron microscopy. The purified HE protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. HE protein released from the viral membrane failed to agglutinate red blood cells. However, it was found to recognize glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of BCV, HEV, and influenza C virus. The purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses.

The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK i cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-0-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation ofa BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.

The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

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.

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-9-0-acetylneuraminic acid (Neu5,9Acz). We compared the ability of the three coronaviruses to recognize 9-0-acetylated sialic acid and found that they all bind to Neu5,9Acz 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 Neu5,9Acz 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.