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Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry. These viruses include important human and animal pathogens, such as influenza, parainfluenza, mumps, corona, noro, rota, and DNA tumor viruses. Attachment to sialic acid is mediated by receptor binding proteins that are constituents of viral envelopes or exposed at the surface of non-enveloped viruses. Some of these viruses are also equipped with a neuraminidase or a sialyl-O-acetyl-esterase. These receptor-destroying enzymes promote virus release from infected cells and neutralize sialic acid-containing soluble proteins interfering with cell surface binding of the virus. Variations in the receptor specificity are important determinants for host range, tissue tropism, pathogenicity, and transmissibility of these viruses.

The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV) and the spike protein of infectious bronchitis virus (IBV). Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

Avian Infectious bronchitis virus (IBV) is a coronavirus that infects chickens via the respiratory epithelium as primary target cells. The binding of coronaviruses to the cell surface is mediated by the viral surface protein S. Recently we demonstrated that alpha2,3-linked sialic acid serves as a receptor determinant for IBV on Vero cells and primary chicken embryo kidney cells. Here we analyze the importance of the sialic acid binding activity for the infection of tracheal organ cultures (TOCs) by different IBV strains. Our results show that alpha2,3-linked sialic acid also serves as a receptor determinant on chicken TOCs. Infection of TOCs by IBV results in ciliostasis. Desialylation induced by neuraminidase treatment of tracheal organ cultures prior to infection by IBV delayed the ciliostatic effect or resulted in partial loss of ciliary activity. This effect was observed with both respiratory and nephropathogenic strains. Inhibition of ciliostasis was also observed when TOCs were pretreated with an alpha2,3-specific neuraminidase. Analysis of the tracheal epithelium for reactivity with lectins revealed that the susceptible cells in the epithelium abundantly express alpha2,3-linked sialic acid. These results indicate that alpha2,3-linked sialic acid plays an important role for infection of the respiratory epithelium by IBV.

The importance of sialic acid for infection by avian Infectious bronchitis virus (IBV) has been analysed. Neuraminidase treatment rendered Vero, baby hamster kidney and primary chicken kidney cells resistant to infection by the IBV-Beaudette strain. Sialic acid-dependent infection was also observed with strain M41 of IBV, which infects primary chicken kidney cells but not cells from other species. In comparison with Influenza A virus and Sendai virus, IBV was most sensitive to pre-treatment of cells with neuraminidase. This finding suggests that IBV requires a greater amount of sialic acid on the cell surface to initiate an infection compared with the other two viruses. In previous studies, with respect to the haemagglutinating activity of IBV, it has been shown that the virus preferentially recognizes alpha2,3-linked sialic acid. In agreement with this finding, susceptibility to infection by IBV was connected to the expression of alpha2,3-linked sialic acid as indicated by the reactivity with the lectin Maackia amurensis agglutinin. Here, it is discussed that binding to sialic acid may be used by IBV for primary attachment to the cell surface; tighter binding and subsequent fusion between the viral and the cellular membrane may require interaction with a second receptor.

Among coronaviruses, several members are able to interact with sialic acids. For bovine coronavirus (BCoV) and related viruses, binding to cell surface components containing N-acetyl-9- O-acetylneuraminic acid is essential for initiation of an infection. These viruses resemble influenza C viruses because they share not only the receptor determinant, but also the presence of an acetylesterase that releases the 9- O-acetyl group from sialic acid and thus abolishes the ability of the respective sialoglycoconjugate to function as a receptor for BCoV. As in the case of influenza viruses, the receptor-destroying enzyme of BCoV is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells and by preventing the formation of virus aggregates. Another coronavirus, porcine transmissible gastroenteritis virus (TGEV) preferentially recognizes N-glycolylneuraminic acid. TGEV does not contain a receptor-destroying enzyme and does not depend on the sialic acid binding activity for infection of cultured cells. However, binding to sialic acids is required for the enteropathogenicity of TGEV. Interaction with sialoglycoconjugates may help the virus to pass through the sialic acid-rich mucus layer that covers the viral target cells in the epithelium of the small intestine. We discuss that the BCoV group of viruses may have evolved from a TGEV-like ancestor by acquiring an acetylesterase gene through heterologous recombination.

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 importance of the receptor-destroying enzyme of influenza C virus for inactivation of inhibitors was analysed. Using three different inhibitors (rat serum, bovine submandibulary mucin and bovine brain gangliosides) inhibition of virus infection was observed only at an inhibitor concentration that was about 100-fold higher than the maximum concentration of inhibitor that could be inactivated by the receptor-destroying enzyme of a given amount of virus. From our data and other observations we conclude that the receptor-destroying enzyme is not required to inactivate inhibitors.

Persistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection. By reconstitution experiments, using bovine brain gangliosides as artificial receptors, the degree of super-infection was markedly increased. Analysis of cellular receptor expression revealed reduced concentrations of sialoglycoproteins in general and a limited presentation of the major receptor gp40. Cocultures of persistently infected and uninfected cells (the latter carrying normal receptor levels) initiated a transient rise in virus titers. This kind of induction of virus synthesis appeared to be mainly receptor-linked, since a receptor-deprived subline, MDCK II, did not give rise to a similar effect. Susceptibility of MDCK II cocultures could be partly restored by ganglioside treatment. In accordance to related virus systems, these findings on influenza C virus suggest a role of cell receptor concentrations in the regulation of long-term persistence.

We have analysed the uptake of influenza C virus and bovine coronavirus (BCV) by a polarized epithelial cell line, Madin-Darby canine kidney (MDCK) cells. Both viruses use N-acetyl-9-O-acetylneuraminic acid as a receptor determinant for attachment to cells. Virus binding assays with immobilized proteins indicated that a glycoprotein of 40 kDa is the major surface protein containing the receptor determinant for the two viruses. MDCK cells grown on filters for permeable support were found to have differential sensitivity to infection by these viruses. Both viruses were able to initiate infection via the apical domain of the plasma membrane, but only influenza C virus also accomplished infection via the basolateral plasma membrane. The resistance of MDCK cells to BCV infection from the basal filter chamber was overcome when the cell polarity was abolished by maintaining the cells in calcium-free medium. This finding indicates that the resistance to basolateral infection by BCV is a property of the cell line and not due to a technical problem related to the use of filters. Our results indicate that two viruses which use the same receptor for attachment to cells may differ in their ability to enter polarized cells. The possible involvement of an accessory molecule in the entry of BCV is discussed.

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.

A synthetic sialic acid, N-acetyl-9-thioacetamidoneuraminic acid (9-ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but-in contrast to the natural receptor determinant, N-acetyl-9-O-acetylneuraminic acid-is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer.

A synthetic sialic acid analog, N-acetyl-9-acetamido-9-deoxy-neuraminic acid, can be used by influenza C virus as a receptor determinant for attachment to cells. In contrast to the natural determinant, N-acetyl-9-O-acetylneuraminic acid, the synthetic sialic acid is resistant to the action of the receptor-destroying acetylesterase of this virus. The sialic acid analog was enzymatically transferred to the surface of Madin-Darby canine kidney cells that are resistant to infection because of a lack of receptors. Influenza C virus was able to infect the modified cells through requiring a 10-fold larger amount of the sialic acid analogue on the cell surface compared to the natural receptor determinant. The quantitative difference is accounted for mainly by a less efficient binding of influenza C virus to the analog. Thus, in our system, inactivation of the receptor by the viral esterase is not required for the initiation of an influenza C virus infection.

Infection of cells by influenza C virus is known to be initiated by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in vitro virus binding assay, we have detected this carbohydrate on several glycoproteins of Madin-Darby canine kidney cells (type I), a polarized epithelial cell line permissive for infection with influenza C virus. Among these proteins, only one was found to be present to a significant extent on the cell surface. This protein, gp40, was characterized as an O-glycosylated (mucin-type) integral membrane protein of 40 kDa, which was predominantly localized on the apical plasma membrane of filter-grown cells. It is a major cell surface sialoglycoprotein in this cell line and was shown to be subject to constitutive and rapid endocytosis. Thus, this glycoprotein can mediate not only the binding of influenza C virus to the cell surface, but also its delivery to endosomes, where penetration occurs by membrane fusion. Other highly sialylated cell surface glycoproteins were also detected but did not mediate influenza C virus binding to a significant extent, indicating that only gp40 contains 9-O-acetylated sialic acids.

Membrane cofactor protein (MCP) has been shown to act as a cellular receptor for measles virus. In previous binding studies we demonstrated a direct interaction between the measles virus H protein and MCP. The binding was shown to be independent of the O-glycans but dependent on the N-glycans of MCP. To elucidate the role of N-glycans for the receptor function of MCP, the effect of the glycosylation inhibitors tunicamycin (TM) and 1-deoxymannojirimycin (DMJ) was analyzed. TM which prevents N-glycosylation has been reported to inhibit the expression of functional measles virus receptors. Here we show that MCP lacking all N-glycans was detectable on the surface of Vero cells, although in a reduced amount. Therefore, the lack of receptor activity cannot be explained by intracellular degradation or defective transport. In the presence of DMJ, a mannosidase I inhibitor, MCP is synthesized with N-glycans of the high-mannose type in contrast to the complex oligosaccharides present on MCP of untreated cells. Both MCP with mannose-rich and MCP with complex N-glycans were recognized by measles virus H protein in an in vitro binding assay. They both could also serve as receptors for the infection of cultured Vero cells, arguing against a direct binding of virus to a carbohydrate moiety within the N-glycans of MCP. We propose that N-linked oligosaccharides are required to maintain a conformation-dependent receptor determinant of MCP.

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.

A model of long term viral persistence has been established by selecting a spontaneous mutant strain of influenza C/Ann Arbor/1/50 virus in a permanent carrier culture of MDCK cells. Infectivity and cell tropism are mainly determined by the multifunctional viral membrane glycoprotein (HEF). HEF analysis was aimed at identifying a putative correlation between sequence and function, i.e. receptor binding, enzymatic activity, antigenicity and rate of infection. The current experimental picture is summarized by the following findings: (i) C/Ann Arbor/1/50 persistent virus carries a modified receptor-binding sequence, (ii) receptor-binding activity is altered, as indicated by a higher efficiency in recognizing low amounts of the receptor determinant N-acetyl-9-O-acetylneuraminic acid, (iii) direct attachment to cell surfaces differs from that of wild-type virus, as measured by slower kinetics of viral elution, (iv) receptor-destroying enzymatic activity is diminished, (v) characteristic features of virion surface morphology are altered or unstable, (vi) persistent-type HEF epitopes are distinguishable by monoclonal antibodies from wild-type and (vii) viral infectivity is intensified for cells bearing a low number of receptors. The sum of these changes highlights a structurally and functionally modified HEF glycoprotein that allows long term viral persistence. In order to clarify which of the described points are required for the persistent viral phenotype, a working concept is presented.

Bovine coronavirus (BCV) initiates infection by attachment to cell surface receptors the crucial component of which is N-acetyl-9-O-acetylneuraminic acid. Inactivation of receptors by neuraminidase treatment and restoration of receptors by enzymatic resialylation of asialo-cells is described as a method to determine (i) the type of sialic acid that is recognized; (ii) the linkage specificity of the viral binding activity; (iii) the minimal amount of sialic acid required for virus attachment. Evidence is presented that both glycoproteins and glycolipids can serve as receptors for BCV provided they contain 9-O-acetylated sialic acid. A model is introduced proposing that after initial binding to sialic acid-containing receptors, the S-protein of BCV interacts with a specific protein receptor. This interaction may result in a conformational change that exposes a fusogenic domain and thus induces the fusion between the viral and the cellular membrane.

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.

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.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.

From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

Influenza C virus and a group of coronaviruses, a typical representative of which is bovine coronavirus, use the same strategy for binding to cells. Surface components containing 9-O-acetylated sialic acid are recognized as cellular receptors by these viruses. In addition to the receptor determinant both virus groups have a receptor-destroying enzyme in common, which has been identified as a sialate 9-O-acetylesterase. Differences are, however, found in the distribution of these activities on the viral surface proteins. The influenza C glycoprotein HEF is a multifunctional protein, which is responsible for receptor-binding, receptor-inactivation and fusion. In the case of coronaviruses these activities are functions of two different glycoproteins, S and HE.

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.

Influenza C virus uses 9-O-acetyl-N-acetylaneuraminic acid (9-O-acetyl-Neu5Ac) as a receptor determinant for attachment to cells. The virus contains an acetylesterase which releases acetyl residues from position C-9 of sialic acid thereby inactivating the receptors. A synthetic sialic acid analogue, 9-N-acetyl-Neu5Ac, was attached to cell surface glycoconjugates by purified sialyltransferase and analyzed for its ability to substitute the 9-O-acetylated sialic acid. Erythrocytes which have been modified to contain either 9-O-acetyl-Neu5Ac or 9-N-acetyl-Neu5Ac were agglutinated by influenza C virus to the same titer. However, in contrast to the 9-O-acetyl group the 9-N-acetyl residue is resistant to cleavage by the viral acetylesterase. This characteristic property (recognition as a receptor determinant by influenza C virus, but resistance against the action of the receptor-destroying enzyme) makes this synthetic analogue a valuable tool to analyze the role of the receptor-destroying enzyme for an influenza C virus infection.

Erythrocytes from chicken of different age were analysed for their agglutinability by influenza C virus, which has been shown recently to use N-acetyl-9-O-acetylneuraminic acid as a high-affinity receptor determinant for the attachment to cells. Only with birds not younger than six days complete agglutination of the erythrocytes was observed. The hemagglutination titer which was initially low reached its maximum value at the age of about 20 days. Sialic acid was isolated from erythrocytes, purified and analysed by colorimetry, thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography-mass spectrometry. The sialic acid content of erythrocytes from one-day old and adult chicken was 21 micrograms and 18 micrograms sialic acid/ml packed erythrocytes, respectively. While N-acetylneuraminic acid was the major type of sialic acid on erythrocytes from both one-day old and adult chicken, N-acetyl-9-O-acetylneuraminic acid was only detected on red blood cells from adult animals accounting for 30-40% of total sialic acid. These results indicate that N-acetyl-9-O-acetylneuraminic acid, in addition to serving as a receptor determinant for influenza C virus, represents a developmental marker on chicken erythrocytes.

Neuraminidases of both viral and bacterial origin have been reported to be unable to destroy the cellular receptor for influenza C virus on chicken erythrocytes, in contrast to the receptors for influenza A and B virus. However, under appropriate conditions neuraminidases from both Vibrio cholerae and Clostridium perfringens were able (i) to make chicken red blood cells resistant against agglutination by influenza C virus and (ii) to reduce the hemagglutination-inhibiting activity of rat serum. Both effects were abolished in the presence of the neuraminidase inhibitor 2,3-dehydro-2-deoxyneuraminic acid (DDN). These results indicate that contrary to previous assumptions sialic acid may very well be an essential component of the receptor for influenza C virus.

Bacterial neuraminidases destroy influenza C virus receptors of chick erythrocytes and inactivate hemagglutination inhibitors: rat alpha 1-macroglobulin (RMG) and bovine submaxillary mucin (BSM). These data indicate that neuraminic acid may be a component of influenza C virus receptor. The inhibiting activity of RMG and BSM is also eliminated by the receptor-destroying enzyme (RDE) of influenza C virus. After inactivation, the inhibitors (RMG and BSM) contain a reduced amount of N-acetyl-9-0-acetylneuraminic acid (Neu5, 9Ac2) and a larger amount of N-acetylneuraminic acid (Neu5 Ac). Transformation of Neu5, 9Ac2 into Neu5 Ac may also occur upon incubation of free neuraminic acid with influenza C virus. These data indicate that the RDE of influenza C virus is neuraminate-O-acetylesterase (N-acyl-9 4-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53). It was shown that inhibition of influenza C virus hemagglutination by RMG and BSM and, apparently, adhesion of the virus to the cell surface involves binding of influenza C virus with Neu5, 9Ac2.

Identification of the receptor-destroying enzyme of influenza C virus as a specific neuraminate O-acetylesterase has suggested that 9-O-acetyl-N-acetylneuraminic acid is an essential component of the cell surface receptor of influenza C virus (Herrler, G., Rott, R., Klenk, H.-D., Muller, H.-P., Shukla, A. K., and Schauer, R. (1985) EMBO (Eur. Mol. Biol. Organ.) J. 4, 1503-1506). In this report, three common sialic acids, N-acetylneuraminic acid (NeuAc), N-glycollylneuraminic acid (NeuGc), and 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) were compared for their ability to mediate attachment of influenza A, B, and C viruses to cells. Human asialoerythrocytes were resialylated to contain the three sialic acids in defined sequence on glycoprotein carbohydrate groups using purified sialyltransferases and corresponding CMP-sialic acid donor substrates. While influenza C virus failed to agglutinate native cells or resialylated cells containing NeuAc and NeuGc, resialylated cells containing 9-O-Ac-NeuAc in three different sialyloligosaccharide sequences were agglutinated in high titer. In contrast, most representative influenza A and B viruses examined preferentially agglutinated cells containing NeuAc and NeuGc and failed to agglutinate cells containing 9-O-Ac-NeuAc. Cells containing 9-O-Ac-NeuAc were sensitive to the action of influenza C virus neuraminate O-acetylesterase which converts 9-O-Ac-NeuAc to NeuAc. This treatment abolished agglutination by influenza C while making the cells agglutinable by several influenza A and B viruses. Finally, the ability of influenza C virus to agglutinate the erythrocytes of various species correlated with the presence of 9-O-Ac-NeuAc. The results provide direct evidence that influenza C virus utilizes 9-O-acetyl-N-acetylneuraminic acid as the primary receptor determinant for attachment to cell surface receptors.

N-Acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) has been shown to be a high-affinity receptor determinant for attachment of influenza C virus to erythrocytes (G. N. Rogers, G. Herrler, J. C. Paulson, and H-D. Klenk, 1986, J. Biol. Chem. 261, 5947-5951). In this report the nature of the cell surface receptor for influenza C virus on tissue culture cells was analyzed. Pretreatment with either neuraminidase or neuraminate 9-O-acetylesterase was found to render LLC-MK2 cells resistant to infection by influenza C virus as evidenced by the failure to detect virus release into the medium by hemagglutination titration. Susceptibility to infection was fully restored after incubation of neuraminidase-treated cells with bovine brain gangliosides known to contain Neu5,9Ac2. These results indicate that (i) Neu5,9Ac2 is the primary receptor determinant required for influenza C virus to attach to tissue culture cells and to initiate infection and (ii) gangliosides containing this type of sialic acid are potential receptors for influenza C virus. Several cell lines which are resistant to infection by this virus were able to release influenza C virus into the medium provided they were incubated with bovine brain gangliosides prior to virus infection. This result indicates that lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus.

The nature of the receptor-destroying enzyme (RDE) of influenza C virus has been elucidated by analyzing its effect on the haemagglutination inhibitors rat alpha 1-macroglobulin (RMG) and bovine submandibulary mucin (BSM), respectively. The inhibitory activity of both compounds is abolished by incubation with influenza C virus. After inactivation, RMG and BSM were found to contain reduced amounts of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) and increased amounts of N-acetylneuraminic acid (Neu5Ac). H.p.l.c. analysis revealed that purified Neu5,9Ac2 is converted to Neu5Ac by incubation with influenza C virus. These results demonstrate that RDE of influenza C virus is neuraminate-O-acetylesterase [N-acyl-9(4)-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53)]. The data also indicate that haemagglutination-inhibition (HI) by RMG and BSM and most likely virus attachment to cell surfaces involves binding of influenza C virus to Neu5,9Ac2.