RESUMO
BACKGROUND: Research in humans has demonstrated that high serum iron (sFe) concentration can predispose to infection, and many infections subsequently result in alterations of host sFe. A decrease in sFe concentration is an early and sensitive indicator of systemic inflammation caused by tissue necrosis, bacterial infections, or endotoxemia in horses. Serum iron parameters in acute equine herpesvirus type 1 (EHV-1) infection have not been evaluated previously. OBJECTIVES: To document the sFe response to EHV-1 infection and to determine whether or not significant differences in sFe concentration exist between EHV-1 infected horses that develop neurologic disease and those that do not. ANIMALS: A total of 14 horses experimentally infected with EHV-1. METHODS: Data were collected as an ancillary data set during a blinded experimental EHV-1 infection. Horses were infected with the rAb4 strain of EHV-1. Temperature, neurologic score, packed cell volume (PCV), and sFe parameters (sFe concentration, % saturation, and total iron-binding capacity) were recorded daily for 2 weeks. Data were evaluated using Wilcoxon signed rank tests and Wilcoxon rank sum tests with Bonferroni corrections. CONCLUSIONS AND CLINICAL RELEVANCE: Serum iron concentration decreases significantly in a biphasic pattern after EHV-1 infection. There was no significant difference in sFe concentration in horses that developed neurologic disease and those that did not in these experimentally infected animals. Serum iron parameters may be useful in monitoring the clinical course of viral infections such as EHV-1.
Assuntos
Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/virologia , Ferro/sangue , Doenças do Sistema Nervoso/veterinária , Animais , Temperatura Corporal , Hematócrito/veterinária , Infecções por Herpesviridae/virologia , Doenças dos Cavalos/sangue , Cavalos , Doenças do Sistema Nervoso/sangue , Doenças do Sistema Nervoso/virologia , Estatísticas não ParamétricasRESUMO
Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/patogenicidade , Herpesvirus Equídeo 4/patogenicidade , Doenças dos Cavalos/virologia , Cavalos/virologia , Animais , Células Epiteliais/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/diagnóstico , Leucócitos Mononucleares/virologia , Mucosa Nasal/virologia , Técnicas de Cultura de Tecidos , Ensaio de Placa Viral/veterinária , Tropismo Viral , Viremia/veterinária , Viremia/virologia , Replicação ViralRESUMO
The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20DeltagE), gI (20DeltagI), or both gE and gI (20DeltagEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088-11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20DeltagE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20DeltagI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20DeltagEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae.
Assuntos
Mardivirus/crescimento & desenvolvimento , Proteínas do Envelope Viral/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Primers do DNA , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Genes Virais , Mardivirus/genética , Mutagênese , Proteínas do Envelope Viral/genéticaRESUMO
The immunogenicity of equine herpesvirus type 1 (EHV-1) strain RacH was compared to a RacH virus in which gene 52 encoding glycoprotein M (gM) was interrupted by insertion of LacZ (HDeltagM-Ins) and a RacH with 75% of gene 52 was deleted and replaced by LacZ (HDeltagM-HS). HDeltagM-Ins failed to produce full-length gM, but the carboxy-terminal portion was still expressed. No gM expression was detected in HDeltagM-HS-infected cells. Mice were immunised once with 1x10(3) to 1x10(5) plaque-forming units (PFU) of RacH or mutant viruses and challenged with virulent RacL11 virus 29 days later. A dose-dependence of protection was observed in RacH-immunised mice, and following immunisation with 1x10(4) or 1x10(3) PFU body weight losses and increased virus titres in lungs were observed after challenge infection. HDeltagM-HS-immunised mice were completely protected even after immunisation with 1x10(3) PFU. Mice immunised with 1x10(3) PFU of HDeltagM-Ins but not the higher doses showed signs of disease after challenge infection.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/prevenção & controle , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Animais , Relação Dose-Resposta Imunológica , Deleção de Genes , Expressão Gênica , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/virologia , Cavalos , Lipídeos de Membrana/genética , Lipídeos de Membrana/imunologia , Camundongos , Mutação , Ensaio de Placa ViralRESUMO
Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.
Assuntos
Alphaherpesvirinae/crescimento & desenvolvimento , Alphaherpesvirinae/ultraestrutura , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Deleção de Genes , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/ultraestrutura , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 1/metabolismo , Herpesvirus Equídeo 1/ultraestrutura , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Herpesvirus Suídeo 1/metabolismo , Herpesvirus Suídeo 1/ultraestrutura , Microscopia Eletrônica , Nucleocapsídeo/metabolismo , Nucleocapsídeo/ultraestrutura , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Montagem de Vírus , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestrutura , Rede trans-Golgi/virologiaRESUMO
Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.
Assuntos
Herpesvirus Equídeo 1/fisiologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Primers do DNA , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/ultraestrutura , Cavalos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Deleção de Sequência , Proteínas do Envelope Viral/química , Ensaio de Placa ViralRESUMO
The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.
Assuntos
Antígenos Virais/fisiologia , Cromossomos Artificiais Bacterianos , DNA Viral/genética , Herpesvirus Galináceo 2/genética , Proteínas do Envelope Viral/fisiologia , Animais , Antígenos Virais/análise , Embrião de Galinha , Clonagem Molecular , Mutação , Proteínas do Envelope Viral/análiseRESUMO
Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatitis-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 gene from a German ChHV isolate was used to generate a digoxigenin-labeled 307-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was detected in paraffin-embedded tissues of five naturally infected tortoises (two Afghan tortoises [Testudo horsfieldii], USA; two Hermann's tortoises [Testudo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many characteristics of alphaherpesvirus but also some characteristics of betaherpesvirus infections. The amino acid sequence of a portion of the ChHV UL5 homolog exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nuclear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia cells and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Importantly, ChHV DNA was detected by ISH in multiple tissues of tortoises originating from different geographic provenances. This indicates a high degree of conservation of the UL5 gene fragment among viruses prevalent in tortoises on different continents. With the described ISH, a molecular biological tool is available for rapid and specific diagnosis of ChHV infections and, more importantly, comparative pathogenetic studies of ChHV isolates from geographically unrelated regions.
Assuntos
DNA Viral/isolamento & purificação , Infecções por Herpesviridae/veterinária , Herpesviridae/isolamento & purificação , Hibridização In Situ/veterinária , Rinite/veterinária , Estomatite/veterinária , Tartarugas/virologia , Animais , Clonagem Molecular , Europa (Continente) , Herpesviridae/genética , Infecções por Herpesviridae/virologia , América do Norte , Reação em Cadeia da Polimerase/veterinária , Rinite/virologia , Estomatite/virologiaRESUMO
The nucleotide sequence of the U(L)10 (glycoprotein M) and the U(L)11 homologs of Marek's Disease Virus 1 strain GA was determined. The U(L)10 open reading frame encodes a type III membrane protein of 424 amino acids that contains eight hydrophobic domains and two consensus N-linked glycosylation sites. The U(L)11 homologous gene encodes an 84 amino acid polypeptide, and contains a highly conserved myristylation site at its aminoterminus. By analysis of infected-cell RNA with strand-specific RNA probes, transcription of both U(L)10 and U(L)11 in infected cells was demonstrated. Coupled in vitro transcription-translation confirmed that the U(L)10 product is a 47 kD N-glycosylated viral protein that aggregated upon boiling, whereas the U(L)11 protein exhibited a size of 12 kD after in vitro translation.
Assuntos
Glicoproteínas/genética , Herpesvirus Galináceo 2/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Patos , Glicoproteínas/química , Glicoproteínas/metabolismo , Herpesvirus Galináceo 2/química , Herpesvirus Galináceo 2/fisiologia , Dados de Sequência Molecular , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismoRESUMO
The equine herpesvirus 1 (EHV-1) modified live vaccine strain RacH is apathogenic for both laboratory animals and the natural host. The apathogenicity of RacH was caused by serial passages of the virus in heterologous cells. When compared to the virulent parental strain RacL11 several changes in the RacH genome occurred. Previous results have shown that the loss of the IR6 gene correlated with the loss of virulence. Additional important mutations were observed within the US2 gene which is directly adjacent to the IR6 gene and within the glycoprotein B (gB) gene. To answer the question whether these mutations contribute to the attenuation of RacH several recombinant EHV-1 were constructed: The mutated genes in RacH were replaced by the wild-type US2 gene or the wild-type gB gene, respectively. In addition, a RacL11 recombinant expressing the mutated (RacH) gB instead of the wild-type gene was generated. All recombinant viruses were tested for virulence using the EHV-1 mouse model. The results were as follows: i) The insertion of the RacL11 US2 gene into the RacH virus did not restore virulence and none of the infected mice showed typical signs of EHV-1-caused disease (symptoms and body weight loss). ii) Exchanging gB genes between RacL11 and RacH did not alter their virulence phenotypes remarkably either. Therefore, it is concluded that attenuation of the EHV-1 vaccine strain RacH is caused solely by the absence of the IR6 gene and protein.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/imunologia , Mutação , Vacinas Atenuadas , Proteínas do Envelope Viral/genética , Vacinas Virais , Animais , Genoma Viral , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/prevenção & controle , Cavalos , Camundongos , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.
Assuntos
Herpesvirus Equídeo 1/genética , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Vírus Defeituosos/genética , Vírus Defeituosos/crescimento & desenvolvimento , Células Eucarióticas/metabolismo , Células Eucarióticas/virologia , Engenharia Genética , Glicosaminoglicanos/biossíntese , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 1/metabolismo , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Ligação Proteica , Proteínas do Envelope Viral/metabolismo , Replicação Viral/genéticaRESUMO
Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.
Assuntos
Genes Virais , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/genética , Animais , Membrana Celular/virologia , Infecções por Herpesviridae/metabolismo , Herpesvirus Equídeo 1/metabolismo , Camundongos , Proteínas Virais/metabolismo , Vírion/metabolismoRESUMO
The ability of recombinant preparations of equine herpesvirus type 1 (EHV-1) glycoprotein D (gD) to elicit specific antibody and T lymphocyte responses in the BALB/c mouse model of respiratory infection was investigated. Recombinant gD (rgD) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli elicited both high titer neutralizing antibody (nAb) and CD4 T cell proliferative responses following subcutaneous or intranasal immunization, but elicited only a weak antibody response after intraperitoneal immunization. Protection against respiratory tract infection with pathogenic EHV-1 RacL11 was observed in mice immunized subcutaneously with GST-gD. Furthermore, the degree of protection correlated to the titer of nAb and the T cell response observed. Finally, GST-gD was more effective in protecting against respiratory RacL11 infection if delivered intranasally. These results confirm that gD plays an important role in eliciting the protective immune response against EHV-1 infection, and indicate that subunit vaccines containing preparations of gD may be very effective if delivered directly to the upper respiratory tract.
Assuntos
Infecções por Herpesviridae/imunologia , Herpesvirus Equídeo 1/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Clonagem Molecular , Feminino , Herpesvirus Equídeo 1/isolamento & purificação , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Infecções Respiratórias/imunologia , Vacinação , Proteínas Virais de Fusão/genéticaRESUMO
Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.
Assuntos
Produtos do Gene env/biossíntese , Produtos do Gene gag/biossíntese , Proteína gp120 do Envelope de HIV/biossíntese , HIV-1/metabolismo , Herpesvirus Humano 4/metabolismo , Glicoproteínas de Membrana/biossíntese , Precursores de Proteínas/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Primers do DNA , Citometria de Fluxo , Produtos do Gene env/isolamento & purificação , Produtos do Gene gag/isolamento & purificação , Genes env , Proteína gp120 do Envelope de HIV/isolamento & purificação , HIV-1/genética , Herpesvirus Humano 4/genética , Humanos , Substâncias Macromoleculares , Glicoproteínas de Membrana/isolamento & purificação , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Precursores de Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera , TransfecçãoRESUMO
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
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.