RESUMO
The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the alpha-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman-Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A-E domains of the palm subdomain of nucleic acid polymerases.
Assuntos
Evolução Molecular , Genoma Viral , Vírus da Gastroenterite Transmissível/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Suínos , Vírus da Gastroenterite Transmissível/classificação , Vírus da Gastroenterite Transmissível/isolamento & purificaçãoAssuntos
Vírus da Gastroenterite Transmissível/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Animais , Proteínas M de Coronavírus , Genoma Viral , Nucleocapsídeo/metabolismo , Vírus da Gastroenterite Transmissível/química , Vírus da Gastroenterite Transmissível/genética , Proteínas do Core Viral/química , Proteínas da Matriz Viral/genética , Vírion/químicaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Vírus da Gastroenterite Transmissível/fisiologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Células COS , Linhagem Celular , Linhagem Celular Transformada , Humanos , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Testes de Neutralização , Análise de Sequência de DNA , SuínosRESUMO
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.
Assuntos
Anticorpos Antivirais/biossíntese , Imunoglobulina A/biossíntese , Camundongos Transgênicos , Leite/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Sequência de Bases , Células COS , Linhagem Celular , Linhagem Celular Transformada , DNA Complementar , Humanos , Imunoglobulina A/genética , Isotipos de Imunoglobulinas , Cadeias alfa de Imunoglobulina/biossíntese , Cadeias alfa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Suínos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Interferência Viral , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Sequência de Bases , Células COS , Linhagem Celular Transformada , DNA Complementar , Humanos , Imunoglobulina A/genética , Imunoglobulina G/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Gastroenterite Transmissível/química , Vírus da Gastroenterite Transmissível/imunologia , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/imunologia , Linhagem Celular , Epitélio , Haplótipos/imunologia , Ativação Linfocitária , Masculino , Micelas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Especificidade da Espécie , Suínos , Porco Miniatura , Linfócitos T/imunologia , Testículo , Vírus da Gastroenterite Transmissível/genética , Proteínas Virais/isolamento & purificação , Proteínas Estruturais Virais/genéticaRESUMO
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.
Assuntos
Capsídeo/análise , Vírus da Gastroenterite Transmissível/química , Proteínas do Core Viral/análise , Proteínas da Matriz Viral/análise , Animais , Linhagem Celular , Octoxinol , Polietilenoglicóis/farmacologia , Suínos , Vírus da Gastroenterite Transmissível/ultraestrutura , Vírion/química , Vírion/ultraestruturaRESUMO
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.
Assuntos
Adenovírus Humanos/genética , Gastroenterite Suína Transmissível/prevenção & controle , Vetores Genéticos , Vírus da Gastroenterite Transmissível/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Adenovírus Humanos/crescimento & desenvolvimento , Animais , Anticorpos Antivirais/sangue , Humanos , Luciferases/genética , Suínos , Vacinas Virais/genética , Replicação ViralRESUMO
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.
Assuntos
Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Epitopos de Linfócito B/imunologia , Proteínas de Escherichia coli , Glicoproteínas de Membrana/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Toxinas Bacterianas/genética , Sequência de Bases , DNA Viral , Enterotoxinas/genética , Epitopos de Linfócito B/genética , Expressão Gênica , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Testes de Neutralização , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium , Glicoproteína da Espícula de Coronavírus , Suínos , Vírus da Gastroenterite Transmissível/genética , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Adenovírus Humanos/genética , Coronavirus/imunologia , Glicoproteínas de Membrana/imunologia , Suínos/virologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Humanos , Dados de Sequência Molecular , Glicoproteína da Espícula de CoronavírusRESUMO
Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.
Assuntos
Adenovírus Humanos/genética , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Gastroenterite Suína Transmissível/prevenção & controle , Vetores Genéticos , Glicoproteínas de Membrana/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular , Cricetinae , Feminino , Gastroenterite Suína Transmissível/imunologia , Humanos , Imunização Passiva , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus , Suínos , Porco Miniatura , Vírus da Gastroenterite Transmissível/genética , Vacinação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Four strong T cell epitopes have been identified studying the blastogenic response of lymphocytes from haplotype-defined transmissible gastroenteritis virus (TGEV) immune miniswine to sixty-one 15-mer synthetic peptides. Three of these epitopes are located on the nucleoprotein (N46, amino acids 46 to 60; N272, amino acids 272 to 286; and N321, amino acids 321 to 335), and one on the membrane protein (M196, amino acids 196 to 210). N321 peptide induced the highest T cell response and was recognized by immune miniswine lymphocytes with haplotypes dd, aa, and cc. T lymphocytes from peptide N321-immune miniswine reconstituted the in vitro synthesis of TGEV-specific antibodies by complementing CD4- TGEV-immune cells. This response was directed at least against the three major structural proteins. The synthesized antibodies specific for S protein preferentially recognized discontinuous epitopes and neutralized TGEV infectivity. These results show that peptide N321 defines a functional T helper epitope eliciting T cells capable of collaborating with B cells specific for different proteins of TGEV.
Assuntos
Anticorpos Antivirais/biossíntese , Epitopos de Linfócito T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Proteínas Estruturais Virais/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Haplótipos , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/síntese química , Peptídeos/imunologia , Suínos , Proteínas Estruturais Virais/químicaAssuntos
Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/imunologia , Adenoviridae/genética , Adenoviridae/patogenicidade , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Clonagem Molecular , Cricetinae , Epitopos/genética , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/prevenção & controle , Genes Virais , Vetores Genéticos , Imunidade nas Mucosas , Dados de Sequência Molecular , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Nódulos Linfáticos Agregados/virologia , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Suínos , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
At the April 1992, mid-term meeting of the International Committee on Taxonomy of Viruses (ICTV) a proposal from the Coronaviridae Study Group (CSG) to include the torovirus genus in the Coronaviridae was accepted. Following another proposal, the arterivirus genus was removed from the Togaviridae but not assigned to another family. The arteriviruses have some features in common with the Coronaviridae but also have major differences. After much debate, culminating in September 1992, it was decided that the CSG would not recommend inclusion of arterivirus in the Coronaviridae. It was agreed that (a) the nomenclature used for coronavirus genes, mRNAs and polypeptides (Cavanagh et al., 1990) should be used for toroviruses, (b) that the small (about 100 amino acids) membrane-associated protein, which is distinct from the integral membrane glycoprotein M, associated with virions of infectious bronchitis (Liu & Inglis, 1991) and transmissible gastroenteritis (Godet et al., 1992) coronaviruses would be referred to by the acronym sM (lower case 's') and (c) that 'pol' (polymerase) should be used as a working term for gene 1, which comprises open reading frames (ORFs) 1a and 1b in both genera of the Coronaviridae.
Assuntos
Arterivirus/classificação , Coronaviridae/classificação , Arterivirus/genética , Coronaviridae/genética , Coronavirus/classificação , Coronavirus/genética , Genoma Viral , Especificidade da Espécie , Togaviridae/classificação , Togaviridae/genética , Torovirus/classificação , Torovirus/genéticaRESUMO
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.
Assuntos
Hemaglutinação por Vírus/fisiologia , Vírus da Bronquite Infecciosa/fisiologia , Glicoproteínas de Membrana/química , Ácidos Siálicos/fisiologia , Vírus da Gastroenterite Transmissível/fisiologia , Proteínas do Envelope Viral/química , Proteínas de Bactérias/farmacologia , Sequência de Carboidratos , Membrana Eritrocítica/metabolismo , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ácido N-Acetilneuramínico , Neuraminidase/farmacologia , Receptores de Coronavírus , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismoRESUMO
Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S proteins of six European PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have the S protein with a similar size to the enteric viruses. NEB72 and TOY56 viruses have 2 and 15 specific amino acid differences with the enteric viruses, respectively. Four of the residues changed are located within the deletion present in the PRCVs and may influence the enteric tropism of TGEV in vivo. A receptor binding site (RBS) used by the virus to infect ST and other cell types might be located between sites A and D of the S glycoprotein, since monoclonal antibodies (MAbs) specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BRI70, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the mutation fixation rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Coronavirus/classificação , Vírus da Gastroenterite Transmissível/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Genes Virais , Masculino , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus , Suínos , Testículo , Vírus da Gastroenterite Transmissível/classificação , Vírus da Gastroenterite Transmissível/isolamento & purificação , Vírus da Gastroenterite Transmissível/fisiologia , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genéticaAssuntos
Adenovírus Humanos , Antígenos Virais/imunologia , Infecções por Coronavirus/veterinária , Vetores Genéticos , Glicoproteínas de Membrana/imunologia , Infecções Respiratórias/veterinária , Doenças dos Suínos/prevenção & controle , Tonsilite/veterinária , Vacinas Sintéticas , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Adenovírus Humanos/isolamento & purificação , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Infecções por Coronavirus/prevenção & controle , Glicoproteínas de Membrana/genética , Infecções Respiratórias/microbiologia , Infecções Respiratórias/prevenção & controle , Glicoproteína da Espícula de Coronavírus , Suínos , Tonsilite/microbiologia , Tonsilite/prevenção & controle , Vacinação , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologiaAssuntos
Adenovírus Humanos , Antígenos Virais/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/imunologia , Vetores Genéticos , Intestinos/microbiologia , Glicoproteínas de Membrana/imunologia , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium , Vírus da Gastroenterite Transmissível/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Clonagem Molecular , Infecções por Coronavirus/imunologia , Cricetinae , Intestinos/imunologia , Dados de Sequência Molecular , Especificidade de Órgãos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Glicoproteína da Espícula de CoronavírusRESUMO
The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.
Assuntos
Antígenos Virais/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Sítios de Ligação , Clonagem Molecular , Epitopos/análise , Epitopos/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Glicoproteínas/genética , Glicoproteínas/imunologia , Filogenia , Plasmídeos , Homologia de Sequência de Aminoácidos , Suínos , Vírus da Gastroenterite Transmissível/genética , Proteínas Virais/genéticaRESUMO
Transmissible gastroenteritis virus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S protein of six PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have S proteins with a size similar to the enteric viruses. NEB72 and TOY56 viruses have in the S protein 2 and 15 specific amino acid differences with the enteric viruses. Four of the residues changed (aa 219 of NEB72 isolate and aa 92, 94, and 218 of TOY56) are located within the deletion present in the PRCVs and may be involved in the receptor binding site (RBS) conferring enteric tropism to TGEVs. A second RBS used by the virus to infect ST cells might be located in a conserved area between sites A and D of the S glycoprotein, since monoclonal antibodies specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor virus which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BR170, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the fixation of mutations with time, that is, the existence of a well-defined molecular clock. A mutation fixation rate of 7 +/- 2 x 10(-4) nucleotide substitutions per site and per year was calculated for TGEV-related viruses. This rate falls in the range reported for other RNA viruses. Point mutations and probably recombination events have occurred during TGEV evolution.