RESUMO
An outbreak of neurological disease occurred in a well-managed riding school. Ataxia and paresis were observed in several horses, five of which became recumbent and were euthanized. Post-mortem analysis revealed scattered haemorrhages along the spinal cord, that were reflected by multiple haemorrhagic foci on formalin-fixed sections, with the thoracic and lumbar segments being the most affected. Pathohistologically, perivascular mononuclear cuffing and axonal swelling, especially in the white matter, were evident. Parallel to the course of disease, alterations in myelin sheets and activation of astrocytes and microglial cells were also observed. Virological findings confirmed an acute equine herpesvirus type 1 infection and virus was isolated from the spinal cord of a 26-year-old mare.
Assuntos
Surtos de Doenças/veterinária , Encefalomielite/veterinária , Marcha Atáxica/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/epidemiologia , Animais , Encéfalo/virologia , DNA Viral/genética , Diagnóstico Diferencial , Encefalomielite/complicações , Encefalomielite/epidemiologia , Feminino , Imunofluorescência/veterinária , Marcha Atáxica/etiologia , Alemanha/epidemiologia , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/epidemiologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/patologia , Doenças dos Cavalos/virologia , Cavalos , Imuno-Histoquímica/veterinária , Masculino , Reação em Cadeia da Polimerase/veterinária , Medula Espinal/virologiaRESUMO
The equine herpesvirus 1 (EHV-1) IR6 protein forms typical rod-like structures in infected cells, influences virus growth at elevated temperatures, and determines the virulence of EHV-1 Rac strains (Osterrieder et al., Virology 226:243-251, 1996). Experiments to further elucidate the functions and properties of the IR6 protein were conducted. It was shown that the IR6 protein of wild-type RacL11 virus colocalizes with nuclear lamins very late in infection as demonstrated by confocal laser scan microscopy and coimmunoprecipitation experiments. In contrast, the mutated IR6 protein encoded by the RacM24 strain did not colocalize with the lamin proteins at any time postinfection (p.i.). Electron microscopical examinations of ultrathin sections were performed on cells infected at 37 and 40 degreesC, the latter being a temperature at which the IR6-negative RacH virus and the RacM24 virus are greatly impaired in virus replication. These analyses revealed that nucleocapsid formation is efficient at 40 degreesC irrespective of the virus strain. However, whereas cytoplasmic virus particles were readily observed at 16 h p.i. in cells infected with the wild-type EHV-1 RacL11 or an IR6-recombinant RacH virus (HIR6-1) at 40 degreesC, virtually no capsid translocation to the cytoplasm was obvious in RacH- or RacM24-infected cells at the elevated temperature, demonstrating that the IR6 protein is involved in nucleocapsid egress. Transient transfection assays using RacL11 or RacM24 IR6 plasmid DNA and COS7 or Rk13 cells, infection studies using a gB-negative RacL11 mutant (L11DeltagB) which is deficient in direct cell-to-cell spread, and studies using lysates of IR6-transfected cells demonstrated that the wild-type IR6 protein is transported from cell to cell in the absence of virus infection and can enter cells by a yet unknown mechanism.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/virologia , Herpesvirus Equídeo 1/metabolismo , Proteínas Nucleares/metabolismo , Nucleocapsídeo/metabolismo , Proteínas Virais/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/patogenicidade , Cavalos , Laminas , Microscopia Confocal , Microscopia Eletrônica , Fatores de Tempo , Proteínas Virais/genéticaRESUMO
In a previous report, the function of the equine herpesvirus 1 (EHV-1) glycoprotein M (gM) homolog was investigated. It was shown that EHV-1 gM is involved in both virus entry and direct cell-to-cell spread of infection (N. Osterrieder et al., J. Virol. 70, 4110-4115, 1996). In this study, experiments were conducted to analyze the synthesis, posttranslational processing, and the putative ion channel function of EHV-1 gM. It was demonstrated that EHV-1 gM is synthesized as an Mr 44,000 polypeptide, which is cotranslationally N-glycosylated to an Mr 46,000-48,000 glycoprotein. The Mr 46,000-48,000 gM moiety is processed to an Mr 50,000-55,000 glycoprotein, which is resistant to treatment with endoglycosidase H, indicating that processing occurs in the Golgi network. EHV-1 gM forms a dimer in infected cells and the virion, as was demonstrated by the presence of an Mr 105,000-110,000 gM-containing band in electrophoretically separated lysates of infected cells and purified extracellular virions. The Mr 105,000-110,000 protein band containing gM was also observed in lysates of cells that had been transfected with EHV-1 gM DNA. The translation of EHV-1 gM is initiated at the first in-frame methionine of the gM open reading frame as shown by transient transfection experiments of full-length gM and a truncated gM lacking the aminoterminal 83 amino acids. Functional expression of EHV-1 gM in Xenopus laevis oocytes together with voltage-clamp analyses demonstrated that gM per se does not exhibit ion channel activity as had been speculated from the predicted structure of the polypeptide.
Assuntos
Herpesvirus Equídeo 1/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Animais , Células COS , Eletroforese em Gel de Campo Pulsado , Glicosilação , Canais Iônicos/metabolismo , Cinética , Metionina/metabolismo , Biossíntese de Proteínas , Proteínas Virais/biossínteseRESUMO
Experiments to analyze the functions of the equine herpesvirus 1 (EHV-1) glycoprotein gB were performed. Cell lines which stably expressed either the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCgB delta, respectively. Using the cell line TCgBf, a gB-negative viral mutant, L11delta gB, was generated by replacing a 2.1-kb BglII-NruI fragment in the EHV-1 strain RacL11 gB with the Escherichia coli LacZ gene. EHV-1 strain RacL11, the modified live vaccine strain RacH, and L11delta gB were used for functional studies. It was shown that: (i) EHV-1 gB is essential for virus growth in vitro since gB-negative L11delta gB exhibited titers of <10 PFU/ml when grown and titrated on noncomplementing cells. (ii) The cell line expressing truncated gB (TCgB delta) did not complement for the growth of L11delta gB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines tested. Since RacH had amino acids 944-980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extreme carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable for virus growth in cultured cells. (iii) Single infected cells but no plaque formation were observed after infection of noncomplementing cells with L11delta gB, demonstrating the requirement of EHV-1 gB for direct cell-to-cell spread of infection. (iv) The attachment of gB-negative L11delta gB virions to target cells was similar to both phenotypically complemented L11delta gB and parent RacL11 virus. (v) L11delta gB viral titers could be enhanced by using the fusogen polyethylene glycol (PEG). The increase of L11delta gB titers by PEG treatment, however, was considerably lower compared to gB-negative pseudorabies virus, suggesting that EHV-1 gB might not be as stringently required for virus penetration as are its homologs in other Alphaherpesvirinae.
Assuntos
Herpesvirus Equídeo 1/fisiologia , Proteínas do Envelope Viral/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Técnica Indireta de Fluorescência para Anticorpo , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/patogenicidade , Cavalos , Dados de Sequência Molecular , Plasmídeos , Polietilenoglicóis , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Pele , Transfecção , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/química , Vírion/fisiologiaRESUMO
Equine herpesvirus 1 (EHV-1) mutants devoid of the open reading frames (ORFs) of either glycoprotein (g) B or M were constructed and tested for their immunogenic potential in a murine model of EHV-1 infection. The mutant viruses were engineered using the virulent EHV-1 strain RacL11 or the modified live vaccine strain RacH by inserting the Escherichia coli LacZ gene into the viral ORFs. RacL11-infected mice showed signs typical of an EHV-1 infection, whereas mice infected with the EHV-1 gB- or gM-negative mutants or with RacH did not develop disease. No difference in the pathogenic potential of RacL11 gB- and gM-negative viruses was observed after application of either phenotypically completed or negative viruses. However, revertant RacL11 viruses in which the gB or gM gene had been restored caused EHV-1-related symptoms that were indistinguishable from those induced by RacL11. Mice that had been immunized with phenotypically negative gB- and gM-deficient EHV-1 were challenged with the RacL11 virus 25 days after immunization. Mock-immunized mice developed EHV-1 disease and high virus loads in their lungs were observed. In contrast, mice developed not exhibit EHV-1-caused disease. It was concluded (i) that deletion of either gB or gM abolished the virulence of strain RacL11 and (ii) that immunization with gB- or gM-negative EHV-1 elicited a protective immunity that was reflected by both virus-neutralizing antibodies and EHV-1-specific T-cells in spleens of immunized mice.
Assuntos
Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Mutação , Proteínas do Envelope Viral/genética , Vacinas Virais , Animais , Infecções por Herpesviridae/imunologia , Herpesvirus Equídeo 1/imunologia , Herpesvirus Equídeo 1/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta/genéticaRESUMO
The diploid IR6 gene (ORF 67) of equine herpesvirus type 1 (EHV-1) is absent in the modified live EHV-1 vaccine strain RacH and is present in a mutated form in the avirulent EHV-1 strains RacM24 and RacM36, such that the IR6 protein fails to form the typical rod-like structures observed for wild-type EHV-1 RacL11. To assess the role of the IR6 protein in EHV-1 replication and virulence, two recombinant RacH viruses, HIR6-1 and HIR6-2, that harbor a single copy of the wild-type IR6 gene were engineered and characterized. It was shown that: (i) HIR6-1 or HIR6-2 virus encoded for an IR6 protein that was capable of forming the rod-like structures typical of cells infected with the wild-type virulent virus strain RacL11. (ii) Whereas the avirulent EHV-1 strains RacH and RacM36 are temperature-sensitive (in that virus replication at 40 degrees versus that at 37 degrees was reduced by as much as 7,500-fold), the HIR6-1 and HIR6-2 viruses, like RacL11 virus, were capable of significant replication at the elevated temperature. (iii) Electron microscopic analyses revealed that cells infected with the HIR6-1 or HIR6-2 virus, like those infected with virulent RacL11 virus, produced and released comparable numbers of virus particles at both 37 and 40 degrees. In cells infected with the RacH virus at 40 degrees, however, release of extracellular particles was inhibited by greater than 90% and relatively few of the particles were enveloped. (iv) Infections of BALB/cA mice revealed that both the HIR6-1 and HIR6-2 viruses, unlike the parent RacH virus, were as virulent as the wild-type RacL11 strain as judged by the criteria of body weight loss, development of clinical signs of EHV-1 infection, virus titers in the lung, and ability to cause viremia. These findings and those of our recent studies indicate that the IR6 protein is a major determinant of EHV-1 virulence and that the IR6 protein may play a role in virus maturation and/or egress.
Assuntos
Herpesvirus Equídeo 1/crescimento & desenvolvimento , Proteínas Virais/fisiologia , Animais , Linhagem Celular , Feminino , Infecções por Herpesviridae , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/patogenicidade , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Recombinação Genética , Inoculações Seriadas , Temperatura , Proteínas Virais/biossíntese , Vírion/metabolismo , Virulência/genética , Replicação ViralRESUMO
Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.
Assuntos
Herpesvirus Equídeo 1/fisiologia , Proteínas do Envelope Viral/fisiologia , Vírion/fisiologia , Sequência de Bases , Linhagem Celular , Cinética , Dados de Sequência Molecular , Mutação , Proteínas do Envelope Viral/genéticaRESUMO
The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growth in vitro. (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.
Assuntos
Herpesvirus Equídeo 1/genética , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/metabolismo , Compartimento Celular , Células Cultivadas , Chlorocebus aethiops , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Replicação ViralRESUMO
Four formulations of the equine herpesvirus type 1 (EHV-1) glycoprotein gp 14 (gB), were tested for their ability to protect mice against intranasal (inas) EHV-1 challenge infection. The preparations tested included (i) a truncated gp14 produced in Escherichia coli or (ii) a truncated gp14 expressed in insect cells by a recombinant baculovirus, (iii) truncated gp14 coexpressed with human immunodeficiency virus type 1 (HIV-1) gag virus-like particles (VLP) in insect cells, and (iv) a gp14-DNA vaccine under the control of the cytomegalovirus immediate early promoter. All antigen preparations and the DNA vaccine elicited a humoral and delayed-type hypersensitivity (DTH) immune response to EHV-1 after intramuscular (im) immunization. After inas immunization, only the VLP-gp14 preparation produced both a good humoral and a prominent DTH immune response; gp14 expressed by insect cells elicited high titers of EHV-1-specific antibodies, whereas gp14 produced in E. coli and the DNA vaccine elicited only low antibody titers. Glycoprotein gp14 expressed by bacteria, however, induced a strong DTH reaction after inas application. Mice were completely protected against EHV-1 challenge infection after both the im and the inas immunization with the VLP-gp14 preparation. Protection was less efficient after immunization with the E. coli and insect cell gp14s as well as after DNA vaccination. Although the transmembrane domain of EHV-1 gp14 was deleted, recombinant gp14 could be demonstrated in insect cell membranes at late times postinfection and aggregated with the VLPs. It is suggested that the transmembrane domain is not required for gp14-association with membranes in that system.
Assuntos
Infecções por Herpesviridae/prevenção & controle , Herpesvirus Equídeo 1/imunologia , Glicoproteínas de Membrana/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Baculoviridae/genética , Linhagem Celular , Feminino , Produtos do Gene gag/análise , Produtos do Gene gag/biossíntese , HIV-1/química , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Hipersensibilidade Tardia , Esquemas de Imunização , Pulmão/virologia , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Vacinas Sintéticas/administração & dosagem , Proteínas do Envelope Viral , Vacinas Virais/administração & dosagem , Redução de PesoRESUMO
More than 50 reference strains and field isolates of equine herpesvirus type 1 (EHV-1) were examined by a touchdown PCR. Primers for specific amplification of EHV-1 DNA were chosen from the terminal and internal repeat regions of the EHV-1 genome where the high-passaged live vaccine strain RacH displays symmetric 850 bp deletions. The positive strand and one negative strand primer were designed to encompass the deletions present in RacH, and the second negative strand primer was designed to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of amplification products of different size: In all EHV-1 reference strains and field isolates, a 495 bp DNA fragment was amplified specifically, whereas a 310 bp fragment was amplified when DNA of the vaccine strain RacH was used as a template. PCR amplification was only obtained in the presence of 8-10% dimethylsulfoxide and when the primer annealing temperatures were decreased stepwise from 72 degrees C to 60 degrees C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay allows fast and sensitive discrimination of the modified live vaccine strain RacH from field strains of EHV-1 since it is applicable to viral DNA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH live vaccine strain in abortions of vaccinated mares.