Biomedical subjects

L Enjuanes

Publications and source records attributed to L Enjuanes.

At least 37 records · Page 2Linked to original sources

PubMed1998

Progress towards the construction of a transmissible gastroenteritis coronavirus self-replicating RNA using a two-layer expression system.

Three transmissible gastroenteritis coronavirus (TGEV) defective interfering RNAs of 21, 10.6 and 9.7 kb (DI-A, DI-B and DI-C, respectively) were isolated. Dilution experiments showed that the largest DI RNA, DI-A, is a self-replicating RNA (replicon), and thus codes for a functional RNA polymerase and all the necessary replication signals. In order to engineer a cDNA encoding the RNA replicon a strategy based on the cloning of DI-C cDNA, followed by the insertion of the sequences required to complete the DI-A sequence has been developed. A cDNA complementary to DI-C RNA was cloned under the control of the CMV promoter (pDI-C-CMV) and rescued with a helper virus. In the ORF 1a of polymerase gene pDI-C-CMV contained a 10 kb deletion and in ORF 1b a 1.1 kb deletion. The consensus sequence corresponding to the deleted regions was cloned, and the deletions in pDI-C-CMV were replaced to yield a complete cDNA clone of DI-A, pDI-A-21-CMV, containing a full-length TGEV polymerase, driven by a CMV promoter. Expression of a functional TGEV polymerase is being investigated.

Animals↗

PubMed1998

Structure and intracellular assembly of the transmissible gastroenteritis coronavirus.

Coronaviruses have been described as pleomorphic, round particles with a helical nucleocapsid as the unique internal structure under the virion envelope. Our studies on the organization of the transmissible gastroenteritis coronavirus (TGEV) have shown that the structure of these viruses is more complex. Different electron microscopy techniques, including cryomicroscopy of vitrified viruses, revealed the existence of an internal core, most probably icosahedral, in TGEV virions. Disruption of these cores induced the release of elongated ribonucleoprotein complexes. Ultrastructural analysis of freeze-substituted TGEV-infected swine testis (ST) cells showed characteristic intracellular budding profiles as well as two types of virions. While large virions with an electron-dense internal periphery are seen at perinuclear regions, smaller viral particles exhibiting compact internal cores of poligonal contours are more abundant in areas closer to the plasma membrane of the cell. These data strongly suggest that maturation events following the budding process are responsible for the formation of the internal core shell, the new structural element that we have recently described in extracellular infectious TGEV virions.

Animals↗

PubMed1998

Isolation of hemagglutination-defective mutants for the analysis of the sialic acid binding activity of transmissible gastroenteritis virus.

The surface protein S of transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that enables the virus to agglutinate erythrocytes. A protocol is described that has been successfully applied to the isolation of hemgglutination-defective mutants. The potential of these mutants for the characterization of the sialic acid-binding site and the function of the binding activity is discussed.

Animals↗

PubMed1998

Interference of coronavirus infection by expression of IgG or IgA virus neutralizing antibodies.

Mouse immunoglobulin gene fragments encoding the variable modules of the heavy (VH) and light (VL) chains of a transmissible gastroenteritis coronavirus (TGEV) neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. Chimeric immunoglobulin genes with the variable modules from the murine MAb and constant modules of human gamma 1 and kappa chains were constructed using RT-PCR. These chimeric immunoglobulins were stably or transiently expressed in murine myelomas and COS cells, respectively. The secreted recombinant antibodies had radioimmunoassay (RIA) titers higher than 10(3) and reduced the infectious virus more than 10(4)-fold. Recombinant dimeric IgA showed a 50-fold enhanced neutralization of TGEV relative to a recombinant monomeric IgG1 which contained the identical antigen binding site. Epithelial cell lines stably-transformed with these constructs and expressing either recombinant IgG or IgA TGEV neutralizing antibodies reduced virus production by > 10(5)-fold after infection with homologous virus, although a residual level of virus production (< 10(2) PFU/ml) remained in less than 0.1% of the cells.

Animals↗

PubMed1998

Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection against neonatal infections of the enteric tract.

Animals↗

PubMed1998

Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk.

Protection against enteric infections can be provided by the oral administration of pathogen-neutralizing antibodies. To provide passive immunity, 18 lines of transgenic mice secreting a recombinant monoclonal antibody (Mab) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. The genes encoding a chimeric Mab with the variable modules of the murine TGEV-specific Mab 6A.C3 and the constant modules of a human IgG, isotype Mab were expressed under the control of regulatory sequences derived from the whey acidic protein, which is an abundant milk protein. The Mab 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species, which does not allow the selection of neutralization escape mutants. Antibody expression titers of 10(6) were obtained in the milk of transgenic mice that reduced TGEV infectivity 10(6)-fold. The antibody was synthesized at high levels throughout lactation. Integration of matrix attachment region sequences with the antibody genes led to a 20- to 10,000-fold increase in the antibody titer in 50% of the transgenic animals. Antibody expression levels were transgene copy number independent and related to the site of integration. The generation of transgenic animals producing virus neutralizing antibodies in milk could provide an approach to protection against neonatal infections of the enteric tract.

Animals↗

PubMed1998

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

Amino Acid Sequence↗

PubMed1998

Two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis.

The intracellular assembly of the transmissible gastroenteritis coronavirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronavirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.

Animals↗

PubMed1997

Two amino acid changes at the N-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism.

To study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10(-9) recombinants per nucleotide and was 3.7-fold higher at the 5'-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5'- and 3'-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV.

Animals↗

PubMed1997

Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies.

Immunoglobulin gene fragments encoding the variable modules of the heavy and light chains of a transmissible gastroenteritis coronavirus (TGEV)-neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. The sequences of MAb 6A.C3 kappa and gamma 1 modules were identified as subgroup V and subgroup IIIC, respectively. The chimeric immunoglobulin genes encoding the variable modules from the murine MAb and constant modules of human gamma 1 and kappa chains were constructed by reverse transcriptase PCR. Chimeric immunoglobulins were stably or transiently expressed in murine myelomas or COS cells, respectively. The secreted recombinant antibodies had radioimmunoassay titers (i.e., the highest dilution giving a threefold increase over the background) higher than 10(3) and reduced the infectious virus more than 10(4)-fold. Recombinant dimeric immunoglobulin A (IgA) showed a 50-fold enhanced neutralization of TGEV relative to a recombinant monomeric IgG1 which contained the identical antigen binding site. Stably transformed epithelial cell lines which expressed either recombinant IgG or IgA TGEV-neutralizing antibodies reduced virus production by > 10(5)-fold after infection with homologous virus, although a residual level of virus production (< 10(2) PFU/ml) remained in less than 0.1% of the cells. This low-level persistent infection was shown not to be due to the selection of neutralization escape mutants. The implications of these findings for somatic gene therapy with recombinant antibodies are discussed.

Animals↗

PubMed1996

Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity.

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5'-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3' end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3' end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

Animals↗

PubMed1996

A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity.

Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at > 70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.

Amino Acid Sequence↗

PubMed1996

Characterization of transmissible gastroenteritis coronavirus S protein expression products in avirulent S. typhimurium delta cya delta crp: persistence, stability and immune response in swine.

The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium delta cya delta crp delta asd chi 3987. Three partially overlapping fragments of TGEV S gene, encoding the amino-terminal, intermediate, and carboxy-terminal end of the protein, as well as the full length gene were inserted into the asd+ plasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS-3, and pYATS-4, respectively, which were transformed into S. typhimurium chi 3987. Recombinant S. typhimurium chi 3987 (pYATS-1) and chi 3987 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragment of the S protein and the full length protein (144 kDa), respectively, showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expressed the S-protein antigen. Since S. typhimurium chi 3987 (pYATS-1) showed a better level of expression and stability in vitro, this recombinant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonization of swine tissues by S. typhimurium delta cya delta crp, a gene conferring resistance to rifampicin was cloned into the chromosome of S. typhimurium chi 3987, generating chi 4509 strain. Both S. typhimurium chi 4509 (pYA292) and chi 4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10-20 post-infection. The expression of the amino-terminal fragment of the S protein diminished the ability of S. typhimurium chi 4509 (pYATS-1) to colonize deep tissues. The recombinant strain S. typhimurium chi 3987 (pYATS-1) induced TGEV specific antibodies in both serum and saliva of orally inoculated swine.

Animals↗

PubMed1996

Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies.

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S + N, S + M, and to a minor extent with M + N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Animals↗

PubMed1996

An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine.

The full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 micrograms per 10(6) cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 micrograms per 10(6) cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S' and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.

Adenoviruses, Human↗

PubMed1996

Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.

Adenoviruses, Human↗

PubMed1996

The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins.

Coronaviruses are enveloped RNA viruses involved in a variety of pathologies that affect animals and humans. Existing structural models of these viruses propose a helical nucleocapsid under the virion envelope as the unique internal structure. In the present work, we have analyzed the structure of the transmissible gastroenteritis coronavirus. The definition of its organization supports a new structural model for coronaviruses, since a spherical, probably icosahedral, internal core has been characterized. Disruption of these cores induces the release of N-protein-containing helical nucleocapsids. Immunogold mapping and protein analysis of purified cores showed that they consist of M and N proteins, M being the main core shell component. This surprising finding, together with the fact that M protein molecules are also located in the virion envelope, indicates that a reconsideration of the assembly and maturation of coronaviruses, as well as a study of potential M-protein subclasses, is needed.

Animals↗

PubMed1996

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-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.

Animals↗